CHALLENGES FOR PLANT NUTRITION

IN CHANGING ENVIRONMENTS

International Workshop and Meeting of the German Society of Plant Nutrition 2012

University of Bonn September 5 – 8, 2012

Book of Abstracts

Table of Contents

Plenary Session: Introductory talks ...... 2

Plenary Session S1: Processes on leaf surfaces ...... 5

Plenary Session S2: Plant water relations ...... 14

Plenary Session S3: Nutrient dynamics in changing environments .... 26

Plenary Session S4: Crop responses to nutrient imbalances ...... 38

Plenary Session S5: Phenotyping and early stress responses ...... 80

Poster Session P1: Fertilization (inorganic) ...... 92

Poster Session P2: Fertilization (organic) / Soil amendments ...... 110

Poster Session P3: Nutrient efficiency / Genomics ...... 131

Poster Session P4: Root physiology / Root-soil interactions...... 148

Poster Session P5: Physiological response to abiotic stress ...... 169

Poster Session P6: Physiological response to nutrient imbalances 182

Poster Session P7: Nutrients and ecosystems / Climate change ... 200

Poster Session P8: Signalling / Quality / Phenotyping ...... 215

1 DGP Meeting September 5-9, 2012

Plenary Session: Introductory talks

2 DGP Meeting September 5-9, 2012 Plant nutrition in a changing environment.

Patrick H. Brown

Department of Plant Sciences, University of California, Davis, CA 95616, US; E-mail: [email protected]

The scientific discipline of plant nutrition is wonderfully broad in its scale and its scope. From the exploration of the function of nutrients as signals and regulators of plant function, to the role of plant nutrients in agricultural productivity and food quality, to the exploration of the effects of nutrient losses on global environments, plant nutrition is a truly integrative discipline and will play a critical role in mans’ ability to adapt to environmental change. Plant nutrition is by design a multi-scale endeavor embracing science from the most fundamental principle to the grandest policy decisions. Thus the term ‘changing environments’ is equally broad in its implications and would include: 1) the explicit changes in the climate that affect plant nutrient relations (CO 2 enrichment, temperature shifts, altered biotic and abiotic stresses), 2) the growing public demand for better environmental stewardship, 3) the need for higher food quality, 4) a recognition of the challenges of diminishing nutrient resources and growing competition for those limited resources, 5) the changing nature of research funding and the loss of support for adaptive and translational research and 6) the changing manner in which agricultural producers and policy makers get their information and make their decisions. As plant nutritionists we clearly find ourselves in a changing environment. Understanding how the various aspects of this changing environment impact upon the field of plant nutrition will inform how we focus our efforts, and how we adapt and extend our research to our colleagues, to agricultural producers, to governments and policy makers and ultimately will determine how ‘useful’ our contributions will be. These issues will be discussed with examples drawn from the literature and from experiences in California and Australia.

3 DGP Meeting September 5-9, 2012 The mineral nutrition of plants and people

Philip J. White

The James Hutton Institute, Dundee, UK; E-mail: [email protected]

Food security is defined as having sufficient, safe and nutritious food to meet the dietary needs of an active and healthy life. This presentation describes the role of plant mineral nutrition in crop production and dietary delivery of mineral elements essential to human wellbeing. Plants require at least 14 mineral elements. Fertilisers containing these elements are often applied to crops to maximise yields. However, the application of fertilisers has both economic and environmental costs. Research on agronomic strategies to improve the efficiency of fertiliser use by crops and genetic approaches to develop crops with improved acquisition and physiological use of mineral elements will be presented. Ultimately, plants provide humans with their minerals. Unfortunately, the diets of over 60% of the world’s population lack essential mineral elements. The concentrations of mineral elements in edible crops can be increased through either agronomic or genetic approaches. Soil or foliar fertilisers can be used to biofortify edible crops with, for example, iodine, selenium, iron and zinc. In addition, if the mineral element is present in the soil, crops can be developed for greater acquisition and accumulation in edible portions. Examples of the variation in mineral composition of edible crops will be given and the potential of crop genetics to deliver varieties to improve the mineral nutrition of humans will be discussed.

4 DGP Meeting September 5-9, 2012

Plenary Session S1: Processes on leaf surfaces

5 DGP Meeting September 5-9, 2012 Functions of and processes on leaf surfaces

Markus Riederer

Julius-von-Sachs-Institut für Biowissenschaften, University of Würzburg, Julius-von-Sachs-Platz 3, Würzburg, Germany; E-mail: [email protected]

6 DGP Meeting September 5-9, 2012 Foliar fertilization of crops

Victoria Fernández

Forest Genetics and Ecophysiology Research Group, School of Forest Engineering, Technical University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain; Email: [email protected]

Foliar fertilisation is an increasingly-used agricultural practice which may help to increase crop yields and quality while contributing to a rational use of fertilizers when applied in combination with soil treatments. However, many factors influencing the effectiveness of foliar sprays and the process of foliar uptake and translocation remain unclear, which hinders the development of reliable foliar fertilization strategies and may lead to variable plant responses to the treatments. In the course of this presentation the current state of knowledge on foliar nutrient sprays will be revised within a physiological, physico-chemical and agronomic context, focusing on the constrains and opportunities of applying nutrients to crop plants via the foliage.

7 DGP Meeting September 5-9, 2012 The role of stomata in foliar fertilization

Thomas Eichert

University of Bonn, Institute of Crop Science and Resource Conservation (INRES), Dept. of Plant Nutrition, Karlrobert-Kreiten-Str. 13, D 53115 Bonn, Germany; E-mail: [email protected]

Foliar fertilization is a common practice to quickly correct nutrient deficiencies, especially under conditions of limited soil nutrient availability. For a long time it was assumed that foliar nutrient uptake is restricted to the cuticle, while stomata were supposed to be impermeable to foliar-applied solutes. In our studies we found evidence that this paradigm needs to be revised (Fernández and Eichert 2009, Eichert and Fernández 2012). Our results indicate that solute uptake through stomata is in fact possible (Eichert and Burkhardt 2001) and that the stomatal penetration pathway can be as important as the cuticular pathway (Eichert and Goldbach 2008). Experiments using N compounds or nm-particles showed that the stomatal pathway enables penetration of substances as large as 40 nm (Eichert and Goldbach 2008, Eichert et al. 2008) whereas our estimations of pore sizes in cuticles of stomata-free, intact leaves yielded diameters of 4-5 nm (Eichert and Goldbach 2008). These results are in accordance with field observations that large molecules (e.g., Fe-chelates) can penetrate stomata-free upper leaf surfaces and that uptake rates of mineral nutrients are frequently larger through the stomata-bearing lower leaf surface than through the upper surface.

References Eichert T, Burkhardt J (2001) Quantification of stomatal uptake of ionic solutes using a new model system. Journal of Experimental Botany 52: 771-781. Eichert T, Fernández V (2012) Uptake and Release of Mineral Elements by Leaves and Other Aerial Plant Parts. In: Marschner’s Mineral Nutrition of Higher Plants, 3rd edition, Academic Press, pp. 71-84 Eichert T, Goldbach HE (2008) Equivalent pore radii of hydrophilic foliar uptake routes in stomatous and astomatous leaf surfaces – further evidence for a stomatal pathway. Physiologia Plantarum 132: 491–502. Eichert T, Kurtz A, Steiner U, Goldbach HE (2008) Size exclusion limits and lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes and water-suspended nanoparticles. Physiologia Plantarum 134: 151–160. Fernández V, Eichert, T (2009) Uptake of hydrophilic solutes through plant leaves: current state of knowledge and perspectives of foliar fertilization. Critical Reviews in Plant Sciences 28: 36-68

8 DGP Meeting September 5-9, 2012 Biofortification of field vegetables with iodine: efficiency of soil and foliar fertilization techniques

Patrick Lawson, Roman Czauderna and Diemo Daum

University of Applied Sciences Osnabrück, Faculty of Agricultural Science and Landscape Architecture E-mail: [email protected]

Introduction Iodine is an essential trace element for the synthesis of thyroid hormones in the human body. An inadequate dietary iodine intake has certain negative effects on human health. According to the World Health Organization almost 2 billion individuals worldwide are currently affected by iodine deficiency (De Benoist et al. 2009). Although the use of iodized table salt has been common for several decades, even in Germany about 40 % of the population still have an insufficient iodine intake (Thamm et al. 2007). Thus, further approaches are required to improve the nutritional supply of this trace element. Recent studies indicate that biofortification of vegetables with iodine could be a suitable strategy for this purpose (Dai et al. 2006, Blasco et al. 2008 and Voogt et al. 2010). Methods proposed so far are based predominantly on the application of iodine salts in the root zone of crops. The objective of the present study was to compare the efficiency of soil and foliar fertilization techniques for the enrichment of iodine in field vegetables.

Materials and Methods Field trials were conducted over two years and included the following vegetable crops: butterhead lettuce, iceberg lettuce, spinach, rocket, white cabbage, broccoli, kohlrabi, radish, onions and carrots. The experiments in 2010 were carried out on a sandy loam soil at the horticultural research station of the University of Applied Sciences Osnabrück. The experiments in 2011 were set up on different soils of four vegetable farms located within a radius of 80 km from Osnabrück. Potassium iodate

(KIO 3) and potassium iodide (KI) were used as iodine fertilizers and applied dissolved in water at different concentrations. Soil fertilization occurred just before planting as drenches at a rate of 1 L m -2. Foliar applications were done once or twice with a hand-held fine spray system (Easy Sprayer Plus, Lehnartz GmbH) and took place 1 - 2 weeks before harvest. The sprayed solution contained the organosilicone adjuvant BREAK-THRU® S 240 (0.05 % v/v) to improve spreading and sticking properties. The trials were performed in a split-plot design or completely randomized block design with 3 or 4 replications per treatment and a gross plot area of 4.5 - 12 m 2, depending on the experimental question and the investigated plant species. After the harvest, the edible parts of the vegetables were thoroughly washed under flowing tap water, air-dried afterwards at 60 °C and then micro -milled. The plant powder was

9 DGP Meeting September 5-9, 2012 digested at 550 °C by an alkaline ashing procedure with KOH adapted from a method described by Ku čera und Krausova (2007). The iodine content was determined with a flow injection analysis system using a catalytic spectrophotometry method (Switala 2001). Soil samples were taken at three depths (0-30, 30-60, 60-90 cm), extracted with a 0.0125 M CaCl 2 solution and subsequently analyzed for iodine by ICP-MS.

Results and Discussion A moderate iodine supply did not affect growth, yield or external quality of the vegetable species investigated in this study. However, for example in butterhead - - lettuce, soil applications of I and IO 3 significantly reduced the biomass production at a fertilizer rate of ≥ 7.5 kg I ha -1 and 15 kg I ha -1, respectively (Fig. 1). Using foliar sprays, yield reductions of more than 10 % were only observed when I - was fertilized at rates higher than 1 kg I ha -1. A stimulation of plant growth by low iodine application rates, as occasionally reported in the literature (Weng et al. 2008, Hong et al. 2008), was not observed.

Soil application Foliar spray 120 120 100 100

80 80 - 60 60 IO 3 40 40 I- Relative Relative yield [%] Relative yield [%] 20 20 0 0 0 1.0 2.5 7.5 15 0 0.5 0.5 1.0 1.0 (1x) (2x) (1x) (2x) Iodine fertilization [kg I ha -1] Iodine fertilization [kg I ha -1]

Fig. 1: Influence of the iodine supply on the relative yield of butterhead lettuce (mean yield of plants from the control plots = 100 %, bars indicate 95 % CI, field trial in 2010)

The iodine content in the edible parts of plants increased with increasing iodine supply, as shown in Figure 2 for butterhead lettuce. In the case of soil applications, - - IO 3 led to a distinctly higher accumulation of iodine in the plant matter than I . The targeted iodine enrichment (50 – 100 µg I 100 g -1 FM) was obtained with a fertilizer -1 -1 - - rate of 7.5 kg I ha and 15 kg I ha using IO 3 and I , respectively. Similar differences between both iodine species were also observed in other field vegetables. However, some crops accumulated significantly less iodine in the harvested organs. The lowest iodine content was found in onions and carrots. Kohlrabi, radish and iceberg lettuce - reached or closely approached the targeted iodine level when IO 3 was fertilized at a rate of 7.5 kg I ha -1. In contrast to our findings, studies on vegetables grown in 10 DGP Meeting September 5-9, 2012 - - hydroponic systems have shown that roots absorb I at a higher rate than IO 3 (Zhu et al. 2002, Blasco et al. 2008 and Voogt et al. 2010). It is assumed that the heavier - molecular weight and higher valency of IO 3 reduce its uptake by plants (Mackowiak - - and Grossl 1999). However, in soils IO 3 may be better phytoavailable, because I is easily released from terrestrial environments through the production of volatile organoiodides (Dai et al. 2006). In the second trial year, the formerly treated sandy loam soil was again cultivated with butterhead lettuce and radish without repeating the iodine fertilization. The iodine content in these crops was of the same order as in plants from the unfertilized plots, - except where IO 3 was applied at the highest rate in the previous year. In accordance with this result, it was found that the CaCl 2-extracted iodine content in the soil (depth 0 – 90 cm) was almost at the ambient level in all treatments at the beginning of the - new growing season. Thus, even applied IO 3 was largely lost from the soil within one year, possibly by leaching below the root zone.

Soil application Foliar spray 600 600 FM] FM] IO - IO - -1 -1 500 3 500 3 - - I I 400 400

300 300

200 Target area 200 Target area 100 100

Iodine content g [µg100 I Iodine content g [µg100 I 0 0 0 1.0 2.5 7.5 15 0 0.5 0.5 1.0 1.0 (1x) (2x) (1x) (2x) Iodine fertilization [kg I ha -1] Iodine fertilization [kg I ha -1] Fig. 2: Influence of the iodine supply on the iodine content of butterhead lettuce (bars indicate 95 % CI, field trial conducted in 2010)

In contrast to soil applications, foliar sprays with I - usually resulted in a higher iodine - accumu-lation in the edible parts of plants than IO3 treatments. In butterhead lettuce, the targeted iodine content was obtained at a fertilizer rate of 0.5 kg I ha -1 and 1.0 kg -1 - - I ha applied as I and IO 3 , respectively (Fig.2). Thus, in comparison with the soil treatment, the foliar fertili-zation technique was much more efficient for the iodine biofortificaton of butterhead lettuce. Spinach and rocket have shown a similar response to foliar sprays with iodine salts. Thorough washing did not affect the iodine - - content of the vegetables, indicating that I and IO 3 were actually absorbed and had not only adhered to the surface of the leaves. In contrast to the leafy vegetables mentioned before, iceberg lettuce and white cabbage were proved to be unsuitable for foliar sprays, since the applied iodine remained in the outer leaves, which are

11 DGP Meeting September 5-9, 2012 usually removed at harvest. Furthermore, broccoli and kohlrabi have shown a very low accumulation potential for iodine. Obviously, iodine was hardly translo-cated from the absorbing leaves to other plant organs (inner leaves, tubers or inflores-cences). This confirms previous studies, indicating that iodine is mainly transported in the xylem and only slightly in the phloem (Blasco et al. 2008 and Voogt et al. 2010). In conclusion the results of our investigation demonstrate that foliar iodine sprays preferably with I - are a suitable method for increasing the iodine content of certain leafy vegetables 10 to 50-fold of the initial levels without inflicting phytotoxic effects or reducing crop yields. However, a sufficient spreading of the applied solution on the harvested plant parts is crucial for the efficiency of this biofortification approach, since retranslocation of iodine in plants appeared to be very limited.

Acknowledgment We thank the cooperating growers J. Biewener, W. Mählmann, S. Stegemeier and A. Wehmeyer for providing their fields to conduct our trials. This study is supported by the Bundesministerium für Bildung und Forschung in the scope of the program IngenieurNachwuchs ( Grant No. 17N0210).

Literature Blasco, B., Rios, J. J., Cervilla, L. M., Sánchez-Rodrigez, E., Ruiz, J. M. and Romero, L. (2008): Iodine biofortification and antioxidant capacity of lettuce: potential benefits for cultivation and human health. Ann. App. Biol.152, 289–299. Dai, J.-L., Zhu, Y.-G., Huang, Y.-Z., Zhang, M. and Song, J. L. (2006): Availability of iodide and iodate to spinach ( Spinacia oleracea L.) in relation to total iodine in soil solution. Plant Soil 289, 301– 308. De Benoist, B., McLean, E. and Andersson, M. (2009): Iodine deficiency: The extent of the problem. In: Preedy, V. R., Burrow, G. N. and Watson R.R. (eds.): Comprehensive Handbook of Iodine – Nutritional, Biochemical, Pathological and Therapeutic Aspects. Academic Press, Amsterdam, pp 461–467. Hong, C.-L., Weng, H.-X., Qin, Y.-C., Yan, A.-L. and Xie, L.-L. (2008): Transfer of iodine from soil to vegetables by applying exogenous iodine. Argon. Sustain. Dev. 28, 575–583. Ku čera, J. and Krausová, I. (2007): Fast decomposition of biological and other materials for radiochemical activation analysis: A radiochemical study of element recoveries following alkaline-oxidative fusion. J. Radioanl. Nucl. Chem. 271, 577–580. Mackowiak, C. L. and Grossl, P.R. (1999): Iodate and iodide effects on iodine uptake and partitioning in rice ( Oryza sativa L.) grown in solution culture. Plant Soil 212, 135–143. Switala, K. (2001): Determination of Iodide in 0,2 M Potassium Hydroxide by Flow Injection Analysis. QuikChem® Method 10-136-09-1-A. Lachat Instruments, 6645 West Mill Road, Miwaukee, WI 53218-1239 USA, 11 pp. Thamm, M., Ellert, U., Thierfelder, W., Liesenkötter K.-P. and Völzke, H. (2007): Jodversorgung in Deutschland – Ergebnisse des Jodmonitorings im Kinder- und Jugendgesundheitssurvey (KiGGS). Bundesgesundheitsbl. Gesundheitsforsch. Gesundheitsschutz 50, 744–749. Weng, H.-X., Yan, A.-L., Hong, C.-L., Xie, L.-L., Qin Y.C. and Cheng, C. Q. (2008): Uptake of different spezies of iodine by water spinach and its effect to growth. Biol. Trace Elem. Res. 124, 184– 194. Voogt, W., Holwerda, H. T. and Khodabaks, R. (2010): Biofortification of lettuce with iodine - the effect of iodine form and concentration in the nutrient solution on growth, development and iodine uptake of lettuce grown in water culture. J. Sci. Food Agric. 90, 906–913. Zhu, Y.-G., Huang, Y.-Z., Hu, Y. and Liu, Y.-X. (2003): Iodine uptake by spinach ( Spinacia oleracea L.) plants grown in solution culture: effects of iodine species and solution concentrations. Environment International 29, 33–37.

12 DGP Meeting September 5-9, 2012 Chaotropic anions of the Hofmeister series promote stomatal uptake of aqueous solutions

Burkhardt J., Basi S., Pariyar S., Hunsche M.

University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

With the recent visualisation of stomatal nanoparticle uptake, a 40-year-old paradigm ended. Based on water surface tension and on the assumption of clean, hydrophobic leaf surfaces, the paradigm disallowed stomatal liquid water transport. Real leaves are not clean. The impact of hygroscopic leaf surface particles on hydrophobicity and water surface tension was investigated. Droplets containing NaCl, NaClO 3,

(NH 4)2SO 4, glyphosate, a surfactant, or various combinations thereof were evaporated on stomatous abaxial and astomatous adaxial surfaces of apple leaves. Photosynthesis, necrosis, and biomass were measured. Observed using an environmental scanning electron microscope, NaCl and NaClO 3 crystals on hydrophobic tomato cuticles underwent repeated deliquescence/efflorescence cycles. Photosynthesis depression, necrosis, and biomass reduction were stronger for abaxial than for adaxial treatments. Cuticular hydrophobicity decreased and salts spread out and crystallised into a dendritic form: NaClO 3 during the second cycle and NaCl during the fifth cycle. Stomatal uptake was confirmed. Stomatal activation for diffusional transport occurs due to deliquescence/efflorescence cycles and is ion- specific, pointing to the Hofmeister series. Chaotropic chlorate ions reduce surface tension and form continuous extensions into the stomata more effectively than kosmotropic chloride ions, likely explaining the defoliant action of NaClO 3 and the protection of coastal plants from sea-spray.

13 DGP Meeting September 5-9, 2012 Plenary Session S2: Plant water relations

14 DGP Meeting September 5-9, 2012 The hydraulic life of dead wood: biophysics of water transport in xylem

Maciej A. Zwieniecki

Arnold Arboretum of Harvard University, Boston, MA, USA and Department of Plant Sciences, UC Davis, Davis, CA, USA; E-mail: [email protected]

Introduction The lack of mobility in plants is often interpreted as a sign of their passivity in the face of environmental variation. This view is perhaps most firmly entrenched with regard to water transport through the xylem in which water flows through the lumen of cells that are “dead” ( i.e., lack any cytoplasm or nucleus) at maturity. However, recent work demonstrates that a number of active, physiological processes may be involved in maintaining the transport capacity of this essential pathway. Here I discuss work relating to both embolism repair and the effect of ion concentrations on xylem hydraulic properties as examples of such dynamic processes.

Embolism repair The co-occurrence of tension reconciliation and embolism refilling in the xylem of transpiring plants remains an active research area. Significant progress has been made in the analysis of the physical conditions associated with the refilling process (1-6), in its in vivo imaging (7, 8), and in the determination of the principal role that xylem parenchyma cells play in supplying both energy and water for refilling (9-18). However, despite these efforts we are still missing a comprehensive understanding of the cellular biology, including signaling paths and metabolic processes, involved in the plant response to an embolism that result in stem water transport restoration (1, 12). Recent analysis of the transcriptome response to embolism formation in stems of Populus trichocarpa has revealed the complexity of genetic activity that is associated with the process of refilling (19) . The transcriptome analysis confirmed that aquaporins play an important role in the facilitation of water transport during the recovery from an embolism as well as that carbohydrate metabolism is necessary to override the preexisting energy gradients. This study also suggested that initializing refilling activity could be linked to a reduction in oxidative stress that is followed by a massive response of a wide range of transporter encoding genes (19). This new transcriptome data combined with physiological observations to provide a strong basis to formulate a set of new hypotheses describing metabolic and membrane level activity of parenchyma cells during refilling that allow for the generation of an osmotic gradient and the facilitation of water transport.

15 DGP Meeting September 5-9, 2012 Ionic Effects on Xylem Hydraulic Resistance The traditional view of the xylem as a series of inert tubes considers the xylem to have only two states: a vessel can be water-filled and thus able to transport water with a fixed resistance or it can be embolized and thus unable to transport water under tension. However, the hydraulic resistance of the xylem is not fixed, but instead varies in response to the ionic concentration of the transported fluid (20-22). The mechanism underlying these changes in resistance appear to result from the swelling/deswelling of hydrogels located in bordered pit membranes (23, 24). With increasing concentrations of ions, these hydrogels are hypothesized to shrink, increasing the porosity of the pit membrane and thus decreasing its resistance to water flow. These changes are both reversible and repeatable, suggesting that plants could actively modulate their xylem resistance by altering the ionic concentration of the fluid in the xylem.

The functional significance of ion-mediated changes in xylem resistance still remains to be explored. However, the effect of ions on xylem hydraulic properties occur in most species so far examined, suggesting that it may play an important role (23). In particular, the ability to enhance flow through the xylem by increasing the ionic concentration of the xylem sap may allow plants to compensate for losses in conductive capacity due to cavitation. It could also allow plants to effect short-term, dynamic changes in the hydraulic architecture of the xylem and thus to prioritize water delivery to those leaves currently experiencing the most favorable conditions for photosynthesis (25-27). Such a mode of autonomous regulation could be appropriate for fine-tuning water transport through a highly branched system. Such active redistribution would be analogous to changes in blood flow patterns in vertebrates resulting from constriction of capillaries. Further studies focusing on in vivo changes in xylem sap ion concentration and xylem properties are needed to understand the significance of this newly recognized mechanism. What is clear, however, is that plants have the ability to rapidly modify their hydraulic properties, opening the door to understanding how they may utilize this to enhance their capacity to optimize water delivery to leaves. Literature 1. Zwieniecki MA & Holbrook NM (2009) Confronting Maxwell's demon: biophysics of xylem embolism repair. (Translated from English) Trends Plant Sci 14(10):530-534 (in English). 2. Holbrook NM & Zwieniecki MA (1999) Embolism repair and xylem tension: Do we need a miracle? (Translated from English) Plant Physiol 120(1):7-10 (in English). 3. Berndt ML, McCully ME, & Canny MJ (1999) Is xylem embolism and refilling involved in the rapid wilting and recovery of plants following root cooling and rewarming? A cryo-microscope investigation. (Translated from English) Plant Biology 1(5):506-515 (in English). 4. Buchard C, McCully M, & Canny M (1999) Daily embolism and refilling of root xylem vessels in three dicotyledonous crop plants. (Translated from English) Agronomie 19(2):97-106 (in English).

16 DGP Meeting September 5-9, 2012 5. Hacke UG, Stiller V, Sperry JS, Pittermann J, & McCulloh KA (2001) Cavitation fatigue. Embolism and refilling cycles can weaken the cavitation resistance of xylem. (Translated from English) Plant Physiol 125(2):779-786 (in English). 6. McCully ME, Huang CX, & Ling LEC (1998) Daily embolism and refilling of xylem vessels in the roots of field-grown maize. (Translated from English) New Phytol 138(2):327-342 (in English). 7. Brodersen CR, McElrone AJ, Choat B, Matthews MA, & Shackel KA (2010) The Dynamics of Embolism Repair in Xylem: In Vivo Visualizations Using High-Resolution Computed Tomography. (Translated from English) Plant Physiol 154(3):1088-1095 (in English). 8. Holbrook NM, Ahrens ET, Burns MJ, & Zwieniecki MA (2001) In vivo observation of cavitation and embolism repair using magnetic resonance imaging. (Translated from English) Plant Physiol 126(1):27-31 (in English). 9. Secchi F & Zwieniecki MA (2011) Sensing embolism in xylem vessels: the role of sucrose as a trigger for refilling. (Translated from English) Plant Cell Environ 34(3):514-524 (in English). 10. Secchi F & Zwieniecki MA (2010) Patterns of PIP gene expression in Populus trichocarpa during recovery from xylem embolism suggest a major role for the PIP1 aquaporin subfamily as moderators of refilling process. (Translated from English) Plant Cell Environ 33(8):1285-1297 (in English). 11. Salleo S, Lo Gullo MA, Trifilo P, & Nardini A (2004) New evidence for a role of vessel- associated cells and phloem in the rapid xylem refilling of cavitated stems of Laurus nobilis L. (Translated from English) Plant Cell Environ 27(8):1065-1076 (in English). 12. Nardini A, Lo Gullo MA, & Salleo S (2011) Refilling embolized xylem conduits: Is it a matter of phloem unloading? (Translated from English) Plant Sci 180(4):604-611 (in English). 13. Nardini A, Ramani M, Gortan E, & Salleo S (2008) Vein recovery from embolism occurs under negative pressure in leaves of sunflower (Helianthus annuus). (Translated from English) Physiol Plantarum 133(4):755-764 (in English). 14. Salleo S, LoGullo MA, DePaoli D, & Zippo M (1996) Xylem recovery from cavitation-induced embolism in young plants of Laurus nobilis: A possible mechanism. (Translated from English) New Phytol 132(1):47-56 (in English). 15. Salleo S, Raimondo F, Trifilo P, & Nardini A (2003) Axial-to-radial water permeability of leaf major veins: a possible determinant of the impact of vein embolism on leaf hydraulics? (Translated from English) Plant Cell Environ 26(10):1749-1758 (in English). 16. Salleo S, Trifilo P, & Lo Cullo MA (2008) Vessel wall vibrations: trigger for embolism repair? (Translated from English) Funct Plant Biol 35(4):289-297 (in English). 17. Trifilo P, Gasco A, Raimondo F, Nardini A, & Salleo S (2003) Kinetics of recovery of leaf hydraulic conductance and vein functionality from cavitation-induced embolism in sunflower. (Translated from English) J Exp Bot 54(391):2323-2330 (in English). 18. Trifilo P, Lo Gullo MA, Salleo S, Callea K, & Nardini A (2008) Xylem embolism alleviated by ion- mediated increase in hydraulic conductivity of functional xylem: insights from field measurements. (Translated from English) Tree Physiol 28(10):1505-1512 (in English). 19. Secchi F, Gilbert ME, & Zwieniecki MA (2011) Transcriptome Response to Embolism Formation in Stems of Populus trichocarpa Provides Insight into Signaling and the Biology of Refilling. (Translated from eng) Plant Physiol 157(3):1419-1429 (in eng). 20. Zwieniecki, M. A., P. J. Melcher, and N. M. Holbrook. (2001b). Hydrogel control of xylem hydraulic resistance in plants. Science 291:1059–1062. 21. Zimmermann, M. H. (1983). Xylem structure and the ascent of sap . Springer-Verlag, Berlin. 22. van Ieperen, W., U. van Meeteren, and v. G. H.. (2000). Fluid ionic composition influences hydraulic conductance of xylem conduits. J. Exp. Bot 51:769–776. 23. Zwieniecki, M. A., P. J. Melcher, and N. M. Holbrook. (2001a). Hydraulic properties of individual xylem vessels of Fraxinus americana . J. Exp. Bot 52:1–8. 24. Lee J.N., Holbrook, N.M., Zwieniecki M.A. (2012). Ion induced changes in the structure of bordered pit membranes. Frontiers in Plant Sciences 3:55. doi: 10.3389/fpls.2012.00055 25. Zwieniecki M.A., Orians C., Melcher P.J. and Holbrook N.M. (2003). Ionic control of lateral exchange of water between vascular bundles in tomato. Journal of Experimental Botany 54:1399-1405. 26. Zwieniecki M.A., Melcher P.J., Field T.S., and Holbrook N.M. (2004). A potential role for xylem- phloem interactions in the hydraulic architecture of trees: effects of phloem girdling on xylem hydraulic conductance. Tree Physiology . 24:911-917. 27. Boyce C.K., Zwieniecki M.A., Cody G.D., Jacobsen C., Wirick S., Knoll A.H. and Holbrook N.M. (2005). Evolution of xylem lignification and hydrogel transport regulation. Proceedings of National Academy of Science, USA. 101:17555-17558. 17 DGP Meeting September 5-9, 2012 What HR-CT imaging can teach us about xylem structure and function

Ken Shackel 1, Craig Brodersen 2, Eric Lee 3, Andrew McElrone 3, Brendan Choat 4, Ronald Philips 5, Steven Jansen 6 and Mark Matthews 3

1Department of Plant Sciences, Davis, CA, 2University of Florida, Lake Alfred, FL, 3Department of Viticulture & Enology, University of California, Davis, CA, 4Australian National University, Canberra, 5Department of Chemical Engineering and Materials Science, Davis, CA, 6Institute of Systematic Botany and Ecology, Ulm University, Ulm. E-mail: [email protected]

Introduction It is well established that plant xylem is composed of a complex and interconnected system of vascular elements, but little is known about how the three-dimensional (3D) organization of this network influences properties such as plant hydraulics (Tyree & Zimmermann, 2002), and few studies have measured the spatial distribution, orientation, or frequency of connections between the vascular elements. High- resolution X-ray computed tomography (HRCT) is a diagnostic imaging technique with a micrometer-range resolution that provides continuous serial sections through plant tissue in any orientation. HRCT imaging is based on the same principles as medical CT systems, but a high intensity, focused X-ray source results in decreased section thickness and image acquisition time. Both two-dimensional (2D) and 3D HRCT have been employed in the cursory analysis of plant-related materials, including the soil and root interface (Aylmore, 1993; Heeraman et al., 1997), fruit development (Verboven et al., 2008), paleobotanical anatomy (DeVore et al., 2006), and the anatomy of wood and vascular tissue (Stuppy et al., 2003; Maeda & Miyake, 2009). Fromm et al. (2001) and Steppe et al. (2004) successfully used HRCT to study wood density; however the image resolution was insufficient for analyzing intervessel connections. Recent advances in synchrotron HRCT technology have improved image resolution and signal to noise ratio sufficiently so that vessel networks can be visualized to explore the implications of network organization. The analytical potential of HRCT in exploring xylem organization is even greater than its 3D visualization capabilities, as intervessel connections can be assigned 3D coordinates that can be exported and used in model simulations.

Materials and Methods Grapevines (V. vinifera L. ‘Chardonnay’) were grown from grafted cuttings in 7.6 l pots in a glasshouse. For dry stem analysis, internode sections from current year stems were excised and dehydrated in a drying oven at 70C for 48 h. A silicone resin (Rhodorsil RTV 141; Bluestar Silicones Corp., East Brunswick, NJ, USA) was injected via a microcapillary tube into a single vessel following the methods described by Choat et al. (2006). This resin-filled vessel acted as a fiduciary marker for 18 DGP Meeting September 5-9, 2012 orientation purposes in future dissection and HRCT imaging experiments. Internode sections (5–7 mm) were scanned at the Lawrence Berkeley National Laboratory Advanced Light Source X-ray microtomography facility (Berkeley, CA, USA). The beamline configuration was similar to the standard setup for this technique developed by Kinney & Nichols (1992), using an X-ray energy of 15 keV. The stem sample was mounted on an air-bearing stage and rotated 180degrees in the X-ray beam in increments of 0.125 degree, yielding 1440 different 2D images per sample. Samples were scanned in absorption mode, and the reconstructed images were obtained following normalization and the application of a filtered back-projection algorithm. Raw 2D tomographic projection images were reconstructed using Octopus 8.3 software (Institute for Nuclear Sciences, University of Ghent, Belgium) to create a 3D, 16-bit series or ‘stack’ of tagged image file (.TIF) format files (Dierick et al., 2004). Each TIF image was composed of 3D pixels (volumetric pixel elements, or ‘voxels’), where intensity was based on X-ray attenuation. Each voxel was assigned an x, y and z coordinate in 3D space. All images were processed with Avizo’s ‘Edge- Preserving Smoothing’, an edge-preserving smoothing filter in Avizo 6.2 software (VSG Inc., Burlington, MA, USA), to increase contrast between plant tissue and vessel lumen. Relative mass attenuation coefficients were then calculated using Octopus software to verify the density values present in the HRCT images to distinguish between plant tissue and air- or water filled vessels.

Results and Discussion A typical cross-sectional area of the grapevine stem could be scanned at a rate of c. 1 cm of stem h –1. The high-density silicone resin was easily distinguished from the air-filled lumen of surrounding vessels in both the SEM and HRCT micrographs. Intervessel scalariform pits could not be resolved in the HRCT images at the resolution used to capture entire stem section data. To empirically establish when intervessel connections were present, a series of experiments were conducted in which a stem section was imaged by HRCT, and then dissected to extract a subsample containing the fiduciary resin marker, which was then observed by SEM. In this way, we directly observed whether intervessel pits were present for specific vessel pairs in known locations in the HRCT images. An analysis of 363 vessel pairs showed that vessels with boundaries separated by < 14 um in HRCT images were classified as interconnected (i.e. sharing intervessel pits). HRCT revealed that the proximity of adjacent vessels was not constant along their length, and intermittent vessel connectivity was frequently observed. An analysis of xylem conduits in grapevine stem confirmed the suggestion by Stevenson et al. (2004) that vessel diameter is greater in Dorsal/Ventral zones (D/V) than in the lateral zones that give rise to lateral shoots, leaves, tendrils, and fruit. It

19 DGP Meeting September 5-9, 2012 was also clear that the distribution of vessel diameters did not follow the typical pattern of large diameters early followed by smaller diameters later, towards the epidermis. The largest vessels appeared in an intermediate position in the D/V zones. Smaller diameter vessels in the lateral zones were more closely spaced and interconnected than large-diameter vessels in the D/V zones. In these young, current-season V. vinifera stems, most intervessel connections were oriented radially (i.e. outward from the stem center) rather than in a tangential plane between vessels. V. vinifera rays are multiseriate (Sun et al., 2006), and this limits the potential for tangential movement of vessels and, hence, water, solutes, and even xylem-dwelling pathogens in young shoots. Vessels were observed passing through rays establishing trans-ray connections, although this was rare. This noninvasive technology was recently used to show the refilling process in individual embolized vessels of grapevine (Brodersen et al., 2010). No evidence of tissue damage was visible in the stems used here, nor has any evidence developed in the plants used previously (Brodersen et al., 2010), although this has not been the case in all species tested (Kim & Lee, 2010). Using this technology we have also developed a spatially explicit hydraulic model of xylem function (Lee et al, submitted), demonstrating that reverse flow in some vessels may be expected as a result of the patterns of vessel interconnections and the positions of restrictions (end-walls) in the pathway. Grapevine is also well known for its long, wide vessels (Zimmermann & Jeje, 1981), and the potential for significant reductions in hydraulic conductance due to the spread of embolisms (Choat et al., 2010). These and other properties of the xylem network are currently under study.

Literature Aylmore L. (1993). Use of computer-assisted tomography in studying water movement around plant roots. Advances in Agronomy 49: 1–54. Brodersen CR, McElrone AJ, Choat B, Matthews MA, Shackel KA. (2010). The dynamics of embolism repair in xylem: in vivo visualizations using high resolution computed tomography. Plant Physiology 54: 1088–1095. Choat B, Brodie T, Cobb AR, Zwieniecki M, Holbrook N. (2006). Direct measurements of intervessel pit embrane hydraulic resistance in two angiosperm tree species. American Journal of Botany 93: 993–1000. DeVore M, Kenrick P, Pigg K, Ketcham R. (2006). Utility of high resolution X-ray computed tomography (HRXCT) for paleobotanical studies: an example using London clay fruits and seeds. American Journal of Botany 93: 1848–1851. Dierick M, Masschaele B, Van Hoorebeke L. (2004). Octopus, a fast and user-friendly tomographic reconstruction package developed in labview. Measurement Science and Technology 15: 1366–1370. Fromm J, Sautter I, Matthies D, Kremer J, Schumacher P, Ganter C. (2001). Xylem water content and wood density in spruce and oak trees detected by high-resolution computed tomography. Plant Physiology 127: 416–425. Heeraman D, Hopmans J, Clausnitzer V. (1997). Three dimensional imaging of plant roots in situ with X-ray computed tomography. Plant and Soil 189: 167–179.

20 DGP Meeting September 5-9, 2012 Kim K, Lee S. (2010). Synchrotron X-ray imaging for nondestructive monitoring of sap flow dynamics thorugh xylem vessel elements in rice leaves. New Phytologist 188: 1085–1098. Kinney J, Nichols M. (1992). X-ray tomographic microscopy (XTM) using synchrotron radiation. Annual Review of Materials Science 22: 121–152. Lee Fehler! Textmarke nicht definiert. EF, Brodersen CR, McElrone AJ, Philips RJ, Shackel KA, Matthews MA. Analysis of HRCT-derived xylem network reveals reverse flow in some vessels. (submitted to Journal of Experimental Botany, 2012) Maeda E, Miyake H. (2009). A non-destructive tracing with an X-ray micro CT scanner of vascular undles in the ear axes at the base of the lower level rachis-branches in japonica type rice (Oryza ativa). Japanese Journal of Crop Science 78: 382–386. Steppe K, Cnudde V, Girard C, Lemeur R, Cnudde JP, Jacobs P. (2004). Use of X-ray computed microtomography for non-invasive determination of wood anatomical characteristics. Journal of tructural Biology 148: 11–21. Steppe K, Lemeur R. (2007). Effects of ring-porous and diffuse-porous stem wood anatomy on the hydraulic parameters used in a water flow and storage model. Tree physiology 27: 43. Stevenson J, Matthews M, Rost T. (2004). Grapevine susceptibility to Pierce’s disease I: relevance of hydraulic architecture. American Journal of Enology and Viticulture 55: 228–237. Stuppy W, Maisano J, Colbert M, Rudall P, Rowe T. (2003). Threedimensionalvanalysis of plant structure using high-resolution X-raycomputed tomography. Trends in Plant Science 8: 2–6. Sun Q, Rost T, Matthews M. (2006). Pruning-induced tylose development in stems of current-year shoots of Vitis vinifera (Vitaceae). American Journal of Botany 93: 1567. Tyree M, Zimmermann M. (2002). Xylem structure and the ascent of sap. New York, NY USA: Springer Verlag. Verboven P, Kerckhofs G, Mebatsion H, Ho Q, Temst K, Wevers M, Cloetens P, Nicolai B. (2008). Three-dimensional gas exchange pathways in pome fruit characterized by synchrotron X-ray computed tomography. Plant Physiology 147: 518–527. Zimmermann M, Jeje A. (1981). Vessel-length distribution in stems ofsome American woody plants. Canadian Journal of Botany 59: 1882–1892.

21 DGP Meeting September 5-9, 2012 Effects of ambient aerosol particles on the water relations of crop plants

Shyam Pariyar 1, Heiner E. Goldbach 1, Tomas Eichert 1, Mauricio Hunsche 2, Jürgen Burkhardt 1

1Plant Nutrition Group, 2Horticultural Science Group, INRES-University of Bonn, Germany; E-mail: [email protected]

Introduction Plant surfaces are a major sink for aerosol deposition. The deposited particles are mostly hygroscopic and may become deliquescent within the humid leaf boundary layer due to plant transpiration. These highly concentrated solutions may then interact with leaf surfaces and thus creating a water sink in close vicinity to the stomata, thereby influencing plant water relations (Burkhardt, 2010). The ecological role of deposited aerosol particles has frequently been studied by applying additional particles to the foliar surface (Thompson et al., 1984; Hirano et al., 1995; Burkhardt et al., 2001b). It has been reported that the deposition of hygroscopic particles may lead to an increase in transpiration (Beasley, 1942; Hirano et al., 1995, Burkhardt, 2001a). The leaf surfaces can however, be already contaminated with significant amounts of atmospheric deposits (Pariyar et al., 2012), we assessed the influence of particle deposition on gas exchange and transpiration by particle exclusion experiments.

Materials and Methods Bean ( Vicia faba ) and sunflower ( Helianthus annuus ) plants were grown in greenhouses ventilated with almost particle free (FA) or with ambient air (AA). The greenhouses were located within the city with typical concentrations of ambient aerosols between 10,000 and 30,000 cm −3. The plants were grown either in well- water soil or in hydroponic solution. The reaction of leaf gas exchange to stepwise decreasing humidity was measured at 400 ppm CO 2 by using a porometer (Li-cor 6400), while water use efficiency (WUE) was calculated as WUE = A/E. Sap flow, 13 C isotopic ratio, osmotic and plant water potential were determined.

Results and Discussion Hydroponically grown FA sunflowers and FA beans showed 20–40% lower stomatal conductance (gs) and transpiration (E) compared to AA plants under otherwise identical conditions. Leaf-area related transpiration from sap flow measurement of soil-grown sunflowers was about 20–30% lower for FA plants, partially due to lower night time values. Midday water potentials as well as osmotic potentials of FA plants were higher compared to the respective AA plants. However, pre-dawn water

22 DGP Meeting September 5-9, 2012 potentials did not differ. Reduced transpiration of FA plants with stable photosynthesis (A) was observed for beans and can be explained by the “hydraulic activation of stomata”, where deposited hygroscopic aerosols form liquid water connections along the stomatal surfaces (Burkhardt et al., 2009), thereby forming a second, liquid-water type of stomatal transpiration. Additionally, the hygroscopic particles on the leaf surface may form the outer end of a ‘wick’ (Burkhardt, 2010), which may provide a way for water movement through a liquid phase. This “hydraulic activation” may increase stomatal transpiration, with the overall effect depending on the number of affected stomata. Simultaneously decreased transpiration and photosynthesis were observed for sunflower and point to a smaller stomatal aperture of FA plants. These results show for the first time an impact of natural aerosol particles on plant water relations. Particulate air pollution may thus decrease the WUE and possibly affect the drought tolerance of plants.

Literature Beasley, E.W., 1942. Effects of some chemically inert dusts upon the transpiration rate of yellow Coleus plants. Plant Physiology 17, 101–108. Burkhardt, J., 2010. Hygroscopic particles on leaves: nutrients or desiccants? Ecological Monographs 80, 369–399. Burkhardt, J., Hunsche, M., Pariyar, S., 2009. Progressive wetting of initially hydrophobic plant surfaces by salts – a prerequisite for hydraulic activation of stomata? In: The Proceedings of the International Plant Nutrition Colloquium XVI, Davies, California, USA, http://escholarship.org/uc/item/2m09483mi. Burkhardt, J., Kaiser, H., Kappen, L., Goldbach, H.E., 2001a. The possible role of aerosols on stomatal conductivity for water vapour. Basic and Applied Ecology 2, 351–364. Burkhardt, J., Koch, K., Kaiser, H., 2001b. Deliquescence of deposited atmospheric particles on leaf surfaces. Water, Air and Soil Pollution, Focus 1, 313–321. Eichert, T., Burkhardt, J., 2001. Quantification of stomatal uptake of ionic solutes using a new model system. Journal of Experimental Botany 52, 771–781. Eichert, T., Kurtz, A., Steiner, U., Goldbach, H.E., 2008. Size exclusion limits and lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes and water-suspended nanoparticles. Physiologia Plantarum 134, 151–160. Hirano, T., Kiyota, M., Aiga, I., 1995. Physical effects of dust on leaf physiology of cucumber and kidney bean plants. Environmental Pollution 89, 255–261. Pariyar, S., Goldbach, H.E., Eichert, T., Hunsche, M., Burkhardt, J. (in Press). The exclusion of ambient aerosols changes the water relations of sunflower ( Helianthus annuus ) and bean ( Vicia faba ) plants. Environ. Exp. Bot. (2012), doi:10.1016/j.envexpbot.2011.12.031. Thompson, J.R., Mueller, P.W., Flückiger, W., Rutter, A.J., 1984. The effect of dust on photosynthesis and its significance for roadside plants. Environmental Pollution Series a-Ecological and Biological 34, 171–190.

23 DGP Meeting September 5-9, 2012 Molecular and functional characterization of a recently identified class of plant aquaporins: the X intrinsic proteins

Gerd P. Bienert, Manuela D. Bienert, François Chaumont

Institut des Sciences de la Vie, Université catholique de Louvain, Louvain la Neuve, Belgium. E-mail: [email protected]

Introduction In plants, the movement of water and small neutral solutes across diverse membranes is reliant on the aquaporin (AQP) protein family. AQPs fulfil crucial roles in a variety of physiologically processes, like water conductance, gas and nutrient uptake and translocation, metalloid homeostasis and signal transduction. Recently, a novel, phylogenetically distinct AQP subfamily, the X intrinsic proteins (XIPs) has been discovered in silico in the genomes of moss and a wide variety of eudicots but not in Arabidopsis and all so far sequenced monocots (Danielson and Johanson 2008, Gupta and Sankararamakrishnan 2009). Surprisingly, XIPs were also identified in several fungi and a protozoan species and are therefore, in contrast to all other plant AQP subfamilies, not plant specific. Being just discovered, very little is known about XIPs from the different organisms.

Materials and Methods Various XIPs from plants and fungi were cloned and analyzed on the genomic, transcriptional and phylogenetic level. Transport, complementation and survival assays with the heterologously expressed XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae yeast mutants were performed. NtXIP expression is monitored on the transcriptional level via analysis of tobacco plants containing NtXIP1;1 -promoter-GUS reporter-gene and qPCR and, on the translational level, by Western blotting using anti-XIP antibodies. Nicotiana tabacum leaf discs were infected with Agrobacterium tumefaciens carrying the genetic constructs either leading to a 1) constitutive NtXIP1;1 α over-expression, 2) constitutive 10His-tagged NtXIP1;1 α over-expression 3) silenced expression of both NtXIP1;1 splice variants, 4) YFP protein-tagged NtXIP1;1 α and NtXIP1;1 β over- expression or 5) NtXIP1;1 -promoter-GUS reporter-gene expression. Calli were regenerated and selected on antibiotic-containing growth media. Antibiotic resistant seedlings were obtained for all constructs. Transgenic NtXIP1;1 -promoter-GUS and YFP protein-tagged NtXIP1;1 α and NtXIP1;1 β tobacco plants were analyzed by (confocal-)microscopy. Transgenic lines over-expressing or silenced for NtXIP1;1 will be challenged with different biotic and abiotic stresses to reveal the physiological function of XIPs in planta .

Results and Discussion XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A

24 DGP Meeting September 5-9, 2012 conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA- processing mechanism that results in two splice variants (NtXIP1;1 α and NtXIP1;1 β) (Bienert et al. 2011). When transiently or stably expressed in tobacco plants, YFP protein-tagged NtXIP1;1 α and NtXIP1;1 β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1 -promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in tissues facing the environment (epidermal and subepidermal cell layers) in all above-ground tissues (Bienert et al. 2011). Using XIP antibodies and splice-variant-specific qPCR primers, we are currently generating a detailed expression map both on the gene and the protein level. Heterologous expression of both XIP splice variants in X. laevis oocytes and S. cerevisiae cells demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for small solutes, such as ammonia and water, was very low or undetectable (Bienert et al. 2011). These results suggest that XIPs do not play a role in plant water homeostasis. The important role of conserved amino acid residues in XIPs determining their substrate specificity was analyzed using a mutational approach. Interestingly, Fusarium oxysporum , a fungal pathogen infecting the Solanaceae species, has also a XIP encoding gene, which we are currently characterizing. Transgenic lines partly and fully silenced for NtXIP1;1 were obtained and homozygous lines are currently selected. Excitingly, several T0 lines over-expressing NtXIP1;1 α either non-tagged or tagged with 10His under the control of two different constitutive promoters show a striking phenotype resembling a nutrient deficiency one. The developing phenotype in several independently transformed and regenerated plants clearly indicates that neither the transformation event nor the construct design can be responsible for the phenotype alone but that the ectopic expression of NtXIP1;1 α causes this phenotypic disorder. At the moment we are examining potentially underlying physiological reason for this phenotype. Our data suggest that XIPs function in the transport of uncharged nutrients/solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers. These features turn XIPs into potential targets for the directed usage in nutrient uptake or allocation processes.

Literature Bienert, G.P., Bienert, M.D., Jahn, T.P., Boutry, M. and Chaumont, F. (2011) Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates. Plant J. 66:306-317. Danielson, J.A. and Johanson, U. (2008) Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens . BMC Plant Biol. 8, 45. Gupta, A.B. and Sankararamakrishnan, R. (2009) Genome-wide analysis of major intrinsic proteins in the tree plant Populus trichocarpa : characterization of XIP subfamily of aquaporins from evolutionary perspective. BMC Plant Biol. 9, 134.

25 DGP Meeting September 5-9, 2012 Plenary Session S3: Nutrient dynamics in changing environments

26 DGP Meeting September 5-9, 2012 Challenges for plant nutrition in sub-Saharan Africa

Ken E. Giller 1, Linus Franke 1, Shamie Zingore 2, and Bernard Vanlauwe 3

1Wageningen University, Wageningen, Netherlands; 2International Plant Nutrition Institute, Nairobi, Kenya; 3International Institute of Tropical Agriculture, Nairobi, Kenya. E-mail: [email protected]

Introduction Although intensive sedentary agriculture is a fairly recent phenomenon in much of sub-Saharan Africa, having evolved over the past 100 years, it is often practiced on the oldest land surfaces in the world. Many soils are continuously cropped with minimal inputs of organic manure or fertilizer, and in the cases where fertilizers are used these are commonly only NPK fertilizers, or in many cases only N and P. Perhaps not surprisingly, a multitude of plant nutrient deficiencies are commonly observed and appear to be increasing in frequency.

Materials and Methods A wide array of field experiments will be reported where nutrient deficiencies have been observed in maize, soyabean, cassava and highland banana in Kenya, Rwanda, Nigeria, Uganda and Zimbabwe. The limiting nutrients have been determined by plant and soil analysis, backed up by missing-nutrient pot trials.

Results and Discussion Nutrient deficiencies of calcium, magnesium, sulphur and zinc are widespread, as well as molybdenum in more isolated cases. Soil maps are useful but inadequate tools to indicate the likelihood of nutrient deficiencies. Problem soils are often found in specific fields of farms, presenting a fine, granular mosaic of fields. Deficiencies of potassium appear to be widespread in addition to pervasive problems of soil nitrogen and phosphorus supply. Africa can be seen as a plant nutritionists’ paradise, but how these problems will be addressed is a thorny problem, particularly due to the cost of fertilizer and the lack of appropriate fertilizer blends on the market. The problems are becoming more acute and deserve greater attention of the scientific community.

Literature

Brodrick, S.J., Sakala, M.K., Giller, K.E., 1992. Molybdenum reserves of seed, and growth and N 2 fixation by Phaseolus vulgaris L. Biol. Fert. Soil. 13, 39-44. Brodrick, S.J., Amijee, F., Kipe-Nolt, J.A., Giller, K.E., 1995. Seed analysis as a means of identifying micronutrient deficiencies of Phaseolus vulgaris L. in the tropics. Tropical Agriculture 72, 277- 284. Zingore, S., Delve, R.J., Nyamangara, J., Giller, K.E., 2008. Multiple benefits of manure: The key to maintenance of soil fertility and restoration of depleted sandy soils on African smallholder farms. Nut. Cycl. Agroecosyst. 80, 267-282. Zingore, S., Murwira, H.K., Delve, R.J., Giller, K.E., 2007. Soil type, historical management and current resource allocation: three dimensions regulating variability of maize yields and nutrient use efficiencies on African smallholder farms. Field Crop Res. 101, 296-305. 27 DGP Meeting September 5-9, 2012 Effects of land use and soil characteristics on crop production in African wetlands

Dold, C., Changwony, D., Schneider, D., Becker, M.

Bonn University, INRES Plant Nutrition, Karlrobert-Kreiten-Str. 13, 53111 Bonn, Germany; E-mail: [email protected]

African wetlands are increasingly used for agriculture. The sustainability of crop production is likely to depend on the duration and type of land use as well as on environmental and soil characteristics. At Lake Naivasha in the dry savanna zone of Kenya, the water level has continuously declined during the past decades and the newly emerging land is being used by both pastoralists and farmers. These chronosequence of land use were investigated to study the effects of the type and the duration of land use on soil and production attributes. We compared grazed pasture land and small-scale crop production plots over land use durations of 5 – 30 years. Using field and greenhouse studies, solid and solution phase soil parameters were related to production and productivity gaps of maize as well as to biomass production, species composition and forage quality of pastureland. With increasing land use duration, Corg and Ntot but also soluble C decreased, thus negatively affecting plant growth, and differentially affecting the main yield-limiting factors in maize. Probably soil characteristics also affected plant growth. Besides soil moisture availability, strongest relations to crop performance were observed with Ntot (r = 0.88), POC: (r = 0.87) and pH (r = 0.79). Consequently, the addition of mineral N and P as well as supplementary irrigation produced largest maize yield gains in areas that are used since < 20 years. Implications for future wetland management will be discussed.

28 DGP Meeting September 5-9, 2012 Genotypic differences in radiation use efficiency in relation to nitrogen efficiency of oilseed rape

A. Ulas 1*,2 , F. Wiesler 3, W.J. Horst 1 and G. Schulte auf‘m Erley 4

1Institute for Plant Nutrition, Leibniz University Hannover; 2Soil Science and Plant Nutrition Dept., University of Erciyes, Kayseri, Turkey; 3Agricultural Analysis and Research Institute (LUFA), Speyer; 4Institute of Plant Nutrition and Soil Science, Christian Albrechts University Kiel E-mail: [email protected]

Introduction Nitrogen supply can affect plant growth and productivity by altering both leaf area and photosynthetic capacity. At the crop level, photosynthetic capacity can be expressed as radiation use efficiency (RUE) (Muchow and Sinclair, 1994) which is defined as the quantity of biomass produced per mega joule (MJ) of radiation intercepted by the crop (Monteith, 1972). In terms of solar radiation, oilseed rape ( Brassica napus L.) has a natural advantage compared to other annual field crops, because it has the longest growing period of about 350 days from sowing to maturity which allows high radiation interception (RI) of the canopy. However, many authors have shown that the RUE of the oilseed rape crop varies within a wide range, from 1.0 to 4.0 g MJ -1 according to developmental stage and environmental conditions (Leach et al., 1989; Habekotté, 1997). The aim of this study was to test if high N efficiency in rapeseed cultivars can the attributed to either improved RUE or RI. N-efficient rape cultivars are characterized by a high N uptake activity during reproductive growth (Schulte auf’m Erley et al., 2011), which was associated with an efficient and vigorous root growth (Ulas et al., 2012). Our hypothesis was that high root growth at limiting N supply is caused by a high radiation interception.

Materials and Methods Two field experiments were carried out in the 2000/2001 and 2001/2002 cropping periods at the experimental station of the Institute of Plant Breeding, University of Göttingen near Göttingen in Northern Germany. The experiments were laid out in a split plot design with N rates as main plots and cultivars as sub-plots with four block replicates. The N fertilization levels were no N fertilization (N0: soil N min ) and 240 kg -1 N ha minus the soil N min contents (N2). Two contrasting oilseed rape cultivars, cv. Apex as “N-efficient” and cv. Capitol as “N-inefficient”, were compared. Intermediate harvests were performed at the beginning of shooting (BS), beginning of flowering (BF), and end of flowering (EF). At maturity (MA), one complete plot (11.25 m 2) was harvested with a combine harvester. A solar system was installed on the field to measure continuously the incoming global radiation (kW m -2 s-1) above canopy level and the fractions that were reflected, intercepted and transmitted at canopy and soil

29 DGP Meeting September 5-9, 2012 level from BS till MA using tube and dome solarimeters. No harvests were performed in the plots, in which the solarimeters were installed. After conversion into Mega joule (MJ) the values were summed up for three different growth intervals: i) BS-BF, ii) BF- EF, iii) EF-MA. To calculate the RUE (g MJ -1) for each growth interval the dry matter production (g m -2) determined for each growth interval was divided into cumulative RI at the same growth interval. Because seeds contain energy-rich oil, seed dry matter was converted to ‘green crop units’ by multiplying each gram of oil by a factor of 2.17, which was derived from the data reported by Habekotte (1997).

Results and Discussion The cumulative RI was usually higher at non-limiting N (N2) than limiting N (N0) supply throughout the whole investigation period (Tab. 1). The N-efficient cv. Apex intercepted more radiation than the N-inefficient cv. Capitol from BS until MA at N0 in 2001 and 2002. On the other hand, except the difference at BS-BF in 2002, no considerable differences in RI were found between the cultivars at N2. Cv. Apex intercepted almost 12% and 9% at BS-BF, 17% and 5% at BF-EF, 12% and 3% at EF-MA and 13% and 6% at BS-MA more radiation than cv. Capitol at N0 in 2001 and 2002, respectively. The leaf area is the major source of RI and carbon assimilation of the oilseed rape crop from re-growth in early spring up to end of flowering (Grosse et al., 1992). However, since cv. Apex did not have a higher leaf area than cv. Capitol (Ulas, 2010), the higher interception might have been achieved by a better leaf orientation or leaf shape, which avoids mutual shading within the canopy.

Table 1: Cumulative intercepted radiation at three growth intervals of two oilseed rape cultivars as affected by N supply (N0: soil mineral N, N2: 240 kg N ha -1) in 2001 and 2002. (BS: Beginning of shooting, BF: Beginning of flowering, EF: End of flowering, MA: Maturity).

Radiation interception [MJ m-2] Growth interval Seed yield N uptake Year N Rate Cultivar BS-BF BF-EF EF-MA BS-MA [t ha -1] [kg ha -1] N0 Apex 58.9 87.7 287.9 441.4 3.35 91.8 2001 Capitol 52.0 73.4 253.2 383.0 3.28 93.6

N2 Apex 103.7 94.7 317.7 519.1 5.02 215.8 Capitol 101.2 92.4 314.1 510.9 5.05 187.1

N0 Apex 124.4 145.4 237.1 512.5 3.06 107.8 2002 Capitol 113.0 137.5 229.9 484.6 2.72 97.4

N2 Apex 130.5 152.0 253.6 544.1 4.16 180.0 Capitol 148.8 154.5 257.4 563.6 4.22 202.9

Apart from the high RI, seed yield and total N uptake were higher at N2 than N0 in both field experiments (Tab. 1). The N-efficient cv. Apex produced a higher seed yield than the N-inefficient cv. Capitol at N0 only in 2002 when also the N uptake was

30 DGP Meeting September 5-9, 2012 higher for cv. Apex. At high N supply, cv. Apex had a higher N uptake than cv. Capitol at N2 in 2001, but cv. Capitol had a higher N uptake than cv. Apex in 2002. Seed yield, however, did not vary much between cultivars at N2 in both years. The shoot dry matter (green crop units) increased continuously from BS until MA at both N rates in 2001 and 2002 (Fig. 1). Differences in dry matter production between two N rates were usually small at BS whereas large differences were found at later growth stages. Shoot growth differed between cultivars at both N0 and N2 in 2001 and 2002. In both field experiments cv. Apex showed consistently slow shoot growth particularly between BS and BF, but then rapid shoot growth between BF and EF as compared to cv. Capitol at N0 and N2. These cultivar differences in shoot growth did not correspond to variation in root growth. Total root length in the same field experiments was higher for cv. Apex than for cv. Capitol already during early growth stages (Ulas et al., 2012).

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6 8

6 4

Shoot dry matter [t ha matter [t dry Shoot 4 2 (N0) 2 (N2)

0 0 BS BF EF MA BS BF EF MA Growth interval Growth interval Figure 1: Shoot dry matter (green crop units) of two oilseed rape cultivars (Apex: filled symbols ( ●▲ ), Capitol: open symbols ( ○∆)) as affected by N supply (N0: soil N min , N2: 240 kg -1 N ha including N min ) at different growth stages (BS: Beginning of shooting, BF: Beginning of flowering, EF: End of flowering, MA: Maturity) in 2001 ( ●○ ), 2002 ( ▲∆).

RUE was usually higher at N2 than N0 throughout the study in 2001 and 2002 (Tab. 2). Our results corroborated other studies which stated that RUE of oilseed rape can vary within a wide range, from 1.0 to 4.0 g MJ -1 according to developmental stage (Leach et al., 1989; Habekotté, 1997). Despite of its higher cumulative RI at all growth intervals (Tab. 1), Apex showed lower RUE than Capitol particularly during vegetative growth (BS-BF) in 2001 and 2002 at both N0 and N2. This may be related to a high assimilate allocation to the roots during vegetative growth which was not included into the calculation of RUE. However, opposite to the vegetative stage cv. Apex had a higher RUE than cv. Capitol during reproductive growth (BF-EF) at both N rates in both field experiments. Total RUE from BS to MA at N0 was lower for cv. Apex than for cv. Capitol in 2001 and about equal for both cultivars in 2002.

31 DGP Meeting September 5-9, 2012 Therefore only in 2002 at yield advantage of cv. Apex compared to cv. Capitol was reached, despite the superior RI of cv. Apex. Since also N uptake at N0 was higher for cv. Apex only in 2002, it seems that RUE depended more on N uptake than RI. Also at N2, cultivar differences in RUE were similar to the variation in N uptake, although RI varied in a similar manner.

Table 2: Radiation use efficiency at three growth intervals of two oilseed rape cultivars as affected by N supply (N0: soil mineral N, N2: 240 kg N ha -1) in 2001 and 2002. (BS: Beginning of shooting, BF: Beginning of flowering, EF: End of flowering, MA: Maturity).

Radiation use efficiency [g MJ -1] Growth interval Year N Rate Cultivar BS-BF BF-EF EF-MA BS-MA N0 Apex 1.94 4.28 0.81 1.64 2001 Capitol 3.88 4.05 1.01 1.97

N2 Apex 2.31 5.54 1.39 2.32 Capitol 3.42 4.58 1.00 2.12

N0 Apex 1.00 2.68 1.03 1.48 2002 Capitol 2.25 2.00 0.74 1.44

N2 Apex 2.82 2.85 0.67 1.78 Capitol 3.19 2.41 0.87 1.90

Our results show that the N-efficient cultivar had a higher RI at limiting N supply. This could enable a high assimilate investment in root growth during the vegetative stage, which reduced shoot growth and RUE during vegetative growth, but enhanced N uptake and RUE during reproductive growth.

Literature Grosse F, Leon J, Diepenbrock W (1992): Ertragsbildung und Ertragsstruktur bei Winter-raps (Brassica napus L.). Vergleich zwischen Elternlinien und deren F1- und F2-Generationen J. Agron. Crop Sci. 169, 94-103. Habekotté B (1997): Identification of strong and weak yield determining components of winter oilseed rape compared with winter wheat. European Journal of Agronomy 7: 315–321. Leach JE, Milford GFJ, Mullen LA, Scott T, Stevenson HJ (1989): Accumulation of dry matter in oilseed rape crops in relation to the refection and absorption of solar radiation by different canopy structures. Aspects Appl. Biol. 23: 117-123. Monteith JL (1972): Solar radiation and productivity in tropical ecosystems. Journal of Applied Ecology 9: 747–766. Muchow RC, Sinclair TR (1994): Nitrogen response of leaf photosynthesis and canopy radiation use efficiency in field grown maize and sorghum. Crop Science 34, 721-727. Schulte auf'm Erley G, Behrens T, Ulas A, Wiesler F and Horst W J (2011) Agronomic traits contributing to nitrogen efficiency of winter oilseed rape cultivars. Field Crops Res. 124, 114- 123. Ulas A, Schulte auf'm Erley G, Kamh M, Wiesler F and Horst W J (2012) Root-growth characteristics contributing to genotypic variation in nitrogen efficiency of oilseed rape. J. Plant Nutr. Soil Sci. 175, 489-498. Ulas Fehler! Textmarke nicht definiert. A (2010) Agronomic and Physiological Parameters of Genotypic Nitrogen Efficiency in Oilseed Rape ( Brassica napus L.). PhD Thesis, Leibniz University Hannover. 32 DGP Meeting September 5-9, 2012 Sulfur mineralization of 35-S labeled rice straw under upland and flooded conditions

Christoph Wedde 1, Gerhard Welp 2, Heinrich W. Scherer 1

1Institute of Crop Science and Resource Conservation- Department of Plant Nutrition, University of Bonn, Bonn, Germany; 2 Institute of Crop Science and Resource Conservation – Department of Soil Science, University of Bonn, Bonn, Germany. E-mail: [email protected]

Introduction Sulfur (S) is well known as a yield limiting element in plant nutrition. Also in Chinese paddy soils S deficiency has increased during recent years. Caused by increasing costs for mineral fertilizers the use of rice straw is becoming more attractive (Lefroy et al., 1994), because S derived from rice straw may be an important S source for the rice crop. Theses authors demonstrated that the application of rice straw favored yield formation under flooded conditions. Rice straw contains between 0.05% and 0.10% S (Dobermann and Fairhurst, 2002). 2- S in rice straw has to be mineralized to sulfate (SO 4 ) before it is plant available (Ponnamperuma, 1984). However, the knowledge of S mineralization under various soil moisture regimes and redox conditions is rare. To evaluate the effects of soil water conditions on rice straw mineralization an experiment with two typical Chinese paddy soils and a German Luvisol was 2- conducted. We determined the changes in contents of SO 4 , reduced inorganic sulfur (RIS) and estersulfate (ES) concentrations in unplanted soil during a period of 13 weeks.

Materials and Methods The experiment was established with an Ultisol and an Entisol from Hangzhou, Province Zhejiang, PR China and a Luvisol derived from Loess from North Rhine- Westphalia, Germany. Rice was grown with nutrient concentrations based on Yoshida et al. (1976) containing 360MBq tracer-free 35-S as long as possible. 126 pots (3 soils x 2 treatments x 3 repetitions x 7 sampling dates) were filled with 40 g soil before thoroughly incorporating 0.2 g of finely grounded rice straw per pot. With the rice straw 3428 Bq 35-S per g soil were added. Two different moisture levels (moist at 60% of the maximum water holding capacity (WC) and flooded conditions) were adjusted. The pots were incubated in a dark climate cabinet at 20°C for 1, 2, 4, 6, 8, 10 and 13 weeks after start of incubation. After 10 weeks the flooded treatments were dried to 60% of WC to simulate upland conditions. In the non-flooded treatments soil samples were taken from the whole pot; in the flooded pots samples were taken some millimeter underneath the water layer (upper

33 DGP Meeting September 5-9, 2012 layer) and from the bottom of the pot (deep layer), respectively. After sampling the wet soils were stored frozen. All analyzes were performed with wet samples to avoid 2- changes in sulfur speciation during drying. Water soluble and adsorbed SO 4 and hydrogen-iodine extractable S were extracted based on methods of Shan et al. (1992, 1997), modified by Morche (2008). Reduced inorganic sulfur (RIS) was analyzed according to Burton et al. (2008). 35-S in the extracts was measured after mixing with Perkin Elmer Ultima Gold AB Cocktail solution in a Liquid Scintillation Counter (Perkin Elmer Tri-Carb 2900TR). Estersulfate (ES) contents were calculated 2- by subtraction of water soluble and adsorbed SO 4 from hydrogen-iodine extractable sulfur (Morche, 2008). All results were calculated on a dry matter basis. Statistical analyses were performed with IBM SPSS Statistics (20.0.0). Means and standard errors were calculated with Microsoft Excel 2007.

Results and Discussion Sulfate 2- In the moist treatment (60% WC) 35-S-SO 4 recovery remained rather constant in all soils (Tables 1 and 2). While in the Entisol and Luvisol the recovery of water soluble 2- 2- SO 4 was significantly higher as compared to the adsorbed sulfate, both SO 4 - fractions were almost at the same level in the Ultisol (Tables 1 and 2). Confirming 2- results of Zhou et al. (1999) we found that SO 4 is the main mineralization product under upland conditions.

Table 1 - Percentage of recovered 35-S as water soluble sulfate at selected sampling dates; different letters indicate statistical differences between sampling dates (a-c), treatments and sampling layers (d-e); (p ≤0,05); ANOVA with Tukey-HSD-Test Ultisol Entisol Luvisol weeks 60% WC Deep Upper 60% Deep Upper 60% Deep Upper layer layer WC layer layer WC layer layer 1 25.20bd 38.82ce 31.53cd 41.30ad 45.91cd 19.96bd 39.91ae 1.76ad 1.37ad

10 20.05abe 0.31ad 0.60ad 38.57ae 1.10ad 0.66ad 36.43ae 0.69ad 0.99ad

13 19.01abd 36.89cd 35.87ce 30.21ad 23.33bd 22.95bd 36.22ae 21.94bd 18.54bd

Table 2 - Percentage of recovered 35-S as adsorbed sulfate at selected sampling dates; different letters indicate statistical differences between sampling dates (a-d), treatments and sampling layers (e-f); (p ≤0,05); ANOVA with Tukey-HSD-Test Ultisol Entisol Luvisol weeks 60% Deep Upper 60% Deep Upper 60% Deep Upper WC layer layer WC layer layer WC layer layer 1 24.14ae 15.86bce 13.62cde 3.22bcf 1.52abe 1.03abe 1.90abf 1.45aef 1.05abe

10 24.27af 1.40ae 0.99ae 2.21abe 0.97ae 0.74ae 1.30abe 1.33ae 1.35bce

13 26.64af 17.41cf 17.47ce 0.88ae 1.23de 1.08abe 1.61abe 1.78ae 1.70ce

34 DGP Meeting September 5-9, 2012 2- In contradiction to upland conditions the recovery rate of 35-S-SO 4 decreased significantly in all soils under flooded conditions, followed by an increase after drying to 60% WC after the 10 th week (Tables 1 and 2). Significant differences between the 2- two sampling layers were not detectable but SO 4 -recovery differed significantly between the 1 st and the 10 th week in the Ultisol and Entisol. In the Luvisol significant differences were only detectable after the 13 th week. In the Ultisol the decrease was slightly slower than in the Entisol. The decrease was 2- st fastest in the Luvisol, where 35-S-SO 4 reached a very low level already after the 1 2- week of incubation. This decrease is assumed to be caused by the reduction of SO 4 to sulfide under flooded conditions (Starkey, 1966). Reduced inorganic Sulfur (RIS) While under upland conditions no RIS was detectable, under flooded conditions RIS increased in all three soils from the start of the experiment until the 10 th week (Table 3). After drying to 60% of WC after the 10 th week, RIS contents decreased significantly.

Table 3 - Percentage of recovered 35-S as RIS at selected sampling dates; different letters indicate statistical differences between sampling dates (a-c), treatments and sampling layers (d-e); (p ≤0,05); Student t-Test Ultisol Entisol Luvisol Weeks Deep layer Upper layer Deep layer Upper layer Deep layer Upper layer 1 8.10abd 1.4ae 12.52ad 9.69ad 18.17abd 23.12abd 10 44.01cd 10.19ae 30.97cd 30.58bcd 27.19abd 20.49abd 13 3.75ad 4.91ad 16.53abd 21.88abd 11.86ad 14.07abd

2- The increase of RIS was accompanied by a decrease of SO 4 and the decrease of 2- RIS by an increase of SO 4 . We assume that these changes are caused by the flooded conditions until the 10 th week, which induced a decrease in redox potential as previous studies showed (Ponnamperuma 1985; Islam and Dick, 1998). Therefore 2- th SO 4 was reduced to sulfide. The change to upland conditions after the 10 week caused an oxidation of RIS to sulfate. Organic sulfur At 60 % WC, the ES contents increased over time up to the end of the experiment, but the level was mostly lower than under paddy conditions. In the flooded samples, ES contents increased strongly and decreased again after drying to 60 % WC. We assume that under flooded conditions S from C-bonded forms and/or from S in rice straw is mineralized and then immediately reduced to RIS.

35 DGP Meeting September 5-9, 2012 Table 4 - Percentage of recovered 35-S as estersulfate at selected sampling dates ; different letters indicate statistical differences between sampling dates (a-c), treatments and sampling layers (d-e); (p ≤0,05); ANOVA with Tukey-HSD-Test Ultisol Entisol Luvisol weeks 60% Deep Upper 60% Deep Upper 60% Deep Upper WC layer layer WC layer layer WC layer layer 1 9.61ad 3.79ad 1.95ad 0.82ad 21.50ad 14.09ad 6.81ad 45.29ae 31.60ae 10 16.82ad 68.38ce 44.43bde 23.22ad 50.07bde 56.67be 16.88ad 54.82ae 42.64ae 13 17.82ad 14.80abd 8.64ad 26.15ad 31.28abde 44.36be 20.27ad 22.05ad 41.41ad

Our results indicate that soil moisture strongly affects rice straw mineralization. Nevertheless, even under flooding mineralization proceeds, but the end products of mineralization differ from those under upland conditions. While under upland 2- condition the main form was SO 4 , it was sulfide under flooding. However, even under flooding a transformation into organic sulfur was detectable.

Literature Burton, E. D., Sullivan, L. A., Bush, R. T., Johnston, S. G., Keene, A. F. (2008): A simple and inexpensive chromium-reducible method for acid-sulfate soil. Appl. Geochem. 23: 27. Dobermann, A., Fairhurst, T. H. (2002): Rice straw management. Better crops Int. 16: 7-11. Islam, M. M., Dick, R. P., (1998): Effect of organic residue amendement on mineralization of sulfur in flooded rice soils under laboratory conditions. Commun. Soil Sci. Plant Anal., 29: 955-969. Lefroy, R. D. B., Chaitep, W., Blair, G. J. (1994): Release of sulfur from rice residues under flooded and non-flooded soil conditions. Aust. J. Agric. Res. 45: 657-667. Morche, L. (2008): S-Flüsse und räumliche Veränderungen anorganischer und organischer Schwefelfraktionen im Boden sowie deren An- und Abreicherung in der Rhizosphäre landwirtschaftlicher Kulturpflanzen unter partiellem Einsatz des Radioisotops 35 S. Bonner Agrikulturchemische Reihe, Band 36, Diss. University of Bonn. Ponnamperuma, F. N. (1984): Straw as a source of nutrients for wetland rice. In: Organic matter and rice: International Rice Research Institute, Los Banos, Philippines. Ponnamperuma, F. N. (1985): Chemical kinetics of wetland rice soils relative to soil fertility, In: wetland soils: Characterization, classification, and utilization. International Rice Research Institute, Los Banos, Philippines. Shan, X.-Q., B. Chen, T.-H. Zhang, Li, F.-L., Wen, B., Quian, J. (1997): Relationship between sulfur speciation in soils and plant availability. Sci. Total Environ 199: 237-246. Shan, X.-Q., Bin, C., Long-Zhu, J., Yan, Z., Xiao-ping, H., Shi-Fen, M. (1992): Determination of sulfur fractions in soils by sequential extraction, inductively coupled plasma-optical emission spectroscopy and ion chromatography. Chem. Spec. Bioavail. 4: 97-103. Starkey, R. L. (1966): Oxidation and reduction of sulfur compounds in soil. Soil Science 101 (4): 297- 306. Yoshida, S., Forno, D. A., Cock, J. H., Gomez, K. A. (1976): Laboratory manual for physiological studies of rice. International Rice Research Institute, Los Banos, Philippines. Zhou, W., Li, S. T., Wang, H., He, P., Lin, B. (1999): Mineralization of organic sulfur and its importance as a reservoir of plant-available sulfur in upland soils of north china. Biol. Fertil. Soils 30: 245- 250.

36 DGP Meeting September 5-9, 2012 Invertase activity in reproductive organs of maize limits yield formation under salt stress

Birgit Hütsch, Muhammad Saqib, Tanja Osthushenrich and Sven Schubert

Institute of Plant Nutrition, JLU, Giessen, Heinrich-Buff-Ring 26, Giessen, Germany; E-mail: [email protected]

Breeding for improved sodium exclusion and osmotic resistance of corn ( Zea mays L.) has resulted in significantly better yield formation of newly developed hybrids (Schubert et al., 2009). Results indicate that avoidance of sodium toxicity allows better performance in the second phase of salt stress according to the model of Munns (1993). However, due to incomplete resistance during the first phase of salt stress, plants show stunted growth, although they do not suffer a water deficit and turgor does not limit vegetative growth (De Costa et al., 2007). Relative to their parental ancestor, Pioneer 3906, the new hybrids show better kernel setting, whereas grain filling is hardly affected. In agreement with this, there is no water shortage within the plants and carbon assimilation does not limit grain yield under salt stress. Although kernel weight is increased under salt stress, yield formation is limited by sink strength because kernel setting is inhibited. Few days after flowering, apoplastic sucrose phloem-unloading is required to support the ovaries. Acid invertase activity hydrolyzes sucrose and the resulting hexoses glucose and fructose are prevented from retrieval into phloem. Instead, they are transported via specific hexose carriers into the ovaries. Under salt stress, acid invertase is significantly decreased by salt stress resulting in accumulation of sucrose and abortion of kernels. Acid invertase activity thus appears to be a key factor for kernel setting although it does not explain genotypic differences.

Literature De Costa, W., C. Zörb, W. Hartung und S. Schubert (2007): Salt resistance is determined by osmotic adjustment and abscisic acid in newly developed maize hybrids in the first phase of salt stress. Physiol. Plant. 131, 311-321 Munns, R.: (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ. 16, 15-24 Schubert, S., A. Neubert, A. Schierholt, A. Sümer und C. Zörb (2009): Development of salt-resistant maize hybrids: The combination of physiological strategies using conventional breeding methods. Plant Sci. 177, 196-202

37 DGP Meeting September 5-9, 2012 Plenary Session S4: Crop responses to nutrient imbalances

38 DGP Meeting September 5-9, 2012 Boron functions in plants and animals: a key element for developmental processes

Luis Bolaños 1, Isidro Abreu 1, María Reguera 1,2 , and Ildefonso Bonilla 1

1Departamento de Biología/Facultad de Ciencias/Universidad Autónoma de Madrid, 28049-Madrid, Spain; 2College of Agricultural and Environmental Sciences/Plant Sciences Department/University of California/Davis, USA. E-mail: [email protected]

The requirement of trace amounts of boron (B) for plant growth was early reported by K. Warington (1923) and, during several decades, this micronutrient has been related with most biochemical, physiological or anatomical plant processes. Cell wall stability through borate cross-linking of apiosyl residues of the pectin polysaccharide rhamnogalacturonan II (RGII) is the most convincingly demonstrated role of B (O'Neill et al., 2004). However, the fact that most plant B is required for developing tissues rather than for mature organs (Bell et al., 2002), and the long-standing report that the first effects of B deprivation appear in meristems (Sommer and Sorokin, 1928), support the hypothesis that signaling mechanisms during cell differentiation and organogenesis are highly sensitive to B deficiency. Reports of abnormal tissue differentiation (Behrendt and Zoglauer, 1996) together with increasing evidence that B is involved in control of the expression of some genes important for regulation of cell cycle and cell differentiation (Reguera et al., 2009), point to functions of B related with cell signaling mechanisms during plant development. Furthermore, the description of the ability of boric acid to inhibit the growth of cell lines of prostate and breast cancer (Meacham et al., 2007), or abnormal embryonic development in animals under low B conditions (Rowe and Eckhert, 1999; Reguera, 2009) extend a key role of the micronutrient not only in plant but also in animal cell fate determination and organogenesis (Bonilla et al. 2009). Further progress on this research topic will depend on new methodologies with greater analytical capability and on the use of biological models highly dependent on the micronutrient, as the legume-rhizobia symbiosis that triggers the development of a nodule following a process of organogenesis highly regulated by molecular plant– bacteria interactions (Foucher and Kondorosi, 2000; Oldroyd and Downie, 2008). Boron deficiency has a strong effect on legume–rhizobia symbioses, affecting not only cell wall structure (Bonilla et al., 1997) but also rhizobia–legume cell-surface interactions and cell-to-cell signaling during every symbiotic event: early molecular plant-bacteria recognition (Redondo-Nieto et al., 2001), the infection process

(Bolaños et al., 1996; Reguera et al., 2010a, b), symbiosome and N 2-fixing bacteroid development (Bolaños et al., 2001, Redondo-Nieto et al., 2007), and cell and tissue differentiation during nodule organogenesis (Reguera et al., 2009). Therefore, investigating the effect of B deficiency on the regulation of nodule development had 39 DGP Meeting September 5-9, 2012 shed new light on the biological role of this micronutrient. Recent transcriptome studies show that B-nutrition affects expression of a majority of genes during nodule organogenesis (Redondo-Nieto et al. 2012). To our knowledge, there is no other nutrient deficiency, including Ca2+ , resulting in such an alteration of gene expression. Although it can be partially due to the disturbed pectic network, it cannot fully explain very early effects of low B on the symbiotic interaction. Furthermore, B deficiency also negatively regulates the physiology and development of living forms without cell walls, including animals (Rowe and Eckert, 1999), therefore it is unlikely that altered cell wall stability is the main and/or only cause of such loss of gene regulation. Why then is there such a far-reaching effect? The key role of B on animal physiology is consistent with the fact that low B induced the MAPK pathway in cultured animal cells and that mammalian cell lines mutated in B- transporters stop to develop and proliferate (Park et al., 2004; 2005). Similarly, B- deficient fertilized eggs of zebrafish ( Danio rerio ) suffer uncontrolled cell proliferation during the cleavage stage without cell differentiation (Reguera, 2009). The finding that B in bacteria is a signaling molecule (Chen et al. 2002) and that borate compounds can interact with regulatory proteins (several examples can be found in Goldbach and Wimmer, 2007) make likely that B is able to interact with transcription factors hence explaining such a wide alteration on gene expression, as was hypothesized by González-Fontes et al. (2008). Other possibilities come from the fact that several components of plant cell signaling and signal transduction pathways during organogenesis processes are known to be affected by B-deficiency. Several studies on B effects in plant and animal development and metabolism point to a role of B in extracellular matrices and/or in membrane functions (Brown et al., 2002). There is a well-known relationship between B, phytohormones, and development (Martín-Rejano et al. 2011), albeit this is clearly insufficient to explain the high B requirement for nodule organogenesis or aberrant animal embryo development. Boron has been described as a modulator of host plant-rhizobia interactions by interacting with legume arabinogalactan proteins (Reguera et al., 2010a) and affecting production and structure of rhizobia cell surface polysaccharides (Abreu et al., 2012) important for nodule infection and development. Moreover, several reports described abnormal glycosylation of glycoproteins apparently involved in cell-to-cell signaling during symbiosome development in nodules (Bolaños et al., 2001; Redondo-Nieto et al., 2007), and a similar role of boron during animal organogenesis has been hypothesized (Redondo-Nieto et al., 2008). In a preliminary study, we have detected altered overall protein N-glycosylation as a very early response to B- deprivation during legume nodule and Arabidopsis root or shoot apical meristem development. This consisted in abnormal accumulation of high mannose type N- glycans, typical for the endoplasmic reticulum. As above described, B-deficiency led

40 DGP Meeting September 5-9, 2012 to uncontrolled cell proliferation and aberrant cell differentiation, and interestingly, accumulation of high-mannose N-glycans is characteristic of some breast cancer lines (de Leoz et al., 2011). Therefore, the role of B on glycosylation mechanisms important for organogenesis has also to be deeply explored in more detail. In an exciting report, Ricardo et al. (2004) proposed that borate minerals could play a crucial role in an early ‘RNA world’ of life on Earth by stabilizing cyclic ribose; therefore, the structural and functional stability of microRNAs by borate is a possibility to be tested. Also, inositides or adenylates, which are potential targets of B (Ralston and Hunt, 2001; Bolaños et al., 2004), could be part of signalling pathways affected by B deficiency. Interestingly Ca 2+ prevents altered expression of more than 70% of genes affected by B deficiency and it has been demonstrated that boric acid can prevent proliferation of some tumor cells by affecting the release of Ca 2+ by cyclic ADP ribose (cADPr). This effect is based on the capacity of boric acid to bind to NAD +, the substrate of ADP ribosyl cyclase (Barranco et al., 2008). Therefore, in addition to exploring a primary interaction of B with transcription factors or with signaling and regulatory molecules such as glycans or microRNAs, it will be interesting to determine whether B deficiency affects Ca 2+ influx (as suggested by Koshiba et al., 2010) and ⁄ or also interferes with Ca 2+ -releasing mechanisms that are dependent on adenylates or inositides.

Literature Abreu et al. (2012) Plant and Soil DOI: 10.1007/s11104-012-1229-0 Barranco et al. (2008) Cell Biology and Toxicology 25:309-320. Behrendt and Zoglauer (1996) Physiologia Plantarum 97:321–326. Bell et al. (2002) In: Boron in plant and animal nutrition. Goldbach et al. (eds). KluwerAcademic/Plenum Publishers, 63–86. New York, NY, USA. Bolaños et al. (1996) Plant Physiology 110:1249-1256. Bolaños et al. (2001) Molecular Plant-Microbe Interactions 14:663-670. Bolaños et al. (2004) Plant Physiology and Biochemistry 42:907–912. Bonilla et al. (1997) Plant Physiology 115:1329–1340. Bonilla et al. (2009) In: Plant Physiology. Taiz and Zeiger (eds), web essay 5.2. Sinauer, Sunderland, MA, USA. http://4e.plantphys.net/article.php?ch=e&id=403 Brown et al (2002) Plant Biology 4:205–223. Chen et al. (2002) Nature 415:545–549. Foucher and Kondorosi (2000) Plant Molecular Biology 43:773–786. Goldbach and Wimmer (2007) Journal of Plant Nutrition and Soil Science 170:39–48. González-Fontes et al. (2008) Plant Signaling and Behavior 3:24–26. Koshiba et al. (2010) Plant & Cell Physiology 51:323–327. de Leoz et al. (2011) Molecular & Cellular Proteomics 10: http://mcponline.org/content/10/1/M110.002717.full Martín-Rejano et al. (2011) Physiologia Plantarum 142:170–180. Meacham et al. (2007) In: Advances in plant and animal boron nutrition. Xu et al. (eds). Springer, 299– 306. Dordrecht, The Netherlands. Oldroyd and Downie (2008) Annual Review of Plant Biology 59:519–546.

41 DGP Meeting September 5-9, 2012 O'Neill et al. (2004) Annual Review of Plant Biology 55:109–139. Park et al. (2004) Molecular Cell 16:331–341. Park et al. (2005) Cell Cycle 4:24–26. Ralston and Hunt (2001) Biochemica et Biophysica Acta 1527:20–30. Redondo-Nieto et al. (2001) Australian Journal of Plant Physiology 28:819–823. Redondo-Nieto et al. (2007) Plant Cell and Environment 30:1436–1443. Redondo-Nieto et al. (2008) Plant Signaling and Behavior 3:298–300. Redondo-Nieto et al. (2012) New Phytologist 195:14–19. Reguera (2009) PhD Thesis. Universidad Autónoma de Madrid. Spain. http://digitooluam. greendata.es/R/7KVIDSLCK6QPUMPPLKQVF83AQQR4227Y29APE97EMG9S6FMSQK- 00363?func=resultsjump-full&set_entry=000022&set_number=000065&base=GEN01 Reguera et al. (2009) New Phytologist 183:8-12. Reguera et al. (2010a) Plant Cell and Environment 33:2112–2120. Reguera et al. (2010b) Plant Cell and Environment 33:1039-1048. Ricardo et al. (2004) Science 303:196. Rowe and Eckhert (1999) Journal of Experimental Biology 202:1649–1654. Sommer and Sorokin (1928) Plant Physiology 3:237–260. Warington (1923) Annals of Botany 37:629–672.

42 DGP Meeting September 5-9, 2012 The mechanisms of B-salt-interaction depend on the B supply level

Monika A. Wimmer

Institute of Crop Science and Resource conservation (INRES), Dept. of Plant Nutrition, University of Bonn, Germany; E-mail: [email protected]

Introduction Salt and high boron stresses often occur simultaneously, especially in irrigated fields in arid or semi-arid climates. Raising temperatures, scarcity of low-salt water and the necessity to use marginal lands and effluent water from waste water treatments carrying high salt and B loads for irrigation will likely increase the occurrence of simultaneous salt and high B stresses in agricultural crops. However, in tropical and subtropical areas, salinity may also occur in fields inherently low in B. A large number of reports confirm interactive effects of simultaneous salt and B stress on plant responses, but the nature of this interaction remains unclear, because results are very inconsistent (Wimmer and Goldbach 2012). Variably, the stresses are reported to act in an antagonistic, synergistic, or independent way with respect to yield parameters and shoot B concentrations, even within the same plant species. So far, no comprehensible explanation is available for these contradictory results. We here bring forward the hypothesis that the mechanisms underlying salt-boron interactions depend on the B supply level. Under high B supply, where uptake of B is passive and B transport to the shoot is driven by transpiration, salinity is expected to reduce B uptake and shoot B concentrations; however, under low B supply, where active B uptake is induced, salinity may not have a direct effect on the uptake of B.

Materials and Methods Differently B- and salt-tolerant wheat genotypes were grown hydroponically at two different B supply levels. Plants were grown in a climate chamber at day/night regimes of 25°C/18°C, 14h/10h light, and 50%/70% re lative humidity. A second experiment was conducted in a greenhouse under ambient conditions. The B levels were chosen to represent low to marginal supply (5 µM B), and adequate to high supply (200 µM B), but none of the B treatments caused severe stress for the plants. Plants grown in 5 µM B did not show visible signs of B deficiency, and only one genotype was slightly inhibited in growth. At 200 µM B supply, plants exhibited some signs of B toxicity, visible as chlorotic or necrotic spots in older leaves, and a slight inhibition of root growth, but no reduction in yield. Boron levels were combined with three different salt treatments (0, 50, 100 mM NaCl). All treatments were started on day 15 after germination. After a 10 d treatment 43 DGP Meeting September 5-9, 2012 period, plant growth, leaf area, specific boron uptake rates, shoot B concentrations and transpiration rates were determined. All experiments consisted of 5 replicates, and statistical analysis was performed by the ANOVA procedure of SPSS and a posthoc test for significant differences.

Results and Discussion Whereas transport of B from root to shoot is mainly driven by the transpiration stream, uptake of B into root cells depends on different factors such as membrane composition and functionality (Dordas et al. , 2000), transpiration and water flow (Nable , 1988), as well as expression of aquaporins (Bastias et al. , 2004) and active B transporters (Takano et al. , 2008). Passive diffusion and channel-mediated uptake prevail at higher B supply, whereas active uptake is more important under low B supply (Miwa and Fujiwara , 2010). Accordingly, specific B uptake rates were high and not different between the wheat genotypes at high B supply, but significant differences were observed in the low B treatment (Fig. 1).

6 140 a ABa no salt 120 5 100 mM NaCl ** a a * a 100 ab * a 4 A A ** ab *** A AB 80 3 ** AB b B 60 2 40 B C b B B uptake rate [µg B/g [µg root/d] rate B uptake B uptake rate [µg B/g [µg root/d] rate B uptake 1 B 20

0 0 KF WI S BT IN KF WI S BT IN Fig. 1: Specific B uptake rates of five wheat genotypes supplied with 5 µM B (A) and 200 µM B (B). Means of 5 replicates, error bars represent standard errors, asterisks indicate sig. differences between no salt and salt treatments.

Under high B supply, B uptake rates were always reduced by the salt treatment (Fig. 1), and reductions correlated significantly with decreases in leaf area and shoot B concentrations. However, when B supply was rather low, additional salinity resulted in variable alterations of B uptake rates and no significant correlation with leaf area reductions or shoot B concentrations was observed. These results suggest that under high B supply, salinity directly affects B accumulation by reducing the transpiration- driven water flow and in consequence B uptake and transport into arial plant parts. Under low B supply, passive diffusion is not the decisive factor for B uptake, and consequently salinity does not directly reduce B uptake via its effect on transpiration. Of course other interactions at the cell level can still occur and are not subject of this study.

44 DGP Meeting September 5-9, 2012 Salt-induced growth reductions can have a dual effect on shoot B concentrations. Reduced leaf area can reduce B transport into the shoot due to a reduction in water flow, but a lower shoot biomass can also enhance shoot B concentrations via a concentration effect. In our study, salt-induced growth reductions were rather similar among the genotypes under high B supply. In this case, the amplitude of reduction of B uptake determined the total shoot B concentrations. Under low B supply, salt- induced growth reductions were significantly different among genotypes. In salt- sensitive genotypes, strong growth reductions and a concomittant concentration effect resulted in unchanged or even increased B concentrations under salt stress, and this was not observed in the more salt-tolerant genotypes. The salt-sensitivity of a genotype is therefore a modifying factor of salt/B interactions, especially at lower B levels. Our results confirm an interaction between salt and boron, and its nature was different under low and high B supply for several wheat genotypes. With respect to shoot B concentrations, an antagonistic interaction predominates under higher B supply, whereas synergistic or nonsignificant interactions are more common under lower B levels. It will be discussed how contradictory reports on the nature of salt/boron interactions might in part be related to low levels of B supply chosen as control treatments, and concomittant differences in predominant B uptake pathways.

Literature Bastias, E., Fernandez-Garcia, N., Carvajal, M. (2004): Aquaporin functionality in roots of Zea mays in relation to the interactive effects of boron and salinity. Plant Biol. 6(4): 415-421. Dordas, C., Chrispeels, M.J., Brown, P.H. (2000): Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots. Plant Physiol. 124(3): 1349- 1361. Miwa, K., Fujiwara, T. (2010): Boron transport in plants: co-ordinated regulation of transporters. Ann. Bot. 105(7): 1103-1108. Nable, R.O. (1988): Resistance to boron toxicity amongst several barley and wheat cultivars: A preliminary examination of the resistance mechanism. Plant and Soil 112: 45-52. Takano, J., Miwa, K., Fujiwara, T. (2008): Boron transport mechanisms: collaboration of channels and transporters. Trends in Plant Science 13(8): 451-457. Wimmer, M.A. and Goldbach, H.E. (2012): Boron-and-salt interactions in wheat are affected by boron supply. J. Plant Nutr. Soil Sci. 175: 171-179.

45 DGP Meeting September 5-9, 2012 Root morphological responses to changing nutrient availabilities

N. von Wirén

IPK Gatersleben, Corrensstr. 3, Abt. Physiologie & Zellbiologie, Gatersleben, Germany; E-mail: [email protected]

Nutrients have a profound effect on plant growth and development. Thereby roots show a particularly high plasticity in their morphological response to changing nutrient availabilities. Although such changes are often associated with an increase the foraging capacity of plants, root morphological responses to the deficiency of different nutrients remain poorly characterised in the reference plant Arabidopsis thaliana. One reason for this is the inherent difficulty in obtaining nutrient deficient conditions in agar culture for many of the essential elements. After establishing growth conditions and characterizing the effect of the deficiency of a particular nutrient on the root morphology of A. thaliana, we observed that root traits describing the initiation or elongation of roots of different orders respond specifically to various nutrient deficiencies. To cope with a spatially and temporarily variable availability of micronutrients, targeted lateral root growth towards nutrient-rich patches in the soil plants have evolved adaptive responses to a non-uniform, localized availability of nutrients. Investigating the lateral root response to localized supplies of different nitrogen forms, we observed a stimulation of lateral root initiation under localized ammonium supply, whereas nitrate rather stimulated the elongation (Lima et al., 2010, Plant Cell). With regard to localized supplies of micronutrients, we found a strong, dose- dependent stimulation of lateral root length by Fe. Using different Arabidopsis mutants affected in Fe acquisition or transport and the Fe supplementation via shoots in tetrapartite agar plates, we found that Fe-dependent lateral root elongation responds to symplastic root Fe rather than to external Fe and this is largely independent of the Fe nutritional signals from the shoot. We further identified the auxin importer AUX1 as a major Fe-responsive signaling step required for the observed in the elongation of lateral root cells (Giehl et al., 2012, Plant Cell). We now use this type of root responses to screen for nutrient-responsive genes that interfere with the developmental program determining root system architecture.

46 DGP Meeting September 5-9, 2012 Complementarity in root architecture for nutrient uptake in ancient maize / bean and maize / bean / squash polycultures

Johannes A. Postma 1,2 , Chaochun Zhang 2,3 , Larry M. York 2, and Jonathan P. Lynch 2

1 IBG-2: Plant Sciences, Forschungszentrum Jülich, 52425 Jülich, Germany, 2 Department of Horticulture, The Pennsylvania State University, University Park, PA, USA 16802, 3 Department of Plant Nutrition, China Agricultural University, Beijing 100193, PR China. E-mail: [email protected]

Introduction Intercropping of maize and bean is common practice by small-scale subsistence farmers and has been so in Mesoamerica since ancient history. The ancient polyculture in the Americas included squash and was commonly referred to as the “three sisters”. The maize/bean and maize/bean/squash intercropping systems have both agronomic and dietary benefits. These polycultures can have up to 30% more yield than the average of the monocultures. Climbing beans need support which in the polyculture is easily provided by the maize stalks (Scarry, 2008). Furthermore, weeds, pests and diseases may be suppressed in the more biologically diverse polycultures (Risch, 1981, 1980; Letourneau, 1986). We hypothesized that these polycultures also have positive plant nutritional aspects, not only because of the presence of a nitrogen fixing bean, but also because differences in root architecture result in different, complementary nutrient foraging strategies.

Materials and Methods In order to study belowground niche complementarity among the three sisters we grew maize/bean and maize/bean/squash polycultures and the respective monocultures on a low fertility field in Pennsylvania, USA. We grew these 5 cropping systems on plots with or without phosphorus fertilizer and with or without nitrogen fertilizer in a complete randomized split split plot block design. We studied the vertical rooting profiles of these crops by washing roots from root cores. The relative root volume of each crop in the polyculture cores was determined by using a quantitative genetic technique. Light capture of the crop canopy was determined by measuring the light intensity above and below the crop. We determined shoot biomass at 50 and 80 days after planting and yield at the end of the cropping season using destructive harvest of individual plants. We measured phosphorus and nitrogen content in the shoot biomass to estimate the total uptake of these minerals. Three weeks prior to the destructive harvests, we injected rubidium and strontium into the soil at 10 and 30 cm respectively and determined the rubidium and strontium concentrations in the harvested biomass of each plant species. We also studied the root architectures of these three species both in the field, using the shovelomics technique (Trachsel et

47 DGP Meeting September 5-9, 2012 al., 2010), and in greenhouse studies and used that data for virtual 3D reconstruction of maize, bean and squash root architectures. The reconstruction was done with SimRoot , a functional structural plant model (Postma and Lynch, 2011a, 2011b). SimRoot was used to simulate nitrogen, phosphorus and potassium uptake and biomass production of the individual plants grown in polycultures or monocultures on soils varying each of these three macro nutrients (Postman and Lynch, 2012). Only belowground competition for nutrients among the species was simulated, while aboveground competition for light in the polycultures was simulated as if the individual plants were growing in a monoculture.

Results and Discussion The polyculture crops had greater biomass production than the average of the monocultures under all fertility treatments. We used the mathematical formulas of Loreau and Hector (2001) to partition this increase of biomass into a complementarity effect and a selection effect and found that the increase in biomass is largely due to complementarity of the crops, not because larger species took advantage of the smaller species. Similar to the biomass increases, yields of both polycultures were 10-30% greater than the monocultures on a land equivalence basis (LER). The overall yield was reduced on the low fertility plots, with a large reduction on the low phosphorus plots, but minor reductions on the low N plots. The relative yield increase due to intercropping did not differ significantly between low and high fertility plots. Soil coring for root length density showed that maize rooted more shallow than bean and squash was the deepest rooting crop. Cores from the polyculture fields did not contain more root length than the average of the monoculture plots, despite the greater biomass on the monoculture plots. However, variation in rooting density between shallow and deep layers was much larger in the monocultures, while the polycultures had more homogeneous distribution of roots. The variation in the monocultures crops was mostly larger due to the species effect, however, the polycultures also had, in comparison to the average of the monocultures, more roots in deeper layers (below 16 cm), and thereby less variation in the rooting density between shallow and deep layers. We did not find important architectural differences, such as changes in branching angle, that might explain the deeper rooting of the polycultures, nor did we find large difference in rooting depth of the individual crops. The deeper rooting of the polyculture is explained by the fact that the deeper rooting crops (bean and squash) had grown more roots in the polycultures while the shallow rooting crop (maize) had grown less roots. The intensity with which the crops foraged for rubidium (injected at 10 cm depth) and strontium (injected at 30 cm depth) differed strongly between the crops. Maize took up the most rubidium, while squash took up the most strontium. The relative ratios in which rubidium and strontium were taken up by the individual crops either were not significantly different between mono or 48 DGP Meeting September 5-9, 2012 polycultures, or were stronger in the polyculture, suggesting that nutrient foraging strategies with respect to depth were even more pronounced in the polyculture. Total uptake of nitrogen was the same or greater in the polycultures under all fertility treatments, but uptake of phosphorus by the polycultures was only greater in the high phosphorus fields and not in the low phosphorus fields. SimRoot simulated greater biomass production in the polycultures on low N soils, similar to the field results (Postman and Lynch, 2012). This complementarity effect of the polycultures depended on the simulation of both root architecture and biological nitrogen fixation. The model did not simulate complementarity effects on low P soils. According to the simulation results, the root density is not high enough for significant inter-root competition for phosphorus. Our results confirm reports in the literature that maize/bean and maize/bean/squash polycultures can produce more yield and we show that this is so on both high and low fertility soils. The greater production is not because of a selection effect, in which large species take advantage of the presence of small species, but because the species occupy complementary niches. Several species specific plant traits may explain the niche complementarity effect. We show that aboveground light capture by the polycultures early in the season is greater than that of the average of the monocultures. Belowground we show that the species differ strongly in their root foraging strategies which is apparent from the different root placements by depth, uptake of tracers injected at different depths and the root architectural measurements. The polycultures had more uniform root distribution than the monocultures which, according to the modeling exercise, may explain the increased N uptake in the polycultures (Postman and Lynch, 2012). Therefore, besides the presence of a biological N fixer, belowground spatial niche differentiation may explain greater production on low N soils. The model suggests that both N fixation and complementarity in root architecture are necessary to simulate the complementarity effect. However the model also suggests that more uniform root distribution has no effect on phosphorus uptake as the distances between individual roots are on average much larger than the phosphorus depletion zones around the roots (Postman and Lynch, 2012). Indeed, the polycultures did not take up more phosphorus than the average of the monocultures on low phosphorus soils, had less and not more shallow roots and also did not have more total roots. Therefore increased biomass production by the polycultures can not be explained by belowground niche complementarity, rather in the polyculture maize takes up more phosphorus at the cost of squash and bean. The greater phosphorus use efficiency by maize, as apparent by the much lower P content in the maize shoot compared to bean and squash shoots, causes this shift in phosphorus uptake to translate into greater total biomass production. Our data suggests that shift occurs, not because of

49 DGP Meeting September 5-9, 2012 direct competition for phosphorus, but because maize tended to root more shallow, while squash, and to a certain extend bean, tended to root deeper when they were growing in the polyculture. However, we can not exclude other possible mechanism including possible interactions with the soil biota, e.g. mycorrhizal fungi.

Literature Letourneau, D.K. (1986). Associational resistance in squash monocultures and polycultures in tropical Mexico. Environmental Entomology 15: 285–292. Loreau, M. and Hector, A. (2001). Partitioning selection and complementarity in biodiversity experiments. Nature 412: 72–76. Postma, J.A. and Lynch, J.P. (2011a). Root cortical aerenchyma enhances the growth of maize on soils with suboptimal availability of nitrogen, phosphorus, and potassium. Plant Physiology 156: 1190 –1201. Postma, J.A. and Lynch, J.P. (2011b). Theoretical evidence for the functional benefit of root cortical aerenchyma in soils with low phosphorus availability. Annals of Botany 107: 829–841. Postma J.A. and Lynch J.P. (2012). Complementarity in Root Architecture for Nutrient Uptake in Ancient Maize/Bean and Maize/Bean/Squash Polycultures. Annals of Botany 110: 521–534 Risch, S. (1980). The Population Dynamics of Several Herbivorous Beetles in a Tropical Agroecosystem: The Effect of Intercropping Corn, Beans and Squash in Costa Rica. Journal of Applied Ecology 17: 593–611. Risch, S.J. (1981). Insect herbivore abundance in tropical monocultures and polycultures: An experimental test of two hypotheses. Ecology 62: 1325–1340. Scarry, C.M. (2008). Crop husbandry practices in North America’s eastern woodlands. In Case Studies in Environmental Archaeology, E.J. Reitz et al., eds, Interdisciplinary Contributions to Archaeology. (Springer New York), pp. 391–404. Trachsel, S. et al. (2010). Shovelomics: high throughput phenotyping of maize (Zea mays L.) root architecture in the field. Plant Soil 341: 75–87.

50 DGP Meeting September 5-9, 2012 Calcium signaling in symbioses with plants

Giulia Morieri, Giles E.D. Oldroyd and J Allan Downie

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. E-mail: [email protected]

Introduction Symbiotic interactions between plant roots and nitrogen-fixing bacteria and arbuscular mycorrhizal fungi play an important role in acquisition of nutrients. The rhizobia induce the development of nodules on roots and infect the cells in these nodules, to form organelle-like structures that reduce N 2 and export the resulting ammonia to the plant (Oldroyd et al. 2011). Arbuscular mycorrhizal fungi infect roots and also form intracellular structures that promote the exchange of nutrients, such as N and P from the fungi to the plants (Parniske 2008). The mycorrhizal symbiosis is at least 450 million years old and is found in most extant plant genera, whereas the bacterial nitrogen-fixing symbiosis developed more recently (around 100 million years ago) and is limited to the Fabidae group of plants which includes the legumes (Doyle 2011). There are parallels in the modes of infection by the micro-symbionts and in the signalling pathways activated by signals produced by the rhizobia and fungi. The identification of legume mutants that are defective for both nodulation by rhizobia and infection by mycorhizal fungi led to a realisation that nodulation signalling must have evolved from a more ancient mycorrhizal signalling pathway, which promotes fungal infection of roots (Parniske 2008). More recently it has been shown that there are similarities in the structures of the signals that activate the pathways that promote the development of the mycorrhizal and rhizobial symbioses Maillet et al. 2011) These signals (referred to here as Nod factors or Myc factors) are oligomers of chitin (beta-1,4-linked N-acetyl glucosamine) carrying an N-linked long chain acyl group on the terminal glucosamine residue and a variety of different substitutions on the chitin backbone (Oldroyd and Downie 2008; Maillet et al. 2011). In legumes, Nod factors are recognized by LysM-type membrane receptor-kinases (Radutoiu et al. 2003), which activate a nodulation-signaling pathway that induces nuclear oscillations of calcium (calcium spiking) in and around the nucleus (Oldroyd et al 2011). Mycorrhizal fungi activate a similar response (Kosuta et al. 2011). The calcium spiking is thought to be recognized by a calcium and calmodulin activated kinase (CCaMK), which is required for both nodulation and mycorrhization. Activated CCaMK activates nodulation-associated transcription factors and gain-of-function mutations in CCaMK promote nodule morphogenesis without microbial signaling; this shows that activation of this kinase is sufficient to induce nodule development

51 DGP Meeting September 5-9, 2012 (Oldroyd et al 2011). It is not yet known how specificity is conferred to the mycorrhization and nodulation signaling pathways, which both require CCaMK. In this work we have been analyzing the requirements for legume infection by rhizobia. In addition to activating the calcium-spiking-CCaMK pathway, rhizobial Nod factors induce at least two other responses that are blocked by mutations in Nod- factor receptors, but are not blocked by mutations affecting CCaMK or affecting other genes in the nodulation/mycorrhization common signaling pathway (Miwa et al. 2006a). One of these responses is the induction of root hair deformation, which can be activated by as little as 10 -13 M Nod factor (Oldroyd and Downie 2004). The other response requires much higher levels of Nod factors (around 10 -9 M) and is a calcium influx across the root hair tip (Shaw and Long 2003); this is associated with membrane depolarization and induction of reactive oxygen species (Cardenas et al. 2008; Felle et al. 1998). We propose that this calcium influx and associated responses are required for initiation of the infection programme by rhizobia.

Materials and Methods The methods used are essentially those described previously to image calcium in root hairs using seedlings of Medicago truncatula expressing the calcium sensing yellow cameleon YC2.1 (Miwa et al. 2006 a, b)

Results and Discussion Nod factors purified from WT Sinorhizobium meliloti induced perinuclear calcium spiking with about 50% of root hairs inducing calcium spiking at about 0.5 pM. In contrast about 1000-fold more Nod factor was required to activate a calcium influx in the tips of 50% of the root hairs imaged. Similar experiments were undertaken with Nod factors lacking specific modifications, such as the loss of an O-linked acetyl group on the N-acylated glucosamine. This alteration, which is caused by mutation of nodL in S. meliloti (Demont et al. 1993) greatly reduced the induction of the calcium influx, but did not affect the induction of calcium spiking. Mutation of nodL causes a significant decrease in infection efficiency by S. meliloti (Ardourel et al. 1994) and so we conclude that the calcium influx must play a role in initiation of infection. This result together with similar experiments with other Nod factors allows us to propose a model in which the calcium influx promotes initiation of infection by rhizobia. However, we were unable to identify legume mutants (other than those defective for Nod-factor receptors) in which this calcium influx was blocked. Therefore we undertook an analysis aimed at identifying genes that are induced by the calcium influx. Some candidate genes were identified and the induction of these genes appeared to require both calcium spiking and the calcium influx. This implies 52 DGP Meeting September 5-9, 2012 that the common symbiosis signaling pathway is required for induction of gene expression and then additional genes and enhanced gene expression may be induced as a consequence of the calcium influx.

Acknowledgements We thank our colleagues for their help. This work was funded by the “Nodperception” Marie Curie European Union grant (MRTN-CT-2006-035546) and by the BBSRC.

Literature Ardourel, M., Demont, N., Debelle, F., Maillet, F., de Billy, F., Prome, J.C., Denarie, J., and Truchet, G. (1994). Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses. Plant Cell 6, 1357-1374. Cardenas, L., Martinez, A., Sanchez, F., and Quinto, C. (2008). Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs). Plant J. 56, 802-813. Demont, N., Debelle, F., Aurelle, H., Denarie, J., and Prome, J.C. (1993). Role of the Rhizobium meliloti nodF and nodE genes in the biosynthesis of lipo-oligosaccharidic nodulation factors. J. Biol. Chem. 268, 20134-20142. Doyle, J.J. (2011) Phylogenetic perspectives on the origins of nodulation. Mol. Plant-Microbe Interact. 24, 1289-1295 Felle, H.H., Kondorosi, E., Kondorosi, A., and Schultze, M. (1998). The role of ion fluxes in Nod factor signalling in Medicago sativa . Plant J. 13, 455-463. Kosuta, S., Held, M., Hossain, M.S., Morieri, G., MacGillivary, A., Johansen, C., Antolin-Llovera, M., Parniske, M., Oldroyd, G.E.D., Downie, A.J., Karas, B., and Szczyglowski, K. (2011). Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program. Plant J. 67, 929- 940. Maillet, F., Poinsot, V., Andre, O., Puech-Pages, V., Haouy, A., Gueunier, M., Cromer, L., Giraudet, D., Formey, D., Niebel, A., Martinez, E.A., Driguez, H., Becard, G., and Denarie, J. (2011). Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469, 58-63. Miwa, H., Sun, J., Oldroyd, G.E., and Downie, J.A. (2006a). Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus . Mol. Plant- Microbe Interact. 19, 914-923. Miwa, H., Sun, J., Oldroyd, G.E., and Downie, J.A. (2006b). Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell. Plant J 48, 883-894. Oldroyd, G.E., and Downie, J.A. (2004). Calcium, kinases and nodulation signalling in legumes. Nat. Rev. Mol. Cell Biol. 5, 566-576. Oldroyd, G.E.D., and Downie, J.A. (2008). Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu. Rev. Plant Biol. 59, 519-546. Oldroyd, G.E.D., Murray, J.D., Poole, P.S., and Downie, J.A. (2011). The rules of engagement in the legume-rhizobial symbiosis. Annu. Rev. Genet. 45, 119-144. Parniske, M. (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Rev. Microbiol. 10, 763-755 Radutoiu, S., Madsen, L.H., Madsen, E.B., Felle, H.H., Umehara, Y., Gronlund, M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N., and Stougaard, J. (2003). Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425, 585-592. Shaw, S.L., and Long, S.R. (2003). Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells. Plant Physiol. 131, 976-984.

53 DGP Meeting September 5-9, 2012 Poly(ADP-ribose)polymerases in plant stress tolerance - key regulators or overrated ?

Dagmar Rissel 1,2 , Kristin Peter 2, Kathrin Thor 2, Edgar Peiter 2

1Agrochemisches Institut Piesteritz e.V., 06886 Lutherstadt Wittenberg; 2Plant Nutrition Laboratory, IAEW, Universität Halle-Wittenberg, 06120 Halle (Saale), Germany, City. E-mail: [email protected]

Introduction Poly(ADP-ribosyl)ation is a fast transient posttranslational protein modification. Activated upon DNA strand breaks, poly(ADP-ribose) polymerases (PARPs) add ADP-ribose molecules to nuclear proteins involved in DNA strand break repair. Thereby NAD + is used as a substrate to build up ADP-ribose polymer chains (Schreiber et al. , 2006). PARPs are important players in mammalian DNA strand break repair and genome stability (De Vos et al. , 2012). In the model plant Arabidopsis thaliana three PARP genes (AtPARP1 , AtPARP2 and AtPARP3 ) have been identified (Hunt et al. , 2004). Recent studies have suggested that a reduced PARP expression may increase plant tolerance towards abiotic stresses and alter signalling processes associated with plant drought stress responses (De Block et al. , 2005; Vanderauwera et al. , 2007). Additionally, the application of PARP inhibitors improved the drought stress tolerance in Lemna minor (Geissler et al. , 2011). However, the mechanisms of how PARPs are involved in plant stress responses are still a matter of debate

Materials and Methods To analyze the mechanisms of PARP action in plant stress responses, several homozygous Arabidopsis T-DNA insertional knockout lines were identified for PARP1, PARP2, and PARP3 . Homozygous parp double and triple mutant lines were generated by crossing the individual T-DNA lines to examine functional redundancy within the PARP gene family. The parp single, double, and triple mutant lines were subjected to various abiotic stress treatments under highly controlled conditions. Furthermore PARP gene expression and enzyme activity was monitored by promoter-GUS studies and a PAR dot-blot, respectively. As PARP expression was found to be very high in imbibed seeds, seed germination assays were performed.

Results and Discussion The plant response to salt, oxidative, and osmotic stress was assayed on sterile- grown seedings. Surprisingly, the plant performance was generally not improved in the various parp mutant lines. Compared to the wild type, hardly any alteration in root

54 DGP Meeting September 5-9, 2012 length, and root and shoot weight of parp single, double and triple mutant plants could be found. In mature plants subjected to drought stress, no phenotypical differences between wild type and mutants were apparent. Besides, transpiration of the parp mutants was similar to that of wild type plants. Our results thus indicate that PARPs are no general players in abiotic stress responses. This is in accordance with a largely absent transcriptional activation of the genes by most abiotic stresses. On the other hand, genotoxic stress by UV irradiation induced the expression of PARP1 and PARP2 in leaves of wild type plants. This coincided with a strong increase in PARP enzyme activity. In silico analysis and promoter-GUS studies revealed a high expression of PARP genes in imbibed and germinating seeds. This may be explained by an increased requirement for DNA repair at this growth stage. Supporting a role of the genes in seed viability, parp1 and parp3 mutant seeds displayed a defective germination.

Our results do not support a generally negative impact of plant PARPs on abiotic stress tolerance, and underline their role in the defense of DNA damage stress and in genome stability. We hypothesise that the reported positive effects of PARP inhibitors on plant stress tolerance are due to off-target effects.

Acknowledgements Financial support by the Federal State of Sachsen-Anhalt and SKW Piesteritz is gratefully acknowledged.

Literature De Block M, Verduyn C, De Brouwer D, Cornelissen M (2005). Poly(ADP-ribose) polymerase in plants affects energy homeostasis, cell death and stress tolerance. The Plant Journal 41, 95-106. De Vos M, Schreiber V, Dantzer F (2012). The diverse roles and clinical relevance of PARPs in DNA damage repair: Current state of the art. Biochemical Pharmacology 84, 137-146. Geissler T, Wessjohann LA (2011). A Whole-Plant Microtiter Plate Assay for Drought Stress Tolerance-Inducing Effects. Journal of Plant Growth Regulation 30, 504-511. Hunt L, Lerner F, Ziegler M (2004). NAD - new roles in signalling and gene regulation in plants. The New Phytologist 163, 31-44. Schreiber V, Dantzer F, Ame JC, de Murcia G (2006). Poly(ADP-ribose): novel functions for an old molecule. Nature Reviews Molecular Cell Biology 7, 517-528. Vanderauwera S, De Block M, Van de Steene N, Van de Cotte B, Metzlaff M, Van Breusegem F (2007). Silencing of poly(ADP-ribose) polymerase in plants alters abiotic stress signal transduction. Proceedings of the National Academy of Sciences of the United States of America 104, 15150-15155.

55 DGP Meeting September 5-9, 2012 Sulfur supply improves tomato pathogen resistance

K. Bollig 1, M. Zahn 1, S. S. Myint 2, C. Hogekamp 3, H. Küster 3 and W. J. Horst 1

1Institute for Plant Nutrition / Leibniz University Hannover, Hannover; Germany 2Department of Horticulture / Yezin Agricultural University, Yezin, Myanmar. 3Institute for Plant Genetics / Leibniz University Hannover, Hannover, Germany E-mail: [email protected]

Introduction The soil-borne phyto-pathogenic fungus Verticillium dahliae (V. dahliae ) generates monocyclic vascular wilt disease worldwide in over 200 economically important dicotyledonous host plant species including legumes, vegetables, ornamentals, trees, and weeds (Fradin and Thomma, 2006). Chemical disease prevention is not easy, since infected vascular tissue cannot be reached from outside, and non-chemical disease suppression approaches often show limited long-term effectiveness (Goichoechea, 2009; Larkin et al., 2010). To attenuate the high economic impact on producers resulting from the fungus-induced devastating yield and quality losses, an effective and environmentally acceptable curative strategy needs to be found. One way of controlling this difficult plant disease might be the use of resistant crop varieties, but until today a genome-integrated resistance source against Verticillium ssp. has only been detected and analyzed in the family of Solanaceae . The dominant Ve resistances locus was sequenced and cloned from tomato and encodes a cell- surface receptor for the recognition of V. dahliae and V. albo-atrum race 1 fungal strains (Fradin et al., 2009). Beside this limited genetic defense, no effective fungicide preventing pathogen infestation is currently available and no possibilities to overcome the disease in susceptible plants. Therefore, our current research focuses on the elucidation of a further resistance- promoting strategy. A promising way to constrain the Verticillium wilt disease levels in planta seems to be the vasculature-localized process of Sulfur (S)-enhanced defence (SED), closely linked to reductive S assimilation. Belonging into the group of S- containing defence compounds (SDCs), the inorganic phytoalexin elemental S (S 0) was found to be synthesized in several plant families in response to fungal colonization. Within members of the Solanaceae family resistant tomato and pepper genotypes accumulated high levels of S 0 in vascular tissue upon V. dahliae contact (Williams et al. 2002; Novo et al., 2007). So far, the mode of action and the exact mechanisms and participating genes of S 0 synthesis in the plant vasculature remain a topic of basic plant research. We investigated the potential of supra-optimal S nutrition to activate SED mechanisms limiting fungal growth in tomato plants colonized by V. dahliae. Therefore, we developed a V. dahliae -specific quantitative Real-Time polymerase 56 DGP Meeting September 5-9, 2012 chain reaction (qRT-PCR) assay. The sensitive measurement of fungal genomic DNA (gDNA) in tomato plants with contrasting S nutritional status, and the combined analysis of selected physiological parameter enabled deeper insights into the role of SED and associated SDCs in the tomato-Verticillium pathosystem. Additionally, we investigated the influence of low and supra-optimal S nutrition on the cell-specific localization and accumulation of S by Laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS). The qRT-PCR analysis of S metabolism and SED- related candidate genes in entire tomato hypocotyls and a laser microdissection- based spatial expression analysis studied the tissue-specific contribution of cysteine (Cys) and glutathione (GSH) synthesis-associated genes to SED against V. dahliae in tomato vascular bundles.

Materials and Methods The near-isogenic tomato lines Craigella GR 26 (CGR 26, sensitive, LA3247) and CGR 218 (resistant, LA3428) were cultivated in a climate chamber, for three weeks until fungal inoculation and harvested 7 dpi. A full low-S nutrient solution was applied every second day, and to obtain a supra-optimal S supply plants were additionally supplied with 25.0 mM K 2SO 4. Fungal inoculum was produced from the V. dahliae isolate TomIGZ (GU060637). Plants were infected by a root-clip dip method according to Novo et al (2007). All qRT-PCR assays were performed in a CFX96™ Real-Time detection system. The SYBR Green-based absolute qRT-PCR mix included the isolate-specific V. dahliae primer pair LeVD and 100 ng total gDNA. For the analysis of suberinization free-hand sections were stained with sudan III / IV. Plant height was measured and roots were analyzed with the WinRHIZO Software (Regent Instruments Inc., Canada). The photosynthesis rate was measured with a portable gas-exchange system (LI-6400, LI-COR, Lincoln, NE, USA) and phloem exudates were collected after 2h according to Cakmak et al. (1994) using intact tomato shoots. S-containing metabolites were determined by high pressure liquid chromatography (HPLC) with an Agilent reversed phase column (Eclipse XDB C18, 5 µm, 4.6 150 mm) in an Agilent 1200 infinity series HPLC system (Agilent Technologies Deutschland GmbH, Böblingen, Germany). Total S was measured in a Vario EL CNS analyzer (Dumas Elementar Analysensysteme GmbH, Hanau, Germany). LA-ICP-MS analysis of uniform hypocotyls cross-sections were performed with a solid-state NYAG-laser (UP193 SS, New Wave Research Co. Ltd., Cambridge, UK), and a coupled quadrupole ICP-MS (7500 CX, Agilent technologies, CA, USA) determined the 34 S signal. Hypocotyl vascular tissue was fixed, embedded and cut for laser microdissection pressure-catapulting (LMPC). Cell structures were extracted with the P.A.L.M. Microbeam system including a Capmover (Zeiss Microimaging GmbH, München, Germany). The TargetAmp™ 2-Round aRNA Amplification Kit 2.0 (Epicentre Biotechnologies, Madison, USA) was applied for the in vitro transcription 57 DGP Meeting September 5-9, 2012 of Poly(A) RNA. For analysis of gene expression cDNA was synthesized with the Revert Aid™ H Minus Kit. The SYBR Green-based relative qRT-PCR mix included gene-specific primer pairs and 50 ng of cDNA.

Results and Discussion A V. dahliae -specific absolute qRT-PCR assay enabled the sensitive measurement of fungal gDNA in tomato plants with divers S nutritional status and thus reflected S nutrition-dependent changes in fungal colonization patterns. High S nutrition significantly reduced fungal spread in the stem of both tomato genotypes. Light microscopy visualized the plant’s protective vascular coating reaction of xylem vessels in stained hypocotyl cross sections. Highest numbers of suberin-coated vessels were detected in the sensitive genotype and supra-optimal S supply significantly reduced the number of infected cells in both genotypes. Shoot growth was significantly impaired by a fungal colonization, but a curative effect of supra-optimal S nutrition was not reflected. Also pathogen-affected root growth did not display a distinct alleviating influence of S nutrition in both genotypes. Analysis of photosynthesis rates underlined the role of V. dahliae as a biotic stressor causing wilting disease. Rates of leaf photosynthesis were impeded by fungal infection more in the sensitive genotype and under low S nutrition. However, assimilate transport rates in the phloem sap were enhanced by fungal infection more in the resistant genotype and under high S suggesting a stronger sink for assimilates in infected plant tissues possibly involved in sugar-induced defense. Cys and GSH were influenced by S supply and genotype in tomato hypocotyl tissue. A consumption of Cys and GSH after V. dahliae infection in resistant plants suggested an implication in SED. LA-ICP-MS identified the localization and spatial distribution of S in tomato hypocotyl cross-sections. In the presence of V. dahliae the frequency of S peak-concentrations was particularly high in resistant high S-supplied vascular bundles. This might indicate a more localized accumulation pattern of S 0 which might be decisive for the fungicidal effectiveness. A qRT-PCR expression study with selected S assimilation-related candidate genes possibly contributing to tomato SED mechanisms was performed. The effect of a variable S nutrition in combination with a V. dahliae infection was analyzed within both tomato genotypes. In entire hypocotyl and cortical tissue, genes facilitating the first steps of the S assimilation pathway showed an S demand-driven expression which was negatively influenced by the fungus. Genes belonging to GSH biosynthesis did not respond to the S supplies but were up-regulated more in the resistant tomato genotype due to a V. dahliae infection. Thus, GSH might contribute to a global redox homeostasis. For the identification of tissue-specific changes in the 58 DGP Meeting September 5-9, 2012 expression of S metabolism-related genes vascular cells were extracted by LMPC. GSH synthesis-associated genes showed a strong cell-specific down-regulation, and especially genes directly contributing to Cys biosynthesis were up-regulated. Therefore, Cys seems to be a key metabolite, possibly being a precursor for S 0 and thus locally contributing to vascular SED. In conclusion, the analysis of selected physiological parameters in combination with an absolute quantification of the in planta fungal spread confirmed S nutrition- enhanced resistance of tomato against V. dahliae mediated by SDCs. Additionally, the transcription of S metabolism-related genes revealed a major role of GSH as a antioxidant for global redox-homeostasis and a cell-specific contribution of Cys to SED against V. dahliae in tomato vascular bundles.

Literature Cakmak, I., Hengeler, C. & Marschner, H. (1994). Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants. Journal of Experimental Botany, 45, 1251 - 1257. Fradin, E. F. & Thomma, B. P. (2006). Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum . Molecular Plant Pathology, 7, 71 - 86. Fradin, E. F., Zhang, Z., Juarez Ayala, J. C., Castroverde, C. D., Nazar, R. N., Robb, J., Liu, C. M. & Thomma, B. P. (2009). Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiology, 150, 320 - 332. Goicoechea, N. (2009). To what extent are soil amendments useful to control Verticillium wilt? Pest Managment Science, 65, 831 - 839. Larkin, R. P., Griffin, T. S. & Honeycutt, C. W. (2010). Rotation and cover crop effects on soilborne potato diseases, tuber yield, and soil microbial communities. Plant Disease, 94, 1491 - 1502. Novo, M., Gayoso, C. M., Pomar, F., Lucas, M. M., Ros Barceló, A. & Merino, F. (2007). Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance. Plant Pathology, 56, 998 – 1004. Williams, J. S., Hall, S. A., Hawkesford, M. J., Beale, M. H. & Cooper,R. M. (2002). Elemental S and thiol accumulation in tomato and defence against a fungal vascular pathogen. Plant Physiology,128, 150 – 159.

59 DGP Meeting September 5-9, 2012 Physiological and molecular analysis of the spatial characteristics of aluminum uptake and translocation in roots of buckwheat (Fagopyrum esculentum )

Thomas W. Kirchner 1*, B. Klug 1, A. Erban 2, J. Kopka 2, W.J. Horst 1

1Institute of Plant Nutrition, Leibniz Universität Hannover; 2Max Planck Institute of Molecular Plant Physiology, Golm. E-mail: [email protected]

Introduction Aluminum (Al) toxicity is one of the most important factors limiting plant growth on acid soils. Whereas most plant species adapt to Al-toxic soils by exclusion of Al from uptake and transport to the shoots, buckwheat ( Fagopyrum esculentum ) is capable of both exclusion of Al from uptake by the exudation of oxalate (Zheng et al., 1998) and accumulation of Al in the leaves (Ma et al., 2001, Shen et al., 2002). Recently we have shown that the Al-induced root release of oxalate, Al uptake as well as Al accumulation are spatially not separated in the root apex (Klug and Horst, 2010a). However, localization of Al along the root tip in the root cortex and the central cylinder supported the view that the 10 mm apical root tip is the main site of Al uptake into the symplast of the root cortex, while the sub-apical 10–20 mm zone is the main site of xylem loading through the pericycle and xylem parenchyma cells (Klug et al., 2011). In the present work we studied the transcription of selected genes and applied metabolic profiling to further elucidate the spatial characteristics of Al uptake and translocation in root apices of buckwheat.

Materials and Methods Shoots of buckwheat ( Fagopyrum esculentum Moench) cultivar ´Lifago´ were adventitiously rooted in full nutrient solution and then treated without or with 75 µM Al in simplified nutrient solution for 24 h (Klug and Horst, 2010b). After the treatment the apical two cm of each root tip were cut into four 5-mm segments. Central cylinder and cortex tissues were separated using a conventional wire stripper. The separated root tissues were immediately placed into liquid nitrogen and stored at 80 °C till RNA isolation or extraction of the metabolites. Degenerate primers were designed based on the consensus sequence of the gene sequences of at least three different plant species per gene as nearly related to buckwheat as possible. qRT-PCR was performed according to Fleck et al. (2011), but 84 ng cDNA was used as template and the number of cycles was 44 (168 ng cDNA and 50 cycles in the cortex-central cylinder analysis). An actin gene was used as endogenous control. Relative expression was calculated using the 2 - CT method according to Livak and Schmittgen (2001). GC-MS-based metabolite profiling was performed according to Führs et al. (2012). 60 DGP Meeting September 5-9, 2012 Results and Discussion Among the genes studied, only for Aco , CS (Fig. 1A) and PEPC the response to Al was root tissue-specific. The expression of these genes was increasingly up- regulated by Al towards the distal root sections. In contrast, the expression decreased from the root apex to more distal root sections in the untreated plants (significant Al*tissue interaction). All studied genes were higher expressed in the root cortex compared to the central cylinder. Al treatment enhanced the expression of CS (Fig. 1B), L-GLDH and PEPC in both tissues; however, this increase was only significant for CS and PEPC in the central cylinder.

(A) 2,0 (B) 8 Al* a Al* Tissue n.s. A Tissue** Al*Tissue* a Al*Tissue n.s. 1,5 B 6 a 0 µM Al a A a ab a 75 µM Al A a a A A A 1,0 A A 4

b a 0,5 A 2 b B A Relative gene Relative expression [-]

0,0 0 S1 S2 S3 S4 Cortex Central cylinder

Figure 1: Relative gene expression of the citrate synthase gene in 5-mm root segments (A) and in the cortex and central cylinder (B) of buckwheat root apices. Small letters denote significant differences between segments; capital letters between treatments. Stars represent the result of the ANOVA with *, **, *** for P < 0.05, 0.01, 0.001, respectively. Data represent means ± SE, n=3.

In order to find out which metabolites are responsible for root spatial and root tissue- specific differences in Al response and Al transport, 5-mm root sections and separated root cortex and central cylinder tissues were subjected to metabolic profiling. First, ICA was performed to check if the major variances in the concentrations of metabolites are due to the experimental factors Al treatment and root tissue. In the comparative analysis of the root cortex/central cylinder tissues, the metabolites clearly separately clustered according to root tissue and within the root tissues according to Al treatment (Fig. 2). The ICA of the segmental analysis resulted in a diagonal clustering owing to Al treatment. Within the Al treatments only the apical 5-mm segment separately clustered, whereas the clusters for the three distal segments overlapped.

61 DGP Meeting September 5-9, 2012 The detailed analysis of those metabolites 3 / Central cylinder, -Al / +Al which responded significantly (P < 0.05) to Al / Cortex, -Al / +Al treatment and/or showing a significant 2 Al*root-tissue/segment interaction revealed 1 that almost all metabolites were present in the -Al -Al central cylinder in higher concentrations +Al 0 (fresh weight basis) compared to the cortex. +Al Among the carboxylic acids only citrate was -1 significantly reduced by Al in the cortex. Al component 2 Independent Cortex Central cylinder treatment significantly decreased the -2 concentrations of all four carboxylic acids in -2 -1 0 1 2 Independent component 1 nearly all root segments. The concentrations Figure 2: Independent component analysis under Al treatment were mostly lower in distal (ICA) of the metabolite profiling of the cortex compared to the apical segments. and the central cylinder. ICA was conducted using MetaGeneAlyse at http://metagene Glycolytic metabolites significantly affected alyse.mpimp-golm.mpg.de. showed a comparable response to Al in the cortex and the central cylinder. Al supply decreased the concentrations in the cortex and increased them in the central cylinder leading to a significant interaction. All metabolites showed the highest concentrations in the apical segment independent of the Al treatment. Al treatment generally decreased metabolite concentrations. Oxalate may be synthesized via ascorbate or glycerate. Among the identified metabolites from the oxalate synthesis pathways oxalate and galactose showed an Al-induced decrease in the cortex and increase in the central cylinder (significant Al*root-tissue interaction). The analysis of the apical 5-mm root segments generally showed a decreasing effect of Al on the metabolite concentrations most clearly and consistently in the apical segment. The results of the transcriptomic and metabolomics analysis confirm a major role of oxalate in the root cortex for the detoxification of Al in the root apoplast by root exudation and transport of Al into the central cylinder and of citrate in the central cylinder for the xylem transport of Al to the shoots particularly in distal segments of the root apex.

Literature Fleck, A.T.; Nye, T.; Repenning, C.; Stahl, F.; Zahn, M.; Schenk, M.K. (2011): Silicon enhances suberization and lignification in roots of rice (Oryza sativa). Journal of Experimental Botany 62: 2001–2011. Führs, H.; Specht, A.; Erban, A.; Kopka, J.; Horst, W.J. (2012): Functional associations between the metabolome and manganese tolerance in Vigna unguiculata. Journal of Experimental Botany 63, 329–340. Klug, B.; Horst, W.J. (2010a): Spatial characteristics of aluminum uptake and translocation in roots of buckwheat (Fagopyrum esculentum). Physiologia Plantarum 139, 181–191. 62 DGP Meeting September 5-9, 2012 Klug, B.; Horst, W.J. (2010b): Oxalate exudation into the root-tip water free space confers protection from aluminum toxicity and allows aluminum accumulation in the symplast in buckwheat (Fagopyrum esculentum). New Phytologist 187, 380–391. Klug, B.; Specht, A.; Horst, W.J. (2011): Aluminium localisation in root tips of the aluminium- accumulating plant species buckwheat (Fagopyrum esculentum Moench). Journal of Experimental Botany 62, 5453–5462. Livak, K.J.; Schmittgen, T.D. (2001): Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Ma, J.F.; Ryan, P.R.; Delhaize, E. (2001): Aluminium tolerance in plants and the complexing role of organic acids. Trends in Plant Science 6, 273–278. Shen, R.F.; Ma, J.F.; Kyo, M.; Iwashita, T. (2002): Compartmentation of aluminium in leaves of an Al- accumulator, Fagopyrum esculentum Moench. Planta 215, 394–398. Zheng, S.J.; Ma, J.F.; Matsumoto, H. (1998): High aluminum resistance in buckwheat. I. Al-induced specific secretion of oxalic acid from root tips. Plant Physiology 117, 745–751.

63 DGP Meeting September 5-9, 2012 Ascorbate metabolism in rice genotypes differing in zinc efficiency

Stefanie Höller 1, Mohammad-Reza Hajirezaei 2, Nicolaus von Wirén 2, Michael Frei 1

1Institute for Crop Science and Resource Conservation (INRES) – Plant Nutrition /University of Bonn, Bonn; 2Dept. Physiology and Cell Biology/ Leibniz-Institute for Plant Genetics and Crop Plant Research, Gatersleben. E-mail: [email protected]

Introduction Zinc (Zn) deficiency is one of the most abundant abiotic stress factors worldwide and affects rice growth and yield in many parts of the world. Zn has numerous functions in the cell, such as involvement in protein folding or as a catalytic or co-catalytic factor of thousands of proteins (Broadley et al., 2007). Similar to other abiotic stress factors, Zn deficiency can cause oxidative stress in plants by perturbing the equilibrium between the production and removal of reactive oxygen species (ROS). Excess production of ROS can lead to severe damage of lipids, proteins and DNA (Apel and Hirt, 2004). Several previous studies showed that Zn efficient and inefficient varieties of crops often do not differ in Zn uptake and shoot Zn concentration, suggesting that ROS detoxification could be an important tolerance trait that can be targeted in the breeding of tolerant crops (Hacisalihoglu and Kochian, 2003). In previous studies the recombinant inbred line RIL46 was selected from a rice mapping population, as it showed less symptoms of oxidative stress when grown in low Zn soil than its intolerant parent IR74 (Frei et al., 2010). It was proposed that tolerance to Zn deficiency in RIL46 was associated with enhanced ascorbic acid (AsA) metabolism. AsA levels of both genotypes declined under –Zn conditions while RIL46 had a constitutively higher AsA level than IR74. Based on this previous study we concluded that the AsA metabolism is crucial in controlling excess ROS under Zn deficiency. The predominant biosynthesis pathway of AsA starts from GDP-D- Mannose via L-Galactose (mannose/L-galactose pathway), but there is also evidence that other pathways via uronic acids intermediates exist. One alternative pathway starts from myo -inositol (Lorence et al., 2004) and is mediated by the enzyme myo - inositol oxygenase. AsA can detoxify ROS directly or via the enzymatic network of the ascorbate-glutathione cycle, in which AsA is constantly oxidized to dehydroascorbate (DHA) and recycled by subsequent reduction (Ishikawa et al., 2006). If not recycled immediately, DHA is further degraded to products such as oxalic acid or tartaric acid (Green and Fry, 2005). The aim of this study was to characterize the AsA metabolism in the contrasting genotypes IR74 and RIL46 under Zn deficiency. Based on previous studies we built three hypotheses on the relation between Zn efficiency and AsA metabolism. (1) The reduced AsA content of IR74 under Zn deficiency is due to inhibited or reduced

64 DGP Meeting September 5-9, 2012 biosynthesis of AsA. Here we focused on the analysis of the different biosynthesis steps and their contribution to the AsA level of the two genotypes. (2) Insufficient recycling of AsA and the subsequent degradation explains the decline of the AsA pool of IR74 under –Zn stress. (3) The contrasting tolerance of the two genotypes can be explained by their different ability to maintain AsA poolsize.

Materials and Methods

Seeds were germinated and placed on Styrofoam sheets floating on 0.5 mM CaCl 2 and 10 µM FeCl 3. After 10 days seedlings were transferred to 60 l hydroponics tanks containing half strength nutrient solution without Zn. After one week plants were transferred to full nutrient solution containing either no (-Zn) or 1 µM Zn (+Zn). Shoot samples were taken on three different sampling days: (1) three days after the start of –Zn treatments; (2) when stress symptoms first emerged in the susceptible genotype after 16 days of –Zn treatment; (3) when IR74 had severe stress after 24 days of –Zn treatment. A leaf bronzing score ranging from 0 (healthy leaf) to 10 (dead leaf) was assigned to the three youngest fully expanded leaves of each plant. Samples (ten replicates per genotype / treatment combination per sampling day) were immediately frozen in liquid N and stored at -80°C. RNA for RT- PCR was extracted from 4 replicate samples, respectively using the RNeasy Plant Minikit (Qiagen). One µg of total RNA was reverse transcribed with the GoScript™ Reverse Transcription System and PCR was performed with 100ng cDNA using the GoTaq® qPCR Master Mix (Promega). Gene specific primers were used for analysis of the different isoforms of all annotated biosynthesis genes of the mannose/L-galactose pathway and the myo - inositol pathway. Total and reduced AsA were measured according to Gillespie and Ainsworth (2007). Malonedialdehyde was measured as an indicator of lipid peroxidation under oxidative stress (Hodges et al., 1999). Dehydroascorbate reductase activity was measured according to Hossain and Asada (1984). Zn concentration was measured in dried leaf material at 60°C for 72h by atomic absorption spectrometry (AAS). Amino acids and sugars were analyzed using either a reverse phase chromatography or an ion chromatography system of fluorescence or amperometric detectors at the Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK). Further analysis of ascorbate precursors and degradation products is in progress.

Results and Discussion As expected, IR74 showed severe symptoms of Zn deficiency after 24 days of treatment, which was reflected in substantial lipid peroxidation, while no symptoms were observed in RIL46. Leaf bronzing of IR74 under Zn deficiency began on the second sampling day and increased to around 5 until the third sampling day. Shoot Zn concentration of plants grown in –Zn conditions were below 30 µg g -1, with even 65 DGP Meeting September 5-9, 2012 lower concentrations in RIL46. Since no significant genotypic differences were observed, tolerance of RIL46 to Zn deficiency could not be explained by a higher Zn uptake. Consistent with our previous studies, a significant difference in treatment by genotype interaction was found for total and reduced AsA concentrations. They decreased in IR74 under Zn deficiency after 24 days, while the opposite was found for RIL46. On the third sampling day, RIL46 had an approximately 30% higher AsA concentration than IR74. We tested DHAR enzyme activity but could not find any genotypic differences. This was in line with previous studies suggesting no enhanced enzymatic ROS detoxification in RIL46 (Frei et al., 2010). We further analyzed AsA biosynthesis by monitoring gene expression patterns. Pairwise comparison of gene expression levels showed that four out of seven genes of the mannose/L-galactose–pathway were more highly expressed in RIL46 compared to IR74 in the same treatment and sampling day. In IR74, expression of biosynthesis genes showed no difference between –Zn and control conditions, suggesting that reduced AsA content under Zn deficiency cannot be explained by a decreased biosynthesis. A highly significant treatment by genotype interaction was seen in the expression of myo -inositol oxygenase, which was induced by the –Zn treatment in RIL46 but not in IR74. This suggests that the myo -inositol pathway may contribute to higher AsA levels in RIL46, and therefore to enhanced Zn efficiency under stress conditions. Concentrations of amino acids were measured in addition to substrates of the AsA metabolism. Glycine concentrations increased under Zn deficiency, but more so in IR74, which could be explained by the fact, that glycine serves as a building block for glutathione, an indicator of oxidative stress that was previously shown to be induced under Zn deficiency (Frei et al., 2010). Asparagine content increased during Zn deficiency but no significant genotype by treatment interaction could be detected. Proline accumulated during Zn deficiency in both RIL46 and IR74 until the second sampling day. On the third sampling day it strongly increased in RIL46 but slightly decreased in IR74. Proline has been well characterized as a stress tolerance factor under salt and drought stress (Ashraf and Foolad, 2007), but our data strongly suggest that it is also involved in tolerance to Zn deficiency. In conclusion, enhanced expression of AsA biosynthesis genes in RIL46 could partly explain the increased AsA concentration, supporting our first hypothesis. Moreover, the gene expression data also suggested that the decrease in AsA level in IR74 under Zn deficiency could not be explained by a decrease in biosynthesis, which strengthens the second hypothesis of degradation of the AsA pool. Further ongoing analysis of AsA degradation products will help to interpret the present data. Overall, the data support the third hypothesis that contrasting tolerance is related to differences in AsA metabolism between RIL46 and IR74.

66 DGP Meeting September 5-9, 2012 Literature Apel K., Hirt H. (2004) Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology 55:373-399. Ashraf M., Foolad M.R. (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59:206-216. DOI: 10.1016/j.envexpbot.2005.12.006. Broadley M.R., White P.J., Hammond J.P., Zelko I., Lux A. (2007) Zinc in plants. New Phytologist 173:677-702. DOI: 10.1111/j.1469-8137.2007.01996.x. Frei M., Wang Y., Ismail A.M., Wissuwa M. (2010) Biochemical factors conferring shoot tolerance to oxidative stress in rice grown in low zinc soil. Functional Plant Biology 37:74-84. DOI: 10.1071/fp09079. Gillespie K.M., Ainsworth E.A. (2007) Measurement of reduced, oxidized and total ascorbate content in plants. Nature Protocols 2:871-874. DOI: 10.1038/nprot.2007.101. Green M.A., Fry S.C. (2005) Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O- oxalyl-L-threonate. Nature 433:83-87. DOI: 10.1038/nature03172. Hacisalihoglu G., Kochian L.V. (2003) How do some plants tolerate low levels of soil zinc? Mechanisms of zinc efficiency in crop plants. New Phytologist 159:341-350. DOI: 10.1046/j.1469-8137.2003.00826.x. Hodges D.M., DeLong J.M., Forney C.F., Prange R.K. (1999) Improving the thiobarbituric acid- reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604-611. DOI: 10.1007/s004250050524. Hossain M.A., Asada K. (1984) PURIFICATION OF DEHYDROASCORBATE REDUCTASE FROM SPINACH AND ITS CHARACTERIZATION AS A THIOL ENZYME. Plant and Cell Physiology 25:85-92. Ishikawa T., Dowdle J., Smirnoff N. (2006) Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiologia Plantarum 126:343-355. DOI: 10.1111/j.1399- 3054.2006.00640.x. Lorence A., Chevone B.I., Mendes P., Nessler C.L. (2004) myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiology 134:1200-1205. DOI: 10.1104/pp.103.033936.

67 DGP Meeting September 5-9, 2012 Silicon decreases arsenic concentration in rice grain by limiting arsenite transport

Alexander T. Fleck 1* , Jürgen Mattusch 2, Manfred K. Schenk 1

1Institute of Plant Nutrition, Faculty of Natural Sciences, Leibniz Universität Hannover, Hannover, Germany. 2UFZ, Centre for Environmental Research Leipzig-Halle, Department of Analytical Chemistry, Leipzig, Germany. *corresponding author, E-mail: [email protected]

Introduction Rice can contain high levels of the toxic and carcinogenic metalloid arsenic (As), exhibiting a relevant source of human As intake (Kile et al ., 2007). The high As levels in rice are caused on one hand by the cultivation of paddy rice on flooded fields under reducing conditions, which lead to the mobilization of As (Ponnamperuma, 1984), and on the other hand by an efficient uptake of As(III), which is taken up into the rice root and transported to the xylem by the transporters Lsi1 and Lsi2 that have firstly been identified as transporters of silicic acid (Ma et al ., 2008). The aquaporin Lsi1 also transports the organic As species monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) into rice roots (Li et al ., 2009a). The beneficial element silicon (Si) was shown to reduce As levels in rice shoot and grain, however, underlying mechanisms remain unclear (Guo et al ., 2005, 2009). Furthermore, Si decreased the inorganic As level in grain, while the DMA concentration was increased (Li et al ., 2009b). However, an explanation for the latter could not be given.

Materials and Methods Pot experiments with three Italian rice paddy soils were conducted in the greenhouse from April to August with average temperatures around 28°C. The soils were supplied with 0 (-Si) or 10 g (+Si) silicagel/kg and flooded seven days before rice planting until the harvest. Seeds of rice ( Oryza sativa L., cv. Selenio) were germinated in tap water for seven days, each 35 seedlings were transplanted in a pot and plants were harvested at maturity after 147 days. The soil solution was collected weekly using suction cups and analyzed for Si, As, phosphorus (P) and iron (Fe). The redox potential of the soils was measured weekly using a Pt electrode embedded at a 20 cm depth. Silicic acid in the soil solution and extracted Si from the straw after digesting with HCl and HF were determined photometrically (Novozamsky et al ., 1984). As, P and Fe in the soil solution were measured with ICP-MS 7500c. Dried and milled straw, husk, brown rice, and polished rice were digested in a microwave with HNO 3 and H 2O2 and analyzed for total As with ICP-MS 7500c. As for speciation in polished rice was extracted by digesting dried and milled plant matter in HNO 3 (Huang et al ., 2010) and As species were analyzed using HPLC-ICP-MS. The sum of As species in polished rice was on average 103% of the total As in polished rice. 68 DGP Meeting September 5-9, 2012 Results and Discussion After flooding, the redox potential in all soils decreased, while concentrations of Fe, As, P, and Si in the soil solution increased. The As concentration in the soil solution increased in the same pattern as the Fe concentration, since As(V) is bound to Fe (hydr)oxides and released into the soil solution when these are reduced (Bogdan and Schenk, 2008; Yamaguchi et al ., 2011). Si application to soils increased the concentration of Si, but also of P and As in the soil solution. The latter can be explained by a substitution by silicic acid because it competes with phosphate and arsenate for the same adsorption sites (Waltham and Eick, 2002).

The straw and grain yield increased with Si supply in all three soils, which is in line with reports of beneficial Si effects on rice (Ma et al ., 1989). The Si concentration in straw was enhanced from around 15 mg/g d.m. in the –Si plants to on average 40 mg/g d.m. in the +Si plants. The highest As concentrations were found in the straw, followed by husk, brown rice, and polished rice, which is the same pattern as observed in other studies (Rahman et al ., 2007). The As concentration in polished and brown rice was an order of magnitude smaller than in straw, which is in accordance with previous studies (Bogdan and Schenk, 2008). The Si supply reduced the As concentration in both straw and husk by half, while the As concentrations in brown rice and polished rice were reduced by only 22 %. These Si effects are in line with literature (Guo et al ., 2005; Li et al ., 2009b). The main As species in the soil solution likely was As(III) as reported in several studies with flooded soils (Bogdan and Schenk, 2008; Li et al ., 2009b). The transport of As(III) into the rice roots may be reduced by Si supply, since the expression of Lsi1 and Lsi2 is decreased by Si supply (Ma and Yamaji, 2008). Other authors suggest that the reason for a decreased As concentration in rice is a competitive inhibition of As(III) transport by silicic acid (Guo et al ., 2009; Li et al ., 2009b; Zhao et al ., 2009). However, for Lsi1 this could not be verified (Bienert et al ., 2008), but a competitive inhibition of As(III) transport by Si could take place at Lsi2. The main As species in polished rice was As(III) with a fraction of 70 %, followed by DMA and As(V) with 24 and 6 %, respectively (Fig. 1). As(III) transport into rice grain occurs nearly exclusively via the phloem (Carey et al ., 2010; Zhao et al ., 2012) and As(III) may be loaded into the phloem in the leaves as well as in the stem nodes by xylem-to-phloem transfer. The Si transporter Lsi6, which unloads Si from the xylem in the node, might be involved in the transport processes in the nodes, since it is a homolog of the aquaporin Lsi1 and the selectivity of aquaporins for As(III) seems to be lower than for silicic acid (Yamaji and Ma, 2009; Mitani-Ueno et al ., 2011).

Si application to soil did not affect DMA or As(V) concentration in polished rice, while As(III) concentration was reduced by 33 %, so a decreased As concentration in

69 DGP Meeting September 5-9, 2012 polished rice can be attributed solely to a reduced As(III) concentration.

A As(III) a DMA 300 A As(V) ab A b B 200 a B B a a

A A A a a A a 100 ab A A b b A A A A A A a a a a a a

Polished rice As [µg / d.m.] kg [µg As Polished rice 0 -Si +Si -Si +Si -Si +Si

Soil L Soil G Soil D Fig. 1: Concentration of As species in polished rice grown in three rice paddy soils as affected by Si supply. Different capital and small letters indicate significant difference between Si treatments of a soil and between soils of a Si treatment, respectively; tukey-test with p < 0.05. Bars are SE.

The DMA concentration in the grain is related to the DMA level in the soil solution, since DMA is synthesized in the soil solution from soil microorganisms, while plants are not able to methylate inorganic As (Lomax et al ., 2012). In contrast to As(III), the uptake of DMA is not affected by Si application since DMA is not taken up by Lsi2 but by Lsi1, where a competitive inhibition by silicic acid was not observed. Moreover, a reduced transporter expression by Si supply would not have been effective for DMA because compared to As(III) the DMA concentration in the soil solution is much lower (Bogdan and Schenk, 2008; Xu et al ., 2008) and the affinity of transporters for DMA is much less (Abedin et al ., 2002).

Literature Abedin MJ, Feldmann J, Meharg AA. (2002). Plant Physiology 128, 1120-1128. Bogdan K, Schenk MK. (2008). Environmental Science and Technology 42: 7885–7890. Carey AM, Scheckel KG, Lombi E, Newville M, Choi Y, Norton GJ, Charnock JM, Feldmann J, Price AH, Meharg AA. 2010. Plant Physioogyl 152, 309-319. Guo W, Hou YL, Wang SG, Zhu YG. (2005). Plant and Soil 272, 173–181. Guo W, Zhang J, Teng M, Wang LH. (2009). Journal of Plant Nutrition and Soil Science 172, 867–874. Huang J-H, Ilgen G, Fecher P. (2010). Journal of Analytical Atomic Spectrometry 25, 800. Kile ML, Houseman EA, Breton CV, Smith T, Quamruzzaman Q, Rahman M, Mahiuddin G, Christiani DC. (2007). Environmental Health Perspectives 115, 889–893.

70 DGP Meeting September 5-9, 2012 Li RY, Ago Y, Liu WJ, Mitani N, Feldmann J, McGrath SP, Ma JF, Zhao FJ. (2009a). Plant Physiology 150, 2071–2080. Li RY, Stroud JL, Ma JF, McGrath SP, Zhao FJ. (2009b). Environmental Science & Technology 43, 3778–3783. Lomax C, Liu WJ, Wu L, Xue K, Xiong J, Zhou J, McGrath SP, Meharg AA, Miller AJ, Zhao FJ. (2012). New Phytologist 193, 665-672. Ma JF, Kazuo N, Eiichi T. (1989). Soil Science and Plant Nutrition 35, 347-356. Ma JF, Yamaji N, Mitani N, Xu XY, Su YH, McGrath SP, Zhao FJ. (2008). Proceedings of the National Academy of Sciences 105, 9931-9935. Ma JF, Yamaji N. (2008). Cellular and Molecular Life Sciences 65, 3049–3057. Mitani-Ueno N, Yamaji N, Zhao FJ, Ma JF. (2011). Journal of Experimental Botany 62, 4391–4398. Novozamsky I, van Eck R, Houba VJG. 1984. Communications in Soil Science and Plant Analysis 15: 205–211. Ponnamperuma FN. (1984). In: Kozlowski TT, ed. Flooding and plant growth , New York, NY, USA: Academic Press, 9–45. Rahman MA, Hasegawa H, Rahman MM, Rahman MA, Miah MA. (2007). Chemosphere 69, 942-948. Waltham CA, Eick MJ. (2002). Soil Science Society of America Journal 66, 818-825. Xu XY, McGrath SP, Zhao FJ. (2007). New Phytologist 176, 590-599. Yamaguchi N, Nakamura T, Dong D, Takahashi Y, Amachi S, Makino T. (2011). Chemosphere 83, 925-932. Yamaji N, Ma JF. (2009). The Plant Cell 21, 2878–2883. Zhao FJ, Ma JF, Meharg AA, McGrath SP. (2009). New Phytologist 181, 777–794. Zhao F-J, Stroud JL, Khan MA, McGrath SP. (2012). Plant and Soil 350, 413-420.

71 DGP Meeting September 5-9, 2012 Physiological and molecular analysis of manganese toxicity and manganese leaf-tissue tolerance in rice ( Oryza sativa L.)

Martin Duschyk, Hendrik Führs and Walter J. Horst

Institute of Plant Nutrition, Leibniz University Hannover, Hannover. E-mail: [email protected]

Introduction In many tropical and subtropical areas manganese (Mn) toxicity on acid soils is a problem, which results in yield reduction (Foy, 1984). Liming is often financially not feasible or meaningful. Great differences in Mn tolerance were observed between different species and cultivars (Horst, 1988), which could contribute to develop Mn- tolerant cultivars. Rice ( Oryza sativa L.) is one of the most important staple food crops for human nutrition in many parts of the world. Evolutionary, paddy rice had to adapt to conditions arising from flooded soils with highly reduced conditions. Transport of oxygen from the shoots to the roots is an efficient mechanism to avoid Fe toxicity, since reduced iron is efficiently oxidized at the rice root surface and, thereby, Fe uptake is restricted (Zhang et al., 1998, Kirk, 2003). However, the root oxidizing capacity is sufficient for quantitative Fe but not Mn oxidation, since Mn oxidation requires a higher redox potential than Fe (Mansfeldt, 2004). Hence, rice shoot Mn accumulation is much less restricted, so that rice had to develop efficient mechanisms to tolerate high Mn tissue concentrations. Whereas Mn concentrations of >150 µg (g dry weight)-1 lead to Mn toxicity symptoms in barley (Vlamis and Williams 1964), rice can tolerate up to 5000 µg (g dry weight)-1 without showing any symptoms (Vlamis and Williams, 1964, Führs et al., 2010). However, above a certain threshold also rice suffers from Mn toxicity expressed as typical brown spots (Mn oxid and phenol depositions) on older leaves such as was described in the Mn-sensitive plant species cowpea. These brown spots consist of oxidized Mn and oxidized phenolic compounds (Wissemeier and Horst, 1992) in the cell wall of the epidermis. Greatly enhanced activities of H2O2-producing and H2O2-consuming peroxidases particularly in the apoplastic washing fluid (AWF) suggest that the leaf apoplast is the decisive compartment for the development or avoidance of Mn toxicity in cowpea (Fecht-Christoffers et al., 2006; Fecht-Christoffers et al., 2007). Subsequent studies suggested that apoplastic H2O2-producing peroxidase-enhancing and inhibiting phenols play a major role in modulating genotypic differences and Si-enhanced Mn tolerance (Fecht-Christoffers et al., 2006, Führs et al., 2009). In this study a Recombinant Inbred Line (RIL) population derived from a Mn-sensitive japonica parent (´Azucena´) and a Mn-tolerant indica parent (´IR1552´) showed great variation in leaf Mn threshold concentrations leading to Mn toxicity symptoms. Physiological

72 DGP Meeting September 5-9, 2012 and molecular studies are being conducted to uncover the reasons for the major differences in Mn leaf-tissue tolerance of rice.

Materials and Methods In a first approach a population of 90 individual RILs was grown in a greenhouse experiment in full nutrient solution under semi-controlled environmental conditions (30 °C temperature, 90-100 % relative humidity). Th e plants were pre-cultured until the fifth leaf was fully developed. Subsequently, the Mn concentration of the nutrient solution was increased from 1 µM to 500 µM until the individual RILs developed toxicity symptoms in form of brown spots. The Mn treatment duration ranged between 6 to 14 days for sensitive and tolerant RILs, respectively. In a subsequent experiment four RILs with similar Mn uptake characteristics but great differences in the expression of toxicity symptoms [R20 and R23 (both Mn-sensitive), R90 and R120 (both Mn-tolerant)] were selected for in-depth characterization experiments. These experiments took place in a growth chamber under controlled environmental conditions (16/8 h day /night under 30/25 °C at 90 % relative humidity) in the same nutrient solution system as described for the screening experiment. After pre-culture the Mn concentration in the nutrient solution was increased from 1 µM to 500 µM for ten days or the plants received 1 µM Mn continuously. A treatment for ten days led to the development of toxicity symptoms in the leaves of the sensitive but not the tolerant lines. For quantification of toxicity symptoms the fifth fully developed leaves were excised and transferred to 96% ethanol in order to discolorate the leaves. After bleaching the percentage of the leaf area covered with brown spots was determined using the GSA Image Analyser (Bansemer & Scheel GbR, Rostock, Germany). To study Mn compartmentation and cell-wall Mn-binding capacity of leaves a cell-wall isolation procedure was established based on Rogalla and Römheld (2002). Manganese was determined by inductive coupled plasma - optical emission spectrometry (ICP-OES). Apoplastic washing fluid (AWF) was extracted from leaves and NADH-peroxidase activity, guaiacol-peroxidase activity, the protein and phenol concentration were measured as described by Führs et al. (2010). The apoplastic peroxidase isoenzyme profile was compared using native isoelectric focusing (IEF) (Pre-cast gels, BioRad, Munich, Germany) according to the manufacturer´s instructions,). To find out transcriptomic differences between the different RILs a Microarray approach was carried out by the Helmholtz Institute, Braunschweig (Agilent.SingleColor.15241)

73 DGP Meeting September 5-9, 2012 Results and Discussion The phenotyping for Mn toxicity symptoms of the RILs revealed great differences in Mn tolerance among the population exceeding by far the differences between the parents of the population. No correlation existed between Mn toxicity symptoms and Mn bulk-leaf concentrations. Two tolerant and two sensitive RILs were selected from this population for further in-depth studies. When the RILs were exposed to excess Mn for ten days the bulk-leaf Mn concentrations rose up to 140 µmol/(g dm), but the genotypes did not differ. The cell-wall fractionation showed that over 90% of the leaf Mn is bound in the cell wall. Nevertheless, the Mn concentrations in the AWF increased up to mM concentrations, which is very high in comparison to other plant species. Also the AWF Mn concentrations did not significantly differ between the cultivars. Thus, these results confirm the high Mn tolerance of rice. But neither the cell-wall binding nor the free apoplastic Mn concentration can explain the difference in Mn tolerance between the individual RILs. However, clear differences between the RILs emerged in the apoplastic peroxidase activity. After prolonged and high Mn supply the Mn-tolerant RILs showed lower activities of NADH- and guaiacol-POD than the Mn-sensitive RILs. In order to study, whether the increase in POD activity is due to a changed POD isoenzyme profile a native IEF-PAGE was performed with apoplastic proteins. Indeed, an enhanced release of new, therefore, additional POD isoenzymes as well as an increased abundance of constitutive POD isoenzymes into the apoplast was found particularly on the basic side of the isoenzyme profile of Mn-sensitive but not Mn-tolerant lines after excess Mn treatment. Moreover, the isoenzyme profile differed constitutively between the lines since particularly the sensitive genotype R20 exhibited a more intense POD band on the neutral side of the IEF gel, which was much weaker (R23, R90) or almost absent (R120) in the other genotypes. A transcriptomic approach using microarrays showed that many of the peroxidase genes were upregulated in the sensitive in comparison to the tolerant RILs. In conclusion, like in other plant species also in rice, leaf apoplastic peroxidases appear to be key mediators of Mn sensitivity, even though sensitivity reactions take place at a much higher tissue tolerance level compared to other plant species. Therefore, an in depth characterization of the apoplastic peroxidases and their modulation by Mn and phenols in the apoplast appear promising targets of future studies.

Literature Fecht-Christoffers MM, Führs H, Braun H-P, Horst WJ. 2006. The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance. Plant Physiology 140: 1451–1463.

74 DGP Meeting September 5-9, 2012 Fecht-Christoffers MM, Maier P, Iwasaki K, Braun H-P, Horst WJ. 2007. The role of the leaf apoplast in manganese toxicity and tolerance in cowpea (Vigna unguiculata L., Walp). In: Sattelmacher B, Horst WJ. eds. The apoplast of higher plants: compartment of storage, transport, and reactions. Dordrecht, The Netherlands: Springer, 307–322. Führs et al. (2010) Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare). Annals of Botany 105: 1129-1140,2010 Foy CD (1984) Physiological effects of hydrogen, aluminium, and manganese toxicities in acid soils. In F Adams. ed, Soils acidity and liming, Agron. Monograph 12 ASA-CSSSA, Madison, USA, pp. 57-97 Horst WJ (1988) The physiology of Mn toxicity. In RD Graham, RJ Hannam, NC Uren, eds, Manganese in soils and plants. Kluwer Academic Puplishers, Dodrecht/Boston/London. ISBN 90-247-3758-3, pp 175-188 Kirk GJD. 2003. Rice root properties for internal aeration and efficient nutrient acquisition in submerged soil. New Phytologist 159: 185–194. Mansfeldt T. 2004. Redox potential of bulk soil and soil solution concentration of nitrate, manganese, iron, and sulfate in two Gleysols. Journal of Plant Nutrition and Soil Science 167: 7–16. Vlamis J, Williams DE. 1964. Iron and manganese relations in rice and barley. Plant and Soil 20: 221– 231. Wissemeier AH, Horst WJ. 1992. Effect of light intensity on manganese toxicity symptoms and callose formation in cowpea (Vigna unguiculata (L.) Walp.). Plant and Soil 143: 299–309. Zhang X, Zhang F, Mao D. 1998. Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.). Zinc uptake by Fe-deficient rice. Plant and Soil 202: 33–39

75 DGP Meeting September 5-9, 2012 Dissection of physiological and genetic mechanisms of tolerance to iron toxicity in rice

Michael Frei, Lin-Bo Wu, Katrin Engel,, Mathias Becker

Institute of Crop Science and Resource Conservation (INRES), Plant Nutrition, Rheinische Friedrich Wilhelms-Universität Bonn. E-mail: [email protected]

Introduction Iron toxicity is a mineral disorder unique to paddy rice production, as it occurs only in anaerobic flooded soils, where iron is present in its reduced form Fe(II). Excess Fe(II) is taken up by rice plants and transported to the leaves, where it causes the generation of reactive oxygen species (ROS). The consequences are visible leaf damage (‘leaf bronzing’), stunted growth, and ultimately yield losses. It can affect rice at different growth stages. Iron toxicity at the seedling stage is frequently observed in inland valleys of West Africa and Madagascar, where strong rainfalls during the early cropping season flush large amounts of Fe(II) from adjacent slopes into rice fields. This leads to sudden episodes of iron toxicity (‘pulse stress’). Especially the young seedlings are not able to exclude or dilute the excess iron, which can cause complete crop failure (Becker and Asch, 2005). In contrast, in the inherently iron toxic acid sulfate soils, prevailing in coastal areas of Southeast Asia and Africa, a more gradual accumulation of in-situ reduced iron leads to a build-up of excess iron in the plant tissue (chronic stress), which tends to affect plants later in the growth season. Damage caused by iron toxicity may be alleviated by management practices such as field drainage (Becker and Asch, 2005) or fertilizer applications (Saharawat, 2004), but these are often not practically feasible. Therefore, the breeding of tolerant genotypes provides the most promising option to increase rice production on sites affected by excess iron. Various studies have reported genotype screens and quantitative trait loci (QTL) for iron toxicity tolerance in rice (Wan et al. , 2003; Shimizu et al. , 2005; Dufey et al. , 2009), but there is an obvious lack of agreement in the genotype tolerance rankings reported from different experiments. This may be due to different types of iron toxicity (pulse stress vs. chronic stress) induced in those experiments, additional confounding environmental factors ( e.g . climate), and different genotype-specific crop adaptation mechanisms. Our research is therefore based on the idea that successful breeding for tolerance to iron toxicity requires a fundamental understanding of the tolerance mechanisms, their genetic regulation, and their effectiveness under specific types of iron toxicity. We propose three distinct physiological mechanisms conferring tolerance (i) exclusion of Fe(II) at the root surface; (ii) compartmentation of excess Fe(II) within the root or leaf sheath to prevent damage on leaf blades, and (iii) the detoxification of ROS within 76 DGP Meeting September 5-9, 2012 the leaf symplast. These tolerance mechanisms are dissected by combining genotype screening, phenotyping, genome mapping, and mutant experiments.

Materials and Methods Experiments were conducted in a greenhouse with a photosynthetically active radiation (PAR) of >200 µmol m -2 sec -1, and minimum day / night temperatures set at 28 / 20 °C. Plants were grown in 40 L hydroponic co ntainers accommodating up to 60 plants at the seedling stage. The root zones of plants grown in the same containers were separated by PVC tubes fixed underneath a perforated covering plate into which the plants were strapped with foam wrappings. Seedlings were pre-grown for five weeks and then exposed to iron pulse stresses of 500 to 1500 ppm Fe(II) for up to five days. During the iron treatment, nutrient solutions were bubbled with nitrogen gas for 10 min at 2 h intervals to keep the redox potential below 100 mV and iron in the reduced state, thus avoiding precipitation. Plant material included diverse rice accession that had previously been reported as tolerant or sensitive, two QTL mapping populations (98 Nipponbare-Kasalath backcross inbred lines, and 140 IR29- Pokkali recombinant inbred lines), and mutants for putative tolerance genes. Tolerance was characterized by assigning a leaf bronzing score (LBS) to each leaf, determining the dry weight, and measuring the shoot iron concentrations. The plant material of contrasting genotypes was used for further physiological characterization.

Results and Discussion Screening Based on the screening in hydroponic culture containing excess Fe(II) (500, 1000, and 1500 ppm), diverse rice accessions were grouped into sensitive genotypes, tolerant excluders (low shoot iron concentration), and tolerant includes (tolerant despite high shoot iron concentration). Different varieties were identified representing each of the specific tolerance mechanism described earlier, such as: (i) Pokkali, which is a tolerant excluder, (ii) TOX3107, which is a tolerant includer retaining significant amounts of iron in the leaf sheaths, (iii) CK73, which is a tolerant despite high leaf iron concentration. As expected, the tolerance ranking observed in hydroponic culture war partly contradictory to rankings reported in other studies, especially those conducted on acid sulfate soils, or experiments mimicking chronic iron stress at later growth stages. In contrast, the experimental system used in our studies was suitable for mimicking iron pulse stresses during the seedling stage. Exclusion mechanism The landrace Pokkali was consistently one of the most tolerant genotypes when exposed to a pulse stress of 500, 1000 or 1500 ppm Fe(II). This was consistent with a low shoot iron concentration, indicating that Pokkali represents an iron exclusion

77 DGP Meeting September 5-9, 2012 mechanism. In order to dissect this trait genetically, mapping of quantitative trait loci (QTL) was performed using a population of 140 recombinant inbred lines of Pokkali, and IR29, an iron sensitive modern variety. Leaf bronzing scores (LBS) and dry weight was measured as phenotypic traits. The most promising QTL obtained was located on chromosome 2, in which the Pokkali allele conferred enhanced tolerance in terms of leaf bronzing score. The observed tolerance was due to an iron exclusion mechanism, as suggested by reduced shoot iron concentration in Pokkali, and a recombinant inbred line carrying the Pokkali allele at the QTL position. Further analyses showed that iron exclusion did not consistently occur due to precipitation of iron at the root surface. Therefore, we are currently investigating alternative exclusion mechanisms such as oxidation of iron in the rhizosphere, mechanical barriers to iron uptake (such as lignin or suberin) or enhanced root exudation. Compartmentation mechanism A method was developed to visualize the compartmentation of Fe(II) in rice tissues based on the selective formation of color complexes between Fe(II) and 2,2’ bipyridine, followed by bifocal microscopy (Engel et al., 2012). The method was used to demonstrate that substantial amounts of Fe(II) were retained in the leaf sheath or the apoplast of tolerant lines such as CK73. Moreover, we are currently investigating the involvement of ferritin in tolerance to iron toxicity, as it provides an important non- toxic storage form of iron in plant tissues. The rice genome contains two isoforms of the ferritin gene. While the two ferritin genes have an almost identical coding sequences, they show differential expression under various conditions, including excess iron supply (Stein et al. , 2009). Knockout and activation tagged mutants for both isoforms have been obtained from international seed banks, and homozygous plants identified in our laboratory will be characterized regarding their response to iron toxicity. Tissue tolerance The screening experiments led to the identification of certain genotypes that showed little symptoms despite high shoot iron concentration, such as CK73. We investigated whether this ‘shoot tolerance’ was associated with enhanced antioxidant capacity. Measurements of antioxidant concentrations and enzyme activities suggested that tolerance was correlated with the ability to maintain a high ascorbate pool under stress, possibly due to high activity of ascorbate recycling enzymes. Preliminary experiments using a rice mutant with low ascorbate concentration as compared to its wild type suggested a moderate influence of ascorbate pool size on tolerance to iron toxicity. Conclusions Substantial genotypic differences in tolerance to iron toxicity exist and can be

78 DGP Meeting September 5-9, 2012 exploited in the breeding of tolerant varieties. However, tolerance to iron toxicity is a highly variable trait that depends on numerous environmental factors, and on the growth stage of the rice plants. Our research therefore intends to elucidate specific mechanisms of tolerance and their genetic regulation, which will ultimately allow for the pyramiding of tolerance traits in the breeding of varieties for specific environments.

References Becker, M., Asch, F., (2005). Iron toxicity in rice—conditions and management concepts. Journal of Plant Nutrition and Soil Science 168, 558-573. Dufey, I., Hakizimana, P., Draye, X., Lutts, S., Bertin, P., (2009). QTL mapping for biomass and physiological parameters linked to resistance mechanisms to ferrous iron toxicity in rice. Euphytica 167, 143-160. Engel Fehler! Textmarke nicht definiert. , K., Asch, F., Becker, M., (2012). In vivo staining of reduced iron by 2,2‘ bipyridine in rice exposed to iron toxicity. Journal of Plant Nutrition and Soil Science, doi: 10.1002/jpln.201200096. Shimizu, A., Guerta, C.Q., Gregorio, G.B., Kawasaki, S., Ikehashi, H., (2005). QTLs for nutritional contents of rice seedlings (Oryza sativa L.) in solution cultures and its implication to tolerance to iron-toxicity. Plant and Soil 275, 57-66. Stein, R.J., Ricachenevsky, F.K., Fett, J.P., (2009). Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2). Plant Science 177, 563-569. Wan, J.L., Zhai, H.Q., Wan, J.M., Ikehashi, H., (2003). Detection and analysis of QTLs for ferrous iron toxicity tolerance in rice, Oryza sativa L. Euphytica 131, 201-206. Saharawat , K.L. (2004). Iron toxicity in wetland rice and the role of other nutrients. Journal of Plant Nutrition 27, 1471-1504.

79 DGP Meeting September 5-9, 2012

Plenary Session S5: Phenotyping science and early stress detection

80 DGP Meeting September 5-9, 2012 Metabolic phenotyping: from screening of genetic diversity to systems biology

Yves Gibon

INRA, University of Bordeaux, UMR 1332 Fruit Biology and Pathology, F-33140 Villenave d’Ornon, France; E-mail: [email protected]

Introduction Metabolic phenotyping defined as the evaluation of metabolism via the determination of transcripts, proteins and/or metabolite levels or fluxes is used in approaches ranging from functional genomics to the search for diagnostic markers. Current ‘omics technologies provide comprehensive data about given classes of analytes, but are usually hampered by low throughput and/or high costs. This is particularly problematic when large numbers of individuals or genotypes are evaluated, or when dealing with genotype x environment interactions. A further complication comes from the highly dynamic nature of metabolism, which appeals for careful and exhaustive sampling strategies.

Materials and Methods By exploiting robust biochemical principles and microplate technology, we have developed a series of simple and cheap assays for enzyme activities [e.g., 1,2] and metabolites [e.g., 3,4]. They enable precise analyses of very large numbers of samples at fair costs, and in a wide range of species or tissues.

Results and Discussion To illustrate the versatility of high throughput metabolic phenotyping, two examples dealing with our model system Tomato will be given: - A large population of EMS mutants of Tomato var. Microtom is screened for alterations in enzyme properties (APEMP project financed by Aquitaine Region and INRA). A preliminary calibration experiment consisting in the analysis of several traits and metabolites as well as 20 enzymes in >50 individuals leads to intriguing results. - A systems biology project is running in collaboration with British, German and French groups, in which a model of the tomato fruit is being built by integrating an ecophysiological model (virtual fruit) and a kinetic model of carbon central metabolism (sucrose metabolism, starch metabolism, glycolysis, and organic acid metabolism), throughout development and maturation (FRIM Eranet Erasysbio+). A large dataset has been collected (climatic, ecophysiological

81 DGP Meeting September 5-9, 2012 and biochemical) from >1000 Tomato plants grown under conditions of production and under water- or light limitation, or in different greenhouses. A wide range of metabolic traits ranging from transcript levels to metabolites are being analysed. Whereas producers and gardeners know very well that the environment has a strong effect of tomato fruit quality, our first results suggest that in contrast the programming of metabolism itself is highly reproducible, from truss to truss, treatment to treatment and even greenhouse to greenhouse.

Literature 1. Gibon et al. (2004) Plant Cell 16: 3304–3325 2. Steinhauser et al. (2010) Plant Physiology 153: 80-98 3. Gibon et al (2009) Plant Cell Environment 32: 859–874 4. Hummel et al. (2010) Plant Physiology 154: 357-372

82 DGP Meeting September 5-9, 2012 The CROP.SENSe.net network: linking breeding and phenotyping science

Jens Léon 1, H.E. Goldbach, U. Schurr, J. Post, K. Spoth, K. Schmidt, L. Plümer, W. Amelung, U. Rascher, H. Kuhlmann, M. Rumpf, P. Schulze-Lammers, E.Oerke, G. Noga, H. Schumann, G. Bareth, C. Jung, W. Förstner, U. Schmidthalter, A. Graner, A.B. Cremer, D. Cremer, A. Pohlmeier, L. Weiermüller, D. van Dusshoten, C. Töpfer, C. Buschmann, T. Weyer, U. Uschkerat, et al.

1University of Bonn, Institute of Crop Science and Resource Conservation (INRES), Dept. of Plant Breeding, Katzenburgweg 5, 53115 Bonn, Germany; E-mail: [email protected]

CROP.SENSe.net is a German interdisciplinary competence network for phenotyping and sensor development for plant breeding and precision farming, funded by the German Ministry of Science and Education (BMBF). It involves very diverse disciplines ranging from agriculture to numerical simulation, information technology, botany, geodesie, and remote sensing. It started to become functional in 2010 and is addressing a number of topics in about 35 individual projects as there are:

• Early identification of traits determining yield and yield potential, biotic and abiotic stress, crop quality • High accuracy of early trait prediction from sensor data • Ascertain phenotype from interactions between genotype and environment • Predict performance of genotypes under different environmental conditions using quantitative models • Using advanced methods of data mining and It is focusing on robustness, coping with complexity, trying to achieve greater precision in phenotyping. It involves rapid “on the run” soil assessment and plant data evaluation, working with the “model plants” sugar beet and barley, in selected cases as well with grape vine. In the presentation, examples of recent advances in the network will be presented. Significant improvements have been achieved e.g. with soil data evaluation where considering surface roughness and moisture significantly improved signal to noise ratios in assessing the carbon status of soils. Hyperspectral evaluation (imaging and spectral analysis) helps to assess and identify early fungal infections as well as initial phases of abiotic stresses. A new THz sensor is reliably able to follow changes in tissue water contents at a resolution comparable to gravimetric measurements. The reconstitution of volume and shape information from laser point and stereo images data may help to identify main components of shape variation and is developed for automated recognition of characteristic parameters of shapes. Further examples of recent research will be presented.

83 DGP Meeting September 5-9, 2012 Phenotying - Can the size of a root system be estimated by electrical capacitance measurement ?

R.C. Dietrich, A.G. Bengough, H.G. Jones, P.J. White

The James Hutton Institute, Dundee, UK; E-mail: [email protected]

There is much interest in the use of capacitance measurement to estimate the size of intact root systems. The equipment required is cheap and easy to apply. Many studies have reported good correlations between capacitance and root system mass, and a linear relationship between these variables is predicted by a model that has been proposed by Dalton (Dalton F. N. 1995, Plant and Soil, 173, 157-165). The model was tested on hydroponically grown barley (Hordeum vulgare L. cv. Optic) and on barley grown in soil, respectively. Treatments included: raising whole roots out of solution, excision of roots, letting plants in soil left to dry, before the soil was selectively wetted. Good correlations were found between capacitance and mass for whole root systems. However, partly or fully excision of root parts had negligible effects on the observed capacitance. Overall, capacitance correlated better with the plant dimensions at the rooting media surfaces, than with total root mass. The bulk of the root system did not influence the capacitance - the presence of plant material at the media surface was sufficient. Good correlations required wet soil. The capacitance was lower and correlations poorer and when the top soil was dry. These findings indicate that previously observed correlations between root mass and capacitance are circumstantial. They arise because shoot- and root-dimensions at the soil surface are correlated with total root mass. A new model is proposed.

84 DGP Meeting September 5-9, 2012 Water relations of the cereal leaf and grain as measured by means of a simple, portable NMR sensor

Carel W. Windt

Forschungszentrum Jülich, IBG-2: Plant Sciences, Leo Brandt street 1, Geb. 06.2, 52425 Jülich, Germany; E-mail: [email protected]

Measuring the water relations of plants is as difficult as it is important: without water no CO2 uptake or growth. The largest problem is the lack of suitable methods to non- invasively measure water. The most widely available ones are either destructive, or indirect and imprecise. Nuclear Magnetic Resonance (NMR) does not have these drawbacks. NMR directly detects the plants’ water and organic compounds, and is able to distinguish between liquids and solids. Tracers are not needed. Unfortunately, NMR typically is expensive and difficult to use. Or is it? We here demonstrate an NMR device that can serve as a simple, low cost sensor to measure water- and dry matter content in objects varying from cereal grain to full sized tree trunk. The readings are quantitative and instantaneous. The sensor allows for automated, uninterrupted measurements over periods of weeks, with a time resolution of less than a minute. In cereals we used it to study the diurnal dynamics of leaf and grain water content, stomatal opening and grain filling, in response to short- and long-term osmotic stimuli. The method was surprisingly sensitive. For example, even mild osmotic stimuli caused the grains to behave as semi-elastic communicating vessels with respect to the rest of the plant, losing or gaining significant amounts of moisture. The latter suggests a high apoplastic connectivity between the reproductive and the vegetative parts of the plant, and a role for grains in buffering water.

85 DGP Meeting September 5-9, 2012 Remote phenotyping of the stay-green dynamic of maize from a Zeppelin

Frank Liebisch, David Schneider, Norbert Kirchgessner, Achim Walter and Andreas Hund

Institute of Agricultural Sciences, ETH Zürich, Universitätsstrasse 2, LFW, A 04, Zürich 8092, Switzerland; E-mail: [email protected]

The selection of maize ( Zea mays L.) genotypes with improved stay green is a major breeding aim to maintain photosynthesis during grain filling and ensure high yield. Remote sensing holds great promise for phenotyping of stay green in field macroarrays planted with different genotypes. We investigated if differences in stay- green can be monitored using the normalized difference vegetation index (NDVI), derived from aerial images. A macroarray was planted in a split-plot arrangement with four replications. Plot size (1, to 4 rows) was the whole plot factor, 16 genotypes were the split plot factor. Aerial images were taken at 9 dates throughout the grain filling period 2011 from a Zeppelin NT hovering 300 m above the field. Simultaneously, ground truth measurement, such as leaf greenness (SPAD and rating), were taken. The one-row plots were precise enough to characterize genotypes using remotely sensed NDVI. The temporal dynamic of NDVI values during grain filling correlated with the dynamic of SPAD values. This finding emphasizes the utility of remote sensing with a high temporal resolution to assess the stay-green dynamic. However, NDVI values taken at one point in time were weakly correlated with SPAD values. As reason for this discrepancy, we identified a variation in canopy soil cover as well as strong differences in canopy architecture (e.g. leaf angles). Further efforts are needed to account for this variation to increase precision of remote phenotyping.

86 DGP Meeting September 5-9, 2012 Spectral detection of phenotypic differences in biomass and nitrogen partitioning during grain filling of wheat Klaus Erdle 1,2 , Bodo Mistele 1 and Urs Schmidhalter 1

1Chair of Plant Nutrition/Technische Universität München, Freising; 2German Agricultural Society DLG e.V., Frankfurt. E-mail: [email protected]

Introduction Increasing the grain yield is a continual effort of plant physiologists, breeders and farmers. It is well known that single plant traits play an important role in establishing the final grain yield. These plant traits are in turn influenced by the development of wheat. Spectral remote sensing is well known as a non-destructive method to obtain plant traits of crops. Cultivar differences in yield are strongly related to their efficiency of transferring resources from the leaves and stems to the spike (Fischer, 2011) which is reflected in biomass and N compound partitioning. With spike development, the yield components, spikes per area and grain weight, gain influence on the dry matter content and biomass distribution within the plant. Several parameters linked to the source-sink relationship of wheat have already been assessed so far, however, the traditional screening methods are highly laborious and time consuming. Spectral remote sensing offers a rapid tool to estimate crop productivity before harvesting. In Western Europe, crops are grown in environments characterised by fertile soils and sufficient plant available water in which genetic differences are hard to find (Ferrio et al., 2005). Nevertheless, breeders depend on the evaluation of new crossbreds also in those high yielding environments. Although the final grain yield is known to be a consequence of the interplay between single plant traits, as far as we know, there has yet been no investigation observing source-sink parameters via canopy reflectance measurements during grain filling. Therefore, the objectives of this study were to evaluate the use of spectral indices during grain filling and to distinguish cultivars by their grain-filling characteristics.

Materials and Methods The field experiments were conducted at the research station of the Technische Universität München in southwestern Germany (11°41´ 60´´ E, 48°23´60´´ N). Grain yields in this area vary between 6 and 10 t ha -1. In the experimental field, the soil was sampled before planting, to ensure an adequate supply of P and K. In 2009 six high- yielding winter wheat ( Triticum aestivum L. ) varieties were grown with a nitrogen supply of 160 kg ha -1 split and applied at the stem-elongation (60 kg ha-1), booting (60 kg ha -1) and late-flowering (40 kg ha -1) stages. Biomass sampling was performed at four times during grain filling, at Zadoks growth stages (Zadoks et al., 1974) (ZS) of 75- medium milk, 77 - late milk, 83 - early dough 87 DGP Meeting September 5-9, 2012 and 85 - soft dough. For biomass sampling, two parallel sowing strips (0.12 m 2), twice 0.5 meter were cut at ground level. The plant samples were put into plastic bags and immediately weighed, then the spikes were separated, and the remaining biomass (leaves+stems) was weighed again and then dried. The dry weight (DW in t ha -1) was recorded, and the dry matter content (DM in %) was calculated. N content was measured and the aboveground N uptake (Nup in kg ha-1) of the spikes and leaves+stems was calculated. The relationships of the DW development and N uptake behavior between the spikes and leaves+stems, respectively, were calculated as:

For the final harvest, a combine was used to harvest an equally treated plot located next to the sampled plot. The grain yield (t ha -1) and grain dry matter content (grain DM in %) were recorded. Grain N content (%) was measured. The spectral reflectance of the plot canopy was measured with a bi-directional radiometer with tec5 electronics (tec5, Oberursel, Germany) with a spectral detection range of 400 to 1000 nm (Mistele and Schmidhalter, 2010). This system, simultaneously, detected solar radiation as a reference signal and measured the canopy reflectance with two individual spectrometer devices. The sensor system was mounted on a frame in front of a tractor. The custom- designed tractor was designed with a ground clearance of 0.90 m. The sensor outputs were co-recorded along with the GPS coordinates from an RTK-GPS and averaged per plot. Three well-established and one lately tested vegetation indices were selected for this study: The normalised difference vegetation index (NDVI) (Rouse et al., 1974), the red edge inflection point (REIP) (Guyot et al., 1988), the index R 760 /R 730 (Erdle et al., 2011), and the water index (WI) (Peñuelas et al., 1993).

A one-way analysis of variance (ANOVA, p-value = 0.05) was applied to differentiate the means of the cultivars. To allow for a qualitative ranking of the cultivars, the cultivar means of destructively assessed plant parameters and the spectral indices were normalised using the equation:

where x is the average value of one cultivar´s replications, xmin is the minimum value of all the cultivar means and xmax is the maximum value of all the cultivar means. Conclusively, all the values lie in the range of ≥ 0 and ≤ 1.

88 DGP Meeting September 5-9, 2012 Results and Discussion The cultivars´ spike parameters were best related to the final grain yield (Figure 1).

The R 760 /R 730 index showed good relationships to the spikes dry weight (DW) and the spikes dry matter content (DM) (Figure2). Low index values were associated with the spike parameters being related to increased kernel yield.

Figure 1: Relationships between final grain yield and biomass parameters obtained at Zadoks stages (ZS) 75, 77, 83 and 85. Linear relationships are shown (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).

The REIP was strongly affected by decreasing chlorophyll contents (Guyot et al., 1992), whereas representing the reflectance patterns of structure-influenced NIR wavebands, the R 760 /R 730 index best reflected the fraction of the structural tissues in the spike expressed by spikes DM and spikes DW. Spikes can have considerable influence on spectral assessments in wheat (Shibayama et al., 1986).The water index (WI) resembled the DW index, which expresses the relationship of the biomass accumulation between spikes and leaves+stems already at early grain filling. The DW index at early grain filling mirrors the actual state of the source-sink relationship which is known to be of high importance for yield development (Evans, 1993). Also N uptake parameters were well expressed by spectral measurements. The grain DM content was shown by Calderini et al. (2000) and Slafer et al. (2009) to be a promising trait to assess the physiological maturity of grains and could best be predicted by the spectral indices. Thus, spectral reflectance measurements may be a promising tool for breeders and crop producers to differentiate source-sink related plant traits of wheat cultivars during grain filling.

89 DGP Meeting September 5-9, 2012 Figure 2: Relationships between the index

R760 /R 730 and the biomass parameters a) , spikes dry weight (DW), b) spikes dry matter (DM) and c) final grain dry matter (Grain DM) of the four sampling dates. Significant r 2-values are indicated by p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). Different letters indicate significant differences of the index R 760 /R 730 .

Literature Erdle, K., Mistele, B., Schmidhalter, U., 2011. Comparison of active and passive spectral sensors in discriminating biomass parameters and nitrogen status in wheat cultivars. Field Crops Res. 124, 74-84. Evans, L.T., 1993. Crop evolution, adaptation, and yield. Cambridge University Press, Cambridge, UK. Ferrio, J.P., Villegas, D., Zarco, J., Aparicio, N., Araus, J.L., Royo, C., 2005. Assessment of durum wheat yield using visible and near-infrared reflectance spectra of canopies. Field Crops Res. 94, 126-148. Fischer, R.A., 2011. Wheat physiology: a review of recent developments. Crop Pasture Sci. 62, 95- 114. Guyot, G., Baret, F., Major, D.J., 1988. High spectral resolution: Determination of spectral shifts between the red and the near infrared. Int. Arch. Photogr. Remote Sens. 11, 750-760. Peñuelas, J., Filella, I., Biel, C., Serrano, L., Savé, R., 1993. The reflectance at the 950-970 nm region as an indicator of plant water status. Int. J. Remote Sens. 14, 1887-1905. Rouse, J.W., Haas, J.R.H., Schell, J.A., Deering, D.W., 1974. Monitoring vegetation systems in the Great Plains with ERTS, NASA SP-351., Third ERTS-1 Symposium. NASA, Washington, DC, pp. 309- 317.

90 DGP Meeting September 5-9, 2012

Non-invasive measurements of shoot and root phenotypic traits at the Jülich Plant Phenotyping Center

Fabio Fiorani, Ulrich Schurr

Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich, Leo Brandt- Strasse, 52425 Jülich, Germany, E-mail: [email protected]

Plant phenotyping is an emerging field of research that combines the application of non-invasive sensor technologies to measure canopy traits in the field or in automated plant cultivation systems in semi-controlled environments. Both field and greenhouse based applied research contribute directly to the selection of genotypes with improved performance in particular for resource-limited environments. At the Jülich Plant Phentoyping Center (JPPC), we are developing a suite of protocols for measuring plant architecture and physiological responses to a set of varying environmental conditions (e.g., limited water supply). Our phenotyping platform encompass automated systems for the cultivation and imaging of both monocot and dicot model and crop species in both pots and rhizotrons towards accurate measurements of biomass. In addition, we combine different phenotypic assays in chains that can strengthen the conclusions upon performance. For example, genotypes can be pre-screened for variability in root architecture parameters in artificial systems such as agar and subsequently cultivated in soil-filled rhizotrons. Also, a unique Magnetic Resonance Imaging facility offers the opportunity to reconstruct root systems in 3D and study physiological mechanisms underlying the observed phenotypes. In this presentation I will introduce the JPPC platform by using selected case studies and examples highlighting in particular the assays and technologies that are offered to users under the Transnational Access allowed within the new EU funded infrastructure project, the European Plant Phenotyping Network (EPPN, ). Finally, I will give an overview of the roadmap towards the development of a German national Plant Phenotyping Network.

91 DGP Meeting September 5-9, 2012

Poster Session P1: Fertilization (inorganic)

92 DGP Meeting September 5-9, 2012 Nitrogen Forms and Sulfur Status Affect Nitrogen and Sulfur Absorption in Brachiaria brizantha

Fabiano Daniel De Bona 1 and Francisco Antonio Monteiro 2

1National Wheat Research Center – Embrapa Trigo, Passo Fundo – RS, Brazil; 2College of Agriculture Luiz de Queiroz/University of São Paulo, Piracicaba – SP, Brazil. E-mail: [email protected]

Introduction Nitrogen (N) and sulfur (S) interactions in plant metabolism and growth performance are widely recognized. Evidences point out that this interaction occurs at the N and S uptake processes level and could be affected by the N source supplied and also by plant species. This study aims to investigate the N and S uptake using 15 N and 34 S stable isotopes in Brachiaria brizantha plants treated with N forms and contrasting S supplies (low and high) in nutrient solution.

Materials and Methods Two experiments were performed growing B. brizantha treated with N forms [Low N, ammonium, ammonium+nitrate (30/70%), nitrate] combined to low and high S supplies in nutrient solution (De Bona et al. , 2011) during 15 d before the 15 N or 34 S influx assays. For 15 N influx assays, plants attached to frames were taken from the N forms and S supply pre-treatments and placed in 2.5-L labelled deionized water-based medium 15 15 15 15 containing 2 mM NHCl (10.6 atoms% N), 2 mM K NO 3 (12.15 atoms% N), or 15 15 0.6 mM NHCl + 1.4 mM K NO 3 which was aerated. Plant labelling continued for 3 h after which the roots were washed during 2 min with deionized water to remove + - entrained labelled solution and rapidly exchangeable NH 4 and/or NO 3 . Roots were separated from the shoots. Plant material was dried in a ventilated oven at 65ºC for 48 h. Dried material was ground to a fine powder using a coffee grinder (Model MDR 301, Cadence, BR) and dry ice. Aliquots of 3-4 mg of the powder were weighed into small tin cups for the measurements of 15 N in plant tissue using a mass spectrometer with an automated N analyzer (ANCA-GSL model 20-20, Sercon, Crewe, UK). For 34 S influx assays, intact plants pre-treated with N forms and S supply were placed 34 in 2.5-L labelled deionized water-based medium containing 2 mM K 2 SO 4 (11.5 atoms% 34 S) which was aerated. Plant labelling continued for 2 h. Roots were washed during 2 min with deionized water and separated from the shoots. Plant material was dried and ground following the procedure described for 15 N assays. Aliquots of 500 mg of ground plant material were used for 34 S isotopic measurements. Determination of 34 S atoms% in plant tissue were performed using a mass spectrometer (ATLAS MAT, model CH-4, Berlin, GE). 93 DGP Meeting September 5-9, 2012 Results and Discussion The rate of 15 N influx was not significantly affected by prior N nutrition with N forms and S supply in the B. brizantha plants. Contrary to our results, Prosser et al. (2001) showed a decrease in the N influx (nitrate) when barley plants suffered an S deprivation. Also, Clarkson et al. (1989) working with barley ( Hordeum vulgare ) cultivated in a solution medium containing mixed nitrate and ammonium, found a markedly decrease in the nitrate (45%) and ammonium (36%) influx related to plants deprived of sulphate. Although N uptake was not affected by S deficiency, 15 N influx rates by roots of B. brizantha plants were 75 and 25% higher under mixed composition compared to ammonium or nitrate sole as 15 N sources, respectively. Regarding the S uptake, plants supplied with ammonium solely or in the mixture showed highest 34 S influx potential. Supplying nitrate and ammonium together instead of nitrate solely to S deficient, B. brizantha plants increased the 34 S influx rate by roots twice. While the 15 N influx was not affected in N deficient plants, B. brizantha plants growing under N limitation exhibited a decreased of 34 S absorption rate. According to the exposed results, the supply of N as nitrate and ammonium together can be strongly recommended to B. brizantha plants intending to minimize the effects of S deficiency once that this N mixed source does not impair N uptake and, additionally, enhances the S absorption capacity of the plants.

Acknowledgements To the State of São Paulo Research Foundation (FAPESP) for financial support and for the postdoctoral fellowship to F. D. De Bona. To the Brazilian Government Research Council (CNPq) for financial support.

Literature De Bona, F.D.; Fedoseyenko, D.; von Wirén, N.; Monteiro, F.A. (2011) Nitrogen utilization by sulfur- deficient barley plants depends on the nitrogen form. Environmental and Experimental Botany 74: 237-244. Clarkson D.T.; Saker L.R.; Purves J.V. (1989) Depression of nitrate and ammonium transport in barley plants with dimished sulphate status. Evidence of co-regulation of nitrogen and sulphate intake. Journal of experimental Botany 40: 953-963. Prosser, I.M.; Purves, J.V.; Saker, L.R.; Clarkson, D.T. (2001) Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate. Journal of experimental Botany 52: 113-121.

94 DGP Meeting September 5-9, 2012 Magnesium-Ammonium-Phosphate – Why is it not water soluble but plant available

Bernd Steingrobe and Ricardo Cabeza

DNPW – Plant Nutrition and Crop Physiology, Georg-August-University, GöttingenI. E-mail: [email protected]

Introduction Previous experiments with different P compounds mainly derived from recycling processes had shown that the water-solubility as measured according to the VDLUFA-method to characterize P fertilizers has only a week correlation to the plant availability of these compounds (Cabeza et al. 2011). For instance, only 0.5-1.5% of total P of Magnesium-Ammonium-Phosphate (MAP) was water-soluble but the availability of MAP was comparable to triplesuperphosphate (TSP), which was also confirmed by Johnston and Richards (2003). In contrast, the availability of a modified sewage sludge ash (MSA) with a water-solubility of 6.4% was as low as for phosphate rock (water-solubility about 0.1%). The extraction ratio of the VDLUFA-method (Ostmann, 1995) is narrow (1:50, i.e. 10 g fertilizer in 500 mL water) which is similar to the EU regulation 2003/2003 (extraction ratio 1:100). Hence, a compound with a low solubility reaches easily its equilibrium concentration. However in soil, the equilibration is disturbed by sorption processes. The hypothesis is that MAP becomes completely water soluble at wider extraction ratios and that the steady state situation in the soil facilitates the complete solution of MAP.

Materials and Methods Two extraction experiments were performed with P fertilizers showing large differences in their plant availability. These were in order of a decreasing availability: TSP, three MAPs of different origin, an alkali sinter phosphate (Sinter-P), a modified sewage sludge ash (MSA) and rock phosphate (RP). The experiments were as follows: (I) sequential extraction: 2 g fertilizer was shaken with 500 mL water (ratio 1:250) for 60 min, filtrated, and P concentration in the filtrate was measured. This procedure was repeated seven times with the same fertilizer sample. (II) different wide extraction ratios: 800 mg down to 100 mg of the fertilizers were shaken in 1 L of water (ratios 1:1250 to 1:10000) for 60 min. and P concentration in the solution was measured. Furthermore, three different soils (sand, pH 4.4, loam pH 5.6, loam pH 6.8) were treated with the fertilizers (60 mg P/kg soil) and the development of the P soil solution concentration was monitored during the seven following days. 95 DGP Meeting September 5-9, 2012 Results and Discussion In each of the seven sequential extractions, MAP achieved an equilibrium concentration of about 20 mg P/L. MSA reached a higher concentration in the first extraction (50 mg P/L) which decreased rapidly during the following extractions down to an equilibrium concentration comparable to rock-P (0.1-0.5 mg P/L). Sinter-P equilibrates in all extractions at a concentration of 2 to 3 mg P/L. These results were confirmed by the extractions with a wide ratio. The percentage of P solved increased for MAP nearly linearly with the extraction ratio until nearly 100% of total P had been solved at the widest ratio of 1:10.000. A similar increasing tendency was also visible for sinter-P and even for rock-P, however, at the widest ratio only about 16% and 3% of total P was solved, respectively. In contrast, MSA had the same total solubility of about 16% for each extraction ratio. Hence, the extraction experiments indicate that MAP is completely water soluble when the equilibrium concentration is not reached, yet. MSA obviously contains different P compounds, about 16% is easily water soluble and has a relatively high equilibrium concentration, whereas the other 84% had a solubility comparable to rock-P or even less. The solubility in soil depends on the relation between the equilibrium concentration and soil solution concentration. In all three soils, the fertilization with MAP increased soil solution concentration in a similar manner than TSP fertilization. The soil solution concentration that was reached (0.5 to 1.5 mg P/L) was much lower than the equilibrium concentration of MAP. Hence, due to the sorption capacity of the soil which keeps the soil solution concentration on a comparably low level all the MAP can be solved. On the other hand, soil solution concentration after fertilization with MSA or rock-P remained on a level comparable to the unfertilized treatment (0.1 mg/L) and was in the order of the equilibrium concentration. That means, MSA or rock-P didn't become soluble in the soil.

Literature Cabeza R., B. Steingrobe, W. Römer and N. Claassen (2011) Effectiveness of recycled P products as P fertilizers, as evaluated in pot experiments. Nutrient Cycling in Agroecosystems 91 (2), 173- 184 Johnston A.E. and I.R. Richards (2003) Effectiveness of different precipitated phosphates as phosphorus sources for plants. Soil Use and Management 19 , 45-49 Ostmann, H.-J. (1995) Bestimmung des wasserlöslichen Phosphats, Methode 4.1.7 in: VDLUFA (ed.) Methodenbuch Band II: Die Untersuchung von Düngemitteln. VDLUFA-Verlag, Darmstadt

96 DGP Meeting September 5-9, 2012 Does N fertilization increase wine quality?

M. Pfliehinger 1, I. Smit and O. Löhnertz

1Forschungsanstalt Geisenheim, Von-Lade-Straße 1, 65366 Geisenheim, Germany; E-Mail: [email protected]

N fertilization causes enhanced formation of yeast available nitrogen (YAN) in grapes which is important for fermentation. YAN deficient must may result in lower wine quality and high levels of fertilization (>90 kg N) may result in formation of off- flavours. We performed a field and pot experiment to investigate if moderate N- fertilization promotes the quality of wine. Sensory and chemical quality were analyzed. In the field trial (Riesling) no differences in total N and YAN were found between fertilization levels (0, 60 kg N). One may suggest a non-optimal N efficiency of vines. N fertilization in the pot trial (Mueller-Thurgau, 25, 75 kg N) was directly available to vines (fertigation) and resulted in higher concentrations of YAN in musts. Sensory evaluation of Riesling wines shows a better overall impression from unfertilized vines which could be attributed to higher residual sugar level in those wines. In the pot trial quality of wines was not increased due to higher incorporation of N in must. Although aroma intensity was rated higher (75 kg N) the overall appearance of those wines was not improved. Our results show that a moderate N- fertilization did not improve sensory quality of wine. However, no fertilization may not be considered as an alternative as it can causes nitrogen deficiency over time. N efficiency should be enhanced in viticulture. Additionally to increased YAN further quality substances should be promoted by appropriate viticultural measures.

97 DGP Meeting September 5-9, 2012 Impact of Magnesium leaf-application on specific gene-expression and photosynthesis in leaves of Vicia faba L.

C. Neuhaus 1, C. M. Geilfus 1, C. Zörb 2 and K. H. Mühling 1

1Instituteof Plant Nutrition and Soil Science/Kiel University, 24118 Kiel; 2Institute of Biology/University Leipzig, 04103 Leipzig. E-mail: [email protected]

Introduction Magnesium deficiency results in a loss of yield and yield quality in crop plants. Foliar application of Mg is in discussion to meliorate Mg deficiency. In this study the effect of a Mg foliar application on the leaf photosynthesis rate and on the mRNA abundance of enzymes that depend upon the supply of Mg was tested.

Materials and Methods Faba bean was grown under controlled environmental conditions in nutrient solution containing 0.5 mM or 0.015 mM MgSO 4. Plants growing in low Mg solution were treated thrice with foliar applications of 0 mM, 50 mM or 200 mM MgSO 4. After six weeks, photosynthesis rate was measured with a portable infrared gas exchange system (Licor LI-6400). Old and young leaves were collected for mRNA extraction. Real-time quantitative PCR was performed for measuring the abundance of Vicia faba Mg-chelatase and three different plasmalemma H +-ATPase isoforms ( vha ).

Results and Discussion In result, the application of Mg had no impact on the expression of plasmalemma H +- ATPase-isoforms vha2 , vha4 and vha5 in the older leaves, whereas in the younger leaves the mRNA abundance could be raised to the control level. In contrast Mg foliar application increased Mg concentration, chlorophyll concentration and photosynthesis in the older leaves, whereas this effect was only marginally in young leaves. In conclusion, a heterogeneous effect of the Mg foliar application between young and old leaves was demonstrated. By Magnesium foliar application it was possible to ameliorate the deficiency situation of parts of the plant.

98 DGP Meeting September 5-9, 2012 Manganese status of mainly grown cereals in Turkey

Mustafa Harmankaya 1, Fatma Gökmen Yilmaz 1, Erdo ğan E. Hakki 1, Sait Gezgin 1

1Selcuk University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, Konya, Turkey. E-mail: [email protected]

Introduction Manganese (Mn) deficiency is a serious nutritional problem of crop plants in some regions of the world and may markedly limit in crop yield. However, plant species differ in their susceptibility to Mn deficiency (Sayyari-Zahan et al. 2009). Manganese deficiencies mainly occur on organic soils, high-pH soils, calcareous or heavy limed soils and sandy soils low in organic matter. Most of the agricultural land of Turkey contains relatively high amounts of CaCO 3 and low organic matter resulting in high pH soils. The abundance of manganese oxides and hydroxides in calcareous alkaline soils is the major reason of Mn uptake limitations of plants (McKenzie, 1989). There is a close relationship in between the Mn compounds available and the pH of the soil. While Mn uptake by plants is considerably high at acidic soil, soil characterized by high pH substantially reduces Mn uptakes. Hence, Mn deficiencies are frequently observed in crops grown at soils with high pH (Kacar and Katkat, 1998). Cereal crops represent a major source of minerals and protein in developing world. For example, in most of Central and West Asian countries, wheat provides nearly 50% of the daily calorie intake on average, likely increasing to more than 70% in the rural regions (Cakmak et al., 2004). There is an enormous worldwide effort to enhance the yield and the quality of these strategic products. The purpose of this study was to evaluate the Mn status of mainly grown Anatolian cereals on the basis of yield increases observed upon Mn fortification.

Materials and Methods Fifteen bread wheat, 10 durum wheat, 5 barley, 5 triticale, 1 oat and 1 rye were grown under greenhouse conditions in a Mn-deficient calcareous soil obtained from the Central Anatolia Region. The soil used in the experiment had a sandy loam texture, pH 7.23, a CaCO 3 content of 63.3% and organic matter content of 2.6%. The concentrations of DTPA-extractable Mn, Fe, Zn and Cu, 0.98, 5.71, 0.93 and 1.7 respectively, were measured according to the method described by Lindsay and Norvell (1978). About 10 seeds were sown into each plastic pot containing 0.850 kg soil with (+Mn=.40 mg Mn kg -1 soil) or without Mn fertilization. Manganese was added in the form of MnSO 4.H 2O. Before potting soil was homogenously mixed with a basal −1 −1 treatment of 150 mg N kg soil as Ca(NO 3)2 and 80 mg P kg soil as KH 2PO 4. After emergence, the plants were thinned to 6 seedlings per pot. Plants were watered daily by using deionized water. The treatments were performed as four replicates

99 DGP Meeting September 5-9, 2012 according to completely randomized design in a greenhouse. At stem elongation period, shoots were harvested and dried at 70ºC for the determination of dry weights. The dried plant samples were ground and digested by using 5ml of %65 HNO 3 and 2 ml of 35% H 2O2 with a closed microwave system (Cem-MARS Xpress). Mn concentrations of samples were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES; Vista-Pro Axial; Varian Pty Ltd, Australia). Mn measurements were checked using the certified Mn values in different reference leaf samples received from the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA).

Results and Discussion Mn application affected the crop plants, however, the influences were significantly different between as well as within the species, among different varieties. Upon Mn application, yield increases among the bread wheats varied in between 8.20 to 16.4%, 2.72 to 27.93% among durum wheat varieties and 10.85 to 24.52% among the barley varieties. Overall, bread wheats were seen to increase their yield at 11.36%, durum wheats at 14.36% and barley at 18.90%. On the other hand, triticale varieties were also highly influenced, 11.50 to 36.83% yield increases were observed among different varieties, as was also the case for the oat variety used, with18.04% yield increase, and the rye variety with 23.10% higher yield observed. In conclusion, from the cereal species that were fortified with Mn, rye was the one most favorably influenced species, followed by triticale, barley, oat, durum wheat and bread wheat, in sequence.

Literature Sayyari-Zahan, M.H., Singh Sadana, U. Steingrobe, B. Claassen, N. (2009) Manganese efficiency and manganese-uptake kinetics of raya ( Brassica juncea ), wheat ( Triticum aestivum ), and oat (Avena sativa ) grown in nutrient solution and soil. Journal of Plant Nutrition and Soil Science, 172(3):425 - 434 McKenzie, R.M.,(1989) Manganese oxide and hydroxides p.439-466. In: Minerals in soil environments. (J.B.Dixon and S.B. Weed, eds.) 2nd ed. SSSA Madison, WI. Kacar, B. ve A.V., Katkat, (1998) Bitki Besleme. Uluda ğ Üniversitesi Güçlendirme Vakfı Yayın No:127 Cakmak, I., Torun, A., Millet, E., Feldman, M., Fahima, T., Korol, A., Nevo, E., Braun, H.J. and Ozkan, H., (2004): Triticum dicoccoides: An Important genetic resource for increasing zinc and iron concentration in modern cultivated wheat. Soil Sci. Plant Nutr. 50: 1047-1054. Lindsay, W.L.; Norvell, W.A. Development of a DTPA Soil Test for Zinc, Iron, Manganese and Copper. Soil Sci. Soc. Am. J. 1978, 42, 421–428.

100 DGP Meeting September 5-9, 2012 Winter wheat and winter rape yield after N-S fertilizer application

Martin Kulhánek, Jind řich Černý, Ji ří Balík, Šárka Shejbalová, Filip Vašák

Czech University of Life Sciences, Prague, E-mail: [email protected]

Introduction Sulfur (S) deficiency in European soils was described in many studies. Although S has been known as an essential element for plant growth for over two centuries, S- containing fertilizers were not widely used for wheat and rape crops before 1980s. At that time these crops did not show evidence of S deficiency, because of air pollution from industry, intensive application of mineral fertilizers containing S and application of manure and cropping of plants with lower S uptake in crop rotation on the bigger area (Scherer, 2001, Blair, 2002, Zhao et al., 1999). From the mentioned reasons it is clear that sulfur becoming a limiting factor for the harvest and production quality (Eriksen et al., 2004) Winter wheat and winter rape are the key crops for Czech Republic. Therefore the efficiency of S fertilizing of these crops should be tested. Materials and Methods Precise field experiments were established on 3 sites with winter rape (Hn ěvčeves, Humpolec and Uh řín ěves) and 2 with winter wheat (Hn ěvčeves and Humpolec) under different soil-climatic conditions in the Czech Republic in the years 2008 - 2010. For this experiment, four fertilizing treatments were evaluated. Addition of nitrogen to all treatments was 200 kg N/ha. 0, 10, 20, 40 kg S/ha was added to the wheat. 0, 12.5, 25, 50 kg S/ha was added to the rape. Each treatment was conducted in four replicartions. The size of plot was 12 m 2 for rape and 20 m 2 for wheat, respectively.

Sulfur was applied in the CaSO 4 form (Lovofert®) fertilizer. Harvest was realized using small plot harvestor. The yields of both crops were evaluated. Results and Discussion Wheat has a relatively low S requirement, amounting to about 20 kg S/ha with a grain yield of 8 t/ha (McGrath et al., 1996). Obtained yields ranged between 7.20 and 10.86 t/ha. The highest yields were estimated in the years 2008 and 2010 at the Hn ěvčeves site. The lowest yields were found in 2009 at Humpolec site, where the average reached only 7.28 t/ha. The highest yields were always found in the year 2008. It was due to more favourable rainfall and temperature conditions at both sites. From the point of view of fertilizing treatments comparison, the significantly differences between the yields were found only at the Hn ěvčeves site in the year 2008. There were found the significantly higher yields at the treatments which were fertilized using 20 kg S/ha and 40 kg S/ha, respectively (compared to control non- fertilized treatment). In the other cases we did not found significantly differences between studied treatments, but there are visible some tendencies to the yield increase with increasing S rate. The highest yields in the years 2008 and 2009 at the

101 DGP Meeting September 5-9, 2012 Hn ěvčeves site and in the years 2009 and 2010 at the Humpolec site were always obtained at the treatment D fertilized using the highest S rate. This corresponds with the results of Ryant and H řivna (2004) who also did not found significantly differences between treatments fertilized using nitrogen fertilizer and the treatment fertilized using nitrogen fertilizer together with gypsum, but they found increasing tendencies with increasing S rate. Similar results got Ercoli et al. (2011), who applicated higher

S rates (60 and 120 kg S/ha) in CaSO 4 form to winter wheat together with nitrogen. The second aim of our experiment was also to evaluate the yields of oilseed rape. There were no statistically significant differences between the studied treatments.

Despite that is possible to assume that S-fertilising using CaSO 4 S-form positively influenced the oilseed rape yield. There are visible tendencies: the seed yields on non-S-fertilised treatments are usually the lowest and at the treatment fertilised using the highest S dose (50 kg S/ha) the highest yields were obtained. The highest yields were reached in the year 2010, where we got a range between 6.84 and 7.02 t of oilseed rape seed per ha at the Hn ěvčeves site. The least favourable was the year 2008, where we got the highest yields at the Hn ěvčeves site as well, but the values ranged only between 3.93 and 4.33 t/ha. The lowest yields were usually found at the Humpolec site, where were the worst soil-climatic conditions for oilseed rape cropping. The increasing tendencies in yields are caused with increasing nitrogen efficiency due to the sulfur fertilizing, that is mentioned with many authors (e.g. Scherer, 2001, Salvagiotti et al., 2009, and others). From our experiment is possible to conclude following facts: The highest oilseed rape and winter wheat yields were always found at the Hněvčeves site and the lowest yields were usually observed at the Humpolec site. Increasing S dose led usually to an increase of oilseed rape and winter wheat seed yield, but the differences between the observed treatments were usually not statistically significant. Literature Blair, G.J.: Sulphur fertilizers: A global perspective. The International Fertilizer Society; Proceedings, 498 , 2002: 1-36. Eriksen J., Thorup-Kristensen K., Askegaard M. (2004): Plant availability of catch crop sulfur following spring incorporation. J Plant Nutr Soil Sci 167 (5) : 609-615 Ercoli, L., Lulli, L., Ardunni, I., Mariotti, M., Masoni, A.: Durum wheat grain yield and quality as affected by S rate under Mediterranean Conditions. Europ. J. Agronomy, 35 , 2011: 63–70. McGrath, S.P., Zhao, F.J., Withers, P.J.A.: Development of sulfur deficiency in crops and its treatments. Proc. Fertilizer Soc., No 379. The Fertilizer Society, London 1996. Ryant, P., H řivna, L.: The effect of sulphur fertilization on yield and technological parameters of spring wheat grain. Annales Universitatis Mariae Curie-Sklodowska, Sectio E, Agricultura, 59 (4) , 2004: 1669-1678. Salvagiotti, F., Castellarín, J.M. - Miralles, D.J. - Pedrol, H.M.: Sulfur fertilization improves nitrogen use efficiency in wheat by increasing nitrogen uptake. Field Crops Research, 113 , 2009: 170–177. Scherer, H.W.: Sulphur in crop production – invited paper. Eur. J. Agron., 14 , 2001: 81-111. Zhao, F.J., Hawhesford, M.J., McGrath, S.P., 1999: Sulphur assimilation and effects on yield and quality of wheat. Journal of Cereal Science, 30 , 1999: 1–17.

This research was funded by National Agency for Agricultural Research project No. NAZV QH 81202

102 DGP Meeting September 5-9, 2012 Role of Zn, K and Mg to mitigate the adverse impact of water stress on wheat productivity

M.M. Tawfik, M.O. Kabish, E.M. Abd El Lateef, A. Bahr Amany, T.A. Elewa, B.A. Bakry

National Research Centre, El Bohooth Str. Dokki, Giza 12311, Egypt; E-mail: [email protected]

Water scarcity is one of the most serious issues due to the impact of climate change which expected to aggravate the situation even more. So providing sound solutions for this environmental problem is a vital issue to cope with water shortage and drought as they are the main constraints of crop production. To achieve the aforementioned objectives, two field experiment was carried out at private sandy soil farm in Tamiah, Fayum . Egypt to evaluate the effect of foliar application of (100 ppm

Zn-EDTA), (2.0 % KNO 3) or (50 ppm MgSO 4) on growth, yield, yield attributes and nutritional value of wheat plants cv. Sakha-93 grown under irrigation regime (irrigation at 80 % of the maximum water holding capacity of the soil ( W.H.C ) i.e. normal water supply , 60 % of ( W.H.C. ) i.e. moderate soil moisture stress and 40 % of ( W.H.C ) i.e. soil severe moisture stress. The results revealed that water stress significantly reduced all the tested growth parameter, yield, yield attributes and (DSI) as well as photosynthetic pigments content as compared with unstressed plants (control). However, water stress had a stimulating effect on proline, soluble carbohydrates% cellulose% and crude protein content. The study also indicate that foliar application of Zn, K or Mg had a positive effect on growth parameters, yield , yield attributes and (DSI) as well as photosynthetic pigment content with superiority to K over the two other nutrients.

103 DGP Meeting September 5-9, 2012 Fe and Zn concentrations in potato tubers as affected by the rate and form of nitrogen fertilization

U.Reichert 1, M. Bonierbale 2, W.Amoros 2, E.Salas 2, U. Eckhardt 1, C. Engels 1

1Humboldt University, Department of Plant Nutrition, Albrecht-Thaer-Weg 4, 14195 Berlin, Germany; 2International Potato Center (CIP), P.O. Box 1558, Lima, Peru. E-mail: [email protected]

Introduction To increase micronutrient concentrations in potato tubers through agronomic intervention and genetic selection, the understanding of plant processes that regulate Fe and Zn concentration of potato tubers must be advanced. This may contribute to reduce Fe and Zn malnutrition among rural populations especially in low-income countries. It is well documented that in cereals grain Fe and Zn concentrations are positively correlated with N concentrations, and can be increased by N fertilization (Aciksoz et al., 2011). Within cereal grains, concentrations of N and Zn are substantially higher in the embryo and aleuron than in the endosperm (Cakmak et al., 2010), indicating co-localization of proteins and Zn e.g., in protein bodies. Little is known about N fertilization effects on potato tuber Fe and Zn concentrations. Potato tubers do not contain embryonic and aleuron tissue, and differ from cereals also in the strategy of Fe acquisition. In this study, the effect of the rate and form of N supply on tuber Fe and Zn concentrations was investigated.

Materials and Methods Tuber micronutrient concentrations of different genotypes were measured in a long- term field experiment which is running since 1986 (International organic nitrogen long-term Fertilization experiment), and in a pot experiment with silica sand. In the field experiment with variety Granola , organic fertilizer supply was varied (no organic fertilizer, farmyard manure or crop residues). In addition to organic fertilization also mineral N (calcium ammonium nitrate) application was varied at 4 different rates (0, 60, 100, 150 kg N ha -1) in plots with manure or crop residues, and 2 rates (0, 150 kg N ha -1) in plots without organic fertilization. In the pot experiment three genotypes were used, ( 300048.12 ; Unica; 397078.16 ) which according to CIP´s database differ in tuber Fe and Zn concentrations. Plants were supplied with nutrient solution containing N in the form of NH 4NO 3 at 3 different rates (low N, medium N, high N).

Results and Discussion In both experiments mineral N fertilization increased tuber yield (P < 0.05, data not shown) and N concentration (P < 0.001; Table 1 for field experiment only). In contrast to preceding experiments under different environmental conditions, in this pot experiment tuber micronutrient concentrations did not significantly differ among 104 DGP Meeting September 5-9, 2012 genotypes. This indicates a strong genotype x environment interaction on tuber micronutrient concentrations. Tuber Fe and Zn concentrations in the pot experiment significantly increased with tuber N concentrations (P < 0.001, data not shown). In the field experiment, tuber Fe and Zn concentrations were higher in plants fertilized with crop residues compared to manure. Fe and Zn concentrations in tuber dry mass increased with increasing mineral N fertilization (Table 1), and were positively correlated with tuber N concentrations (P < 0.001). In the field experiment, mineral N fertilization increased leaf Zn concentrations indicating that high N fertilization was associated with increased Zn uptake from soil. Thus, the positive effect of mineral N fertilization on tuber Zn concentrations might be a consequence of higher total Zn uptake. Leaf Fe concentrations, in contrast, were not increased by N fertilization. Therefore, it is suggested that the positive effect of N fertilization on tuber Fe concentrations is related to N effects on tuber sink strength for Fe rather than on Fe acquisition from soil.

Table 1 Effect of fertilization on tuber N concentrations and micronutrient concentrations in tuber and leaf dry mass; leaf concentrations were measured at flowering; ± standard deviation Organic Min. N Tuber concentrations Leaf concentrations fertilization kg ha -1 N g kg -1 Fe mg kg -1 Zn mg kg -1 Fe mg kg -1 Zn mg kg -1 No 0 10 ± 0.7 12 ± 1.4 11 ± 0.8 80 ± 7.2 6 ± 0.6 No 150 17 ± 2.9 16 ± 4.5 14 ± 1.6 71 ± 9.3 12 ± 1.0 Manure 0 11 ± 1.4 11 ± 3.3 10 ± 2.3 67 ± 9.7 6 ± 1.2 Manure 60 11 ± 1.8 11 ± 0.8 10 ± 1.2 69 ± 6.6 8 ± 2.4 Manure 100 12 ± 1.1 12 ± 0.3 11 ± 0.4 66 ± 3.9 10 ± 1.0 Manure 150 14 ± 2.9 16 ± 3.0 12 ± 2.2 79 ± 4.6 12 ± 1.5 Crop Res. 0 11 ± 1.4 14 ± 3.5 11 ± 0.6 65 ± 3.5 7 ± 0.3 Crop Res. 60 15 ± 3.7 15 ± 3.1 13 ± 2.0 64 ± 5.9 8 ± 1.1 Crop Res. 100 17 ± 3.2 17 ± 4.4 14 ± 1.5 71 ± 2.7 10 ± 1.0 Crop Res. 150 19 ± 0.8 19 ± 1.4 16 ± 1.6 75 ± 6.6 13 ± 0.9

Literature Aciksoz S.B., Yazici A., Ozturk L., Cakmak I. (2011). Biofortification of wheat with iron through soil and foliar application of nitrogen and iron fertilizers. Journal of Plant Nutrition and Soil Science 349: 215-225. Cakmak I., Kalayci M., Kaya Y., Torun A. A., Aydin N., Wang Y., Arisoy Z., Erdem H., Yazici A., Gokmen O., Osturk L., Horst W.J. (2010). Biofortification and localization of zinc in wheat grain. Journal of the Agricultural and Food Chemistry 58: 9092-9102.

105 DGP Meeting September 5-9, 2012 Influence of different mineral nitrogen sources (NO3−-N vs. NH4+-N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices–cowpea symbiosis

Benard Ngwene, Elke Gabriel, Eckhard George

IGZ Großbeeren; E-mail: [email protected]

Many studies have reported that the ERM of AM fungi can take up and transfer considerable amounts of N from both organic (1) and inorganic (2) sources to the host plant. However, under some conditions ammonium (NH 4+) supply, compared with nitrate (NO 3-) supply, has been shown to decrease mycorrhizal activity (3). Some authors have argued that the contribution of AM fungi to shoot N content is likely to be small compared with uptake by the roots (4). The case may be different when AM fungal hyphae have access to N sources that are not accessible to roots (5). An understanding of N dynamics between AM fungi and host plants may help to quantify N uptake processes in individual plants, but also to describe plant and AM fungal contribution to global N cycles. Labeled nitrogen (15N) was applied to a soil-based substrate in order to study the uptake of N by arbuscular mycorrhizal (AM) fungal extraradical mycelium (ERM) from − + different mineral N (NO 3 vs. NH 4 ) sources and the subsequent transfer to cowpea plants. The experimental design allowed for the simulation of soil patches containing root-inaccessible, but AM-accessible, N. This was achieved by placing fungal compartments (FC) within the plant growth substrate. The FC substrate was a mixture of wet sieved (particle size < 40 µm) soil and glass beads (Ø 2 mm) that permitted the extraction of almost intact AM fungal mycelium after harvest.

- + The fungus was able to take up both N forms, NO 3 and NH 4 . However, the amount − of N transferred from the FC to the plant was higher when NO 3 was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher 15N + − enrichment when the FC was supplied with 15NH 4 compared with 15NO 3 . The 15N shoot/root ratio of plants supplied with 15NO 3− was much higher than that of plants + − supplied with 15NH 4 , indicative of a faster transfer of 15NO 3 from the root to the + shoot and a higher accumulation of 15NH 4 in the root and/or intraradical mycelium. + - We conclude that AM hyphae may absorb NH 4 preferentially over NO 3 , but export of N from the hyphae after uptake to other parts of the mycelium, the root and the shoot - + may be greater following NO 3 uptake. Perhaps this relates to NH 4 needing to be assimilated into organically-bound N prior to transport.

106 DGP Meeting September 5-9, 2012 The effects of different iron compounds and TKI-Humas treatments on iron uptake and growth of lettuce

Fatma Gökmen Yilmaz, Mustafa Harmankaya, Sait Gezgýn

1Selcuk University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, Konya, Turkey. E-mail: [email protected]

This investigation was conducted in a greenhouse to determine the effects of different iron compounds as well as TKI-Hümas treatments as humic and fulvic acid in differnet doses applied on lettuce plant. The trial was conducted according to the randomized complete parcel design experiment with four replications in 8 application forms. The applications were comprised of the following groups: 1. Control, 2.

FeSO 4. 7H 2O (19% Fe), 3. FeEDDHA (6 %Fe), 4. TKÝ-Hümas with 250 ppm humic + fulvic acid, 5. TKi-Hümas with 500 ppm humic + fulvic acid, 6. FeSO 4. 7H 2O + TKi-

Hümas with 250 ppm humic + fulvic acid, 7. FeSO 4. 7H 2O + TKÝ-Hümas with 500 ppm humic + fulvic acid and 8. TKÝ-Hümas contaning 0.96% Fe. were subject to 8 applications. The iron content of the soil used was 12 ppm. Active (Fe +2 ) and total (Fe +2 +Fe +3 ) iron, chlorophyll a, chlorophyll b, cholorophyll a+b and dry matter were determined. Statistically significant (p <0.01) differences were observed among different treatments and iron sources used. Dry matter yield and active iron contents of the lettuce plants were observed to increase at the rates of 3% -25% and 10-45%, respectively. Key words: FeEDDHA, FeSO 4.7H 2O, humic + fulvic acid, TKI-Hümas, lettuce

107 DGP Meeting September 5-9, 2012 Foliar fertilization of Tempranillo grapevine ( Vitis vinifera L.) with iron products

Felipe Yunta 1, Ignacio Martín 2; Juan José Lucena 3; Natalia Domínguez 2; Ana Benito 2, Agustín Gárate 3

1Departamento de Geología y Geoquímica. Universidad Autónoma de Madrid; 2Servicio de Investigación y Desarrollo Tecnológico Agroalimentario - I.C.V.V. (Instituto de Ciencias de la Vid y del Vino. Ctra. Logroño-Mendavia NA-134 Km. 90); 3Departamento de Química Agrícola. Universidad Autónoma de Madrid, Ctra. Colmenar Viejo, P.K. 15.5. 28049. Madrid. SPAIN. E-mail: [email protected]

Introduction Iron chlorosis is a widespread nutritional disorder in grapevine developed on calcareous soils (Lucena, 2003). Despite the effective application of highly efficient Fe chelates such as Fe-EDDHA to the soil to solve the problem, the cost of the treatments limits their use. An alternative could be the utilization of Fe-fertilizers directly supplied through foliar spray. For this, two experiments were conducted to investigate the foliar application efficiency of different Fe complexes and chelates.

Materials and Methods Two iron chelates (Fe-EDTA and Fe-EDDS), an iron complex (Fe-Lignosulfonate; Fe- LS) and an iron salt (Fe-Sulfate) were foliarly applied to a mature Tempranillo (Vitis vinifera L.) grown on a soil with a high active lime content and compared to an untreated control. Three shoots per vine and five vines per treatment were selected. One hundred mL of each iron treatment (5 mM Fe) were sprayed on mature leaves (Treated) while young leaves (Covered) were covered for assessment of iron translocation. Treatments were sprayed twice two weeks apart. Leaf blades were sampled before the first spray application and at the end of the assay, four weeks later. Sampled leaf blades were washed with a non-ionic detergent and micronutrient content (Fe, Mn) and Fe/Mn ratio were determined. Leaf chlorophyll content was measured weekly using a non-invasive Minolta SPAD chlorophyll meter. Second experiment was designed to evaluate the metal translocation rate into the plant by using labelled micronutrients. Iron and zinc complexes and chelates were prepared from different complexing (LS) and chelating agents (EDDS and EDTA) using labelled 57Fe and 67Zn in 5 mM concentration. A selected leaf was divided in 2 halves, the terminal half was immersed into each treatment solution for 30 s (Treated Leaf = TL) while the other half one was left as control zone to study the short- distance translocation (Non-Treated Leaf = NTL). Long-distance translocation was also evaluated by sampling the new born leaf (New Leaf = NL). This trial was conducted for eight days after immersion time. Increments between SPAD readings just before of treatments and eight days after that were calculated and labelled 108 DGP Meeting September 5-9, 2012 micronutrients were quantified by ICP-MS after leaves washing with non-ionic detergent and organic solvent for removal of leaf cuticular waxes.

Results and Discussion Total iron concentration from leaves sprayed with each iron treatment was significantly higher (Fe-EDDS 183 mg·kg -1; Fe-EDTA 260 mg·kg -1; Fe-LS 200 mg·kg - 1; Fe-Sulphate 340 mg·kg -1) than that of untreated treatment (64 mg·kg -1) being the leaves sprayed with Fe-EDTA and Fe-Sulfate those that shown the highest total Fe concentration and the largest Fe/Mn ratio. Covered leaves from all vines sprayed with Fe treatments (Fe-EDDS, 57 mg·kg -1; Fe-EDTA, 61 mg·kg -1; Fe-LS, 57 mg·kg -1; Fe- Sulphate,60 mg·kg -1) showed significantly higher Fe concentration than untreated treatment (48 mg·kg -1) but only the Fe/Mn ratio for Fe-EDTA (0.45) was significantly higher than that of untreated treatment (0.32). Leaves sprayed with Fe-EDTA shown the highest SPAD readings for Sprayed (38.7) and Covered (38.1) leaves at the end of experiment. The inconsistency between total Fe concentration from the sprayed leaves and SPAD readings in both sprayed and covered leaves suggests that other parameter such as Fe/Mn ratio could be used for assessing of iron translocation rate from mature to young leaves when foliar fertilization was applied. Comparable results were found when labelled 57 Fe and 67 Zn were analysed. Significant differences were found between TL, NTL and NL (TL>NTL>NL) for both labelled metals when sprayed with EDDS showing short-distance translocation. However no translocation effect was found for LS and EDTA (TL>NTL=NL). Morever, when both 57 Fe and 67 Zn content for each treatment was compared, EDTA and EDDS were supplying significantly more 57 Fe (232 mg·kg -1 and 245 mg·kg -1) and 67 Zn (1848 mg·kg -1 and 1773 mg·kg -1) in TL than LS complexing agent and untreated leaf (Control). Therefore EDDS chelating agent had shown significantly higher 67 Zn content in NL than for the rest of the treatments. Results from both experiments agree well with those reported by other authors as Yliviano et al. (2004) and Rodríguez-Lucena et al. (2009): as a recommendation when iron and zinc are foliarly added to correct mild nutritional disorders they should be applied using synthetic chelating agents such as EDTA or EDDS because not only total micronutrient in leaf is significantly increased but also translocation rate to apical zones is improved.

Literature Rodríguez-Lucena, P., T. Tomasi, R. Pinton, L. Hernández-Apaolaza, J.J. Lucena, and S. Cesco. 2009. Plant Soil. 325 (1-2): 53-63. Ylivainio, K., A. Jaakkola, and R. Aksela, 2004.Journal of Plant Nutrition and Soil Science 167: 602– 608. Lucena J.J. 2003. Journal of Plant Nutrition 26: 1969-1984

109 DGP Meeting September 5-9, 2012 Poster Session 2: Fertilization (organic) / Soil amendments

110 DGP Meeting September 5-9, 2012 Nitrogen fertilizing using different fertilizers to the spring barley

Š. Shejbalová, J. Černý, M. Kulhánek, J. Balík, F. Vašák

Department of Agro-Environmental Chemistry and Plant Nutrition, Czech University of Life Sciences in Prague, Czech Republic. E-mail: [email protected]

Introduction Nitrogen is one of the most important nutrients for the production of all crop plants and represents the most applied nutrient to agriculture land. This is because available soil-N supplies are often inadequate for optimum crop production and because commercial fertilizers, manures, and other sources of N are generally easily and economically applied (Follett, 2008). Efficient nitrogen fertilization is crucial for economic barley production and protection of ground and surface waters. Nitrogen fertilizer rate and timing are the major tools available after planting to manipulate barley to produce higher yields per hectare (Alley et al., 2009).

Materials and Methods For the purpose of experiment two characteristics feature were observed: yields (t/ha) and nitrogen content (%) in grain of spring barley. Long-term experiments were established in the year 1996 at four sites with different soil and climatic conditions in the Czech Republic: at colder locations (Humpolec, Lukavec) and warmer locations (Hn ěvčeves and Suchdol). A simple crop rotation contained: potatoes, winter wheat and spring barley. Each year were grown all crops. Treatments of fertilization were repeated in three blocks. The size of experimental plots was 60 m 2. In this research 7 fertilizing treatments for spring barley were evaluated: 1. no fertilization (Control), 2. sewage sludge 1 (SS1), 3. sewage sludge 3 (SS3), 4. farmyard manure (FYM), 5. N in mineral fertilizers (N), 6. NPK (70-30-100) in mineral fertilizers (NPK) and 7. straw of spring barley + N in mineral fertilizers (ST + N). Whole experiment was based on the same nitrogen rate 330 kg N/ha to the crop rotation (of which 70 kg N/ha for spring barley) except Control (0 kg N/ha) and SS3 treatment (990 kg N/ha/crop rotation). Organic fertilizers were applied only to the potatoes in crop rotation. Plant samples were taken after harvest of plants (at maturity).

Results and Discussion Results of experiment are presented as means from years 1997 to 2011. Average values of nitrogen content in grain of spring barley were from 1,48 % (Control) to 1,86 % (SS3). The highest content of nitrogen was occurred after using nitrogen in mineral fertilizers (N, NPK, ST + N) and SS3 treatments. At warmer locations was content of

111 DGP Meeting September 5-9, 2012 nitrogen in grain about 23 % higher after SS3 treatment application and about 17 % higher after N treatment compared to Control (non-fertilized) treatment. At colder locations were increases of nitrogen contents compared to Control treatments about 6 % on SS3 treatment and 7 % on N treatment. The lowest values were measured at the Control treatment at all sites. Lower N contents after application of organic fertilizers (FYM and SS1) compare to N mineral fertilizers treatments were observed. Using nitrogen fertilization supported production of higher yields (Angás, P. et al., 2006, Glendining et al., 1997, McKenzie, 2005). The average yields ranged between 1,96 t/ha (Control) and 5,27 t/ha (NPK). At colder locations were achieved 64 – 74 % higher yields after application of nitrogen in mineral fertilizers: N, NPK, ST + N in compare to the Control treatment. At warmer locations were average yields about 36 % higher after treatment SS3 and about 46 % higher after treatment NPK compare to the Control treatment. After application nitrogen in mineral fertilizers increases of yield were lower at warmer locations than at colder sites. The lowest yields were always observed at the Control treatment. Lower yields were measured after application of organic fertilizers (FYM and SS1) in compare to using mineral fertilizers (N, NPK, ST + N).

Literature Alley, M. M., Pridgen, T. H., Brann, D. E., Hammons, J. L., Mulford, R. L. 2009: Nitrogen Fertilization of Winter Barley: Principles and Recommendations, Virginia Cooperative Extension, Virginia Polytechnic Institute and State University, 4 p. Angás, P., Lampurlanés, J., Cantero-Martínez, C. 2006: Tillage and N fertilization: Effects on N dynamics and Barley yield under semiarid Mediterranean conditions, Soil and Tillage Research, 87, 59-71 Follet, R. F. 2008: Transformatin and Transport Processes of Nitrogen in Agricultural Systems. Nitrogen in the environment, Elsevier, London, 17-26 Glendining, M. J., Poulton, P. r., Powlson, D. S., Jenkinson, D. S. 1997: Fate of 15N-labelled fertilizer applied to spring barely grown on soils of contrasting nutrient status. Plant and Soil, 195, 83-98 McKenzie, R. H., Middleton, A. B., Bremer E. 2005: Fertilization, seeding date, and seeding rate for malting barley yield and quality in southern Alberta. Canadian Journal of Plant Science, 85, 603- 614

This study was supported by The Ministry of Agriculture CR, projects No. QH 81 202 and QH 91 081

112 DGP Meeting September 5-9, 2012 Nutrient supply to optimize N 2 fixation and productivity in grain legumes (faba bean)

Claudia Seehuber, Heiner E. Goldbach and Heinrich W. Scherer

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany. E-mail: [email protected]

Introduction Nutrient supply to grain legumes in organic farming may become limiting for biological nitrogen fixation (BNF), especially under no-till conditions. Thus, a study was carried out to assess the nutrient status of grain legumes in Germany and how fertilizer may help to overcome nutrient constraints in faba bean ( Vicia faba L.) especially on organic farms, as they are more dependent on BNF.

Materials and Methods Experiments were carried out at Klein-Altendorf in Rheinbach and Wiesengut in Hennef, Bonn and 2 organic farms near Mönchengladbach. In addition, 105 leaf samples were collected at various stages, esp. at BBCH 61 (beginning of bloom) by mainly organic farmers throughout Germany. In addition leaf samples were supplied by our project partners and the “Landwirtschaftskammer NRW” to create DRIS (Diagnosis Recommendation Integrated System) norm values. Plant material was analyzed for C, N, S, P, K, Ca, Mg, Mn, Fe, Cu, Zn, B and Mo from dried plant material by standard procedures (elemental analysis, acid digestion, followed by atomic absorption spectrometry, ICP-OES, B spectrometrically by the miniaturized curcumin-method). Soil samples were analyzed for N min (nitrate-N + ammomium-N) by the autoanalyzer in 1 % K 2SO 4 solution. Yield parameters were assessed at harvest.

Results and Discussion In many cases the DRIS indices did not reflect the true nutritional status. As most of the faba beans gave low or average yield, most of the samples could not be used to create the high-yielding, optimal supplied DRIS Norm itself. Independent of that, general problems arose when DRIS Indices are calculated. DRIS does not take account that: 1) Nutrient contents in plants can vary without influencing the nutrient status, growth or yield (especially Fe and Mo), 2) some nutrient relationships are more important than others because of antagonistic behavior (e. g. P/Zn, S/Mo ratio), 3) plants may have combined deficiencies – e. g. resulting in unchanged quotients when 2 limiting nutrients are divided by each other, or a false high nutrient status of a single adequate nutrient in relation to several limiting nutrients which DRIS may address as “normal”, and 4) plants may be abundantly supplied with some nutrients. The CNL (Critical Nutrient Level) appeared to be a better diagnosing tool for our data 113 DGP Meeting September 5-9, 2012 set. Indicating the optimum nutrient range and the inclusion of important nutrient ratios in faba bean leaves at a certain stage such as BBCH 61 – 65 may be superior. As S resulted to be the most limiting element in field experiments in 2010 and in collected samples in 2009, potassium sulphate (35 kg S/ha) had a significantly positive effect on plant growth and yields. In addition it increased the N content in the leaves at bloom from 5.7 % to 6.2 % (DM basis). In all samples N leaf contents as high as 60 g/kg were found in faba beans with an adequate S supply, only (Fig. 1).

Fig. 1: Correlation between S and N content

This indicates that S was the limiting factor for N2 fixation. Sulfate strongly depressed the uptake of molybdate. As result the Mo-contents in the combined fertilization treatment only reached from 1.8 to 5.1 mg/kg compared to Na-molybdate alone (1 kg Mo/ha) with ~ 12 mg/kg. The Mo-fertilization neither had a positive nor a negative effect on growth and yield of the faba beans. The fertilization of 3 kg Mo/ha as sodium-molybdate lead to an extremely high Mo-content in the leaves of 102 mg/kg DM compared to the control with only 1 – 3 mg/kg. The advice for practical agriculture is not to apply more than 1 kg Mo/ha or even less if the field is used for forage production for cattle. Compost may also be suitable to provide sufficient Mo.

Conclusion

Independent of the soil preparation the sulfur supply was in most cases limiting for N 2 fixation of faba bean in organic farming in Germany. Since the atmospheric S input has decreased especially legumes are in need of S fertilization.

Literature Bergmann, W. (1983): Ernährungsstörungen bei Kulturpflanzen – Entstehung und Diagnose. VEB Gustav Fischer Verlag, Jena, 1. Auflage Needham, T.D., Burger, J.A. and Oderwald, R.G. (1990): Relationship Between Diagnosis and Recommandation Intgrated System (DRIS) Optima and Foliar Nutrient Critical Levels; Soil Sci. Am. J. 54: 883-886

114 DGP Meeting September 5-9, 2012 Gypsum amendment to peat substrates reduces P availability

Binner, I. 1 and M. K. Schenk 1

1Institute of Plant Nutrition, Leibniz Universität Hannover, E-mail: [email protected]

Introduction Peat is commonly used in horticultural growing media but it has only a low capability of buffering plant nutrients such as phosphorus. The buffer capacity can be increased by the addition of mineral components, which may contain gypsum, CaSO 4 * 2 H 2O. Gypsum can lead to severe immobilization of phosphate due a precipitation of calcium phosphates. In this study the effects of gypsum amendments on the P availability in peat-substrates and the growth of marigold plants were investigated.

Materials and Methods Marigold ( Tagetis x patula cv. `Disco Flame´) was grown in black peat at pH 5.5 with 0, 5 and 10 g gypsum L -1 substrate, respectively. Substrates were fertilized with 25 mg P L -1 substrate at the beginning of the experiment. Substrate moisture was maintained at 50% (v/v) by weighting and fertigating the pots every second day with a

P-free nutrient solution. Available P in substrates ( PCAT ) was determined by CAT extraction (Alt and Peters, 1992). Additionally the P concentration in the substrate solution ( PSubSol ) was measured and the buffer power ( b) was calculated from

PCAT /PSubSol . Plants and substrates were harvested 28 and 35 days after planting (DAP). Root physiological parameters were adopted from and morphological parameters were determined according to Khandan-Mirkohi and Schenk (2009) (Tab. 1). The mechanistic simulation model NST 3.0 described by Claassen and Steingrobe (1999) was used to predict plant P uptake.

Results and Discussion

At the beginning PCAT in the substrates was not affected by gypsum, while PSubSol was clearly lowered by 50%. The decreased PSubSol in the gypsum treated substrates was also reflected in the P concentration in shoot d.m. of marigold plants, although d.m. yield was not significantly changed. When plant P uptake between 28 and 35 DAP was predicted by the mechanistic simulation model using the calculated buffer power at 28 DAP, the predicted P uptake exceeded the observed plant uptake by far (Fig. 1). The calculated buffer power did not reflect the actual P availability as the CAT extraction strongly overestimated the available P amount in the substrates. If only the dissolved P was considered to be actually plant available, resulting in a b of 0.5, the predicted and observed P uptake agreed fairly well (Fig. 1). Such a low buffer power is characteristic for an unbuffered nutrient such as nitrate. At 28 DAP the calculated

115 DGP Meeting September 5-9, 2012 extension of P-depletion zones around roots exceeded the mean half distance between neighboring roots (Tab. 1). Thus plants exploited 100% of the substrate volume at this time. Tab. 1: Specific model parameters used for simulation of P uptake at 28 DAP. f = impedance factor; P SubSol = substrate-solution P concentration; PCAT r 0 = root radius; r 1 = mean half distance between b = b = 0.5 PSubSol roots; L 0 = initial root length; k = root growth rate;

] I max = maximum uptake rate; K m = Michaelis -1 80 constant; C min = concentration of P in substrate observed 60 predicted solution where uptake equals zero; V 0 = water- uptake rate of root cylinder; ∆x = extension of depletion zone around a root. 40

20 Gypsum [g L -1 substrate] 0 5 10

Substrate parameters plant P[µmol uptake 0 f 0.9 0.9 0.9 0 5 10 ' 0 5 10

-3 Gypsum [g L -1 substrate] PSubSol [µmol cm ] 0.19 0.1 0.08 Plant morphology r [mm] 0.16 0.18 0.17 Fig. 1: Comparison between 0 observed and predicted plant P r1[mm] 1.72 1.73 1.74 uptake at increasing gypsum L [m plant -1] 21.5 21.3 21 0 supply assuming two different P -1 k [m day ] 3.5 2.2 3.3 buffer powers of substrates. Plant physiology -2 -1 -7 Imax [µmol cm s x 10 ] 5.8 5.8 5.8 -3 -3 Km [µmol cm x 10 ] 5 5 5 -3 -4 Cmin [µmol cm x 10 ] 4 4 4 -2 -1 -7 V0 [cm³ cm s x 10 ] 6.8 6.6 6.1

Рx [mm] (at b = 0.5) 4.9 4.9 4.9

Conclusion The experiment revealed that the gypsum amendment led to a P fixation in the substrates which was not reflected by the CAT extraction. This led to an overestimation of the P availability, since the extraction of 1 g substrate with 8 ml CAT-solution dissolved the Ca-phosphates. This disadvantage could be diminished by a narrower extraction ratio.

Literature Alt, D., Peters, I. (1992). Agribiol. Res. 45, 204-214. Claassen, N., Steingrobe, B. (1999). In Rengel, Z. (ed.): Mineral Nutrition of Crops: Fundamental Mechanisms and Implications. The Haworth Press, Inc., New York, London, pp. 327–367. Khandan-Mirkohi, A., Schenk, M.K. (2009). J. Plant Nutr. Soil Sci. 172, 369–377.

116 DGP Meeting September 5-9, 2012 Compost as raw material in potting substrates for ornamental plants

Thi Cam Van Do, Heiner E. Goldbach, Heinrich W. Scherer

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Organic wastes should be recycled from an ecological as well as from an economical point of view. For this reason composting has been increasingly recognized as a viable management method for solid organic wastes aimed at recycling of its end- product as a potting substrate for ornamental plants. In a pot experiment Pelargonium and Salvia as salt-sensitive plants were grown in the mixture of compost and additives with different volumetric ratios (4:1, 1:1, 1:4). Because plants still suffered from the high salt content, in a further experiment the share of compost was reduced and peat incorporated into the mixture. This paper reports plant growth (dry matter yield) and nutrient uptake (N, P, K, Mg, Ca, Na and S) of plants grown in potting substrates based on compost with and without additives as compared to standard soils. While the results of the first pot experiment revealed that the high percentage of compost in the substrate had negative effects on plant growth and nutrient uptake, both parameters significantly increased and almost gained levels of the control in the second pot experiment. Especially, the growth of Salvia was significantly increased. From our result it may be deduced that pure compost cannot be recommended for salt sensitive ornamental plants, but in peat-based substrates plant growth and nutrient uptake is favored, when is the share of compost does not exceed 25%.

117 DGP Meeting September 5-9, 2012 Nutrient recovery from anaerobically treated pig slurry

N. Stamm 1, H. Goldbach 1, J. Clemens 2

1INRES Plant Nutrition/ University of Bonn; 2Gewitra Ltd, Troisdorf. E-mail: [email protected]

Introduction Intensive animal husbandry creates large amounts of wastewater containing nutrients like nitrogen and phosphate. Concentration of animal production especially in periurban areas thus leads to local nutrient overloads of soils and surface water bodies, while there are nutrient deficits in other areas. The long distance transport of those wastes is economically and ecologically not feasible due to their high water contents. An adequate distribution of animal husbandry would solve the problem of nutrient overload, but is on the other hand economically not feasible in the view of an increasing urbanization, either. As easily accessible phosphate is a finite resource which is estimated to last only for the next 40 – 100 years, nutrient separation from bulky wastes might offer a practical solution. P i can be precipitated simultaneously + with NH 4 as struvite (MgNH 4PO 4 · 6 H 2O). Regarding N, struvite is characterized as slow- release fertilizer, with a P efficiency comparable to superphosphate. Below, we report about experiments with anaerobically treated pig slurry. The objectives of this study were to select the most effective Mg- source and to determine further factors affecting struvite precipitation.

Materials and Methods Batch experiments were carried out with anaerobically treated pig slurry with low dry matter content (<1%) using centrifuge tubes (50 mL) and glass bottles (500 mL) with screw caps. The concentration of the initial solute orthophosphate (PO 4-P) was determined before starting the experiment using the molybdenum blue method. Afterwards, MgO was added at varying molar ratios of Mg: P of 1: 1 and 2:1 and shaken horizontally for 1 hour at 170 – 180 rpm. The samples were then allowed to settle down and struvite to be formed for 17 hours. Final PO 4-P concentration was determined and MgO was added a second time at the same ratio. Precipitation was calculated by the decrease of P i in the substrate.

Results and Discussion

Struvite formation reduced the dissolved inorganic P i concentration by 75 % - 80 %

(see Fig.1). The first MgO addition resulted in a Pi reduction in the residue by 55% (control: 10%). After the second addition, further 20 % (control: 5 %) were precipitated. We observed also a reduction of around 15 % in the control sample where no MgO was added. This is most likely due to a spontaneous formation of 118 DGP Meeting September 5-9, 2012 struvite during shaking as air turbulence enhances struvite formation (Ohlinger et al. (1998)). There was no difference between the two molar ratios Mg: P of 1:1 and 2:1 which is in accordance with Nelson et al. (2003) who observed best struvite precipitation at a molar ratio of 1.2 in untreated pig slurry.

Struvite precipitation

0.5 before MgO 17h after 1.MgO 17h after 2.MgO 0.4

0.3

0.2 PO4-P conc. conc. mmol/LPO4-P

0.1

0.0 control 1:1 1:1 2:1 2:1 treatment Figure 1: Struvite precipitation in anaerobically treated pig slurry after one and two additions of MgO in different ratios (Mg: P of 1:1 and 2:1).

Conclusions

Up to 80 % of soluble P i could be precipitated from biogas slurry as struvite. This corresponds to about 27 % of total slurry P and may become a means to reduce local nutrient overload and simultaneously collect P (plus equimolar ratios of N) in a transportable form. Additional solid separation can recover remarkable amounts of the remaining P o.

Literature Munch, E.V. and Barr, K. (2001): Controlled struvite crystallization for removing phosphorus from anaerobic digester side- streams. Water Res. 35, 151-159 Nelson, N.O., Mikkelsen, R.L. and Hesterberg, D.L. (2003): Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg: P ratio and determination of rate constant. Biores. Tech. 89, 229-236 Ohlinger, K.N., Young, T.M. and Schroeder, E.D. (1998): Predicting struvite formation in digestion. Water Res. 32, 3607 – 3614

119 DGP Meeting September 5-9, 2012 Release of greenhouse gases after fermentation residue application on ten different soils in a labor

Friedhelm Herbst, Bend Apelt, Wolfgang Gans

Universität Halle, Betty-Heimann-Str. 3, 06120 Halle (Saale), Germany; E-mail: [email protected]

In a laboratory experiment it was examined how much and which greenhouse gases were released from soils of ten regions of Germany after fermentation residue application. Soils were from: (1) Halle (lS), (2) Bad Lauchstädt (Lö), (3) Speyer (lS), (4) Weihenstephan (L), (5) Cunnersdorf (sL), (6) Dornburg (L), (7) Dedelow (lS), (8) Ascha (sL), (9) Güstrow (lS), (10) Hohenschulen (sL). After filling the soil into glass vessels, the fermentation residue solution were dripped onto the surface and mixed with the soil. The residue (consisting of cow slurry, corn silage and manure) had the following characteristics: 8.8% dry matter, pH 7.9, 0.304% ammonia-N, 0 % nitrate-N, 0.563 % total-N. Ammonia was absorbed in washing bottles using 2% boric acid and determined potentiometrically using a pH meter. N 2O, CO 2 and CH 4 were quantified by gas chromatography. The main results of the study were as follows: NH 3 was emitted from only seven soils. The emission, which lasted between five and nine days, was highest from the Speyer soil, followed by the Bad Lauchstädt and Gülzow soils. The Speyer soil also released the highest amounts of N 2O. However, N 2O emission lasted for a longer time, i.e. up to 32 days. The CO 2 measurements indicated only minor differences between the ten soils. CH 4 was released from all soils, but only during the first seven hours after residue application. The Speyer soil released the highest amounts of CH 4.

120 DGP Meeting September 5-9, 2012 Impact of yellow water on germination of different crops

Judith Schmidt, Ute Arnold

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Introduction Due to limited natural nutrient resources, secondary resources are becoming more important as potential fertilizers to feed an increasing world population and compensate the decline of arable land. Alternative concepts, e.g. “new sanitary systems” propose the reuse of separated waste water. Most nutrients can be gained by separating urine from the residual waste water as these are mainly excreted in urine. Despite a rather low hygienic risk - pathogens are rarely present in urine - pharmaceutical residues, a high salt content and a high pH may hamper its safe and proficient use. In a number of field trials the efficiency of yellow water as a fertilizer has likewise been proofed, however, few and contradictory has been reported on its impact on germination. To clarify the impact of urine, resp. its components, germination experiments were conducted with different crops. Besides yellow water, several drugs frequently present in urine - 17 α-Ethinylestradiol (hormone), Carbamazepine (anticonvulsant), Diclofenac (anti-inflammatory agent), Atenolol (β-adrenergic blocker), and Verapamil (calcium channel blocker), different pH (5, 7, 9, 11) and different salt solutions - NaCl, (NH 4)2SO 4, NH 4NO 3, NH 4Cl, K 2HPO 4 - were added to seeds in different concentrations.

Materials and Methods Seeds of wheat ( Triticum aestivum ), barley ( Hordeum vulgare ), fava bean ( Vicia faba ), and sunflower ( Heliantus anuus ) were placed on cotton wool in 4 replicates per variant. In a first test, yellow water in different dilutions (yellow water directly, dilutions 1:10 and 1:100) was added to the seeds. Experiments with diluted yellow water of different pH and water spiked with different concentrations of one drug followed.

Furthermore, several “urine-composing” salts (NaCl, NH 4, NO 3, K2HPO 4) were used; concentrations were according to the electric conductivity of yellow water (resulting in

0.2 molar solutions) and 10 times lower. Germination rate was determined five times within the first 10 days after seeding.

Results and Discussion Highest germination rates were achieved with diluted yellow water (1:100) - slightly higher than for those seeds watered with tap water. However, yellow water without

121 DGP Meeting September 5-9, 2012 dilution inhibited germination completely. Pharmaceuticals – even 100-times higher concentrated than usually found in urine – did not cause any negative effect on plant germination. High pH (pH 9, 11) reduced the germination of wheat - this reduction was larger in diluted yellow water than in tap water of the same pH. Sunflower, in contrary, was not affected significantly by different pH. Strongest impact on germination was found when applying different salts in concentrations comparable to salt concentration of yellow water. Plant responses differed according to the salt type and plant species. However, even for concentrations 10 times lower than in yellow water effects were visible. Regarding the potential use of yellow water as a fertilizer, salts were identified as the critical component for germination. In field experiments carried out in parallel, no inhibition on germination of wheat could be seen in parcels where yellow water was applied. Nevertheless, to assure adequate germination, direct contact of seeds with undiluted yellow water should be avoided.

Literature Blume S.,Winker M. 2011.Three years of operation of the urine diversion system at GTZ headquarters in Germany:user opinions and maintenance challenges. Water Science & Technology 64, pp. 579-589. Schürmann B., Everding W., Montag D., Pinnekamp J. 2012. Fate of pharmaceuticals and bacteria in stored urine during precipitiation and drying of struvite. Water Science & Technology 65, pp. 1774-1780.www.saniresch.de Winker, M., Dimova, D., Ritter, K., Otterpohl, R., Clemens, J. 2010. Effect of five pharmaceutical substances contained in urine on the germination of cress and cereal seedlings. IWA Conference Sustainable solutions for small water and wastewater treatment systems, Girona, Spanien. Winker M., Vinneras B., Muskolus A., Arnold U., Clemens J (2009): Fertiliser products from new sanitation systems: Their potential values and risks. Bioresource Technology, Vol. 100, Issue 18, 4090-4096 Wohlsager S., Clemens J., Nguyet P.T., Arnold U.(2010): Urine Storage and Nutrient Load of a Sanitary Separation System in Southern Vietnam, Water Environment Research, Volume 82, No 9

122 DGP Meeting September 5-9, 2012 Using Yeast Estrogen Screen (YES) assay for analysis of estrogenic compounds in soil and plant material after application of urine as fertilizer

Maria Alejandra Arias, Anh Hong Le, Ute Arnold, Heiner Goldbach

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Human urine contributes less than 1% of domestic wastewater, but it is responsible for more than 80% of N, 50% of P and 70% of K in wastewater. These characteristics make it a potential source of nutrients for plants. However, urine also can contain hormone and pharmaceutical residues, which can represent a risk if these compounds are taken up by the plants, especially the ones for human consumption. The extraction method has been developed and YES assay has been used to analyze estrogenic residues in plant and soil samples, after application of human urine as fertilizer.

Primary results of extraction recoveries of 17 β-estradiol (E2) in soil and wheat grain was 107.6% (±19.5%) and 96.2% (±19.2%) respectively. This means that the extraction method could be replicated for similar material. Since it has been observed that the chlorophyll interferes in the analysis of the estrogenic compounds, further experimentation is taking place in order to test other plant parts such as leaves and stems.

123 DGP Meeting September 5-9, 2012 NILE – New Irrigation system for Low and Efficient water use in agriculture

Jens Wester 1, Manfred Trimborn, Heiner Goldbach

1Institute of Crop Science and Resource Conservation (INRES) Rheinische Friedrich-Wilhelms- University, Bonn. E-mail: [email protected]

Introduction Water is one of the most important growing factors for every plant. Especially in arid and semi-arid areas irrigation is essential for an efficient plant production. But with an increasing demand of water it is more and more important to economize the usage and to protect soil from salinization.

The HYDRIP technology is an innovative irrigation system which combines subsurface drip irrigation with a humidity retention agent and a microbe stimulating additive, to keep water in an efficient way in the root zone and to minimize salinization of the top soil. The soil conditioner will be placed in the soil together with the irrigation tubes to build up a reservoir that protects water especially in sandy soils against deep percolation or ineffective evaporation. In a Bmbf founded Eurostars joint research project (NILE) we investigated different soil conditioner and plant additives on their properties concerning their water holding effects with the aim to find an optimized combination and application rate for the Hydrip-System.

Materials and Methods For the first analysis ten different mineral, organic and synthetic soil conditioner were chosen: Axis ®, Betasoil ®, Alginit ®, Perlite, Zeolith, Bentonit, rock flour, coconut fiber, peat and geohumus ®. The water holding capacity (WHC) were tested with pure soil conditioner (100%) and in different mixtures (30% and manufacturer’s suggestion) with a sandy soil form our experimental field („Uedorfer Krume“) that was mixed with an additional amount of 30 % quartz sand (0,2-0,63 mm) to achieve a nutrient-poor sandy soil. A greenhouse experiment was conducted to simulate a depot of a soil conditioner and one dropper within a single pot. The 10 different soil conditioners and a control treatment with 5 replications were created with an automatically irrigation system. The pots (Kick-Brauckmann) were filled with 11 kg of the soil/sand mixture with a core of a soil conditioner and planted with “Lolium multiflorum” which were harvest 5 times to analyze fresh and dry matter and nutrient uptake.

124 DGP Meeting September 5-9, 2012 Results and Discussion The results of the WHC analysis show very high differences especially between the varieties “manufacturer’s suggestion” and “pure” (100%). Some soil conditioners like geohumus or coconut fiber lose their water holding capacity if they are mixed in the soil. The reason might be the limited free pore volume in the soil that reduces the maximal water uptake of the particle of these soil conditioners. So some of our results are inconsistent with manufacturers suggests that their product work in the soil (up to a depth of 50 cm) with their maximum swelling property. The fresh matter harvest of L. multiflorum in the pot experiment is an indicator for the water availability during the five growing periods (figure 1). We found no significant differences so fare, but some trends are visible. For example, alginit ® and betasoil ® raised their positive effects from harvest to harvest in relation of some other soil conditioners. This long-term effect is interesting for the Hydrip-System which should be used for several years. Other soil conditioners like peat lost their positive qualities during the experiment period.

Figure 1: The fresh matter [g] of the grass after five cuts in relation of the different varieties; control1 = overhead irrigation. Error bars show the standard deviation, n=5.

The greenhouse experiment is not yet finished. We are still looking for the water balance, nutrients in the leakage water, salt content in the top soil and nutrient uptake of the plants.

125 DGP Meeting September 5-9, 2012 Charcoal effects on soil properties and herbal vegetation of temperate beech forests at historical charcoal production cites

Dominik Hegenberg, Sita Eschemann, Timo Adolphs, Nils Borchard, Bodo-Maria Möseler

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

The use of charcoal as a soil amendment is currently being discussed in relation to its effects on increasing both nutrient and water retention in soils. We studied the effect of charcoal on soil and vegetation parameters on historic (>60 years) charcoal production sites under beech forest in Germany. Sites differed in terms of soil attributes (acidic and base-rich soils) and species richness (species-poor Luzulo- Fagetum and species-rich Hordelymo-Fagetum) beech forests. The soil concentrations of plant-available N, P, K, and Mg tended to be higher in soils of historic charcoal producing sites than in adjacent control soils. Also physical parameters such as bulk density and water-holding capacity were improved with charcoal. While this coal-related improvement in soil properties was not reflected in plant species richness or species composition, larger numbers of individuals with a high demand in base saturation and available N were observed on the coal-amended sites. We conclude that charcoal appears to have positive effects on soil quality that are not or only poorly reflected in the response of the natural vegetation.

126 DGP Meeting September 5-9, 2012 Soil amendments to alleviate rhizotoxicity and nutritional disorders in maize on heavy metal contaminated soil

Holger Beßler, Christof Engels

Institute of Plant Nutrition, Humboldt-Universität zu Berlin, Albrecht-Thaer-Weg 4, 14195 Berlin, Germany. E-mail: [email protected]

Introduction In the surrounding land of Berlin, sewage has been deposited on agricultural land for decades and has caused high loads of organic pollutants and heavy metals in the irrigated soils. Today, cultivation of crops for food and feed production on these former sewage farms is often prohibited due to high risk of Cd toxicity for humans. Cultivation of energy crops provides an alternative to use this land but establishment of productive stands is challenging due soil contamination with various potentially phytotoxic HM (Cu, Ni, Zn). To advance the design of a tailored energy crop system, the potential of six soil amendments to enhance crop growth on HM contaminated soil was evaluated in this study.

Materials and Methods Zea mays (L. cv. Torres) was grown on uncontaminated soil (–HM), HM contaminated soil from a sewage farm (+HM), and +HM amended with: (g fresh mass kg -1 soil) charcoal (10), hydrothermal carbonized wood (10), slurry (15), lime and slurry (1.2 g CaO kg -1), peat (3) or zeolite (25) in a pot experiment. Soils were fertilized (mg kg -1 soil: NO 3-N 2 x 100, P 160, K 200, Mg 60), irrigated to a water content of 25 % (w/w; 65 % water capacity) and incubated for seven days before sowing of maize. Plants were harvested 23 days after sowing to determine dry mass of shoots and roots, mineral nutrient concentrations in plants and concentrations of NH 4NO3-soluble Zn, Cu and Ni in soils.

Results and Discussion

Concentrations of NH 4NO 3-soluble Cu and Zn in +HM exceeded legally threshold values for agricultural soils and were higher than in –HM (Table 1). Maize grown on – HM developed well without symptoms of nutritional disorders. When grown on +HM, root and shoot biomass decreased (Table 2). Primary, seminal and crown root length was strongly reduced which agrees with well-documented inhibition of root elongation by excessive external concentrations of Cu, Zn and Ni (rhizotoxicity). In addition, young leaves showed chlorosis and suboptimal concentrations of Fe (Table 2). Suboptimal Fe concentrations were associated with increased concentrations of Zn, Cu and Ni, indicating an antagonism between Fe and Zn, Cu and/or Ni leading to Fe deficiency.

127 DGP Meeting September 5-9, 2012 -1 Table 1: Concentrations of NH 4NO 3-soluble heavy metals (HM; mg kg soil) and pH of uncontaminated soil (–HM), HM contaminated soil (+HM), or HM with various amendments. Different letters within a row indicate significant (P<0.05) treatment effects. Threshold values according to BBodSchV (mg kg -1 soil): Zn 2, Cu 1, Ni 1.5. HTC: hydrothermal carbonized wood; Sl: Slurry

–HM +HM +HM with amendment Slurry Peat HTC Zeolite Lime+Sl. Charcoal Zn 1.2 a 4.6 bc 5.2 c 4.8 bc 3.7 b 4.6 bc 0.2 d 2.2 a Ni 0.2 a 0.6 b 0.7 b 0.6 b 0.5 b 0.6 b 0.1 a 0.1 a Cu 0.3 a 3.9 c 4.1 c 3.9 c 3.5 c 3.6 c 3.8 c 1.8 b pH 5.4 a 5.2 c 5.2 c 5.3 b 5.4 a 5.3 b 6.7 e 5.7 d

Amendment of +HM with slurry, peat, htc or zeolite did not decrease the concentrations of NH 4NO 3-soluble Zn, Ni and Cu (Table 1). Consequently, symptoms of rhizotoxicity and Fe deficiency were not alleviated and plant growth was not enhanced by these amendments (Table 2). Liming strongly increased the soil pH, and decreased the concentrations of NH 4NO 3-soluble Zn and Ni (Table 1). This was associated with less severe symptoms of rhizotoxicity, increased concentrations of Fe and decreased concentrations of Zn in young leaves, less severe chlorosis and enhanced plant growth (Table 2). Similar to liming, amendment of +HM with charcoal alleviated symptoms of rhizotoxicity, reduced chlorosis and enhanced plant growth

(Table 2). Charcoal amendment decreased the concentrations of NH 4NO 3-soluble Zn, Ni and Cu, but increased soil pH only slightly (Table 1).

Table 2: Dry mass of roots and shoots (g plant -1), and concentrations of Fe, Zn, Ni and Cu in young leaves (mg kg -1 dry mass) of maize grown at –HM, +HM and +HM with various soil amendments.

–HM +HM +HM with amendment Slurry Peat HTC Zeolite Lime+Sl. Charcoal Root 1.1 a 0.3 c 0.3 c 0.4 c 0.3 c 0.2 c 1.0 ab 0.8 b Shoot 3.9 a 1.4 b 1.5 b 1.7 b 1.2 b 1.2 b 3.4 a 3.2 a Fe 84 a 46 bc 46 bc 44 c 34 c 46 bc 62 b 51 bc Zn 51 a 80 b 65 ab 68 b 64 ab 64 ab 42 a 53 a Cu 7 a 15 c 12 bc 12 bc 12 bc 13 bc 13 bc 9 ab Ni <0.2 a 4.1 b 0.5 a 0.4 a 1.2 a 1.4 a 0.3 a <0.2 a

Our data show that in the short term liming and charcoal amendment decrease HM mobility in soil and enhance plant growth. In the long term, however, liming might stimulate soil organic matter mineralization, and thus increase HM mobility. Therefore it is suggested that charcoal amendment to HM polluted soil may be an effective measure to establish productive energy crop stands, which avoids an increase in HM mobility in the long term.

128 DGP Meeting September 5-9, 2012 Growth of nine grassland species on heavy metal contaminated soil amended with biochar

Anna Adam, Holger Beßler, Christof Engels

Institute of Plant Nutrition, Humboldt-Universität zu Berlin, Albrecht-Thaer-Weg 4, 14195 Berlin, Germany. E-mail: [email protected]

Introduction In the urban hinterland of Berlin, sewage has been deposited on agricultural land for decades, and has caused high loads of organic pollutants and heavy metals (HM) in the irrigated soils. Today, production of food and feed on these former sewage farms is often prohibited due to high risk of Cd toxicity for humans. Alternative concepts for land use have to consider soil contamination with various potentially phytotoxic HM (Cu, Ni, Zn), and the risk of pollutant transfer from soil to hydrosphere. Establishment of grassland for bio-energy production might lead to phytostabilization of polluted soils, and simultaneously provide some economic yield. To advance the design of a tailored grassland community, HM resistance of nine species was assessed. In addition, it was tested if plant growth on HM contaminated soil can be increased by soil amendment with biochar.

Materials and Methods Six grass species ( Calamagrostis epigejos [Ce], Dactylis glomerata cv. Revolin, Festuca rubra cv. Lifalla [Fr], Lolium mutliflorum cv. Fabio [Lm], Lolium perenne cv. Trivos [Lp], Phleum pratense cv. Fidanza [Pp]) and three species of legumes ( Lotus corniculatus [Lc], Trifolium pratense cv. Milvus [Tp], Trifolium repens cv. Liflex [Tr]) were cultivated for 34 to 41 days in a pot experiment on uncontaminated soil (–HM), contaminated soil from a sewage farm (+HM), and +HM amended with biochar (10 g biochar kg -1 soil). Soils were fertilized (mg kg -1 soil: N 100, P 160, K 200, Mg 60) and soil water content was maintained at 65 % water capacity.

Results and Discussion When grown on –HM, all species developed well without symptoms of nutritional disorders. When grown on +HM, young leaves became chlorotic, particularly in D. glomerata , L. perenne and P. pratense . Shoot and root biomass decreased, whereby large species differences in the extent of growth inhibition existed. In the grasses C. epigejos , D. glomerata, F. rubra, L. perenne, P. pratense and the legumes L. corniculates, T. pratense growth was reduced by 86 to 97% (Table 1), indicating low HM resistance. In contrast, growth of L. multiflorum and T. repens was only reduced by 61 and 75%. In legumes, the number of nodules g-1 root fresh mass was severely reduced, particularly in L. corniculata . 129 DGP Meeting September 5-9, 2012 Table 1: Shoot and root dry mass of grass and legume species grown on uncontaminated (–HM), contaminated (+HM), or biochar amended +HM soil (+HMc). Different letters within a row indicate significant (P < 0.05) treatment effects; see text for species names. Treat- Grass species Legume species ment Ce Dg Fr Lm Lp Pp Lc Tp Tr Shoot dry mass (g pot -1) –HM 1.15a 2.25a 1.06a 3.36a 1.63a 2.15a 2.15a 1.90a 1.68a +HM 0.09b 0.05b 0.02b 1.18b 0.09b 0.07b 0.35b 0.29b 0.44b +HMc 0.22c 0.10c 0.04c 1.85ab 0.32c 0.37c 1.13c 1.16c 1.31c

Root dry mass (g pot -1) –HM 0.46a 0.84a 0.35a 1.42a 0.80a 0.94a 0.79a 0.59a 0.54a +HM 0.03b 0.02b 0.01b 0.28b 0.02b 0.06b 0.09b 0.10b 0.14b +HMc 0.08c 0.03b 0.01b 0.49b 0.09c 0.08b 0.42a 0.36c 0.41c

Amendment of +HM with biochar increased the soil pH, and decreased the concentrations of NH 4NO 3-soluble Cu, Ni and Zn in soil (Table 2). In comparison to +HM-plants, in +HMc plants leaf chlorosis were less severe, and number of nodules g-1 root fresh mass strongly increased in T. repens and T. pratense . Shoot and root growth were increased by biochar amendment in all species (Table 1). In the more HM resistant species, shoot biomass formation on biochar amended +HM was only 20 to 39% lower than on –HM.

-1 Table 2: Soil pH and concentrations of NH 4NO 3-soluble heavy metals (mg kg soil). Treatment pH Cu Ni Zn –HM 5.5 0.3 <0.1 0.7 +HM 5.3 3.2 0.3 3.2 +HMc 5.7 2.0 0.2 1.9

Our data suggest that selection of HM resistant species and biochar amendment can help to establish productive grassland stands even on soil which is highly contaminated with several HM.

130 DGP Meeting September 5-9, 2012

Poster Session P3: Nutrient efficiency / Genomics

131 DGP Meeting September 5-9, 2012 Role of transcription factors for genotypic differences in nitrogen deficiency-induced leaf senescence in oilseed rape ( B. napus )

Fabian Köslin-Findeklee 1, Salma Balazadeh 2, Bernd Müller-Röber 2 and Walter Horst 1

1Institute of Plant Nutrition/Leibniz University Hannover, Hannover; 2Institute of Biochemistry and Biology/University of Potsdam, Potsdam. E-mail: [email protected]

Introduction Winter oilseed rape is the most important oil crop in European agriculture. However, this crop greatly contributes to a high cropping-system nitrogen (N) balance surplus. Previous work suggested that N efficiency (high grain yield under N-limiting conditions) was related to delayed leaf senescence (stay green) because of maintained photosynthesis into the reproductive growth stage (Wiesler et al., 2001). Leaf Senescence is a complex and highly coordinated process and constitutes the final stage of leaf development. As well as being a developmental process, senescence can be induced by sub-optimal environmental conditions such as nutrient limitation, especially N deficiency. Leaf senescence is accompanied by changes in the expression of many genes with regulatory functions (Buchanan- Wollaston et al., 2002; Lim et al., 2007). Among these, transcription factors (TFs) are key regulators of the gene expression network. Materials and Methods A nutrient solution experiment was performed with the genotypes NPZ-1 (N-efficient) and NPZ-2 (N-inefficient) differing in leaf senescence (stay green) and yield formation under N-limited conditions. The plants were grown after a four weeks pre-culture for another 7 days under N deficiency conditions (0.1 mM N). The senescence status of the 3 rd leaf was determined using photosynthesis rate as indicator. Additionally, the expression of the senescence-associated cysteine proteinase SAG12-1 and a set of 36 Arabidopsis thaliana TFs was measured by qRT-PCR in the leaves at the beginning and end of the deprivation period. The 21 NAC, 9 WRKY and 6 MYB TFs are known as SAGs or involved in the response to reactive oxygen species (ROS). Results and Discussion After 7 days of N deprivation the photosynthesis rate declined significantly, but no genotypic differences appeared (Fig. 1). However, the gene encoding SAG12-1 was highly up-regulated and clear genotypic differences existed confirming later onset of senescence in the N-efficient genotype NPZ-1 (Fig. 2). Half of the 36 tested TFs were up-regulated in N deprived leaves - 10 NAC, 6 WRKY and 2 MYB (Fig 3.). The senescence delaying NAC TF ANAC042 also called Jungbrunnen1 (JUB1) was up- regulated, but there were no genotypic differences in the expression. The transcription of the senescence-enhancing NAC genes ANAC074, ANAC087 and ANAC092 was strongly up-regulated in response to N deprivation and genotypic

132 DGP Meeting September 5-9, 2012 differences occurred. ANAC092 also called ORE1 is a regulator of many SAGs and responsive to hydrogen peroxide (Balazadeh et al., 2010). WRKY genes are involved in the regulatory network of stress responses. WRKY006 is a positive regulator of SIRK a receptor-like protein kinase, whose expression is strongly induced during leaf senescence (Robatzek et al., 2002). MYB TFs are involved in the synthesis of anthocyanin pigments. MYB090 regulates the Pigment 2 (PAP2) synthesis and was up-regulated in NPZ-2 in agreement with the accumulation of anthocyanins in the shoot and leaves in hydroponic culture under N deprivation in NPZ-2. Anthocyanins protect the plant from UV-radiation stress and inhibit photo-oxidation.

12 3000 NPZ-1 Gen ns ] 2500

-1 NPZ-2 DAT *** s 10 2000 -2 ns Gen*DAT ns m 1500 2 8 1000 500 ns 6 20

4 15

10 2 5 Photosynthesis rate [µmol CO [µmol rate Photosynthesis SAG12-1 of expression relative Linear

0 0 0 7 NPZ-1 NPZ-2 Days after N deprivation Genotype Fig. 1 Photosynthesis rate of NPZ-1 and NPZ-2 grown at 2.0 mM N Fig. 2 Linear relative expression of SAG12-1 in senescing leaves for 4 weeks followed by 7 days at 0.1 mM N. Error bars represent 7 days after N deprivation. The data are shown relative to day 0. the SD. Significant differences *** P<0.001, ns = non-significant. Error bars represent the SE. 350 NPZ-1 300 NAC WRKY MYB NPZ-2 250 (10) (6) (2) 200 150 100 50 40

30

20 Linear relative expression relative Linear 10

0 MYB015 MYB090 ANAC016 ANAC042 ANAC055 ANAC074 ANAC079 ANAC083 ANAC087 ANAC092 ANAC100 ANAC102 WRKY006 WRKY030 WRKY033 WRKY040 WRKY071 WRKY075

Transcription factor Fig. 3 Linear relative expression of the detected NAC, WRKY and MYB TFs in senescing leaves 7 days after N deprivation. The data are shown relative to day 0. () Number of detected TFs. Error bars represent the SE.

The results confirm that a range of TFs are involved in the regulatory network of the leaf senescence process and their expressions reflect genotypic differences in leaf senescence induced by N deprivation. Literature Balazadeh et al. (2010). Salt-triggered expression of the ANAC092-dependent senescence regulon in Arabidopsis thaliana . Plant Signaling & Behavior 5: 733-735. Buchanan-Wollaston et al. (2002). The molecular analysis of leaf senescence – a genomics approach. Plant Biotechnology Journal. 1: 3-22. Lim et al. (2007). Leaf senescence. Annual Review of Plant Biology. 58:115–36. Robatzek et al.(2002). Targets of At WRKY6 regulation during plant senescence and pathogen defense. Genes & Development. 16: 1139-1149. Wiesler et al. (2001). Nitrogen efficiency of contrasting rape ideotypes. In: Horst, W.J. et al. (eds.): Plant nutrition – Food security and sustainability of agro-ecosystems. Dordrecht: Kluwer Academic Publishes. 92:60-61.

133 DGP Meeting September 5-9, 2012 Role of leaf-inherent and root-localized factors for genotypic differences in nitrogen deficiency-induced leaf senescence in oilseed rape ( Brassica napus L.)

Martin A. Becker, Andreas Meyer, Fabian Köslin-Findeklee and Walter J. Horst

Institute of Plant Nutrition / Leibniz University Hannover, Hannover. E-mail: [email protected]

Introduction Winter oilseed rape is the most important oil crop in European agriculture, but one with the highest nitrogen (N) balance surpluses. This may negatively impact the environment. To reduce N balance surpluses, genotypes with improved N use efficiency (high grain yield under N-limiting conditions) have to be developed. Previous work suggested that increased nitrogen efficiency was related to delayed leaf senescence which leads to a longer “stay-green” status. Leaf senescence is a highly controlled process induced by the developmental age of the plant, but also by N starvation or by other growth-limiting conditions. It was hypothesized that delayed leaf senescence is beneficial to yield formation because of maintained photosynthesis into the reproductive growth stage (Wiesler et al. 2001). A longer assimilation period may not only directly benefit reproductive growth but also indirectly by increasing root growth and nitrogen uptake. The aim of the study was to clarify if genotypic differences in leaf senescence under N deficiency are due to leaf- inherent and/or root-localized factors?

Materials and Methods Based on an experiment comparing N deficiency-induced leaf senescence with the senescence of detached leaves two pairs of genotypes NPZ-1 (N-efficient) & NPZ-2 (N-inefficient) and Apex (N-efficient) & Capitol (N-inefficient) were selected. Plants of each pair were reciprocally, self- and non-grafted . After four weeks pre-treatment at optimal N supply, the plants were starved in N (0.1 mM N) or cultured under optimal conditions (4.0 mM N) for up to 12 days. The 3 rd leaf – counted from the bottom to the top – was harvested and analyzed. During the treatment period the senescence status of the leaf was non-destructively measured using SPAD. Additionally, the expression of the senescence-associated cysteine proteinase SAG12-1 was measured by qRT-PCR.

Results and Discussion At optimal N supply the SPAD values were constant in all genotypes (Fig. 1). Whereas under N starvation declined SPAD values were measured. The non-grafted plants and the self-grafted controls showed the genotypic differences in SPAD as expected. The N-efficient genotypes NPZ-1 and Apex had higher SPAD values after 134 DGP Meeting September 5-9, 2012 12 days N starvation than the N-inefficient genotypes NPZ-2 and Capitol. For the reciprocal grafting variants, NPZ-1/NPZ-2 (root/shoot) showed lower SPAD values in comparison to NPZ-2/NPZ-1. The reciprocal grafts of the second pair of genotypes (Apex&Capitol) reacted in the same way.

50 NPZ-1&NPZ-2ANOVA Apex&Capitol ANOVA *** 0.1 mM N Variant ** 0.1 mM N Variant N *** N *** 4.0 mM N 4.0 mM N 40 Variant*N + Variant*N ***

30

SPAD 20

10

0 Apex NPZ-1 NPZ-2 Capitol Apex/Apex Apex/Capitol Capitol/Apex NPZ-1/NPZ-1 NPZ-2/NPZ-2 NPZ-1/NPZ-2 NPZ-2/NPZ-1 Capitol/Capitol

Variant Fig. 1 SPAD values of non-grafted, self-grafted and reciprocal grafted plants of NPZ1&NPZ-2 and Apex&Captiol 12 days after N starvation (0.1 mM N) or grown under optimal N supply (4.0 mM N) . Significant differences p-value = 0.10 +; 0.05 *; 0.01 **; 0.001 ***. The error bars showed the SD. The relative expression of SAG12-1 confirmed the results (Fig. 2.) However, the SAG12-1 expression was much more sensitive then SPAD and genotypic differences become clearer. The low expression in the leaves of the non- and self-grafted plants of NPZ-2 was due to the advance stage of the senescence (Fig. 3)

250000 160000 NPZ-1&NPZ-2 Apex&Capitol 140000 200000 120000

150000 100000

80000 100000 60000

50000 40000

20000

Linear relative expression ofSAG12-1 0

Linear relative expression of SAG12-1 of expression relative Linear 0 Apex

NPZ-1 NPZ-2 0 5 10 15 20 25 Capitol

Apex/Apex SPAD low N (0.1 mM N) Apex/Capitol Capitol/Apex NPZ-1/NPZ-1 NPZ-2/NPZ-2 NPZ-1/NPZ-2 NPZ-2/NPZ-1

Capitol/Capitol Fig. 3 Correlation between SPAD and linear relative expression of SAG12-1 12 days after N starvation (0.1 mM N). Variant Fig 2. Linear relative expression of SAG12-1 12 days after N starvation (0.1 mM N). The data are shown relative to the high N control (4.0 mM N). The error bars showed the SE. The results suggest that the shoot is responsible for the development of leaf senescence induced by N deficiency. It can be assumed that genotypic differences in leaf senescence under N deficiency are due to leaf-inherent factors. To identify these factors, further research is required.

Literature Wiesler, F., Behrens, T., Horst, W.J. (2001). The role of nitrogen-efficient cultivars in sustainable agriculture. Optimizing Nitrogen Management in Food and Energy Production and Environmental Protection: Proceedings of the 2nd International Nitrogen Conference on Science and Policy.

135 DGP Meeting September 5-9, 2012 Genotypic variation of leaf nitrogen retranslocation between oilseed rape lines and hybrids

Li Wang, Gunda Schulte auf’m Erley, Karl H. Mühling

1Institute of Plant Nutrition and Soil Science/Kiel University, 24118 Kiel; E-mail: [email protected]

Oilseed rape hybrids were found more N-efficient than lines. Results from a preliminary field experiment suggest that this is not caused by higher N uptake or higher total biomass production but probably higher N translocation to the grains. A higher N translocation to the grains might rely on a better N retranslocation from dying leaves. To investigate genotypic variation in leaf N retranslocation capacity between lines and hybrids, a hydroponic experiment was conducted with four rape hybrids and the four corresponding father lines. The plants were cultivated under N sufficient conditions (2 mM) for 4 weeks and half of the plants were harvested. Then the remaining plants were subjected to N deficient conditions (0 mM) and harvested. Fully expanded leaves of certain positions were sampled separately at the first harvest, and the leaves from the same positions were collected after being shed from the plant because of N deficiency. Thus, maximal N retranslocation capacity from the leaves could be determined. Results were used to determine if hybrids generally have a higher leaf N retranslocation and if this is related to differences in leaf N accumulation during leaf growth, to N demand of growing tissues or to the translocation processes itself.

136 DGP Meeting September 5-9, 2012 Nitrogen use efficiency of some Turkish bread wheat genotypes

Fatma Gökmen Yilmaz 1* , Nesim Dursun 1, Sait Gezgin 1,

1Selcuk University Faculty of Agriculture Department of Soil Science and Plant Nutrition, Kampus, 42079, Konya, Turkey. E-mail: [email protected]

Introduction Nitrogen (N) is often the most limiting nutrient for crop productivity in many region of the world. N fertilizer is one of the main inputs for cereals production systems. Worldwide, nitrogen use efficiency (NUE), which can be defined as the product of uptake efficiency 'total N uptake/applied N through fertilizer' and utilization efficiency 'yield/total N uptake', for cereal production including wheat is approximately 33% (Raun and Johnson, 1999). At low N rates, uptake efficiency is dominant compared to utilization efficiency, whereas utilization efficiency is relatively more important than uptake efficiency at high N rates. Genetic variation has been reported to be important on wheat for nitrogen use efficiency (Ortiz-Monasterio et al. 1997, Dhugga and Waines 1989). In our study where hexaploid Turkish wheat varieties were evaluated in terms of nitrogen use efficiency. Significant genotypic differences were also observed for Nitrogen physiological efficiency (NUE), Nitrogen uptake efficiency (NupE) and Nitrogen utilization efficiencies (NutE). Materials and Methods The trial using randomized complete parcel design with three replications, was conducted within a controlled (heat, light and relative humidity) greenhouse using N- deficient soil obtained from the Central Anatolian Region. The slightly alkaline -1 (pH=8.09) soil was clay loam, low in NO 3-N (30.0 mg kg ) and organic matter (0.95 %).Other soil test parameters were; available P=10.4 mg kg -1, Zn=0.30 mg kg -1, K=0.92 meq 100 g -1 and EC= 0.82 dS/m. Experiment with 39 wheat cultivars (Ahmeta ğa, Altay 2000, Atilla 12, Atlı 2002, Ba ğcı 2002, Bayraktar 2000, Bezostaya 1, Bolal 2973, Da ğda 94, Demir 2000, Ekiz, Eser, Gelibolu, Gerek 79, Göksu 99, Gün 91, Harmankaya, Đzgi 2001,Karahan 99, Kate A-1,Kınacı 97, Kıraç 66, Konya 2002, Mızrak 98, Momtchil, Odeskaya, Pehlivan, Prostor, Saraybosna, Seval, Sönmez 2001, Sultan 95, Tekirda ğ, Tosunbey, Türkmen 98, Uzunyayla 98, Yakar 99, Yıldız 98 and Zencirci 2002) was conducted within pots. Seeds were sown in each plastic pot containing 3 kg of oven-dried soil. N application included 75 mg kg -1 of N at the time of sowing and also at tillering followed by 150 mg kg -1 of N addition during -1 stem elongation, overall 300 mg kg N, as NH 4NO 3 application/pot. Other nutrient elements used were in the form of their respective available commercial fertilizers

(TSP, FeSO 4.7H 2O and ZnSO 4. 7H 2O). Soil moisture levels were monitored throughout the growing period by weighing three randomly selected pots for each treatment everday. When needed, the required amount of water was supplied. Plants were harvested after 50 days of germination. The samples were oven-dried to a constant weight at 70 ̊C for 72 hours. Samples were analyzed for total N content 137 DGP Meeting September 5-9, 2012 using a LECO C/N analyzer according to the Dumas Combustion Method (AACC, 2004). Data on dry matter production, nitrogen concentration and nitrogen efficiency parameters were collected. The terminology of NUE parameters is in accordance with Moll et al., (1982). NupE =N t / N supply where N t is total plant N uptake and N supply is the sum of the soil N content and N fertilizer. NutE =P w / N supply where P w is plant dry weight. NUE =P w / N t. El Bassam’s (1998) method was used to determine the differences between efficiencies of the genotypes (physiological, utilization and uptake efficiencies). Accordingly, 39 varieties used were divided into 3 different groups, namely high, medium and low performance groups. Results and Discussion Significant differences between the wheat genotypes used were observed in terms of dry matter production, nitrogen concentration and nitrogen efficiency parameters, within the ranges of 2.65 -4.82 g pot -1, 3.25-5.20 % and 103.9-226.6 mg pot -1, respectively. Nitrogen uptake efficiency reflects the efficiency of the crop in obtaining N from the soil. Wheat genotypes differed in NupE, significantly. Yakar-99 bread wheat variety was observed to provide the highest uptake efficiency (68.2). According to Moll et al. (1982), variation in NUpE could be separated from grain yield variation. In addition, Lopez-Bellido (2001) indicated that crop N uptake is directly related to, the NUpE while Lee et al., (2004) indicated that NUpE was positively correlated with plant dry matter, leaf area index and leaf nitrogen content. Results demonstrated that there was a positive correlation between NUpE and nitrogen uptake of wheat varieties used. Wheat genotypes presented differences for NutE. Gerek-79 bread wheat variety was found to exhibit the highest utilization efficiency (14.5) while the variety Karahan-99 was observed to provide the highest physiological efficiency (30.8). In conclusion, significant differences between bread wheat genotypes were observed for NUE, NUpE, NutE when evaluated in terms of nitrogen use efficiency. Field trials should be conducted using the bread wheat genotypes of high nitrogen use efficiency (Gerek 79, Bezostaya 1, Altay 2000, Bayraktar 2000, Kate A-1, Đzgi 2001, Sönmez 2001 and Karahan 99). Literature Dhugga, K.S. and Waines, J.G. 1989. Analysis of nitrogen accumulation and use in bread and durum wheat. Crop Sci. , 29: 1232-1239. El Bassam, N. 1998. A concept of selection for ‘low input’ wheat varieties. Euphytica, 95-1010. Lee, H.J, Lee, S.H, Chung, J.H. 2004. Variation of nitrogen use efficiency and its relationships with growth characteristic in Korean rice cultivars. In Proc. 4 th International Crop Science Congress, Australia. López-Bellido, R.J. and López-Bellido, L. 2001.Effects of crop rotation and nitrogen fertilization on soil nitrate and wheat yield under rainfed Mediterranean conditions.Agronomie 21: 509-516. Moll, R.H., Kamprath, E.J. and Jackson, W.A. 1982. Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J. , 74: 562-564. Ortiz-Monasterio, R., J.I., Sayre, K.D., Rajaram, S. and McMahon, M. 1997. Genetic progress in wheat yield and nitrogen use efficiency under four N rates. Crop. Sci. , 37(3): 898-904. Raun, W.R., and G.V.Johnson. 1999. Improving nitrogen use efficiency for cereal production. Agron. J. 91:357–363.

*This study was a part of Gökmen Yilmaz’s PhD thesis.

138 DGP Meeting September 5-9, 2012 How to improve salt-resistant maize genotypes genetically - application and future prospects

Philipp T. Eitenmüller

Institute of Plant Nutrition, JLU Gießen; E-mail: [email protected]

Two salt-resistant maize (Zea mays L.) genotypes with similar growth in the first phase of salt stress, show different strategies of salt resistance. While SR 03 maintains cell wall acidification under salt stress to keep cell walls extensible. SR 12 changes the composition of its cell wall to maintain extensibility. Combining both strategies in one genotype should further enhance cell wall extensibility and increase salt resistance in the first phase of salt stress. To combine both strategies in one genotype, SR 03 and SR 12 where crossed. The resulting F2 material is very heterogenous. To obtain a line with superior properties for further breeding purposes, the inbred progeny will be selected recurrently over seven generations for the attribute of increase of leaf length in the first phase of saltstress. The first cycle of selection has been completed and shows improvement of salt resistance in the first phase of salt stress. The increase of leaf length compared to SR 03 as control value shows an improvement of 50% for the selected plants. After obtaining a superior line, this will be tested for maintenance of cell-wall acidification and cell-wall composition. The combination of the two different strategies may improve the resistance in the first phase of salt stress in a synergistic way.

139 DGP Meeting September 5-9, 2012 Effects of drought and salt stress on kernel setting of maize

Stephan Jung, Christina Ander, Sven Schubert

Insitute of Plant Nutrition Giessen, Heinrich-Buff-Ring 26-32, IFZ, 35392 Gießen Germany; E-mail: [email protected]

Maize is considered a sensitive crop with regard to drought and salt stress conditions, while it is a major food crop at the same time. In order to develop more resistant varieties, it is essential to understand the underlying physiological mechanisms of these environmental constraints. In the present study, various maize varieties were tested for drought resistance in a container experiment. Kernel setting as a sensitive parameter is usually decreased in maize plants when drought or salt stress occurs, while kernel weight often increases as adjustment to reduced kernel setting. Of all varieties tested, one genotype showed no reduction in kernel setting under drought. Therefore this variety was tested in a second container experiment under drought- and salt stress conditions. The results show that kernel setting was decreased by 20% under drought stress compared to the corresponding control, while there was no significant change at 11 dS m-1 of salt stress. In conclusion, this variety is an interesting genotype with regard to kernel setting and thus is noteworthy for further physiological studies and the development of drought and salt-resistant maize genotypes. Keywords: drought stress, salts stress, Zea mays, kernel setting

140 DGP Meeting September 5-9, 2012 Early leaf growth rates reflect adaptation of maize genotypes to temperature regimes

Katrin von Arx, Frank Liebisch, Niclas Freitag and Achim Walter

Institute of Agricultural Sciences, ETH Zürich, Universitätsstrasse 2, LFW, A 04, Zürich 8092, Switzerland; E-mail: [email protected]

Maize is cultivated from tropical to temperate regions around the world. The identification of tolerance to high and low temperatures in early growth stages may support breeding programs and crop research concerned with climate change. In our study we measured leaf growth of three Zea mays genotypes in three different climate conditions (cold, temperate summer and constantly warm). The tested genotypes were a tropical line (Penjalinan), a European Hybrid (Fabregas) and the cold tolerant genotype Z7. Leaf growth has been measured with Rotational Resistance Transducers (RRTs) on leaf two. We investigated the growth on absolute parameters (length, duration), growth pattern (evolution of growth rates) and short term changes (day/night transitions). We found that the total leaf growth and duration differed between the genotypes reflecting their temperature adaptation. Growth patterns were generally similar, but showed differences of the fitted sigmoidal function parameters (plateau length and slope) between genotypes and climate conditions. Reactions to short term temperature changes were similar between genotypes. Our results show that early maize growth can be used as an indicator for temperature preference and adaptation of maize genotypes. To identify genotypic differences the measurement of the whole growth period was superior to the short term temperature changes. The here presented method could be used for early and cheap recognition of cold or heat tolerance in maize.

141 DGP Meeting September 5-9, 2012 Iron exclusion as a tolerance mechanism to iron toxicity in rice: genetic and physiological aspects

L. B. Wu 1, G. Gregorio 2, P. Oldgee 1, M. Becker 1, M. Frei 1

1Institute of Crop Science and resource Conservation (INRES), Plant Nutrition, Rheinische Friedrich Wilhelms-Universität Bonn, 2International Rice Research Institute (IRRI), Philippines; E-mail: [email protected] Introduction Iron is an essential element in photosynthesis, respiration, and nitrogen metabolism in rice, Oryza sativa (L.). However, excess amounts of Fe(II) are toxic as they lead to the generation of renieceactive oxygen species (ROS) in leaf tissue, which can irreversibly damage cell structural components and impair physiological processes (Becker and Asch, 2005). The typical visual symptom associated with these processes is leaf bronzing. Plants may adapt to Fe toxicity by excluding Fe from uptake into the plants. This exclusion mechanism could be due to (a) oxidation of Fe (II) in rhizosphere forming root plaque (b) impermeable barriers in roots preventing excess iron taken up into plants or (c) binding of iron in insoluble complexes in the rhizosphere, for example by exudation of organic acids. In this study, we aimed at genetically dissecting iron exclusion traits in rice and investigating their physiological basis. Materials and Methods A population of 124 recombinant inbred lines from IR29 (intolerant)/Pokkali (tolerant) were grown hydroponically in the greenhouse. Genotyping data (100 SSR markers) were available from previous experiments (Thomson et al., 2010) Five-week old seedlings were exposed to iron stress of 1,000 ppm Fe(II) with N 2 bubbling for 15 min every 2h to keep iron in the reduced state. The leaf bronzing score (LBS) and dry weight of each line were taken as phenotypic traits. The parent lines (IR29 and Pokkali) and the tolerant RIL FL510 were used for further experiments, in which iron concentrations in different plant compartments, as well as root lignin and suberin concentration were determined. Root oxidation was investigated using a 0.75% agar solution containing 2mg.l -1 methylene blue as a redox indicator (Chabbi et al., 2000). Results and Discussion QTL mapping QTL mapping was conducted using leaf bronzing score as phenotypic data. Using various mapping approaches, the most consistent QTL was located on chromosome 2, between the markers RM13197 and RM13332, which could explain 16.9% of the phenotypic variance. The favorable allele was from Pokkali. Exclusion mechanism study The parent lines (IR29 and Pokkali) and FL510, which carried the favorable allele at the QTL position, were grown to study shoot iron concentration, root plaque formation, and mechanical barriers in root. Leaf bronzing score (LBS) was measured 142 DGP Meeting September 5-9, 2012 on the second and fifth day of treatment. The leaf bronzing scores after two days and five days were significantly higher in IR29 than in the tolerant genoytypes, confirming the previous results in an independent experiment. The shoot Fe concentrations were 1.8 and 2.1 times higher in IR29 than in Pokkali and FL510, respectively. While the total amount of iron taken up by Pokkali was significantly higher (1.7-fold) than in IR29 due to substantially higher biomass, the opposite was true for FL510, where total iron uptake was even lower than in IR29. Together, these data suggest that the tolerance QTL determined represents an exclusion mechanism. Lignin and suberin concentration in roots of contrasting lines were tested, however, no significant differences were found, suggesting that other factors were responsible for the observed differences in iron uptake, such as oxidation of iron in the rhizosphere. However, no consistent differences in root plaque formation between the contrasting lines were observed. The methylene blue staining experiments suggested that the root oxidative power was highly related to the amount of root hairs and root tips, which diffuse oxygen from the aerenchyma into rhizosphere to oxidize ferrous iron to ferric iron. However, root plaque formation (especially in nutrient solution) mostly occurs on secondary roots and adventitious roots, which provide a physical support for iron plaque. This could explain the apparent lack of correlation between root plaque formation and iron exclusion. We are currently conducting some experiments to quantify root hairs of contrasting lines. Another hypothesis we are currently investigating is that root exudates stimulated by iron toxicity could lead to the formation of insoluble iron complexes. For example, the solubility of the tridentate complex composed of ferrous iron and citric acid is poor in water, and is resistant to biodegradation(Francis et al., 1992). Thus, high citrate exudation could lead to the formation of ferrous citrate complexes that are precipitated to exclude iron from uptake into plants. Experiments to test this hypothesis are currently ongoing. Conclusion The observed tolerance of IR29/Pokkali recombinant inbred lines was due to an iron exclusion mechanism, which was not related to root mechanical barriers such as lignin and suberin, but rather to oxidation and/or precipitation of iron outside the root. Literature Becker M., Asch F. (2005) Iron toxicity in rice-conditions and management concepts. Journal of Plant Nutrition and Soil Science 168:558-573. DOI: 10.1002/jpln.200520504. Chabbi A., McKee K.L., Mendelssohn I.A. (2000) Fate of oxygen losses from Typha domingensis (Typhaceae) and Cladium jamaicense (Cyperaceae) and consequences for root metabolism. American Journal of Botany 87:1081-1090. Francis A.J., Dodge C.J., Gillow J.B. (1992) Biodegradation of metal citrate complexes and implications for toxic-metal mobility. Nature 356:140-142. Thomson M.J., de Ocampo M., Egdane J., Rahman M.A., Sajise A.G., Adorada D.L., Tumimbang-Raiz E., Blumwald E., Seraj Z.I., Singh R.K., Gregorio G.B., Ismail A.M. (2010) Characterizing the Saltol Quantitative Trait Locus for Salinity Tolerance in Rice. Rice 3:148-160. DOI: 10.1007/s12284-010-9053-8.

143 DGP Meeting September 5-9, 2012 Expression of aluminium tolerance genes in triticale

Neele Wendler 1* , Emmanuel Delhaize 2, Peter R. Ryan 2, Walter J. Horst 3, Hans P. Maurer 4, Karl H. Mühling 1

1Institute of Plant Nutrition and Soil Science/ Kiel University, Kiel; 2CSIRO Plant Industry, Black Mountain Laboratories Canberra; 3Institute of Plant Nutrition/ Leibniz University, Hannover; 4Plant Breeding Institute/ University Hohenheim. E-mail: [email protected] *present address: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben

Introduction Acid soils are one of the major plant growth limiting factors worldwide (Granados et al. 1993). Plant growth on acid soils is primarily inhibited due to direct effects of aluminium (Al) on root growth. Here the main injury occurs in the first ~ 3-5 mm of the root tips. The inhibited and damaged roots are then unable to maintain sufficient nutrition for the plant, which leads to nutrient deficiencies, reduced shoot growth and finally a diminished yield. To cope with the problem of Al toxicity, breeding of Al resistant crops is a viable approach. Al resistance of cereals relies mainly on the exudation of organic acids (OA) such as citrate and malate to the rhizosphere (Delhaize et al. 1993). In the rhizosphere the OAs bind Al and this complex is harmless to plant roots (Delhaize et al. 1993). There are two known families of membrane proteins: the MATEs (multi-drug and toxin extrusion) and the ALMTs (Al 3+ -activated malate transporter) that confer Al resistance in cereals. In both wheat and rye there are genotypes that possess genes for the MATE as well as the ALMT protein family. Within Al resistance cultivars of cereals rye shows the highest Al resistance. Triticale is a hybrid between rye and wheat and it is still not well understood whether the Al resistance is due to the rye and/or the wheat resistance genes. Therefore the aim of the project was to investigate expression patterns of the wheat (TaALMT1 and TaMATE1) and the putative rye (ScALMT and ScMATE) resistance genes in triticale in comparison to its parental lines.

Material and Methods To investigate the pattern of Al resistance in triticale, two different triticale lines have been chosen, which were generated by crossing two different wheat lines differing in Al resistance with the same Al resistant rye inbred line. Seeds of all genotypes were grown in Al treatment with 40µM Al and a control treatment with 0 µM Al, respectively. The seedlings root length was measured on the day they were planted and again after four days growth in the two treatments. A reverse trancriptase qPCRs for the Al resistance genes was performed at RNA samples extracted from each genotype after four days of treatment.

144 DGP Meeting September 5-9, 2012 Results and Discussion Root growth measurements indicated that both triticale lines had a similar resistance level as the resistant wheat parent. Furthermore both triticale lines did not reach the high Al resistance of the rye parent. The findings indicate that expression of Al resistance in triticale does not simply rely on additive gene effects derived from the rye and wheat parents. The qPCR results showed that there was no change in the expression pattern of the wheat genes in triticale compared to their wheat parents. By contrast there were changes in the expression of the investigated rye genes in triticale compared to the rye parent. The observed suppression of the rye genes seemed to depend on the expression level of the homolog wheat genes and it might explain the resistance pattern found in triticale.

Literature Delhaize, E., Ryan, P. R., Randall, P. J. (1993) Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiol . 103 : 695–702. Granados G, Pandey S, Ceballos H. (1993) Response to selection for tolerance to acid soils in a tropical maize population. Crop Sci . 33 : 936–940.

145 DGP Meeting September 5-9, 2012 Sulfur-enhanced plant defense against Verticillium dahliae in a tomato introgression-line population

Inga-Mareike Bach 1, Katharina Bollig 1 and Walter J. Horst 1

1Institute of Plant Nutrition, Leibniz Universität Hannover, Herrenhäuserstraße 2, Hannover, Germany. E-mail: [email protected]

Introduction Verticillium dahliae (V. dahliae ) is a soil-borne fungus, which spreads in vascular plant tissue and induces wilt disease (Fradin and Thomma, 2006). Presently available fungicides are not effective in protecting plants against fungal colonization. Therefore, Supra-optimal sulphur (S) fertilization might promote S-enhanced plant defense (SED) mechanisms, which were detected in Solanaceae and are thought to prevent in planta V. dahliae spread (Williams et al., 2002; Novo et al. 2007). Additionally, genes conferring increased resistance (R gens) of tomato against the Verticillium -wilt disease might also be involved in V. dahliae defense in this IL set. Therefore, the aim of this study was the identification of V. dahliae -sensitive and resistant introgression lines (ILs), to analyze existence and chromosomal localization of SED. Additionally, the expression of known R genes and downstream signalling compounds involved in V. dahliae defense has been analyzed. In this study an IL- population originating from the introgression of defined chromosomal segments of the wildtype (WT) Solanum habrochaites into the cultivated recurrent parent (RP) Solanum lycopersicum (Monforte and Tanksley, 2000) was used. The influence of supra-optimal plant S nutrition on fungal colonization was determined in the whole IL population to further select for SED-capable ILs. In WT, RP and selected IL plants, the expression of the resistance gene Ve1 , which encodes an extracellular receptor, and the expression of Rar1 , a co-chaperon mediating fungal effector-triggered plant immunity, was monitored (Fradin et al., 2009; Schulze-Lefert, 2004).

Material and Methods V. dahliae-infected ILs, WT and RP plants were cultivated in the greenhouse at supra-optimal (25 mM SO 4-S) S supply . A quantification of the fungus by absolute qRT-PCR was performed with a V. dahliae -specific primer pair. Based on this general IL-screening, a resistant and a sensitive IL were selected and together with WT and

RP plants cultivated in the greenhouse at low (0.016 mM SO 4-S) and supra-optimal

(25 mM SO 4-S) S supply in the presence or absence of V. dahliae . The fungal colonization was determined by an isolate-specific absolute qRT-PCR. Physiological parameters including leaf total S concentration, shoot height, root length, photosynthetic rate, and phloem sugar-transport rate were analyzed. A relative qRT- PCR assay was performed with primer pairs for the R-gene Ve1 and the signal 146 DGP Meeting September 5-9, 2012 transduction-facilitating gene Rar1.

Results and Discussion The set of tomato ILs was analyzed for V. dahliae resistance with an absolute qRT- PCR assay. According to the level of fungal gDNA four significantly different groups including a susceptible group, two intermediate groups and a highly resistant group were identified. In the intermediate groups the WT was among the more susceptible lines showing a higher fungal colonization in comparison to the RP. Hence, possible SED-mediating gene loci seem to be inherent in the RP genomic background. Based on the global V. dahliae screening, a resistant and a sensitive IL were selected for further analysis under variable plant S fertilization and V. dahliae infection. The resistant IL showed lowest fungal concentrations of about 0.1 pg ng -1 at low and supra-optimal S supply. Both parental lines had similar levels under S deficiency, whereas the fungal colonization was reduced by high S supply in the RP but increased in the WT. Highest concentration of V. dahliae could be measured in the sensitive IL at low S status, but supra-optimal S nutrition reduced fungal gDNA to a level comparable to the RP. This indicates an in-planta fungicidal effect of externally applied S against V. dahliae being existent in the RP and not in the WT. Rar1 was less expressed in the WT but highest transcript levels were detected in the resistant IL at high S supply together with a fungal colonization. This underlines the signal- transduction supporting role of Rar1 in V. dahliae defense of ILs being further enforced by high S supply. WT plants showed lowest expression of Ve1 , which was only slightly induced by high S supply and fungal colonization. In contrast Ve1 transcript levels were equal and high in the RP, sensitive and resistant IL in response to V. dahliae . Supra-optimal S supply further increase Ve1 expression in the infected resistant IL. Thus, a beneficial effect of enhanced S-nutrition could also be demonstrated in the resistant IL for participants of the V. dahliae elicitor-triggered plant immunity response.

Literature Fradin, E.F., Thomma, B.P.H.J. (2006). Molecular Plant Pathology, 7, 71-86. Fradin, E.F., Zhang, Z., Juarez Ayala, J.C., Castroverde, C.D.M., Nazar, R.N., Robb, J., Liu, C.-M., and Thomma, B.P.H.J. (2009). Plant Physiology, 150, 320-332. Monforte, A.J., Tanksley, S.D. (2000). Genome, 43, 803-813. Novo, M., Gayoso, C. M., Pomar, F., Lucas, M. M., Ros Barceló, A. & Merino, F. (2007). Plant Pathology, 56, 998 – 1004. Schulze-Lefert, P. (2004). Current Biology, 14, R22-R24. Williams, J.S., Hall, S.A., Hawkesford, M.J., Beale, M.H., and Cooper, R.M. (2002). Plant Physiology, 128, 150-159.

147 DGP Meeting September 5-9, 2012 Poster Session P4: Root physiology / Root-soil interaction

148 DGP Meeting September 5-9, 2012 Influence of calcium content and pH on plant available phosphorus in two luvisols

Holger Lemme 1, Heinz-Josef Koch 1

1Institut für Zuckerrübenforschung, Göttingen. E-mail: [email protected]

Introduction In agricultural soils Calcium (Ca) and pH value play an important role for the availability of nutrients. Our study aimed (i) to quantify the effect of liming on plant available phosphorus (P) depending on the period between liming and plant nutrient uptake and (ii) to investigate the suitability of several soil analysis methods (EUF, CAT, CAL) to predict the amount of plant available P. Additionally, the two effects of liming, namely the addition of Ca 2+ ions and the increase of the pH, on the P- availability were investigated.

Materials and Methods Two loessial soils (Göttingen, Ochsenfurt) were mixed with three different additives

(Burnt lime (CaO), Gypsum as β Hemihydrate ( β CaSO 4 * 0,5 H 2O) and Soda lye (NaOH); Table 1), adjusted to a water content of 40% of the maximum water holding capacity (WHC max ) and incubated at 12 °C for 24, 8 or 4 weeks. Afte r incubation 1 kg of soil per treatment was taken, dried and ground (<1 mm) for soil analyses. The remaining incubated soil (1 kg) was used in a pot experiment in the greenhouse. Ten sugar beet seeds were planted in each pot and thinned to 5 plants after emergence.

The soil water content was kept between 60% and 80% of WHC max by frequently watering with de-ionized water. The growing conditions were: 18 °C, 4,5 klux m -2 -1 artificial light for 12 h per day, nitrogen (N) fertilization (250 mg N kg as NH 4NO 3) and fungicide application (Tachigaren). After 10 weeks of growing the total plants (beet + leaf) were harvested, dried (105 °C), weigh ted and ground (<0,5 mm). The soil pH was measured and the extractable P content was analyzed by Electro-

Ultra-Filtration (EUF), CAT (CaCl 2+DTPA), and CAL extraction.

Tab. 1: Amounts of the additives Burnt lime, Gypsum and Soda lye added to the soil based on equal amounts of OH - and Ca 2+ ions in three levels (low, medium, high). ------Additives ------Level OH - Ca 2+ Lime Gypsum # Soda lye ## [mmol kg -1 DM] [g kg -1 DM] [t ha -1] [g kg -1 DM] [t ha -1] [ml kg -1 DM]

Low 53.5 26.8 1.5 2.8 3.9 7.3 54 Med 142.7 71.3 4.0 7.5 10.4 19.4

High 285.3 142.7 8.0 15.0 20.7 38.8 # ## Gypsum as β Hemihydrate ( β CaSO 4 * 0,5 H 2O); (1 Molar); DM = dry matter

149 DGP Meeting September 5-9, 2012 The plant material was digested with HNO 3 and H 2O2 at 220 °C in microwave pressure vessels and also analyzed for its P content using ICP-OES. The P uptake was calculated from the total plant yield and the P content of the plant material. Both parameters, P content and P uptake, were used to evaluate the plant availability of P.

Results and Discussion The effect of the soil type was significant for all investigated parameters, whereas no differences were found between the incubation time. There were no relevant interactions between soil, incubation time and additive; therefore, the mean values across soil type and incubation treatments are given below. The pH value increased from 6,9 in the control up to 8,9 in the Lime High treatment, Gypsum had no effect on the pH. The Ca content (EUF) of the soil increased by adding Lime and Gypsum. The extractable Ca in the Gypsum treatments was higher than in the Lime treatments, although the amounts of Ca added were equivalent. The total sugar beet plant yield increased by the addition of Lime, Gypsum and Soda lye compared to the control by 60, 60, and 191%, respectively. In the Lime treatments, P uptake und P content of the plants increased with increasing pH, whereas it remained constant with the increasing Ca content of the Gypsum treatments. The extractable P (EUF, CAL) of the soil increased with the addition of Lime and Soda lye, whereas the CAL and EUF method detected considerably (5,2 - 9,1 mg (100 g) -1) and slightly (3,2 - 5,5 mg (100 g) -1) more P in the soil, respectively, than the plants were able to take up. A close correlation between CAL-P and P uptake (CAL: r = 0,78) and between EUF-P and P uptake of the plants (EUF: r = 0,79) was observed, when all treatments were included. The CAT extracted P achieved a close correlation to P uptake only for the Gypsum treatments. Both, EUF and CAL method were able to indicate plant available P very well. But the portion of plant available P on the total P varied depending on the analysis method. Furthermore, the P availability increased with increasing pH of the soil, even at an initial pH close to seven. The reason for this could be an enhanced desorption of P from the surfaces of Iron- (Fe) and Aluminum- (Al) oxides or an enhanced solubility of Fe- and Al-phosphates (Blume et al., 2010). In contrast to Blume et al., 2010, high Ca contents had no decreasing effect on P availability. These results require further validation.

Literature Blume, H.-P.; Brümmer, G.W.; Horn, R.; Kandeler, E.; Kögel-Knabner, I.; Kretzschmar, R.; Stahr, K.; Wilke, B.-M.; Thiele-Bruhn, S.; Welp, G. (2010): Scheffer/Schachtschabel. Lehrbuch der Bodenkunde. (16. Auflage) Spektrum Akade mischer Verlag, Heidelberg. 569 S (S. 414 - 417).

150 DGP Meeting September 5-9, 2012 Functional analysis of candidate genes for P-starvation induced root hair growth

M. Hinrichs, M. Bremer, M. K. Schenk

Institute of Plant Nutrition, Leibniz Universität Hannover. E-mail: [email protected]

Introduction Phosphate starvation enhanced in Brassica carinata root hair length which was accompanied by an upregulation of leucine-rich/ extensin protein (LRX1) and leucine- rich repeat receptor-like protein kinase (LRR) and a downregulation of hydroxyproline-rich glycoprotein (HRGP). To analyze the function of these genes in root hair growth, the RNAi mechanism was established to knock down genes in an unsequenced organism. The Agrobacterium rhizogenes mediated gene transfer was used to produce transgenic roots containing an RNAi vector for silencing genes of interest (GOI). A transformation protocol for A. rhizogenes mediated gene transfer was established for B. carinata . Successfully transformed roots were selected by using dsRed protein in vivo. Downregulation of the GOI was verified by quantitative real-time PCR. The quantitative transcription levels of GOI will be demonstrated and the effect of gene silencing on root hair growth during P-starvation will be discussed.

Materials and Methods A RNAi vector based on pK7GWIWG2(II):dsRed (LIMPENS et al., 2005) carrying the dsRed reporter gene was constructed using site directed cloning of the genes of interests (GOI). For all RNAi- experiments the strain Arqua1 was used (QUANDT et al., 1993) containing the RNAi- construct was generated by the Gateway-Cloning-System (Invitrogen). Hypocotyls of 7 days old seedlings were infected with Arqua1 with an adjusted method from VIEWEG et al. (2004). 4 weeks after inoculation dsRed positive roots were microscopically selected at wavelength 550 nm and all other roots were removed. Subsequently plants were cultivated in full nutrient solution and nutrient solution without phosphate. After 6 days of cultivation, 1,5 cm root tips were collected for RNA isolation and transcript level was determined by the 2 - -Ct -method. Gene expression of dsRed was used to ensure that harvested roots were transgenic.

Results and Discussion The inoculation method, described in VIEWEG et al. 2004 and LIMPENS et al. (2004) resulted in 30% transformation efficiency, which is in the expected range. By real time PCR it was shown that the transcript level of our GOI was reduced. 151 DGP Meeting September 5-9, 2012 Transcript level as determined by real- time PCR of the GOI, in this case LRR, was upregulated under P- starvation in wildtype plants but not in transgenic plants. This confirmed a very successful downregulation of the GOI using RNAi (Figure 1). The RNAi method offers a chance to investigate the functionality of specific genes in B. carinata .

Figure 1: Relative expression level of leucine rich receptor kinase (LRR) of wildtype and transgenic plants as affected by P- starvation

Literature Limpens E., Ramos J., Franken C., Raz V., Compaan B., Franssen H., Bisseling T. and Geurts R. (2004): RNA interference in Agrobacterium rhizogenes - transformed roots of Arabidopsis and Medicago truncatula . Journal of Experimental Botany, Vol. 55, pp. 983-992 Limpens E., Mirabella R., Fedorova E., Franken C., Franssen H., Bisseling T. and Geurts R. (2005): Medicag o N 2-Fixing Symbiosomes Acquire the Endocytic Identity Marker Rab7 but Delay the Acquisition of Vacuolar Identity. The Plant Cell vol.21 no. 2811-2828 Quandt H.-J., Pühler A., Broer I. (1993): Transgenic Root Nodules of Vicia hirsuta : A Fast and Efficient System for the Study of Gene Expression in Indeterminate-Type Nodules. MPMI Vol. 6, No. 6 p. 699- 705 Vieweg M. F., Fru hling M., Quandt H. J., Heim U., Ba umlein H., Pu hler A., Ku ster H., and Perlick A. M. (2003): The Promoter of the Vicia faba L. Leghemoglobin Gene VfLb29 Is Specifically Activated in the Infected Cells of Root Nodules and in the Arbuscule-Containing Cells of Mycorrhizal Roots from Different Legume and Nonlegume Plants. MPMI Vol. 17, No. 1, pp. 62-69

152 DGP Meeting September 5-9, 2012 Is mineralization of Na-hexaphytate affected by soil characteristics?

Alexandra Wening, Bernadeta Strochalska, Diedrich Steffens and Sven Schubert

Justus-Liebig-Universität Gießen, Heinrich-Buff-Ring 26-32, IFZ, Institut für Pflanzenernährung, 35392 Gießen, Germany; E-mail: [email protected]

In upper layers of arable soils, 20% to 80% of the total phosphorus (P) concentration are organically bound P, which can be used by many plant species. The bioavailability of Na-hexaphytate (C 6H6O24 P6Na 12 ), which was used as a source of organic P, is not be covered by various routine soil-testing methods. In soil incubation experiments, the effect of five soil types (vertisol, podzol, luvisol topsoil, luvisol subsoil, ferralsol) on the mineralization of applied Na-hexaphytate (100 mg P kg -1 soil) in comparison to treatments with Ca(H 2PO 4)2 (100 mg P kg -1 soil) and without P under controlled conditions (16 h at 21°C , 8 h at 16°C, 50% water-holding capacity) was investigated. After 71 d, only the two soils with a higher pH (luvisol topsoil and luvisol subsoil) showed a net mineralization of phytate. After this period, glucose (10 mg kg-1 soil) was added as a further treatment in order to increase the microbial activity for higher mineralization of Na-hexaphytate. The incubation was continued under the same conditions mentioned above until day 140. Surprisingly, in all treatments the addition of glucose resulted in immobilization of P. Obviously, the increasing biomass took up and included the CAL-extractable soil P. In order to check the results of the first part of the experiment another incubation experiment has been started with the addition of CaCO 3 to test if the pH value is responsible for increased mineralization of Na-hexaphytate.

153 DGP Meeting September 5-9, 2012 Phosphate ageing in a luvisol amended with Fe and Al oxides

Imran Ashraf, Diedrich Steffens and Sven Schubert

Institute of Plant Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, Email: [email protected]

Introduction Phosphorus (P) availability is one of the major problems in acid and calcareous soils. In acid soils, P is adsorbed at surfaces such as Fe oxides and Al oxides. These oxides adsorb P more strongly than other soil P adsorbents e.g. clay minerals (Burnham and Lopez-Hernandez 1982). In the presence of amorphous Fe and Al oxides, this adsorbed P further reacts with these amorphous oxides, resulting as occluded P. This occluded P fraction is highly unavailable to plants. Soil P adsorption and P occlusion are the major causes of low P availability in acid soils i.e. Ferralsols. These soils are high in crystalline and amorphous Fe and Al oxides. When these soils are fertilized with P, most of P is adsorbed to these crystalline oxides and then becomes occluded by amorphous oxides, termed phosphate-ageing (Parfitt et al., 1975). The objectives of our research were to understand this process of phosphate- ageing and to know the kinetics of P-adsorption and P-occlusion by Fe oxide and Al- oxide minerals.

Materials and Methods A soil incubation experiment was performed to study the role of these mineral oxides on phosphate ageing in a Luvisol (subsoil). These minerals were artificially prepared according to a method described by Schwertmann and Cornell (2000). The Luvisol used was analyzed for physical and chemical characteristics. The pH value of this soil was 7.5 and we adjusted two other pH levels, i.e. 5.2 and 4.6, by addition of HCl. There were two P levels i.e. with P (P+) and without P (P-). In P+ treatments, 200 mg

P/kg soil were applied as KH 2PO 4. Fe oxides and Al oxides were added as P adsorbents (300 mmol kg -1 soil). Soil was incubated for 1 week, 3 months and 6 months at 25°C. Soil water-content was maintained a t 60% water-holding capacity throughout the incubation period. After completion of the incubation period, soil was analyzed for pH, CAL-extractable P (CAL-P), oxalate-extractable Fe and Al, dithionite-extractable Fe and Al, and P fractions. P-fractionation was done using the method described by Chang and Jackson.

Results and Discussion After 1 week, the CAL-P contents were reduced in treatments with Fe and Al oxides. This was due to adsorption of P by these oxides. CAL-P (Calcium-Acetate-Lactate- extractable P) is regarded as plant-available P. Adsorbed P and occluded P fractions 154 DGP Meeting September 5-9, 2012 are not extractable by the CAL method. The CAL-P data show that CAL-P contents were significantly higher in treatments with Fe oxides than in treatments with Al oxides at pH 7.5 and 5.2 when P was applied. At pH 4.6, it was vice versa but the difference was non-significant. Similar CAL-P data were found after 3 months and 6 months incubation. The CAL-P contents were decreased with time in all treatments. In P treatments, the difference in CAL-P content was significant in the first 3 months while it was non-significant in the last 3 months. In P- treatments, the differences were non- significant. Data from P-fractionation show that most of applied P was present as water-soluble P and adsorbed P. The adsorbed P was significantly increased with time in the first 3 months. The occluded P fraction was very small in all treatments when P was applied. The differences were non-significant with time. In P- treatments, occluded P was too low to be extractable. Data from oxalate-extractable Fe and Al, dithionite- extractable Fe and Al show that almost all oxides were in crystalline from. Due to low content of amorphous oxides, the occluded P fraction was very small.

Literature Burnham, C.P. and Lopez-Hernandez, D. (1982). Phosphate retention in different soil taxonomic classes. Soil Sci. 134, 376-380. Parfitt, R. L., Atkinson R. J., Smart R. St. C. (1975). The mechanism of phosphate fixation by iron oxides. Soil Sci. Soc. Am. J. 39:837–841. Schwertmann, U. and Cornell, R.M. (2000). Iron Oxides in the Laboratory, Preparation and Characterization. Chap. 5.2.1. WILEY-VCH, Weinheim.

155 DGP Meeting September 5-9, 2012 The influence of potassium and calcium on the mobilization of non- exchangeable ammonia

Patrick Beuters 1, Doris Vetterlein 2, Heinrich W. Scherer 1

1Institute of Crop Science and Resource Conservation – Plant Nutrition, University of Bonn, 2Helmholtz Centre for Environmental Research, Department of Soil Physics Halle/Saale. E-mail: pbeuters@uni- bonn.de

Introduction + It is well established that non-exchangeable NH 4 is involved in the N dynamics of soils and may be an important component of the N fertility status of arable soils. + However, it is assumed that the diffusion of NH 4 -ions out of the clay mineral interlayers is influenced by K +- and Ca 2+ -ions.

To evaluate the effect of these two cations the following hypothesis were formulated: 1. Ca² + expands the clay minerals and favors the release of non-exchangeable NH 4-N. + 2. K contracts the clay minerals, so that NH 4-N is fixed between the clay interlayers and is not available for plants.

Materials and Methods In a special compartment system (Vetterlein and Jahn, 2004), in which the root compartment was separated from the bulk soil by a nylon net (mesh 30 µm), alfalfa was grown for 49 days. The root mat formed along the nylon net was regarded as the root surface. The soil was a Haplic Luvisol from the depth of 45-75 cm. The clay minerals of the 15 soil, of the first 29 mm measured from root surface, were labelled with NH 4-N. To investigate the influence of K + and Ca 2+ , the following treatments were established: Control (without plants and without additional K+ and Ca 2+ ), Control (with plants but also without additional K+ and Ca 2+ ), plus K (with plants); plus Ca (with plants) and a treatment plus K and plus Ca (with plants). After the growing season the plants were harvested and separated into roots and shoots. In both fractions N and 15 N were analysed. The soil was cut into slices in defined distances to the root surface, in order to detect a depletion zone of non- exchangeable NH 4-N if present.

Results and Discussion While the total N content of the shoots and also the roots was nearly identical at all treatments, differences in the content of 15 N were found: 15 N content was high in the control and in the plus Ca treatment and significantly lower in the treatment receiving

156 DGP Meeting September 5-9, 2012 K addition ( plus K treatment and plus K -plus Ca treatment.

Independent of the treatment labeled non-exchangeable NH 4-N was lowest in the vicinity of the roots, while it increased up to a distance of 10 mm from the root surface. The decline in the distance from 10-20 mm is assumed to be caused by a 15 diffusion of NH 4-N in the adjacent unlabeled soil. Between the treatments the 15 content of non-exchangeable NH 4-N exhibits significant differences (Fig. 1): In the control (without plants) the content persists on the same level until 10 mm from the root surface, while in the control with plants and the treatment plus Ca the content of 15 non-exchangeable NH 4-N was depleted. Both treatments show a similar behavior. 15 Close to the root surface the amount of non-exchangeable NH 4-N was only half of 15 the amount of the control without plants. The release of non-exchangeable NH 4-N was blocked by K (treatment plus K) and the blocking effect of K could not be overcome by Ca (treatment K and plus Ca). So, one part of the hypothesis could be + proved: K contracts the clay minerals and hinders the diffusion of NH 4-N out of the interlayers. The expanding effect of Ca could not be proven as Ca addition did not further increase soil solution Ca concentration. The blocking effect of K + could not be overcome by Ca 2+ .

15 Fig.1: Content of NH 4 in different distances from the root surface as a function of the application of K and Ca to the soil.

Literature Vetterlein D. and R. Jahn. 2004. Combination of micro suction cups and time-domain reflectometry to measure osmotic potential gradients between bulk soil and rhizosphere at high resolution in time and space. Eur. J. Soil Sci. 55:497-504.

157 DGP Meeting September 5-9, 2012 The effect of nitrate supply on nitrogen fixation in Medicago truncatula

Beke Köster, Stefan Kremer, Ricardo Cabeza, Rebecca Liese, Vanessa Baumgarten, Hagen Zeng, Klaus Dittert and Joachim Schulze

Department of Crop Science, Section of Plant Nutrition and Crop Physiology, Georg-August University, Goettingen. E-mail: [email protected]

Introduction Medicago truncatula (Jemalong A17) serves as a model legume for research on symbiotic nitrogen fixation. It is a longstanding knowledge that nitrate supply impairs nodulation of legumes and inhibits the activity of existing nodules. A decrease in nodule activity was shown to occur within a timeframe of one day at a nitrogen concentration of 5 mM in the nutrient solution. The long-term effect of nitrate eventually results in premature senescence of the nodules. Apart from this relatively clear picture, it is difficult to continuously follow the nitrogenase activity after nitrate supply, since most existing measurement systems can be applied only short term and most often with a destructive effect on nodules. With a comparatively elaborated system it is however possible to follow nitrogenase activity indirectly and continuously by monitoring the concomitant evolution of hydrogen which is an indispensable byproduct of the enzyme activity. Furthermore, the mechanism of the nitrate impact on nodules is under discussion, in particular it is an open question whether the nitrate effect is a local or a systemic one. The objective of this study was to continuously follow nitrogenase activity on a whole root/nodule system after nitrate application and as well the reaction of nodules to nitrate application in a split-root system, where only one part was treated with nitrate.

Materials and Methods Plants were grown in a N free but otherwise full nutrient solution and inoculated with the Sinorhizobium meliloti strain 102F51 (Fischinger et al. , 2010). The measurement of nitrogenase activity was based on the determination of H 2 evolution of the nodules. This system provides a reliable method for continuously following nodule activity without disturbing its physiology (Fischinger and Schulze, 2010). Measurements were made during a period of 24h after nitrate application. Nitrate was applied at a - concentration of 5 mM as KNO 3 . Nodule components were measured by HPLC.

Results and Discussion The measurement of Nitrogenase activity after nitrate application to the whole root system revealed that the nodules did apparently not react over a period of several hours. After that time lag a relatively steep decline occurred that stabilized on a lower

158 DGP Meeting September 5-9, 2012 but still measureable level. When nitrate was applied during daytime, the decline in nitrogenase occurred not before several hours of light on the following day. These results support a systemic effect of nitrate on nodule activity that might be connected to light depending nitrate reduction in shoots after nitrate application. We present data on amino acid concentration in leaves and nodules at various points in time after nitrate application. In contrast to these measurements, the nodules in the nitrate treated compartment of a split-root system did react more quickly to the nitrate. Surprisingly the nodules of the non-treated part of the split-root system showed an increase in activity in the same timeframe as the decline of the treated root part. Apparently, nodules can have regulatory effects on other nodules of the same plant. That might be governed by the N demand of the whole plant. Overall these results show that apparently local and systemic effects on nitrogenase activity occur after nitrate application.

Literature Fischinger SA, Hristozkova M, Mainassara Z-A, Schulze Fehler! Textmarke nicht definiert. J. (2010). Elevated CO 2 concentration around alfalfa nodules increases N 2 fixation. Journal of Experimental Botany 61, 121-130. Fischinger SA, Schulze J. (2010). The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake. Journal of Plant Physiology 167, 1112-1115.

159 DGP Meeting September 5-9, 2012 Leghemoglobin and ferredoxin mRNA abundance in different developmental stages of Pisum sativum

P. Pourmoyyed 1, M. Schultz 2 and H. W. Scherer 1

1Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Germany; 2Institute of Molecular Physiology and Biotechnology of Plants (IMBIO), University of Bonn, Germany.

Introduction Symbiotic nitrogen fixation (SNF) as a significant process in leguminous involves numerous vital proteins such as Leghemoglobin (Lb) and Ferredoxin (Fd). Leghemoglobins are most abundant hemoproteins in plant nodules and sustain low

O2 concentration which is sufficient for respiration of bacteroids and insufficient for nitrogenase inactivation. Pisum sativum contains two types of Lb (A and B) with different function in SNF. Lb A (Pslb5 gene) has a higher O2 binding affinity than Lb B (Pslb120 gene) and expresses in all stages of nodule development but Lb B just is restrictively expressed in early stage of SNF (1). Ferredoxin proteins are soluble electron carriers and act as an unique electron acceptor from photosystem I, also is a + main electron donor in nitrogenase complex for reduction of N 2 to NH 4 . In pea, Fd is a single copy gene with different expression between different tissues. Herein, we tried to investigate the variation of these two proteins at the transcriptional level in a whole life cycle of pea. Material and methods In pot experiments, peas inoculated with Rhizobium leguminosarum biovar : viciae , were cultivated in perlite medium under natural condition under greenhouse conditions. Seedlings were fertilized with nutrient solution in optimum concentration for all micro and macro elements. Plants were harvested in the vegetative and generative phase, (in flowering and seeding step) and separated in nodules, roots, and leaves (6 biological replicates per phase) and stored at -80º C.Total RNA was extracted from nodules, roots, and leaves (QIAGEN RNeasy kit, Hilden, Germany). DNA digestion was done using DNaseI. Integrity and purity of RNA was checked in formaldehyde agarose gels and using Nanodrop (Thermo, Wilmington, U.S.A). Total RNA (1 g) was reverse transcribed using an equal mixture of random hexamer and oligo dT primers. Abundance of Pslb 5-10, Pslb 120, and Fd mRNA were quantified using SYBR Green dye in real-time PCR (Applied Biosystems 7300, ABI, Foster City, California USA). Relative quantities were normalized based on geometric mean of 3 most stable reference genes (PP2A, Transfact II , and Histone H3) selected from a panel of 7 reference genes using geNorm (2). General linear model or non parametric test was done in SPSS 19 to exclude significant differences ( P < 0.05).

Discussion and Conclusion In higher plants, symbiotic Lbs are earliest nodulin genes induced by Nod factor and are crucial for nitrogen fixation (3). Also different isoforms of ferredoxin in nodules,

160 DGP Meeting September 5-9, 2012 roots and leaves are essential in SNF and photosystem I. Herein, we demonstrate that in Pisum sativum expression of leghemoglobin and ferredoxin genes are changing in different developmental stages of life cycle. In nodules, expression of PsLb5 and Pslb120 genes significantly decreased from vegetative to generative phase, confirming the critical function of Lb in young plants in the vegetative phase to fix enough nitrogen (4). Afterwards reduction may happen caused by NH 4NO 3 inhibition or nitrosative stress that modifies Leghemoglobin heme group to nitrated heme and converting red active nodules to green senescent nodules in old plants during generative phase (5,). Indeed, Pslb5 mRNA showed a decline from flowering to seed formation although we could not detect any parallel effects for Pslb120 which is an evidence of expression of this gene almost in early stage of nodulation and helps Lb A to increase O 2-binding and to keep free oxygen condition inside of nodules (1). Ferredoxin mRNA showed a differential expression in nodules, roots, and leaves. In nodules, Fd significantly decreased from vegetative to the generative phase that could be due to a reduction of the nitrogen fixation rate. In roots, Fd mRNA abundance declined from vegetative to the generative phase as a trend. In leaves, plant growth stage had no effect on Fd mRNA expression. This stability and high expression of single ferredoxin gene in leaves could be caused by post transcription and light regulation of this gene and has different isoforms in leaves and roots similar to Arabidopsis photosynthetic Fd (leaf type) which supporting more photosynthetic activity and non photosynthetic Fd (root type) ( 6). In conclusion, leghemoglobin expression is mainly time dependent while expression of ferredoxin is more related to tissue type and post transcriptional steps. Literature Kawashima K, Suganuma N, Tamaoki M, Kouchi H. (2001). Two Types of Pea Leghemoglobin Genes Showing Different O2-Binding Affinities and Distinct Patterns of Spatial Expression in Nodules. Plant Physiology 641. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034. Heidstra R, Nilsen G, Martinez-AbarcaF, van KammenA, BisselingT. (1996). Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation. New Phytol133: 25–43. Ott T, van Dongen JT, Gunther C, Krusell L, Desbrosses G, Vigeolas H,Bock V, Czechowski T, Geigenberger P, Udvardi MK .(2005). Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Curr Biol 15: 531– 535. Navascués J, Rontoméa CP, Gayb M, Marcosc M, Yangd F,Walkerd, F. A, Desboise A, Abiánb J, Becanaa M. (2012). Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules. Proc Natl Acad Sci. Hank e GT, Kimata-Ariga Y, Taniguchi I, Hase T. (2004). Apost genomic characterization of Arabidopsis ferredoxins. Plant Physiol 134(1): 255.-264.

161 DGP Meeting September 5-9, 2012 A study on the nature of the shoot derived inhibitor of nodulation

Vanessa Baumgarten, Ricardo Cabeza, Rebecca Liese, Beke Köster, Klaus Dittert and Joachim Schulze

Department of Crop Science, Section of Plant Nutrition and Crop Physiology, Georg-August University, Goettingen. E-mail: [email protected]

Introduction Legumes do have an intricate root-shoot-root signaling system to adapt nodule number to the whole plant N demand. Mutations or other disturbances in this signaling system result in the formation of huge numbers of nodule per plant (supernodulating mutants). An unregulated nodule formation overloads the plants ability to support shoot and nodule growth at the same time. Nod-factors induce the formation of a small peptide which travels to the shoot and activate a chain reaction resulting in the formation of a substance that moves to roots and inhibits further nodule development. The substance is called ´shoot derived inhibitor (SDI)` and is partially characterized through a system of phloem feeding in plants that do not form SDI due to mutations in the reaction chain that forms SDI. SDI is heat and RNase stable, of relatively low molecular mass and cross active between legumes. The objective of the work is to establish a method to identify the chemical nature of SDI.

Materials and Methods Plants of Medicago truncatula (Jemalong A 17) and a supernodulating mutant (Mtsunn) were used. Mtsunn is mutated in a receptor kinase that is part of the signaling system that leads to the formation of SDI. The line forms up to 1000 nodules per plant. The plants were grown in nutrient solution culture (Sulieman and Schulze, 2010). To establish a bioassay for the activity of SDI an initially for soybean plants developed system for petiole feeding of solution into the phloem was used and adapted for Medicago truncatula (Lin et al., 2011).

Results and Discussion The objective of the study is to identify the chemical nature of SDI. For that purpose the first step is the establishment of a bioassay that allows to determine whether or not a leave extract or a fraction of a leave extract contains SDI. The assay than consists in feeding that solution into the phloem of the mutant and study whether or not that feeding reduces nodulation. An existing method for petiole feeding of soybeans was adapted to “branch feeding” in Medicago truncatula . The method proved valid in that feeding an asparagine solution was connected with the uptake of asparagine and as well an impact on nodule activity. Feeding the leave extract of the 162 DGP Meeting September 5-9, 2012 wild type in the branches is supposed to reduce the nodule number in the mutant. In that way, the activity of differently treated leave extract fractions can be tested and selected for further comparative HPLC-Metabolomic research to identify SDI.

Literature Lin YH, Lin MY, Gresshoff PM, Ferguson BJ. (2011). An efficient petiole-feeding bioassay for introducing aqueous solutions into dicotyledonous plants. Nature Protocols 6, 36-45.

Sulieman S, Schulze J. (2010). g-Aminobutyric acid (GABA) is involved in up-regulating nodule N 2 fixation efficiency in the model legume Medicago truncatula . Plant Cell and Environment 33, 2162-2172.

163 DGP Meeting September 5-9, 2012 Silicic acid nutrition enhances formation of casparian bands in Si- accumulating and non-accumulating plant species

Sascha Schulze 1, Alexander T. Fleck 1, Fritz Waßmann ², Lukas Schreiber ² and Manfred K. Schenk 1

1Institute of Plant Nutrition / Leibniz Universität Hannover; 2Institute of Cellular and Molecular Botany, Department of Ecophysiology / Rheinische Friedrichs Universität, Bonn. E-mail: [email protected]

Introduction Silicon (Si) is a quasi-essential element for higher plants, improves plant vitality and helps plants to overcome biotic and abiotic stress (Epstein, 2009). Furthermore, in adventitious rice roots silicic acid nutrition enhances formation of exodermal casparian bands consisting mainly of suberin and lignin (Fleck et al ., 2011). Maize and especially rice are known for high shoot Si accumulation, whereas onion accumulates no Si in the shoot (Epstein, 1999). Suberin consists of both, a polyaliphatic and a polyphenolic domain, arranges covalent linkage to the primary cell wall carbohydrates and forms a polyester-networking (Schreiber et al ., 2005).

Materials and Methods Rice ( Oryza sativa, c. Oochikara), maize ( Zea mays, c. Helix) and onion ( Allium cepa, c. “Hercules I hybrid”) were cultivated in nutrient solution with Si supply (+Si; 30 mg L-1, applied as silicagel) or without (-Si) in a growth chamber. Si concentrations in roots and shoots were photometrically measured after HF/HCl extraction. Formation of exodermal casparian bands in adventitious roots were visualized by staining freehand cross-sections with berberine-aniline blue and microscopically examined under UV light. The level of casparian band formation was allocated to one of four different rating stages. Exodermal cell walls were enzymatically digested and extracted to remove carbohydrate-rich material. Residual exodermal suberin was quantified by gas chromatographic analysis (GC-FID) coupled with mass spectrometry (GC-MS) after BF 3 transesterification reaction and derivatization (Schreiber et al ., 2005).

Results and Discussion Si nutrition increased the shoot Si concentrations in rice and maize, while the shoot Si concentration of onion was unaffected by Si supply as expected (Fig. 1 A). However, Si concentrations in roots were doubled by Si supply in all plants. The high Si accumulation in the shoot of rice and maize is due to Si transporters (Mitani et al ., 2009), which are obviously not present in onion. Si supply increased formation of casparian bands in exodermal cell walls in all plants independent of

164 DGP Meeting September 5-9, 2012 shoot Si concentration (Fig. 1 A). The concentration of aliphatic suberin was not affected by Si supply, while the concentration of phenolic suberin, mainly consisting of ferulic acid and p-coumaric acid, was decreased by Si supply in rice and maize. In onion no p-coumaric acid could be detected in the phenolic suberin fraction and this may be the reason that no effect of Si supply could be observed (Fig. 1 B). Decreased phenolic suberin in rice and maize could be the result of a Si mediated polymerization of phenolic compounds, such as lignin, which were not determined by the BF 3 assay. Interaction of Si with phenols is described in literature (Fang and Ma, 2006; Law and Exley, 2011). A B ShootShootSi Si concentration concentration(mg (mg Si/ Si/g g dm dm)) (max) 4 0.4 29.6 0.3 7.7 0.5 0.3 0.4 29.6 0.37.7 0.5 0.3 5 -Si nutrition -Si nutrition +Si nutrition +Si nutrition 4

3 3

2

in the exodermis 2

Rating of casparianbands 1 suberin / cm²) concentration(µg

(min) 1 0 Rice Maize Onion phenolic aliphatic phenolic aliphatic phenolic aliphatic Rice Maize Onion Fig. 1 (A): Influence of silicic acid nutrition on shoot Si concentrations and formation of casparian bands in rice, maize and onion. Formation was rated to one to four different stages: 1 = no formation of casparian bands visible, 2 = 0-25%, 3 = 25-50%, 4 = 50-100% formation of casparian bands in anticlinal exodermis cell walls, n=400, error bars show SE. (B) Influence of silicic acid nutrition on phenolic and aliphatic suberin fractions in rice, maize and onion, n=4, error bars show SE.

Literature Epstein E. (1999). Annu Rev Plant Physiol Plant Mol Biol 50, 641-664. Epstein E. (2009). Ann Appl Biol 155, 155-160. Fang JY, Ma XL. (2006). Journal of Zhejiang University Science B 7, 267-271. Fleck AT, Nye T, Repenning C, Stahl F, Zahn M, Schenk MK. (2011). J Exp Bot 62, 2001-2011. Law C, Exley C. (2011). BMC Plant Biology 11, 112. Mitani N, Chiba Y, Yamaji N, Ma JF. (2009). The Plant Cell 21, 2133-2142. Schreiber L, Franke R, Hartmann KD, Ranathunge K, Steudle E. (2005). J Exp Bot 56, 1427-1436.

165 DGP Meeting September 5-9, 2012 Mechanisms of B alleviating Al toxicity

Min Yu

Department of Horticulture, 18 Jiangwanyilu, Foshan 528000, China; E-mail: [email protected]

We present some proofs that B deactivates Al activity by alkalizing root surface especially the transition zone of root tip as well as by crosslinking with newly produced pectin thus helps prevent Al from entering root cells and loading to shoot, B crosslinking with newly produced pectin RGII directly inhibits Al binding in cell wall pectin Long-term Al toxicity induced chlorosis and growth arrest in both shoot and root which was mitigated by B addition due to less Al accumulation in cell wall. We found that majority of Al is bound in chelated pectin of cell wall and exogenous B reduces the adsorption of Al in cell wall samples especially with chelated pectin disclosing the function of B in crosslinking with newly produced pectin RGII directly inhibits Al binding in cell wall pectin. B promote the alkalization of root transition zone thus alleviates Al inhibiting in cell/root elongation is in accordance to acid growth theory. Root transition zone with less elongation has a notably higher pH value (¨SpH 0.3-0.5) than elongation zone. The peak of pH value in transition zone is reduced by Al exposure and it disappears under prolonged Al exposure for 24 h. B is very important to sustain such a higher pH value. Thus an interesting healthy white interval between meristem and elongation zone appears by hematoxylin staining. B undoubtedly alleviates Al toxicity since it is the transition zone that is most sensitive to Al toxicity by maintaining a higher pH value.

166 DGP Meeting September 5-9, 2012 Boron deficiency response in Arabidopsis : alterations in plant architecture development.

Abreu I. 1, L. Bolaños 1 and I. Bonilla 1

1University Autónoma de Madrid, Madrid, España. E-mail: [email protected]

Introduction Boron is a micronutrient essential for plants, whose only known function in these organisms is to promote the dimerization of Rhamnogalacturonan II through the crosslinking of two apiose residues (O’Neill et al. 2001). However, this function seems to be insufficient to explain all the alterations found in B-deficient plants, which include alterations in the hormone signaling, sugar, and RNA metabolism, among others (Maschner 1995; Bonilla et al 2009). Efforts to investigate boron physiology are focused on finding borate transporters (Takano et al, 2008), on describing the transcriptional changes which follow B deficiency (reviewed by Camacho-Cristóbal et al. 2011), on identifying new B ligands (Reguera et al 2010; Wimmer et al. 2009), and finally on unravelling the hypothetical B-deficiency signaling mechanism (Kasajima et al 2010; Martín-Rejano et al. 2011), using Arabidopsis thaliana as biological model. One of the first responses to B deficiency is cessation of root growth (Maschner 1995), which is a phenomenon common to diverse mineral deficiencies (López-Bucio et al. 2003). However there was a gap in the knowledge of the genetic network which controls the root development that difficult shed new light on the effects of mineral deficiencies. This question is now being solved (Perilli et al. 2012; Stahl & Simon 2010) and then several scientists try to connect mineral deficiencies with sensing mechanism and alterations in the root architecture (Gutierrez 2012; Péret et al. 2011). In this work we present preliminary results about the effect of B deficiency on the regulation of Arabidopsis root development. Materials and Methods A. thaliana ecotype Col-O seeds were surface sterilized, stratified, and grown vertically in plates of Murashige & Skoog (MS) solid media containing 0.8% (w/v) agarose and 2% (w/v) sucrose in a chamber at 20ºC with 16:8 h light:dark cycle.

Plants grew in +B (10 µM H 3BO 3) or -B plates, or alternatively transferred from +B to -B plates after 7 days post-germination (dpg).To eliminate B traces from solutions and media, the boron-binding specific resin Amberlite ® IRA7-43 (Sigma) was used. Roots of developing seedlings were stained with lugol or fuschin basic to reveal columella amyloplasts and xylem, respectively. Genes selected for expression analysis through RT-PCR were: a member of SAND protein family (AT2G28390); NIP5;1 (AT4G10380); and CRE1/WOL/AHK4 (AT2G01830). Results and Discussion Boron deficiency In Arabidopsis directly germinated on -B plates caused a reduction in primary root growth that was detectable after 2 dpg. This was accompanied by an 167 DGP Meeting September 5-9, 2012 increased number of abnormally long root hairs. These alterations in root architecture were similar to those previously described by Takano et al (2006). This first approach did not allow discarding between direct or secondary effects of B-deficiency, therefore we performed transference experiments in which +B developed seedlings were transferred to +B or to –B deficient plates and allow grow. Again a strong inhibition in the primary root growth, appearance of lateral roots, and development of numerous long root hairs were features of apical root growth after transferring to –B plates. These effects of B deficiency were more severe than previously described (Martin- Rejano et al. 2011), perhaps because we eliminated most boron traces using Amberlite ® IRA7-43 resin. The appearance of long root hairs close to the root tip under B deficiency suggested an alteration of regulation of cell division and differentiation. Furthermore, lugol and fuschin basic staining confirmed an abnormal development of protoxylem and the apparently not differentiation of columella cells. Therefore we are performing studies of gene expression approach in order to delimitate the genetic networks involved in root development affected by the availability of B. The more clear effect to date is alteration of expression of CRE1/WOL/AHK4 . This gene encodes a two-component protein with a cytokinin response and a histidine kinase domains (Inoue et al. 2001; Mähönen et al. 2000; Suzuki et al., 2001). This result is potentially relevant, because to our knowledge there is no previous reported connection between B deficiency and cytokinin mediated signaling. Overall described changes in root architecture and cell identity determination suggest a high sensitivity to B deprivation of regulation of root organogenesis. Moreover, it has recently been described a connection between auxins and cytokinins in vascular differentiation (Bishopp et al. 2011), therefore considering our preliminary results, we could also hypothesize the existence of a mechanism of signaling boron deficiency stress, involving hormones like cytokinins, auxins, and perhaps ethylene (Martin- Rejano et al. 2011), and a WRKY transcription factor as reported by Kasajima et al. (2010). Literature Bishopp et al. (2011). Current Biology. 21:917-926. Bonilla et al. (2009). Plant Physiology 5 th Ed. http://5e.plantphys.net/article.php?ch=&id=403 Camacho-Cristóbal et al. (2011) Plant Science. 181: 85-89. Gutiérrez. (2012) Science. 336: 1673-1675. Inoue et al. (2001) Nature. 409: 1060-1063. Kasajima et al. (2010) Physiologia Plantarum. 139: 80-92. López-Bucio et al. (2003) Current Opinion in Plant Biology. 6: 280-287. Mähönen et al. (2000) Genes & Develpoment. 14:2938-2943. Marshner (1995) Mineral nutrition of higher plants, 2ª Ed. San Diego, California. Academic Press. Martín-Rejano et al. (2011) Physiología Plantarum. 142: 170-178. O'Neill et al. (2001) Science. 294: 846–849. Péret et al. (2011). Trends in Plant Science. 16: 442-450. Perilli et al. (2012) Current Opinion in Plant Biology. 15:17-23. Reguera et al. (2010) Plant, Cell and Environment. 33: 1039-1048. Stahl & Simon. (2010) Current Opinion in Plant Biology. 13:53-58. Suzuki et al. (2001) Plant Cell Physiology. 42: 107-113. Takano et al. (2008) Trends in Plant Science. 13: 451-457. Takano et al. (2006) The Plant Cell. 18: 1489-1509. Wimmer et al. (2009) Plant Cell Physiology. 50: 1292-1304.

168 DGP Meeting September 5-9, 2012

Poster Session P5: Physiological response to abiotic stress

169 DGP Meeting September 5-9, 2012 Getting to know more about calcium signatures and components involved in their generation

K. Thor, A. Janssen, E. Peiter

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

Calcium performs different roles in the plant. It contributes to stabilizing cell wall and plasma membrane, but also is an important signalling component in plant stress responses, a second-messenger. In the course of fulfilling this function, calcium influxes from the apoplast or from internal stores create specific patterns of oscillations, so-called calcium signatures, which lead to specific downstream responses. Unravelling these patterns and the mechanisms of their generation is of great interest to a broad community of plant scientists. The method of choice for detecting calcium signatures on single-cell level is the use of genetically encoded fluorescent reporters such as Yellow Cameleon (YC). Stomatal guard cells in epidermal strip preparations have become a model system for this kind of measurements. We use this method to dissect the responses of plants to different stimuli from abiotic and biotic stress situations, such as cold or mannitol, plant hormones like abscisic acid, bacterial elicitors like flg22, and others. Wild type plants transformed with YC help us to detect new signatures, while mutant lines for putative elements of the Ca2+ signal-generating machinery are analysed to find out if these genes are involved in a specific response. Moreover, by using inhibitors of specific channel types, the source of the calcium influx is determined and hints on the channels involved are obtained. The poster will show examples of our current work.

170 DGP Meeting September 5-9, 2012 The role of calcium-permeable low-affinity cation transporters in abiotic stress tolerance of crops

R. Happeck, K. Köhler, L. Freitag, E. Peiter

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

The transport of cations across cellular membranes plays an important role in the tolerance of plants to abiotic stress factors. For instance, the tolerance of plants to salt stress depends substantially on their ability to avoid the uptake of Na+ ions, to exclude them, or to store them in specialized compartments, such as the vacuole. In addition, all stress factors cause stress-specific elevations of the Ca 2+ activity in the cytosol or within organelles of plant cells. These Ca 2+ signals are triggered by the activation of channel proteins, they are crucial for the initiation of the stress response because they induce specific mechanisms of resistance. In this project monocot- specific candidate genes from wheat and barley encoding cation transporters are examined, which are likely to be involved in salt stress tolerance and/or in the generation of calcium signals. To test those hypotheses, the genes are being functionally analysed in yeast mutants to quantify the affinity and the selectivity of the transport proteins. In these experiments we could identify a new protein from barley which shows transport activities for monovalent cations such as K +, Na + or Li +. Furthermore expression analysis reveals an upregulation of the genes during K + deficiency and salt stress. To get information about the role of the transporters in uptake and distribution of K +, Na + and Ca 2+ transgenic barley and Arabidopsis thaliana plants are being generated.

171 DGP Meeting September 5-9, 2012 pH signaling in the apoplast of Vicia faba L. fulfills requirements of a transducing signaling element

Christoph-Martin Geilfus 1, Christian Zörb 1,2 , and Karl-Hermann Mühling 1

1Institute of Plant Nutrition and Soil Science/ Christian Albrechts University, 24118 Kiel, Germany, 2Institute of Biology, Botany/ University Leipzig, 04103 Leipzig, Germany.E- mail:. [email protected]

Introduction The extracellular space of plants plays a principal role in their communication with the outer world, because it builds a frontier between the environment and the cell (Hoson, 1998). Stress in general causes the extracellular space (apoplast) transiently to alkalize, an event that is interpreted to be a pH signal of great significance for plants to be able to survive in the field under a variety of stressful conditions.

Materials and Methods The technique of microscopy-based ratio-imaging was used for live monitoring of leaf apoplastic pH dynamics in living Vicia faba L. plants by using an H+-sensitive fluorescence probe.

Results and Discussion Transient alkalinizations were triggered by stressing the roots with NaCl. The novelty of this report is that this real time in vivo technique enabled non-invasive contentious live pH recordings revealing that the NaCl-induced apoplastic alkalinization fulfills requirements of a transducing signaling element. Moreover, we demonstrated that the identical stress treatment triggered different levels of alkalinizations throughout the various leaf apoplastic compartments, and that, thus, the free proton concentration asymmetrically peaked throughout the leaf apoplast.

Literature Hoson T. (1998). Apoplast as the site of response to environmental signals. J. Plant Res. 111, 167- 177.

172 DGP Meeting September 5-9, 2012 Determination of oxidative stress in pea lines as influenced by various concentrations of boron

Tijen Demiral, M. Hamurcu, E. E. Hakki, C. Ozdemir, A. Tamkoc, Z. Z. Avsaroglu, S. Gezgin

Harran University, Department of Biology, Turkey; E-mail: [email protected]

Agricultural regions that contain inadequate or toxic levels of boron (B) in soil have serious problems with yield and quality of many crops. Plants deploy some biochemical and molecular mechanisms to be able to cope with boron deficiency and toxicity. Higher plants alleviate the damages of boron toxicity and deficiency via enhanced activities of antioxidant enzymes. Having rich amino acid and protein content, pea constitutes one of the most important species of legumes and contributes major food resource for human as an alternative to meat. Therefore, in the present study, we investigated impacts of various concentrations of B (0, 2, 20 and 200 mg kg-1) on antioxidative enzymes (SOD, POX, CAT, GR and APX) activities, lipid peroxidation level and proline accumulation of four different pea lines. S2 line was derived from Pisum sativum L., and lines 602, Taş,kent and Özkaynak were derived from Pisum arvense L. Pea plants at three-leaf stage were subjected to different B concentrations for a week and harvested 3 and 7 days after B treatment. All pea lines showed different responses in terms of antioxidant enzyme activities and lipid peroxidation level. Proline accumulation seemed not enough to provide osmotic adjustment in all pea plants but its reactive oxygen species scavenging ability could not be ruled out. Therefore, deficient and toxic concentrations of B seemed to induce oxidative stress in all pea lines employed.

173 DGP Meeting September 5-9, 2012 Does high light intensity affect growth performance of maize in the first phase of salt stress?

Franziska Faust, Stefan Hanstein and Sven Schubert

Institute of Plant Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, Email: [email protected]

Soil salinity is a worldwide problem and causes growth reduction of salt-sensitive plants such as maize. In order to overcome this problem salt-resistant maize hybrids (SR hybrids) have been developed in our institute. However, for further improvement of salt resistance it is necessary to understand the physiological mechanisms of salt resistance, especially in the first phase of salt stress, which is characterized by osmotic stress. According to the acid-growth theory, salt resistance in one hybrid (SR 03) can be partially ascribed to maintained apoplast acidification by plasmamembrane H+-ATPase in the first phase of salt stress. Hydroponic cultivation under high light resulted in higher shoot fresh and dry weight of maize. We assume that high light intensity reduces growth reduction and maintains plasmamembrane H+-ATPase activity in the first phase of salt stress. Therefore, in this study plants of the hybrids SR 05 and SR 12 are grown in hydroponic culture under high-light conditions in the presence of 1 mM NaCl (control) or 100 mM NaCl (salt stress). For determination of plasmamembrane H+-ATPase activity membrane vesicles are isolated by means of ultrazentrifugation. The plasmamembrane H+-ATPase activity is characterized by photometric determination of hydrolytic activity, proton-pumping activity and maximum of established pH gradient.

174 DGP Meeting September 5-9, 2012 Calcium supply effects on apoplastic and cytosolic pH and Ca 2+ in Vicia faba under NaCl stress

S.H. Morgan 1,3 , C.M. Geilfus 1, S. Lindberg 2 and K.H. Mühling 1

1Institute of Plant Nutrition and Soil Science, Kiel University, Kiel, Germany; 2Botany Department, Stockholm University, Stockholm, Sweden; 3Plant Botany Department, Faculty of Agriculture, Cairo University, Egypt. E-mail: [email protected]

Introduction Salt stress impacts the prevailing apoplastic ion activity, as well the cytosolic ionic homeostasis. Under salinity a stabilized low apoplastic pH according to the acid- growth-theory [1], and cytosolic pH and Ca 2+ homeostasis may avoid accumulation of toxic ions and compile cellular signals [2]. This can help the plant toward salt resistance, but the beneficial calcium effects on those mechanisms are still not clear.

Materials and Methods Vicia faba L. cv. Fuego was grown in hydroponics at 1 or 100 mM NaCl and/or extra calcium addition (0 and 5 mM CaSO 4). After 7 d and 21 d, the dry weights of shoots were detected. Moreover, in the second uppermost leaves, apoplastic pH, pH apo , and 2+ 2+ Ca , [Ca ]apo , were detected in the intact leaves using microscopy-based ratio imaging technique [3] and the dextran form of Oregon Green and Calcium Green dyes, respectively. Protoplasts were isolated from the second uppermost leaves by 2+ 2+ an enzymatic method [4], and the cytosolic pH, pH cyt , and Ca , [Ca ]cyt , were detected using the acetoxy methyl-ester form of BCECF and Fura dyes, respectively, and epi-fluorescence microscopy [4].

Results and Discussion The extra calcium addition improved plant growth under salinity. Salinity induced 2+ 2+ alkalization of pH apo and pHcyt , but increased [Ca ]apo and decreased [Ca ]cyt . 2+ Contrariwise, the extra Ca addition reduced the pH apo alkalization caused by 2+ salinity, and increased pH cyt alkalization. At the same time [Ca ]apo was clearly 2+ decreased, and [Ca ]cyt increased, after 21d of salt stress. Thus, this may be a new explanation how calcium can improve plant growth under salt stress. Acidification of the apoplast, alkalinization of the cytosol and amelioration of the cytosolic calcium activity, can improve cell expansion and salt resistance toward normal growth conditions [1,2], as part of the plant signalling process under stress [2].

Literature 1 - Hager A (2003) J Plant Res 116:483-505. 2 - Kader MA, Lindberg S (2010) Plant Signal Behav 5: 233-238. 3 - Geilfus CM, Mühling KH (2011) Front Plant Sci 2: 13. 4 - Kader MA, Lindberg S, Seidel T, Golldack D, Yemelyanov V (2007). Physiol Plant 130: 99-111. 175 DGP Meeting September 5-9, 2012 Physiological responses of a medicinal plant to salt stress: Lepidium sativum L

Farah Chebil *, Arafet Manaa *, Hela Ben Ahmed

Unité d’Ecophysiologie et Nutrition des plantes. Département de Biologie. Faculté des Sciences de Tunis. Campus Universitaire. 1060 Tunis .* equal Contribution. E-mail: [email protected]

Introduction Lepidium sativum L. commonly called Garden cress is a polymorphic species known for its medicinal value: leaves are antiscorbutic, diuretic and stimulant. In many countries, this specie is grown in arid and semi-arid regions where soil salinization is an important problem. Salinity is one of the major abiotic stresses which adversely affect the crop growth and yield. Reduction in growth may result from salt effects on physiological processes, biochemical reactions or a combination of such factors (Munns and Tester 2008). Little information is available regarding the behaviour of Lepidium sativum to salt stress. The present study was focused to assess the tolerance of this medicinal plant to salinity during its vegetative growth stage.

Materials and Methods The experiment was conducted in a growth chamber with controlled temperature (at 25 °C day/ 20°C night), and relative humidity 60 an d 80 % respectively at the day and night. Seedlings of Lepidium sativum were grown into 2.5 L polyethylene pots with fully nutrient solution. Two weeks later, five concentrations of NaCl were applied: 0 (control), 50, 100, 150 and 200 mM. After 20 days plants were harvested for physiological and metabolic analysis.

Results and Discussion Dry weight of plants decreased at all the levels of sodium chloride added. For the whole plant, the reduction was about 45% compared to control. Dry matter deposition was more diminished in roots than aerial parts.

Table . Biomass production sensitivity index NaCl Whole Leaves Stems Roots in Lepidium sativum exposed to salt. (mM) Plant Negative values (-) correspond to a reduction in the dry matter compared to control. 50 -10 -14 -34 -12 100 -33 -51 -53 -44 150 -45 -59 -63 -52 This sensitivity of the root system seems 200 -45 -62 -57 -48 to be a feature of Lepidium sativum , generally it is the aerial organs, which manifest the depressive effect of salt. These results are in agreement with the study of Ben Ahmed et al. (2008) on Setaria verticillata . According to the author, the sensitivity to salinity observed at the root

176 DGP Meeting September 5-9, 2012 system of Setaria results in a decrease in biosynthetic activity. Reduction in dry weight production was associated to a high accumulation of Na + and Cl - and a significant reduction of K + content in shoots. Limitation in the K + uptake was observed in most other Brassicaceae as Arabidopsis thaliana (Munns and Tester 2008). The growth reduction observed was probably induced by the decline in K + uptake. Indeed, analysis of the relationship between biomass production and the amount of K + in the roots and shoots of plants subjected to NaCl showed that growth of these organs is highly correlated with the amount of potassium transported and accumulated in them.

Aerial parts Na+ 40 Aerial parts Roots 10 Roots 8000 Cl- 30 8 6000 6 20 4000 4 10

2000 Proline,µmol/g FW 2

0 Sugars, mg,g-1 FW

Na+, Cl-Na+, content (µeq,gDW) 0 0 50 100 150 200 0 0 50 100 150 200 0 50 100 150 200 NaCl (mM) NaCl (mM) NaCl (mM)

Figure . Na +, Cl - contents in aerial parts, Proline and sugars contents in aerial parts and shoots of Lepidium sativum exposed to salt treatment.

Under salt stress, plants have involved complex mechanisms allowing for adaptation to osmotic and ionic stress caused by salinity. These mechanisms include osmotic adjustment by accumulation of compatible solutes (Krasensky and Jonak, 2012). In Lepidium sativum , proline content in shoots and roots was greatly increased after salt treatment. The accumulation of this solute in the different organs was proportional to the amount of salt added to the medium. Proline was known to be an osmoregulatory compound involved in maintaining the water balance of plant exposed to salinity. The presence of NaCl in the medium induces also an increase in total soluble sugar contents. Sugars are necessary to stabilize cell membranes and macromolecules (Krasensky and Jonak, 2012). Under salinity an increase in the protein levels was registered in the two organs. The use of proteins as indicators of salt tolerance depends on the species and even cultivar (Munns and Tester 2008). On the basis of these results it was concluded that Lepidium sativum can be classified as a moderately tolerant plant to salt stress. The significant accumulation of compatible solutes in plant was probably associated with osmotic adjustment.

Literature Ben Ahmed, H., Manaa, A. and Zid, E. (2008). Tolérance à la salinité d'une poaceae à cycle court : la sétaire ( Setaria verticillata L.). Comptes Rendus Biologies 331, 164-170. Krasensky, J. and Jonak, C. (2012). Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. Journal of Experimental Botany 63, 1593-1608. Munns, R. and Tester, M. (2008). Mechanisms of Salinity Tolerance. Annual Review of Plant Biology 59, 651-681.

177 DGP Meeting September 5-9, 2012 Spermine – modulator of barley drought stress tolerance via stomatal closure?

N. Nowak, L. Freitag, H. Beschow, E. Peiter

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

Worldwide, drought is the main yield-limiting factor in crop production. Drought resistance is therefore a major goal for crop research. Polyamines play a central role in abiotic stress tolerance of plants in general and drought tolerance in particular. Common polyamines in plants are putrescine, spermidine and spermine. Recent studies on the model plant Arabidopsis show that spermine is of particular importance for stress tolerance. The aim of this project is to investigate the role of spermine in drought tolerance of barley (Hordeum vulgare). Two barley genes encoding proteins possibly mediating of the synthesis of spermine and one gene encoding a putative spermidine synthase have been identified, and full-length cDNA sequences have been obtained. To determine the involvement of those genes in stress responses, transgenic barley lines in which those genes are overexpressed or silenced are currently being generated. Stomata are key regulators of transpiration, and the role of polyamines in stomatal regulation of barley is studied in this project. In epidermal strip assays spermine strongly inhibited the opening and induced the closure of stomata. We hypothesise that spermine may alter [Ca2+]cyt and thereby act as regulator for guard cell K+ channels. A central aspect of current investigations is therefore the involvement of polyamines in calcium signalling.

178 DGP Meeting September 5-9, 2012 Effects of induced drought and different shade levels on leaf gas exchange of Musa spp.

Mario Pilz, Jürgen Burkhardt, Daniel Neuhoff, Pablo Siles, Oscar Bustamante, Charles Staver

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Several climate scenarios predict that extreme weather events like droughts will become more frequent and severe. Average precipitation may decrease or at least rainfall patterns could become less predictable. Since agroforests tend to offer better resilience to reduced precipitation they may play an important role in reducing the vulnerability of the rural poor to extreme weather events. Thus the evaluation of plant performance under prolonged drought conditions is essential to predict responses of ecosystems to climate change. In Central America, bananas are commonly grown in agroforests by small scale farmers. This study was conducted in Musa spp. agroforests with Coffea arabica associated with Erythrina spp. shade trees at two spots: one at CATIE, Costa Rica consisting of an agroforest with four levels of shade (full sun, 25%, 50% and 75% shading), the other one near Matagalpa, Nicaragua where drought conditions were established through roof structures and drainage. We studied the effect of continuous soil moisture depletion and different shading on the photosynthetic performance at leaf level and compared the responses of two banana cultivars: Gros Michel (Musa acuminata Colla cv.) and Bluggoe (Musa acuminata x balbisiana Colla cv.). Gas exchange measurements of light and CO2 response curves and chlorophyll fluorescence were carried out. The preliminary results show varying responses of the two cultivars concerning acclimation to shade levels and drought resistance. Furthermore we looked beyond the simple effect of reduction in light availability and available water capacity. The results provide insight into improving resilience and productivity of small scale banana agroforests regarding climate change

179 DGP Meeting September 5-9, 2012 CO 2 enrichment during grain filling differently affects C and N allocation, N uptake and N use efficiency in old and modern spring barley cultivar

Komi Egle, Wolfgang Merbach, Edgar Peiter, Heidrun Beschow

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

The presented study examined the responses of an old and a modern barley cultivar to increased pCO2 during the reproductive stage and to N fertilization at anthesis. Until 33 days after sowing (DAS), plants were cultivated under natural conditions in a wire cage. At 34 DAS, plants were exposed to ambient (360 µmol mol-1) or elevated pCO2 (720 µmol mol-1) and grown in growth cabinets until harvest. At anthesis plants were supplied with 15N-labelled fertilizer solution. After harvest at milk ripeness, dry matter, C and N accumulation, N uptake from fertilization, and N use efficiency (NUE) were determined. While total and ear dry matter were increased only marginally in the modern cultivar, the old one responded very strongly to the pCO2 elevation. N accumulation displayed a different pattern than C accumulation. Elevated pCO2 did not alter significantly total N accumulation, 15N uptake and translocation by the modern cultivar but caused a significant increase in N accumulation in the ear and a significant decrease in stem and roots, as well as a significant increase of 15N uptake in the old cultivar. We conclude from our findings that the old cultivar was source-limited, whereas the modern cultivar was sink-limited. Further analyses of C and N metabolism (carbohydrate and N fractions in vegetative parts, transport to the grain, activities of enzymes) are necessary to recognize the relevance of sink and source strength during grain filling at future pCO2 increases.

180 DGP Meeting September 5-9, 2012 Impacts of drought stress on VOC emissions from tomato, sugar beet and holm oak

Tina Kasal, Lina Hacker, Iida Pullinen, Einhard Kleist, Heiner Goldbach

Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Plants can emit a broad spectrum of VOCs such as terpenoids, aromatic VOCs originating from the phenyl propanoid pathway, and green leaf volatiles (C6 alcohols and aldehydes). The emissions of aromatic VOCs and green leaf volatiles are induced by biotic and abiotic stress factors and also the emissions of terpenoids can change due to the impacts of stress. Our task was to check the impact of drought stress on VOC emissions as drought is a common stress for plants in the field or in greenhouses. Furthermore the expected change in global climate suggests increased durations and frequencies of drought stress periods in the future. Hence the plant’s responses to drought stresses will play an important role in future plant breeding and climate modeling. Our work focusses on in vivo quantification of VOC emissions through GC MS to detect drought stress in three different groups of plants; vegetables, crops and trees. For each group we used representative species: tomato, sugar beet and holm oak. To assess the impacts of the drought stress we checked visual symptoms, water content of soil and transpiration. We here present the results of our first measurements indicating that drought stress causes a reduction of de-novo terpenoid emissions.

181 DGP Meeting September 5-9, 2012 Poster Session P6: Physiological response to nutrient imbalances

182 DGP Meeting September 5-9, 2012 The plant vacuolar Two Pore Channel 1: Regulator of cation homeostasis or generator of calcium signa

J.-P. Maaß, T. Peiter-Volk, K. Thor, E. Peiter

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

The plant vacuolar Two Pore Channel 1: Regulator of cation homeostasis or generator of calcium signals? The slow vacuolar (SV) channel, which is encoded by the TPC1 gene, is probably the best described ion channel in plants. With a calculated density of one channel per µm2, these channels form the largest conductance across the vacuolar membrane. Early electrophysiological analyses revealed permeabilities for Na +, K +, Mg 2+ , and also for Ca 2+ . The plant vacuole represents the cell’s major store for cations and can make up as much as 90% of the whole cell. For this reason TPC1 has been hypothesised to play a central role in the homeostasis of K + and Mg 2+ . On the other hand, the vacuole is also an inexhaustible pool for Ca 2+ ions, which function as second messengers. In addition, the TPC1 channel can be activated by an increase in [Ca 2+ ]cyt. As a consequence of both permeablity to and activation by Ca 2+ , the channel may be able to initiate or alter Ca 2+ signals by releasing Ca 2+ from the vacuole. Here we show that first retranslocation experiments with wild type and tpc1 knock-out plants do not point to a role of TPC1 in K + and Mg 2+ homeostasis. Based on these findings we are currently conducting further examinations concerning the putative function of TPC1 in the Ca 2+ signalling network. Those experiments include aequorin- and cameleon-based measurements of [Ca 2+ ]cyt as well as electrophysiological patch-clamp analyses.

183 DGP Meeting September 5-9, 2012 Influence of P nutritional status on expression of P transporters and P uptake rates in rice

Melanie Bremer 1 and Manfred K. Schenk 1

1Institute of Plant Nutrition, Leibniz Universität Hannover, E-mail: [email protected]

Introduction Phosphate starvation increases transcription of high-affinity P transporters and resupply of P decreases transcription level of transporters within 24 hours (Karthikeyan et al. 2002). However, the effect of this downregulation on the dynamics of P uptake is unknown. In this study, the influence of P nutritional status on the transcription of four P transporters ( PT1 , PT2 , PT3 , and PT6 ) was determined simultaneously to P uptake after resupply of P in rice ( Oryza sativa ). Selection criteria for the examined P transporters were the upregulation in response to P starvation and the expression in the root (Ai et al. 2009a). Within the root, PT2 was expressed in the stele whereas PT6 expression was found in epidermal and stele cells (Ai et al. 2009b). The other P transporters were not further specified.

Materials and Methods Rice ( Oryza sativa L. cv. Selenio) plants were starved for P for 0, 1, 3, and 5 days. P uptake rates were calculated from depletion in the nutrient solution considering the linear phase of depletion to 5 µM P. For RNA isolation, roots were harvested 0, 120, 240, 360, and 480 min after resupply of P and realtime analysis was carried out using the 2 -∆∆ Ct method of relative quantification.

Results and Discussion Starvation of P did not affect dry matter yield, but decreased P concentration in shoots and roots. The expression levels of P transporters (Fig. 1) and of P uptake rates (Fig. 2) were increased with increasing duration of P starvation. However, the transcription was enhanced by the factors 3 to 50 depending on the specific P transporter whereas the uptake rates increased only 3-fold suggesting a post- translational regulation. Also, Bayle et al. (2011) reported about multiple levels of post-translational regulation thus affecting the activity of high affinity P transporters in Arabidopsis thaliana . However, P uptake rates in P starved roots remained constantly high up to 375 min after readdition of P whereas the transcription level of P transporters already declined within 120 min as shown for 5 days starved plants (Fig. 3). Thus, the P uptake rates cannot be explained solely by the gene expression of P transporters. It is suggested

184 DGP Meeting September 5-9, 2012 that P uptakes did not decrease since proteins of P transporters were not degraded during the duration of the experiment. About protein turnover of P transporters nothing is known in literature. In summary, transcription analysis has to be supplemented with examinations on the protein level to understand the physiology of P uptake.

100 0.050

0.045 PT1 )

PT2 -1 PT3 0.040

PT6 min

-2 0.035 0.030 3 days 10 0.025 5 days 1 day 0.020

0.015

0.010 0 days rel. transcription level (x-fold) transcriptionrel. level 1 cm uptakeratesP (µg 0.005

0.000 0 days 1 day 3 days 5 days 0 120 240 360 480 time of P starvation time after resupply of P (min)

Figure 1: Transcription level of P transporters Figure 2: Uptake rates over time after resupply as affected by duration of phosphate starvation of P as affected by duration of P starvation

1.6

1.4 PT1 PT2 1.2 PT3 PT6 1.0

0.8

0.6

0.4 Figure 3: Transcription kinetics of P transporters 0.2 (PT1 , PT2 , PT3 , and PT6 ) after 5 days of P (x-fold) level transcription rel. 0.0 starvation in rice 0 120 240 360 480 time after resupply of P (min)

Literature Ai P., Sun S., Zhao J., and Xu G. (2009a) Regulation and function of Pht1 family phosphate transporters in rice. The Proceedings of the International Plant Nutrition Colloquium XVI, UC Davis. Ai P., Sun S., Zhao J., Fan W., Xin W., Guo Q., Yu L., Shen Q., Wu P., Miller A.J., and Xu G. (2009b) Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. The Plant Journal 57, 798-809. Bayle V., Arrighi J.-F., Creff A., Nespoulous C., Vialaret J., Rossignol M., Gonzalez E., Paz-Ares J., and Nussaume L. (2011) Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation. The Plant Cell 23, 1523-1535. Karthikeyan A.S., Varadarajan D.K., Mukatira U.T., Paino D’ Urzo M., Damsz B., and Raghothama K.G. (2002) Regulated expression of Arabidopsis phosphate transporters. Plant Physiology 130, 221-233.

185 DGP Meeting September 5-9, 2012 Nitrogen fixation of Medicago truncatula at sufficient and deficient P supply

Ricardo Cabeza 1,2 , Saad Sulieman 2, Beke Köster 2, Rebecca Liese 2, Vanessa Baumgarten 2, Joachim Schulze 2

1Departamento de Ingeniería y Suelos/Universidad de Chile, Santiago de Chile; 2Plant Nutrition and Crop Physiology/University of Göttingen, Göttingen. E-mail: [email protected]

Introduction Medicago truncatula (Mt) serves as a model for research on symbiotic nitrogen fixation. However, the model proved to be difficult to be grown in nutrient solution mainly due to an apparent small window of optimal P supply. Pre-experiment showed that nitrogen fixation appeared to be affected by a comparatively low supply of P when given in a discontinuously way (daily doses). Apparent P toxicity symptoms appeared at a supply as low as about 3 µmol per day and plant. Mt grown on the basis of nitrate nutrition showed much less sensitivity to higher P concentration (up to 50 µM). When the system was changed to a flowing nutrient solution supply, nitrogen fixing plants could cope with substantially higher P concentrations and showed improved growth and nitrogen fixation. Using the flow through system, the objective of our experiments was to establish levels of sufficient and deficient P supply. Furthermore, we conducted experiments studying the question whether P depletion affected nitrogen fixation initially through an impact of P deficiency on shoots (less assimilate supply of the nodules) or a direct effect through P starvation of the nodule tissue.

Materials and Methods Plants were grown in nutrient solution culture in glass tubes that allowed separate gas exchange measurement of the root/nodule part of the plant (Fischinger and Schulze, 2010). The nutrient solution was N free (except for the nitrate treatment (5 to 50 µM) and the plants were inoculated with an efficient Sinorhizobium meliloti strain (102F51). The nodule activity was measured during P depletion through the detection of H 2 evolution of the nodules (Schulze et al., 2006). P concentration in leaves and nodules was determined through spectro-photometrical method.

Results and Discussion Symptoms of apparent P toxicity appeared at nitrogen fixing plants above a concentration of 5 µM P in the nutrient solution, while nitrate fed plants could cope with concentration up to 50µM. Most apparently high P concentrations in or around Medicago truncatula nodules affect their nitrogen fixation activity since the symptoms resembled nitrogen deficiency and P concentration in the leaves were about 2% and

186 DGP Meeting September 5-9, 2012 thus not at normally toxic levels. The depletion experiments clearly showed that P deficiency initially affects nitrogen fixation through an impact on shoots. The arguments in that line are: 1. When plants were supplied at levels of P that limited nitrogen fixation, the P concentration in shoots was strongly reduced, while still at normal levels in nodules. 2. P depleted plants showed a strong reaction in nitrogen fixation when the leaves were partially darkened while this does not occurred at sufficient P levels.

Literature Fischinger SA, Schulze J. (2010). The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake. Journal of Plant Physiology 167, 1112-1115. Schulze J, Temple G, Temple SJ, Beschow H, Vance CP. (2006). Nitrogen fixation by white lupin under phosphorus deficiency. Annals of Botany 98, 731-740.

187 DGP Meeting September 5-9, 2012 Activation of the ascorbate-glutathione cycle in Guinea grass forage grown under sulfur limitation

Fabiana Schmidt 1, Luisa Carvalho 2, Francisco Antonio Monteiro 3 and Sara Amâncio 4

1College of Agriculture Luiz de Queiroz/University of São Paulo, São Paulo; 2Institute of Agronomy/Technical University of Lisbon, Lisbon. E-mail: [email protected]

Introduction Increasing the supply of sulfur (S) to a plant improves growth due to the larger concentration of glutathione (GSH), which makes the antioxidative system more efficient, thereby boosting the plant’s capacity for resisting environmentally stressful conditions and increasing production. While the properties of the antioxidant GSH present in plants are beneficial in adverse conditions, studies have not yet assessed the effect of GSH on tropical grass Guinea grass (Panicum maximum cv. Tanzania; Poaceae) a forage species recommended for tropical pasturelands.

Materials and Methods An experiment was carried out to investigate the effects of S rates on ascorbate- glutathione (asc-glut) cycle. Sulfur was applied at 0.10, 1.00 and 1.45 mmol L -1 (low, intermediate and high S supply). Hydrogen peroxide (H 2O2), ascorbate (AsA), dehydroascorbate (DHA), reduced (GSH) and oxidized (GSSG) glutathione concentrations and activities of key enzymes in the AsA-GSH cycle were measured. Plant material was harvested and plant growth measured every 7 days until the final harvest at day 28. Shoot and root fresh and dry matter, shoot height and length of the primary root were measured weekly and leaf area at final harvest. Specific leaf area (SLA); leaf weight ratio (LWR); root weight ratio (RWR), and shoot:root ratio were measured. Chlorophyll (total, a, b, and a:b ratio) was determined in diagnostic leaves (the two most recently expanded leaf laminae ). In leaf and root samples glutathione

(GSH and GSSG), ascorbate (AsA and DHA), soluble protein and H 2O2 were quantified. The activities of the ROS-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) and the asc-glut cycle enzymes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) were determined. Results were statistically evaluated to compare the S rates at each harvest (days of treatment). Significant differences were identified by Tukey’s test at a significance level of 5%.

Results and Discussion Growth parameters were significantly increased by S supply and intermediate S availability was the optimum concentration, given that increasing S supply from that

188 DGP Meeting September 5-9, 2012 point did not result in significant increases in the parameters quantified. Plant growth was significantly reduced under low S availability, affecting dry matter production and its distribution in the plant (shoot:root ratio). Soluble protein was also higher in plants grown with intermediate and high S availability. In addition to showing lower chlorophyll concentrations, plants grown with low S availability accumulated ROS in the form of H 2O2. There is no doubt that increased ROS formation under conditions of nutritional stress caused by S limitation induces responses to protect against oxidative stress. The asc-glut cycle is the main pathway for the efficient removal/control of ROS and is important for maintaining cellular homeostasis ( Noctor and Foyer, 1998 ). The accumulation of GSH, the most important anti oxidative metabolite, was stimulated by supplying S at intermediate concentrations, but 14 days of S limitation also increased GSH concentration in leaves, certainly as a mechanism to protect against oxidative damage. The increased concentration of glutathione in the roots of S-deficient plants may be related to the transfer of GSH from leaves to roots via phloem (Foyer et al., 2001). GR showed high activity in the leaves and roots of plants grown with low S availability, demonstrating its efficiency in maintaining the cellular homeostasis of reduced GSH. The activity of SOD, APX and CAT also increased in plants under low S availability, indirectly confirming the presence of ROS. Although CAT has a low affinity for H 2O2, in conditions of oxidative stress, when the capacity of APX for eliminating H 2O2 is exceeded, CAT can act directly for its elimination (Mittler, 2002). In conclusion, low S availability slowed growth and caused a decline in plant dry matter production and protein content. Sulfur limitation induced the activity of enzymes that contribute to the regeneration of GSH and AsA, in order to overcome oxidative stress. In addition, plants grown with low S availability showed an increase in GSH synthesis and its transfer to the roots. Finally, it is possible to conclude that the highest S concentration applied in the present study was above the optimum threshold, an important result for S fertilization in grazing pastures with this species.

Acknowledgements To the State of São Paulo Research Foundation (FAPESP) for financial support and for the scholarship to F.Schmidt. To the Brazilian Government Research Council (CNPQ) for financial support. CBAA is funded by Foundation for Science and Technology (FCT) (PestOE/AGR/UI0240/2011).

Literature Foyer C.H.; Theodoulou F.L.; Delrot S (2001) The functions of inter- and intracellular glutathione transport systems in plants. Trends in Plant Science 6: 486-492. Noctor G.; Foyer C.H. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Molecular Biology 49:249–279. Mittler R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7:405- 409.

189 DGP Meeting September 5-9, 2012 Amino acids composition in a tropical grass as related to sulfur availability

Francisco Antonio Monteiro 1, Fabiana Schmidt 1 and Fabiano Daniel De Bona 2

1College of Agriculture Luiz de Queiroz/University of São Paulo, São Paulo; 2 National Wheat Research/Embrapa-trigo. E-mail: [email protected].

Introduction Animals exclusively fed with grasses from natural or cultivated pastures characterize a large part of the Brazilian beef cattle production. Considering the nutritional status of grasses, sulfur (S) interacts with nitrogen (N) for proteins synthesis and plant growth. In plants supplied with adequate nutrient rates, the N:S ratios varies within a narrow range, reflecting the abundance of S in amino acids and proteins (Crawford et al., 2000). This close relationship between S and N makes it clear that S should be included in programs of balanced plant nutrition in order to guarantee adequate forage resources. In the case of forage plants, understanding the metabolic and nutritional changes caused by S deficiency is important because suboptimum concentration of essential amino acids and/or the accumulation of secondary products of the assimilatory pathways of N and S can diminish the nutritional value and dry mass production of forage. This study examined the effect of S rates in the production and assessed changes in amino acids concentrations in leaves tissues of Guinea grass ( Panicum maximum cv. Tanzania).

Materials and Methods Guinea grass plants were grown during the austral summer in a greenhouse, in 3.6L plastic pots containing ground quartz as an inert substrate. Sulfur was supplied in the Hoagland and Arnon (1950) nutrient solution to simulate suboptimal, adequate and excessive S rates for the plants. Five rates were tested for Guinea grass (0.10; 0.55; 1.00; 1.45 and 1.90 mmol L -1). Plants were harvested on two occasions during the experiment. The shoot of the grass was harvested 35 days after transplanting (by cutting 5 cm above the root-shoot transition) and subsequently after 30 days of regrowth (by cutting at ground level). Samples of diagnostic leaves (the two most recent fully expanded leaf laminae) were sampled at both harvests in order to quantify amino acid and soluble protein concentrations. The plant material collected during the harvests was dried in a forced-ventilation oven at 65ºC until it reached constant mass, and subsequently weighed on a precision balance in order to quantify shoot dry mass production. Dry diagnostic leaf samples were separated and ground in a Wiley mill for subsequent determination of total S, total N, N-nitrate and S-sulfate concentrations in plant tissues. Statistical analysis was performed by using the statistical software SAS version 9.01 (SAS Institute Inc., Cary, NC, 2004). 190 DGP Meeting September 5-9, 2012 Results and Discussion This study provided clear evidence of the interference of S-limitation with processes of N assimilation and metabolism, and with the incorporation of N into proteins in Guinea grass plants. S-deficient plants also showed a high N:S ratio in leaf tissue (60:1) and an accumulation of nitrate and free amino acids, reflecting metabolic imbalance in plants. The increase in non-assimilated N (N-nitrate) and free amino acids is attributed to the partial interruption of the metabolic pathways of N assimilation and incorporation, which is illustrated by the low concentration of soluble protein and reduced production of dry mass of Guinea grass plants grown at suboptimal rates of S. The total free amino acids content was about 70-80% higher in S deficient plants (0.10 mmol L -1) than in plants supplied with higher S rates. Under S limitation accumulations of asparagine, alanine, glycine, serine and threonine were observed, as well as a low level of cysteine and methionine (S containing amino acids). With the increase in the S supply, the concentrations of many no-S amino acids were decreased. Increasing the supply of S to Guinea grass plants gradually corrected the imbalance in the N:S ratios in plant tissues, resulting in increased dry mass production and nutritional value of the forage, as shown by increased concentrations of S-sulfate, total S, S-containing amino acids (methionine and cysteine) and soluble protein. Concentrations of cysteine and methionine in forage plants are extremely important and beneficial for animal diet. Unlike animals, plants can assimilate inorganic S as sulfate and reduce it to sulfite in the synthesis of S- containing amino acids (Leustek et al., 2000). As a result, forage plants represent one of the main sources of the S-containing amino acids that are an essential part of ruminant diets. In conclusion, low S availability caused a nutritional imbalance with N, as illustrated by a high N:S ratio and high concentrations of N-nitrate and free amino acids in tissues of S-deficient plants. In S-limited Guinea grass plants, amino acid composition was dominated by asparagine. In addition, increasing S supply regulates the N:S ratio to approximately 20:1, providing N and S concentrations that are more suitable for protein synthesis and forage production in Guinea grass plants.

Literature Crawford N.; Kahn M.L.; Leustek T.; Long S.R. (2000) Nitrogen and sulphur. In: Buchanan B.B.; Gruissem W.; Jones R.L. (Eds.), Biochemistry and molecular biology of plants p.786–849. Hoagland D.; Arnon D.I. (1950) The water culture method for growing plants without soil 347p. Leustek T.; Martin M.N.; Bick J.A.; Davies J.P. (2000) Pathways and regulation of sulfur revealed through molecular and genetic studies. Annual Review of Plant Physiology and Plant Molecular Biology 51:141–165. SAS Institute Inc., SAS OnlineDoc® 9.1.2. JMP. (2004) Statistics and Graphics Guide, version 5.

191 DGP Meeting September 5-9, 2012 Are Mg deficiency-induced decreases of photosynthesis and carbohydrate accumulation dependent on sink/source ratio?

K. Mohammed and C. Engels

Division of Plant Nutrition, Humboldt University, Berlin; E-mail: [email protected]

Introduction Mg is essential for the functioning of many enzymes and plays a fundamental role in both, light and dark reactions of photosynthesis. Accordingly, Mg deficiency decreases the photosynthetic rate. In Mg-deficient plants, leaf concentrations of non- structural carbohydrates are increased (Cakmak et al. 1994). This sugar accumulation often precedes any loss of chlorophyll content or photosynthetic activity (Hermans and Verbruggen 2005), suggesting that the reduction of photosynthesis is a response to increased sugar levels. Leaf sugar accumulation in Mg deficient plants may be due to inhibition of phloem loading (Cakmak et al. 1994) or low utilization of assimilates in sink organs (Fischer et al. 1998). In this study, we reduced the source/sink ratio of tomato plants by shading of basal leaves to investigate if Mg deficiency-induced increase of sugar accumulation and decrease of photosynthesis in source leaves can be prevented.

Materials and Methods Tomato plants were grown in nutrient solution. One month after sowing in half of plants the 6 basal leaves were shaded (about 50% of total leaf area). Then shaded and unshaded plants were grown either with optimum or without Mg supply. When slight symptoms of Mg deficiency became visible (15 days after start of treatment, DAT) photosynthetic rate ( A) of a young unshaded source leaf (leaf No. 9) was measured every 3 days. Plants were harvested 24 DAT, and sugar, starch and Mg concentrations were measured in leaf 9.

Results and Discussion Shading of basal leaves reduced total plant biomass by 20-30%, irrespective of Mg supply (data not shown). In plants which were well supplied with Mg, the concentrations of soluble sugars and starch in leaf 9 were lower in shaded than in unshaded plants (Table 1), indicating that shading-induced decrease of the source/sink ratio reduced accumulation of non-structural carbohydrates (NSC) in young source leaves. In Mg deficient plants, starch concentrations in leaf 9 were similarly reduced by shading as in Mg sufficient plants (Table 1). However, in contrast to Mg sufficient plants, in Mg deficient plants soluble sugar concentrations in leaf 9 were higher in shaded than in unshaded plants. The accumulation of soluble sugars 192 DGP Meeting September 5-9, 2012 in Mg deficient plants was associated with a marked increase of the ratio of fructose to glucose plus sucrose (Table 1). This indicates a disturbance of sugar metabolism, which led to high accumulation of soluble sugars in young source leaves of Mg deficient plants even under conditions when starch accumulation was reduced by shading. In plants which were well supplied with Mg, shading of basal leaves did not increase the rate of net photosynthesis (A) in leaf 9 (Table 1). Possibly, A was near to its maximum capacity, irrespective of the source/sink ratio and leaf carbohydrate concentrations. In unshaded Mg deficient plants, A in leaf 9 was similar as in Mg sufficient plants despite of decreased Mg- and increased soluble sugar concentrations (Table 1). In shaded Mg deficient plants, in contrast, A was strongly reduced. This reduction of A in shaded in comparison to unshaded Mg deficient plants was associated with further decrease of Mg concentrations of Mg and strong reduction of chlorophyll concentration (Table 1).

Table 1 Effect of Mg supply and shading of basal source leaves (leaf no 1 - 6) on starch, fructose (Fru), glucose (Glu), sucrose (Suc), Mg and chlorophyll concentrations (Chl) and photosynthetic rate (A, µmol m-2 s-1) of an apical source leaf (no 9); different letters within a row indicate significant (Tukey-Kramer’s test, P<0.05) treatment effects. Sta Fru Glu Suc Mg Chl Treatments A mmol m-2 leaf area +Mg 34a 8c 7a 3c 12.3a 0.81a 23a Unshaded -Mg 34a 12b 5a 6b 1.9b 0.71a 17a +Mg 18b 4d 2b 1c 9.6a 0.71a 18a Shaded -Mg 24ab 22a 5a 9a 1.3c 0.46b 3b

In conclusion, reduction of source/sink ratio through shading of basal leaves did not prevent Mg deficiency-induced sugar accumulation in young source leaves. This finding is in agreement with Mg deficiency-induced inhibition of phloem loading. The decrease of A in Mg deficient plants was not directly related to sugar accumulation but occurred only after leaf Mg-concentrations fall below a critical value, and was associated with a reduction of chlorophyll concentrations.

Literature Cakmak, I., Hengeler, C. and Marschner, H. (1994). Changes in phloem export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants. Journal of Experimental Botany 45 : 1251–1257. Fisher, E. S., Lohaus G., Heineke, D.,Heldt, H. W. (1998). Magnesium deficiency results in accumulation of carbohydrates and amino acids in source and sink leaves of spinach. Physiologia Plantarum 102: 16-20. Hermans, C. and Verbruggen, N. (2005). Physiological characterization of Mg deficiency in Arabidopsis thaliana . Journal of Experimental Botany 56 : 2153–2161.

193 DGP Meeting September 5-9, 2012 Boric Acid Treatment Alters the Hormonal Status of Pea Lines

Mehmet Hamurcu 1, Tijen Demiral, Erdogan E. Hakki, Canan Ozdemir, Ahmet Tamkoc, Zeynep Zuhal Avsaroglu

1Selcuk University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, Konya, Turkey; E-mail: [email protected]

Different pea lines were hydroponically grown in a growth chamber until three-leaf stage and then exposed to 1/5 Hoagland solution containing 0, 2, 20, and 200 mg L-1 boron for a week. Pea lines employed were S2 line (derived from Pisum sativum L.), and lines 602, Taþkent and Özkaynak (derived from Pisum arvense L.). Plants were harvested on 0, 3 and 7 days after B treatments and the ratios of phytohormones in the leaves and roots were studied. Higher ABA contents were determined in the leaves of Özkaynak, after B treatments on day 3, possibly due to elevated synthesis of ABA in leaves or due to higher level sensitivity of this genotype to B. However roots are the main site for the biosynthesis of ABA in plants, the highest level of B treatment (200 mg L-1) remarkably decreased ABA content in roots of all pea lines on day 7, which might suggest either reduced aldehyde oxidase activity or transport to leaves. GA3 levels in leaves of Taþkent significantly increased after exposure to B treatments on days 3 and 7. B treatments enhanced GA3 content in roots and leaves of Özkaynak on day 7. GA3 content was slightly reduced in roots of S2 line on day 7 by B applications. B treatments decreased IAA content in roots of S2 and Özkaynak, respectively, on days 3 and 7 and also IAA/ABA ratio in roots of S2 on day 3. IAA/ABA ratio in roots of Özkaynak was increased after 7 days of B treatments. It seems that B stress affects the synthesis or metabolism of ABA, GA3 and IAA in pea plants.

194 DGP Meeting September 5-9, 2012 The Effects of Boron on the Anatomy of Pisum sativum L. and Pisum arvense L.

Canan Ozdemir 1, Mehmet Hamurcu 2, Tijen Demiral 3, Erdogan E. Hakki 2, Ahmet Tamkoc 4, Sait Gezgin 2

1Celal Bayar University, Faculty of Art and Science, Department of Biology, Manisa, Turkey, 2Selcuk University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, Konya, Turkey, 3Harran University, Faculty of Science, Department of Biology, Sanliurfa, Turkey, 4Selcuk University, Faculty of Agriculture, Department of Field Crops, Konya, Turkey. E-mail: [email protected]

Introduction Excess boron stress is one of the most significant abiotic stress that affects every aspect of plant physiology and metabolism. The physiological effects of boron toxicity include reduced root cell division decreased shoot and root growth (Lowatt and Bates, 1984; Nable et al., 1990., Liu et al., 2000., Demiray et al. 2011), decrease in leaf chlorophyll, inhibition of photosynthesis, lower stomatal conductance (Lowatt and Bates, 1984), deposition of lignin and suberin (Ghanati et al., 2002), reduced proton extrusion from roots (Roldan, 1992), increased membrane leakiness peroxidation of lipids and altered activities of antioxidation pathways (Karabal et al., 2003). The mechanisms of the toxicity revolve around the possible disruption of cell wall development due to the excess binding to apiose (furanoid sugar in the rhamnogalakturonan II complex), or metabolic disruption due to binding to ribose, either as the free sugar or as a component of key compounds such as RNA, ATP, NADP or NADH. During excess boron stress, high concentrations of boron enters the cells and accumulates to a concentration that induces ionic and osmotic stress in plants (Reid et al., 2004). This study aimed to investigate the effects of toxicity and deficiency of boron (B) on anatomical structure of a genotype of Pisum sativum L. (line S2) and 3 genotypes of Pisum arvense L., namely lines 602, Taskent and Özkaynak.

Materials and Methods Anatomical research was carried out with paraffin method on harvested samples being kept in 70% ethanol. Cross-sections of roots, stems and leaves were prepared by using microtome. Sections for microscopic observation were prepared by dehydration through a series of fixation in ethanol using the standard double-stained technique of safranine and fast green stains. Measurements were taken with an ocular micrometer under a light microscope (Leica DM3000 motorized). Data for anatomical features of plants were recorded at 0, 3 and 7 days after exposure to 0, 2, 20 and 200 mg kg -1 B levels. Leaf thickness, root area, parenchyma tissue, and vascular tissue were recorded to see the influence of B on the anatomical features of pea plants. 195 DGP Meeting September 5-9, 2012 Results and Discussion Anatomical characteristics of pea plants showed differences depending on the plant variety, B level applied and the time of exposure to B stress. Excess concentrations of B generally caused a decrease in the root vascular tissue. On the other hand, boron deficiency caused an increase in the stem and leaf parenchyma tissue. A consistent increase was recorded in sclerenchyma thickness in both stem and root tissues of pea plants when treated with 20 mg kg -1 B but the increase was relatively greater under 200 mg kg -1 B treatment. These results led us to conclude that either boron deficiency or toxicity affect the anatomy of pea plants.

Literature Ghanati F, Morita A, Yokota H (2002). Induction of suberin and increase of lignin content by excess boron in tobacco cells. Soil Sci. Plant Nutr. 48: 357-364. Hatice Demiray, Aylin E. Dereboylu, Filiz Altan and Ali Zeytünlüo ğlu African Journal of Biotechnology Vol. 10 (69), pp. 15545-15551, 7 November, 2011 Karabal E, Yücel M, Ökte HA (2003). Antioxidant responses of tolerant and sensitive barley cultivars to boron toxicity. Plant Sci. 164: 925-933. Liu D, Jiang W, Zhang L, Li L (2000). Effects of boron ions on root growth and cell division of broadbean ( Vicia faba L.). Isr. J. Plant Sci. 48: 47-51 Lowatt CJ, Bates LM (1984). Early effects of excess boron on photosynthesis and growth of Cucurbita pepo . J. Exp. Bot. 35(152): 297-305. Nable RO, Cartwright B, Lance RC (1990). Genotypic differences in boron accumulation in barley: Relative susceptibilities to boron deficiency and toxicity. Genetic Aspects of Plant Mineral Nutrition eds El. Bassam N, Dambroth M, and Loughman B. Dordrecht Kluwer Academic Publishers: pp. 243-251. Reid RJ, Hayes JE, Post A, Stangoulis JCR, Graham RD (2004). A critical analysis of the causes of boron toxicity in plants. Plant Cell Environ. 25: 1405-1414. Roldan M, Belver A, Rodriguez-Rosales P, Ferrol N, Donaire JP (1992). In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots. Physiol. Plantarum, 84: 49-54.

196 DGP Meeting September 5-9, 2012 Effect of excess manganese on the apoplastic proteome of rice RILs differing in manganese tolerance

S. Weiß 1, M Duschyk 1, H.-P. Braun 2 and W. J. Horst 1

1Institute of Plant Nutrition, Faculty of Natural Sciences, Leibniz University Hannover, Herrenhäuser Str. 2, 30149 Hannover, Germany, 2Institute of Plant Genetics, Faculty of Natural Sciences, Leibniz University Hannover, Herrenhäuser Str. 2, 30149 Hannover, Germany. E-mail: [email protected]

Introduction Rice typically grows in soils with low redox potential and thus high plant Mn availability. Although Mn is an essential micronutrient, Mn excess leads to decreasing growth and crop yields (Foy et al., 1978). There is a great intra- and interspecific variability in Mn tolerance (Horst, 1988). In the initial stages of Mn toxicity plants develop typical brown spots on older leaves which can be used as a reliable parameter to classify different species and cultivars according to their Mn tolerance. This fact was used to generate a recombinant inbred line population by crossing a Mn-sensitive and a Mn-tolerant parent. From this population two genotypes were selected: a Mn-sensitive and a Mn-tolerant line to analyze the apoplastic peroxidase proteome. Increased activities of H 2O2-consuming and H 2O2-producing peroxidases indicate that the apoplast is a major compartment for the interaction of Mn with the plant tissue (Fecht-Christoffers et al., 2006). For proteomic analysis the apoplastic peroxidase isoenzyme profile was compared using native isoelectric focusing. The increase in peroxidase activity correlates with an enhanced release of additional peroxidase isoenzymes into the apoplast as revealed by the occurrence of an additional peroxidase band on the basic side of the isoenzyme profile of the Mn- sensitive but not the Mn-tolerant genotype after Mn excess treatment.

Materials and Methods A recombinant inbred line population consisting of 92 individuals was derived from a Mn-tolerant (IR 1552) and a Mn-sensitive (Azucena) parent. The population has been characterized for the Mn tolerance level. Two genotypes were selected according to their difference in Mn uptake and expression of typical Mn toxicity symptoms in form of brown spots. R 20 showed low uptake of Mn and high expression of Mn toxicity symptoms whereas R 120 displayed high uptake of Mn and no expression of Mn toxicity symptoms. Therefore, R 20 was classified as Mn-sensitive whereas R 120 was characterized as Mn-tolerant. The two genotypes were pre-cultured at 1 M Mn until the fifth leaf was fully developed. Then the plants received 500 M Mn for

5 days. Apoplastic washing fluid (AWF) was extracted from leaves and H 2O2- consuming and producing peroxidase activities as well as the Mn concentrations in the bulk leaf and the AWF were measured. In addition, the apoplastic peroxidase 197 DGP Meeting September 5-9, 2012 isoenzyme profile was compared using native isoelectric focusing (Pre-cast gels according to the manufacturer´s instructions, BioRad, Munich, Germany). Bands were excised from the gel and analyzed via Nano-LC-MS/MS to identify their peptide composition.

Results and Discussion Both genotypes displayed similar Mn concentrations in the leaf as well as in the AWF but only the Mn-sensitive genotype R 20 showed enhanced apoplastic H 2O2- consuming and producing peroxidase activities under Mn excess conditions whereas the tolerant genotype remained nearly unaffected. The increase in peroxidase activity results from an enhanced release of additional peroxidase isoenzymes into the apoplast as revealed by the appearance of an additional peroxidase band on the basic side of the isoenzyme profile of the Mn-sensitive but not the tolerant genotype after Mn excess treatment. The analysis of excised gel bands by Nano-LC-MS/MS revealed the additional peroxidase isoenzyme band consisting of peroxidase 2 and peroxidase POC1. Future works will focus on the question if the found peroxidases are also higher expressed at the transcriptomic level using qRT-PCR gene expression analysis.

Literature Fecht-Christoffers MM, Führs H, Braun HP, Horst WJ. (2006). The role of hydrogen peroxide- producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance. Plant Physiology 140: 1451-1463 Foy CD, Chaney RL, White MC. (1978). The physiology of metal toxicity in plants. Annual Review of Plant Physiology 29: 511-567 Horst WJ. (1988). The physiology of Mn toxicity. In: Graham RD, Hannam RJ, Uren NC, eds. Manganese in soils and plants. Dordrecht: Kluwer Academic Publishers, 175–188

198 DGP Meeting September 5-9, 2012 An intracellular manganese transporter required for submergence tolerance of plants

B. Meier, X. Chen, T. Peiter-Volk, K. Peter, A. Mustroph, E. Peiter

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

Manganese (Mn) is a micronutrient that is involved in many physiological processes due to its function as a catalytic site or cofactor of enzymes. The defense of reactive oxygen species is a key process where a Mn-containing enzyme, the Mn superoxide dismutase, plays an important role. In order to prevent a toxic accumulation of Mn in the cytoplasm cells regulate Mn homeostasis by employing mechanisms for translocation, sequestration and exclusion. Transport proteins are also responsible for the correct distribution of the micronutrient to its targets. Here we present the functional analysis of a transporter belonging to the CDF family. Visualization of fluorescent fusion proteins indicated an intracellular localization of the transporter. Heterologous expression of the gene in a Mn-sensitive yeast mutant restored the growth on excess Mn, which is in agreement with the function as a Mn transporter. Plant histochemical GUS studies showed a promoter activity in the vascular system of roots and shoots. In roots, expression was specific to the pericycle. Despite the likely involvement of this gene in Mn homeostasis, its expression was not altered by excess Mn in the growth medium. Interestingly, T-DNA knockout mutant seedlings were less sensitive to high Mn than the wild type. In addition, the mutant showed to be hypersensitive to post-anoxia conditions, suggesting a role of this transporter in ROS defense.

199 DGP Meeting September 5-9, 2012 Poster Session P7: Nutrients and ecosystems / Climate change

200 DGP Meeting September 5-9, 2012 A Simplified Recommendation for Nitrogen Fertilisation in a Wheat/Maize Double Cropping System in th

Tobias Hartmann

Universität Hohenheim, Fruwirthstraße 20, Institut für Kulturpflanzenwissenschaften, 70599 Stuttgart, Germany; E-mail: [email protected]

Currently, Farmers in the North China Plain (NCP) are fertilising up to 600 kg (N) ha- 1 a-1 in a winter-wheat/summer-maize double cropping system, resulting in a continuous accumulation of nitrogen in the soil profile, and an increased risk of nitrogen leaching and the emission of climate relevant gases. Field experiments showed that nitrogen fertilisation in these double cropping systems can be reduced by up to 50% compared to farmers practice without significant reductions in the yield of either maize or wheat. Further, significant yield losses of unfertilised maize could only be observed in the third year of the experiment. Based on these results, we propose a simple and straightforward nitrogen fertilisation recommendation for a winter-wheat/summer-maize double cropping system in the NCP, which emphasis the application of nitrogen to the winter-crop, while reducing input for the summer-crop, when mineralisation in the soil covers crop nitrogen demand. Using the crop- simulation model Hermes, we will evaluate this nitrogen fertilisation recommendation for effects on plant-growth, yield and possible nitrogen losses, and compare the results to farmers fertilisation practices, zero nitrogen treatments, as well as fertiliser recommendations based on Nmin – methods.

201 DGP Meeting September 5-9, 2012 Boron in crop production systems: Focus on legumes and forage mineral quality

Lisbeth Linse 1* , Sigrun Dahlin 2, Elisabet Nadeau 3 and Ingrid Öborn 1

1 Department of Crop Production Ecology, Swedish University of Agricultural Sciences (SLU), Uppsala. 2 Department of Soil and Environment, SLU, Uppsala. 3 Department of Animal Environment and Health, SLU, Skara. *E-mail: [email protected]

Introduction The essentiality of boron (B) for growth and reproduction of plants including legumes and grasses is well established, while there is no consensus about the role of B in animal and human metabolism. A literature review has been carried out, that deals with B from an agricultural and food systems perspective (Linse et al. 2011).

Legumes have a high demand for B, for all steps in the symbiosis with N 2-fixing rhizobia. Boron is also beneficial for the legume/mycorrhizal fungi symbiosis. In cropping systems relying on biological processes, e.g. organic farming, legumes are necessary in the crop rotation. Legumes and grasses in mixed swards contribute to the cropping systems with improved soil structure, soil fertility, pest and weed control (Linse et al. 2011). The aim of this study was to evaluate the influence of B nutrition on (i) yield, (ii) botanical composition of mixed swards, (iii) plant macro- and micronutrient concentrations, and (iv) differences in B use efficiency in lucerne varieties. The study is a part of a more extensive project “Micronutrient management strategies in agriculture - how to utilize site specific and local resources to produce high quality products”. This project explores how (i) soils can be managed, (ii) plant species selected, and (iii) local recycling products used, to supply an optimum micronutrient profile in food and feed crops (Öborn et al. 2011; Watson et al. 2012).

Materials and Methods In the south-west of Sweden, in an area with humid climate and geological conditions with expected low B content in the soils, two field studies with foliar application of B have been carried out in: (1) the first year of organic ley (red/white clover and grass) with and without liquid manure fertilization, and (2) a lucerne variety study which is ongoing (2010-2012). (1).Three trials, two in the same field with and without cattle slurry (15 -20 ton/ha) and the third without slurry. The soil texture was coarse and crop rotation was organically managed with a large proportion of legumes. The first year of ley (red/white clover and grass) was the study crop. The design was randomized block including the main effects of trial and treatment and its interaction using 4 blocks per treatment nested

202 DGP Meeting September 5-9, 2012 within trial. Pairwise comparisons between treatment means were done using Tukey´s test ( P < 0.05). Treatments were foliar application of 0g B ha -1 and 450g B ha -1 after the first cut, when the crop covered 50% of the soil surface. The harvests from the second cut were analyzed for yield, botanical composition and macro- and micronutrients in soil (N, P, K, Mg, Ca, Cu, B and OM), and in the individual plant species (N, P, K, Ca, Mg, Na, S, Mn. Cu, Zn, B, Fe and Al) on paired soil-plant samples. (2) One trial with 9 varieties of lucerne and one of red clover. Treatment was the same B doses as in (1) applied after the last cut year 1 and after every cut (3 yr -1) during year 2 and 3. Chemical analyzes as above plus Mo.

Results and Discussion Results from the field study (1) showed that (i) the crops were not B deficient, e.g. red clover with B conc. in a range of 27-42 mg kg -1, in the plots with 0g B ha -1 (sufficient values ~ 25 mg kg -1) (ii) there were significant differences in forage DM yield between the three trials ( P < 0.0001), but the B treatment did not affect yield, (iii) B treatment tended to increase the white clover proportion, when averaged over trials (P = 0.071), and (iv) averaged over trials, the B concentrations in soil and plants were increased by the B treatments ( P < 0.01), while concentrations of the other nutrients were not affected. There were significant differences in the concentrations of other nutrients between the different trials. The probable reasons that a soil content as low as 0,4 mg kg -1 hws B, in the unfertilized plots, was sufficient for legumes are: (i) A high amount of OM (7-8 %), the main source of B on light textured soils, (ii) a pH suitable for B uptake (6,2-6,4) and (iii) a humid climate. Data from study (2) are under evaluation and will be presented at the conference.

Literature Linse L, Öborn I, Sinclair AH. (2011). The role of boron in biological systems with focus on legumes in crop production. A review. In: GL Perkins (Ed). Boron: Compounds, Production and Applications. NOVA Science Publishers, Inc. 557-580. (ISBN 9781617617607). Watson CA, Öborn I, Edwards AC, Dahlin AS, Eriksson J, Lindström BEM, Linse L, Owens K, Topp CFE, Walker RL. (2012). Using soil and-plant properties and farm management practices to improve the micronutrient composition of food and feed. Journal of Geochemical Exploration doi 10.1016/j.gexplo.2012.06.015. Öborn I, Bertilsson J, Campbell CD, Coull MC, Dahlin S, Edwards AC, Eriksson J, Frankow-Lindberg B, Hillier S, Lindström B, Linse L, Lumsdon DG, Ramezanian Bajgiran A, Shand CA, Sinclair AH, Walker RL, Watson CA, Wivstad M. (2011). Micronutrient management strategies in agriculture - how to utilize local and site specific resources to produce high quality products? 11th International Conference on the Biogeochemistry of Trace Elements (ICOBTE), Florence, 3-7 July 2011

203 DGP Meeting September 5-9, 2012 Response of rice genotypes to soil pH and moisture regimes

Maya Subedi, Christine Kreye, Mathias Becker

Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Aerobic rice is a system where adapted rice cultivars are grown in non-flooded and non-saturated soil. It may be an alternative cultivation technique to conventional lowland rice in water scarce areas. However, when grown in soils of relatively high pH it may suffer from micronutrient deficiencies. We tested 5 rice genotypes in a pot experiment during the vegetative stage for their response to 2 soil moisture regimes (aerobic and flooded) at 3 pH levels (approximately 5.0, 6.5 and 7.5). The genotypes comprised improved upland cultivars, Apo, IR78877-208-B-1-2 and CT6510-24-1-2, the improved Oryza sativa lowland cultivar IR72, and the traditional Oryza glaberrima upland cultivar CG14. Across genotypes and soil pH levels, aboveground biomass was 36% higher under flooded than under aerobic conditions. Under aerobic conditions, the reduction in biomass tended to be least in IR72 (34%), and reached 56-70% in other genotypes. Soil flooding increased the uptake of Fe by 47%, while the uptake of Zn was highest under aerobic conditions at low pH. In high pH aerobic soil, average shoot micronutrient concentrations were 91 mg Fe kg-1, and 24 mg Zn kg-1. Fe and Zn content and uptake tended to be highest in IR72 and IR78877-208- B-1-2. Contrary to our expectation, the lowland genotype IR72 outperformed the upland adapted genotypes during the early vegetative stage under aerobic, high pH soil conditions.

204 DGP Meeting September 5-9, 2012 The response of banana root systems to a tree shade gradient in Costa Rica

Charlotte Dreiseidler, Jürgen Burkhardt, Oscar Bustamante, P. Siles, Charles Staver, E. P. Valdivia

Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Throughout Latin America, smallholder farmers plant a wide range of Musa species in association with shade-grown coffee, for national markets and home consumption. In a collaborative GIZ-funded project Bioversity International aims at improving farmers returns in terms of production and income. To assess the partitioning of nutrients within four Musa cultivars, aerial biomass and root systems of three plant replications were examined in minimal, 25%, 50% and 75% shade of Erythrina poeppigiana. Musa root systems (roots >1mm diameter) were studied by large (0.4m distance of plant) and small (0.4, 0.8, 1.2m distance) soil samples, and by estimating the total root system out of these soil samples. Both root systems and aerial biomass decline in high shade (e.g. AAB ‘Curraré‘ root biomass: 1.1kg in 25% to <0.2kg in 75%). The abundance of tree and coffee roots is uncorrelated to Musa roots, with correlation coefficients <0.2. Contrarily, Musa roots moderately correlate to its aerial biomass, with correlation coefficients 0.3 to 0.61. Shoot-root ratios decrease from minimal to 25% shade (e.g. AAA ‘Gros Michel‘ (GM) 4.7 to 3.7) and increase in high shade (e.g. GM 2.1 in 50% to 3.0 in 75%), thus reveal a small tendency in favour of aerial biomass. Therefore, light deficiency in particular reduces Musa root biomass, instead of interspecific root competition. Bananas probably discriminate their root system in favour of an enhanced light-capturing shoot, yet hard evidence still lacks.

205 DGP Meeting September 5-9, 2012 Photosynthesis of three dessert banana cultivars along an altitudinal gradient

P. Siles, M. Deras, O. N. Matute, O. Bustamante, C. Staver, J. Burkhardt 1

1Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Bananas are grown by small holder farmers in agro-forestry systems in a wide range of climatic conditions throughout mid and high altitude zones of Latin America. In this study the leaf photosynthesis of three cultivars was measured at intermediate (1000masl) and high altitude (m asl). Gros Michel (AAA) is the preferred cultivar for national dessert banana markets, but is susceptible to Fusarium wilt. The two hybrids FHIA 17 and FHIA 23 (AAAA) are dessert bananas reported to be resistant to Fusarium wilt and are potential substitutes in Fusarium infested fields. Gas exchange measurements of light and CO2 response curves were conducted to estimate the parameters of a biochemical model of photosynthesis An (Farquhar et al., 1980) - maximum carboxylation rate (Vcmax), potential light-saturated electron transport rate (Jmax) and day respiration (Rd). The results showed higher rates of An at mid altitude. The estimated parameters Vcmax, Jmax and Rd were dependent on leaf temperature, but also differed between locations when standardized at 25°C suggesting an additional effect of site factors such as soil fertility and water availability. FHIA 17 and FHIA 23 had higher rates of An than Gros Michel. Vcmax and Jmax standardised at 25°C were highly correlate d with leaf specific area. The FHIA cultivars with a thicker leaf blade presented higher photosynthesis capacity. Understanding changes in photosynthetic parameters for banana are crucial for modeling long-term photosynthesis.

206 DGP Meeting September 5-9, 2012 Analysis of estrogenicity in different matrices by YES assay

Le Thi Anh Hong 1, Joachim Clemens 2

1Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; 2Gewitra Ltd, Troisdorf; E-mail: [email protected]

Endocrine disrupting compounds (EDCs) or Endocrine disruptors are of global concern since many years due to their negative impact to the wildlife and human health. The main sources of EDCs in the environment are humans and animals. EDCs can appear in several environmental matrices such as: wastewater, surface water, manure, sludge, sediments, etc. The analysis of EDCs is a challenge because of their low concentration in the environment. Furthermore there is a big variety of EDCs. Several analytical methods are available with different advantages and disadvantages. Additionally, the method should be selected according to their purpose. For example, for a monitoring program with a large amount of samples the method should be rather simple and robust. This study used the YES assay developed by Routledge and Sumpter (1996). Matrices analysed were surface water, river sediments, liquid and solid phase of septage slurry and biogas slurry, and vermicompost. The recovery rates were 89.7% ± 15.5%, 79.2% ± 10.7%, 83.5 % ± 6.6%, and 86.8% ± 6.1% in distilled water, vermicompost, liquid phase and solid phase of septage slurry, respectively.

207 DGP Meeting September 5-9, 2012 Chain length and pH effects on uptake of perfluorinated compounds (PFC) in maize (Zea mays)

J. Krippner 1, S. Falk 2, S. Schubert 1, S. Georgii 2, H. Brunn 3, T. Stahl 2

1 Institute of Plant Nutrition, Faculty of Agricultural Sciences, Nutritional Sciences and Environ- mental Management, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany,2 Hessian State Laboratory, Glarusstr. 6, 65203 Wiesbaden, Germany, 3 Hessian State Laboratory, Schubertstr. 60, 35392 Giessen, Germany. E-mail: [email protected]

Introduction Perfluorinated organic compounds (PFC) have been detected globally in environmental matrices and appear to be ubiquitous in foodstuff and animal feed. Few systematic studies have been carried out to determine the uptake of PFC into plants and those have been limited to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS).

Materials and Methods

Uptake of PFC differing in chain length (C 4 to C 10 ) by maize plants was measured at various pH values (pH 5, 6, 7, 8). The maize was grown in containers of 40 L nutrient solution under controlled conditions in a climate chamber (16 h at 26°C, 8 h at 18°C, 50% relative humidity and 500 µE m -2 s-1 light intensity). The pH value of the root medium was maintained for a week using an automatic pH-stat system. PFC were added at a concentration of 100 µg L -1 root medium. The PFC mixture was made up of seven perfluorocarboxylic acids (C 4 to C 10 ) and three perfluorosulfonic acids (C 4,

C6 and C 8). To indentify cross-contamination an additional container was maintained with control plants (blank value control) which received identical nutrient solution but no PFC mixture. One week after addition of PFC plants were harvested and weighed. After appropriate sample preparation the PFC concentrations in maize were measured using LC-MS/MS .

Results and Discussion Perfluorocarboxylic and perfluorosulfonic acids showed distinct differences in uptake depending on the chain length of PFC. A possible influence of pH of the root medium on uptake of perfluorinated compounds will be discussed.

208 DGP Meeting September 5-9, 2012 Hygroscopic leaf surface particles and the hydraulic activation of stomata

Burkhardt, Jürgen 1, Kaiser, Hartmut 2, Pariyar, Shyam 1 and Hunsche, Mauricio 1

1Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, D-53115 Bonn; 2Botanical Institute, University of Kiel, Am Botanischen Garten 9, 24118 Kiel, Germany; E-mail: [email protected]

Hygroscopic particles on leaves are strong water vapour sinks close to stomata; they are ubiquitous and may reach the amount of leaf waxes. The influence of added or excluded particles on stomatal functioning was assessed. The apertures of 50 to 75 stomata of elder and bean leaves were recorded together with gas exchange during opening and closing reactions, before and after treatment with NaNO 3 or KH 2PO 4 particles, respectively. Both salts caused higher leaf conductance at the same degree of stomatal opening. Bean and sunflower plants were grown in ventilated greenhouses supplied with either ambient (AA) or filtered air (FA). At 400 ppm CO 2 , transpiration of FA plants was lower compared to AA plants. When CO 2 was doubled, FA plants stabilized transpiration and increased photosynthesis, while AA plants stabilized photosynthesis and decreased transpiration. On hydrophobic tomato cuticles, NaCl and NaClO 3 particles underwent repeated deliquescence/efflorescence cycles within an environmental scanning electron microscope and spread out in dentritic form. This mechanism may eventually establish the ‘hydraulic activation of stomata’, i.e. continuous thin water connections linking apoplast and leaf surface along the stomatal walls and enabling bidirectional stomatal transport of liquid water, solutes, and hydraulic signals. Aerosols may therefore have important ecophysiological functions.

209 DGP Meeting September 5-9, 2012 Greenhouse gas emissions from anaerobic digestion plants in Germany

Nguyen Thanh Phong 1, Birte Mähl 2, Carsten Cuhls 2, Joachim Clemens 2

1Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; 2Gewitra Ltd, Troisdorf; E-mail: [email protected]

This study investigated emissions of CH 4, N 2O and NH 3 from nine anaerobic digestion plants that treat biowaste. The treatment is in form of mechanical pre- treatment, anaerobic digestion followed by a composting with or without intensive aeration. The exhaust gases from the mechanical and anaerobic steps are treated by biofilters. The emission sources at the plants consisted of biofilters, combined heat and power units (CHP), liquid digestate treatment systems (LTS) and open composting windrows of the solid digestate. In average, the biofilters removed 22% of total organic carbon (TOC), 52% of non methane volatile organic carbon (NMVOC) and 55% NH 3, whereas N 2O concentrations increased by 31%. For CH 4 the biofilters had only a small removal effect (7%). Overall, the emission factors were 0.4-16kg

(Mg biowaste)-1 for CH 4, 7-170g (Mg biowaste)-1 for N 2O and 41-6032g (Mg biowaste)-1 for NH 3. Open composting windrows of solid digestate resulted in high emissions of CH 4 and N 2O. Intensive aeration of the solid digestate could reduce greenhouse gas emissions.

210 DGP Meeting September 5-9, 2012 Denitrification potential of different soils and its impact on N 2O isotopomer signatures

Jan Reent Köster 1, Mehmet Senbayram 2, Klaus Dittert 2, Reinhard Well 3, Anette Giesemann 3, Anna Techow 4, Antje Herrmann 4, Karl H. Mühling 1

1Institute of Plant Nutrition and Soil Science, Kiel University, Kiel; 2Department of Crop Science, University of Goettingen, Goettingen; 3Institute of Agricultural Climate Research, von Thuenen Institute, Braunschweig; 4Institute of Crop Science and Plant Breeding, Kiel University, Kiel; E-mail: [email protected]

Introduction

Nitrous oxide (N 2O) is the most important greenhouse gas and ozone depleting substance emitted from soils. Agricultural N 2O emissions from soils increase after N fertilizer application and emission rates are significantly affected by soil properties. In Schleswig-Holstein, the three major landscape types with agricultural land-use are ‘Marsch’ with clay soils, ‘Hügelland’ with loamy soils, and ‘Geest’ with sandy soils. These soils differ clearly in their texture but also in their chemical properties such as total C content. So far it is still not yet well understood how N 2O producing processes (e.g. nitrification and denitrification) are influenced by physical and chemical soil 15 properties. Recently, the analysis of the intramolecular N distribution of N 2O, i.e. the 15 N site preference (SP), is getting more and more attention as a tool for N 2O source identification (nitrification or denitrification). N 2O production during bacterial denitrification is assumed to result in SP values close to 0 ‰, while N 2O SP from nitrification is expected to be around 33 ‰ (Sutka et al., 2006). In the current study, the denitrification potential (direct measurements of elemental N 2 and N 2O) and the

SP values of emitted N 2O in clay (Marsch) and sandy-loam (Hügelland) soil were investigated in two soil incubation experiments.

Materials and Methods Soils were sampled from unfertilized plots at two sites with different soil type and soil fertility but similar temperate maritime climate (ø 760 and 844 mm, 8.8 and 8.3°C at site with sandy loam and clayey soil, respectively). Soils were air-dried, sieved, and repacked into incubation vessels. Soils were treated with either ammonium nitrate (AN) or potassium nitrate (PN) solution (15 mM N) prior to the experiment. The soil moisture was adjusted to 65 % water holding capacity by vacuum through a ceramic plate at the bottom of the vessels. Then the incubation vessels were sealed.

Atmospheric air was replaced by a He / O 2 mixture in the first experiment with an O 2 concentration of 20 % (v/v) which was then reduced to 5 % and 0 % during the experiment, while in the second experiment the soil was incubated with He only. Incubation was carried out at Hanninghof Research Station for 35 and 10 days in

Exp. 1 and Exp. 2, respectively. CO 2, N 2 and N 2O release was measured online by 211 DGP Meeting September 5-9, 2012 GC. At frequent intervals gas samples were collected from all vessels and δ15 Nbulk 15 and N site preference were analyzed in N 2O by IRMS.

Results and Discussion

Expectedly, CO 2 release was significantly higher from the ‘Marsch’ soil compared to

‘Hügelland’ due to its higher total C content. N 2O and N 2 release in soils applied with

AN was similar from both soils. With PN, N 2O release was significantly higher from the ‘Marsch’ soil than ‘Hügelland’. Emissions of N 2 and soil denitrification potential were slightly higher in ‘Marsch’ soil than ‘Hügelland’, however no significant difference was observed. In both experiments, the main denitrification product was N 2 and the ratio of N 2O / N2 did not differ significantly when comparing soil types. N 2O SP increased gradually after start of the anoxic spell by 5 to 10 ‰ in parallel to a decrease in N 2O / N2 ratio. It may be hypothesized that this increase during the anoxic phase might indicate a strong impact of N 2O reduction on SP. When comparing soil types, the N 2O SP was clearly higher (c. 7 to 10 ‰) with ‘Marsch’ soil compared to ‘Hügelland’.

Literature Sutka, R. L., Ostrom, N. E., Ostrom, P. H., Breznak, J. A., Gandhi, H., Pitt, A. J., and F. Li, (2006). Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances. Applied and Environmental Microbiology 72; 638-644.

212 DGP Meeting September 5-9, 2012 Greenhouse gas emissions from agriculturally used fields after application of biogas waste in Schleswig-Holstein

M. Rohwer 1, K. Dittert 2, A. Pacholski 3, H. Kage 3, K. H. Mühling 1

1Institute of Plant Nutrition and Soil Science, Christian Albrechts University Kiel; 2Department of Crop Science, Plant Nutrition and Crop Physiology, Georg August University Göttingen; 3Institute of Crop Science and Plant Breeding, Christian-Albrechts-University Kiel; E-mail: [email protected]

Introduction The increased use of biogas residues as nitrogen fertilizer in combination with the application of big amounts of mineral nitrogen leads to extensive emissions of gases with high global warming potential from agriculturally used fields (Dittert and Mühling, 2009; Dittert et al. 2009). Because of its huge global warming potential the emission of nitrous oxide (N 2O) is of particular importance. Over the last few years its evolution from nitrification and denitrification processes has been investigated in detail within our research group (Senbayram et al. 2009; Köster et al., 2011). Since the German government aims to reduce greenhouse gas emissions until 2020 by about 40% compared to 1990, further strategies for mitigation have to be developed.

Materials and Methods

The emissions of N 2O have been investigated in a field experiment starting from spring 2011. Maize was cultivated under different forms and amounts of organic and mineral fertilization. Moreover the efficiency of nitrification inhibitors and stabilized mineral fertilizer has been investigated. Gas samples were collected in chambers that were temporarily installed in the field. N 2O concentrations in these samples were determined by gas chromatography and N 2O flux rates were calculated. Meteorological data (air temperature, precipitation…) was also recorded to estimate the weather influence on emission rates of N 2O.

Results and Discussion After application of biogas waste and mineral fertilizer in the spring seasons of two consecutive years the greenhouse gas emissions are compared. During both years the weather varied considerably, especially the amount of rainfall. The higher precipitation in spring 2012 led to a considerable increase in N 2O emissions after application of mineral fertilizer compared to the emission rates in spring 2011. In contrast the N 2O emissions in the organic fertilized plots were not increased by the higher soil moisture. While the application of stabilized mineral fertilizer caused lower

N2O emission rates than the conventional mineral fertilizer during the first two months after fertilization, this effect was not observed for nitrification inhibitors. Nitrification

213 DGP Meeting September 5-9, 2012 inhibitors reduced N 2O emissions more likely later in the year. These results suggest that considering the weather conditions could allow a more target oriented application of stabilized mineral fertilizer with regard to reduction of N 2O emissions.

Literature Dittert, K. and K. H. Mühling (2009) Emission klimarelevanter Spurengase in der intensiven Pflanzenproduktion. J. Verbraucherschutz und Lebensmittelsicherheit 4: 207-211. Dittert, K., M. Senbayram, B. Wienforth, H. Kage and K. H. Mühling (2009) Greenhouse Gas Emissions in Biogas Production Systems. The Proceedings of the International Plant Nutrition Colloquium XVI, 2009, Sacramento, USA. Retrieved from: http://repositories.cdlib.org/ipnc/xvi/1274. Köster J.R., L. Cárdenas, M. Senbayram, R. Bol, R. Well, M. Butler, K. H. Mühling and K. Dittert (2011): Rapid shift from denitrification to nitrification in soil after biogas residue application as indicated by nitrous oxide isotopomers. Soil Biol. Biochem. 43: 1671-1677. Senbayram, M., R. Chen, K. H. Mühling and K. Dittert (2009) Contribution of nitrification and denitrification to nitrous oxide emissions from soils after application of biogas waste and other fertilizers. Rapid Comm. Mass Spec. 23: 2489– 2498.

214 DGP Meeting September 5-9, 2012 Poster Session P8: Signalling / Quality / Phenotyping

215 DGP Meeting September 5-9, 2012 Analysis of chloroplast Ca 2+ signalling networks

Julia Frank, Bianca Baudisch, Tina Peiter-Volk, Edgar Peiter and Sacha Baginsky

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

Calcium is a crucial signalling molecule in plant cells that can influence protein phosphorylation by regulating the activity of different types of Ca 2+ -dependent protein kinases. While its role in signal transduction is well established for the nuclear and cytoplasmic compartments, little is known about the integration of plastids in the cellular Ca 2+ signalling network. This project thus aims to elucidate the plastidic Ca 2+ signalling machinery. Using a “Synapt G2” mass spectrometer, we are pursuing mass spectrometric analyses to detect selected proteotypic peptides in order to identify low-abundance proteins in isolated Arabidopsis thaliana chloroplasts. In addition, selected candidate proteins are being localized by transient transformation of Arabidopsis mesophyll protoplasts and epidermal cells with YFP fusion proteins. Once a set of plastidic Ca 2+ signalling proteins has been established, we will obtain mutant plant lines to further investigate the function of the identified proteins. In parallel, by using plant reporter lines that express plastid-targeted apoaequorin to visualize Ca 2+ transients in the chloroplast, we are in the course of identifying conditions that result in altered [Ca 2+ ]chloroplast. Under such conditions and with the selected mutants, we will set up a quantitative phosphoproteome analysis to identify key targets of Ca 2+ -dependent signalling in chloroplasts.

216 DGP Meeting September 5-9, 2012 Characterization of Arabidopsis homologs to a leguminous Ca 2+ signal modulator

X. Gong, V. Kiep, A. Janssen, E. Peiter, K. Thor

Martin-Luther-Universität Halle Wittenberg, Betty-Heimann-Strasse 3, 06120 Halle, Germany; E-mail: [email protected]

In many plant processes Ca 2+ acts as signalling component. Characteristic oscillations in cytosolic free Ca 2+ ([Ca 2+ ]cyt) thereby induce specific downstream responses. In Arabidopsis, there exists a family of three proteins with homologies to MtDMI1, which in Medicago truncatula is involved in Ca 2+ elevations during nodulation. As all members contain an RCK (Regulator of Conductance of K +) domain, we denominate the proteins RCK Domain Channel (RDC) 1 to 3. We are investigating the role of the RDCs using different approaches from molecular to whole-plant level. EYFP-fusion proteins show subcellular localization of two of the family members, AtRDC2 and 3, to chloroplasts. As they localize to the same compartment, an interaction is likely. Hence, we are currently carrying out a BiFC analysis. Promotor-GUS studies of AtRDC2 and 3 revealed expression of the genes in floral organs, leaves, roots and also in pollen grains. Therefore pollen germination assays have been carried out, which show that the pollen germination rate and the tube length of Atrdc knockout plants are decreased. To get a deeper insight into the function of the proteins and to determine, if they in fact are involved in Ca 2+ signalling or maybe constitute ion channels with other functions, several other approaches like heterologous expression in yeast, phenotypical analysis of plants grown under stress conditions, as well as aequorin measurements of [Ca2+ ]cyt with yeast and plants are under way.

217 DGP Meeting September 5-9, 2012 Effects of Late Sulfur Fertilization on Gluten Protein Composition and Baking Quality of Winter Wheat

Cheng Xue 1, Christian Zörb 2 and Karl H. Mühling 1

1Institute of Plant Nutrition and Soil Science, Kiel University, Hermann-Rodewald-Straße 2, 24118 Kiel, Germany; 2Universität Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany; E-mail: [email protected]

With the decrease of industrial emission of sulfur (S) and not enough attention paid to S fertilization management, wheat S deficiency symptoms may increase. Although at least modest amount of S is required by wheat for optimum growth and yield, baking quality of wheat flour will be reduced with deficient S fertilization. To detect the impact of late S fertilization on wheat gluten composition and baking quality, a three- year field experiment was conducted since 2009 at experimental field station (Rendsburg, Northern-Germany) with two winter wheat cultivars, Akteur (classification E) and Inspiration (classification B). Four S treatments were designed: 0 (S0), 20 (S20), 40 (S40) and 20+20 kg S/ha (late S). Results from 2009-2010 indicated that grain yield, glutathione and S concentration in ripe kernels of both cultivars were significantly increased by S and late S fertilization. Late S increased total storage protein amount and the proportion of glutenin fraction of grain flour. Baking quality related parameters such as loaf volume, sedimentation value were also improved by S and late S fertilization. Further investigation on gluten protein subunits by SDS-PAGE is still in progress. Our first results showed that adequate S together with appropriate N supply has impacts on yield, protein content and gluten protein composition of winter wheat grain. Late S fertilization may change certain proteins, such as gliadin and glutenin subunits, and benefit baking quality.

218 DGP Meeting September 5-9, 2012 Does nitrogen supply change the ratio of isothiocyanate/nitrile in Brassica pekinensis under varying sulfur supply?

C. A. C. Heyer 1, K. Böhlendorf 1, M. Reichelt 2, G. Schulte auf’m Erley 1 and K. H. Mühling 1

1 Institute of Plant Nutrition and Soil Science, Kiel University, Hermann-Rodewald-Straße 2, D-24118 Kiel, 2 Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Straße 8, D-07745 Jena; E-mail: [email protected]

Introduction The secondary Glucosinolates (GLS) are mainly represented in Brassicaceae and consist of a thioglucose unit, a sulfonated oxime unit and a variable side chain with amino acids (H OLST and WILLIAMSON , 2004). They hydrolyze by endogenous thioglucosidase into different hydrolysis products (GLS-HP), such as isothiocyanates (ITC) and nitriles. ITCs are especially known for their chemopreventive effects (BONES and ROSSITER , 1996). Which GLS-HP are synthesized depends for example on pH, epithiospecifier protein and ferrous ions. Nitrogen (N) and sulfur (S) supply are known to influence the synthesis of S containing GLS and GLS-HP (such as ITCs) or S free GLS and GLS-HP (such as nitriles) (GERENDÁS et al., 2008). This experiment was set up to identify the exact amounts of N and S fertilization leading to significant changes in concentration of GLS and GLS-HP. Furthermore, changing pattern ITC/Nitrile ratio affected by N nutrition was analyzed. It was done to settle how N and S fertilization interact with GLS and GLS-HP concentration and pattern.

Material and methods Chinese cabbage ( Brassica rapa L. ssp. pekinensis Yuki) was cultivated in quartz sand (6.6 kg pot -1) with enhanced S (0, 10, 30, 60, 100, 300 and 600 mg pot-1) and fixed N fertilization (1.52 g N pot -1) in Mitscherlich pots using four replications. In a second experiment plants were cultivated with varied N (0.5 and 1.0 g pot -1) and S (0, 300 and 600 mg pot -1) fertilization. After harvest plants were freeze-dried. S and N concentrations were analyzed by elemental analysis, sulfate and nitrate concentration by ion chromatography, GLS concentration and pattern by high performance liquid chromatography. Concentration and pattern of GLS-HP were determinated by gas chromatography.

Results and discussion Sufficient S supply led to higher concentrations of GLS and GLS-HP as compared to increasing amounts of N fertilization. In addition, N and S supply changed pattern of GLS and GLS-HP. With enhanced S fertilization the concentration of aliphatic GLS (S 219 DGP Meeting September 5-9, 2012 containing) increased and indolic GLS (N containing) decreased. The investigation of GLS-HP exhibits higher concentration of ITC under high S and low N fertilization. In an experiment with gradual increases in S supply, enhanced concentration of ITC and decreased concentration of nitriles were shown. This indicates higher ITC/Nitrile ratio under sufficient S supply. In contrast, enhanced N supply decreased the ITC/Nitrile ratio. Thus, we assume that low N and sufficient S supply accumulates bioactive compounds such as ITCs, which was confirmed by the experiment with human cancer cells.

Literature Holst, B.; Williamson, G. (2004): A critical review of the bioavailability of glucosinolates and related compounds. Natural Product Reports 21:425-447. Bones, A. M.; Rossiter, J. T. (1996): The myrosinase-glucosinolate system, its organization and biochemistry. Physiologia Plantarum 97:194-208. Gerendás, J.; Breuning, S.; Stahl, T.; Mersch-Sundermann, V.; Mühling, K. H. (2008): Isothiocyanate Concentration in Kohlrabi (Brassica oleracea L. Var. gongylodes) Plants as influenced by Sulfur and Nitrogen Supply. Journal of Agricultural and Food Chemistry 56:8334-8342. Gerendás, J.; Podestát, J.; Stahl, T.; Kübler, K.; Brückner, H.; Mersch-Sundermann V.; Mühling, K. H. (2009): Interactive effects of sulphur and nitrogen supply on the concentration of sinigrin and allyl-isothiocyanate in Indian mustard (Brassica juncea L.). Journal of Agricultural and Food Chemistry 57:3837-3844.

220 DGP Meeting September 5-9, 2012 Effect of K and Mg on berry shrivel in grapevine

1,2 1 2 2 Kristina Bachteler , Monika Riedel , Nikolaus Merkt , Jens Wünsche

1 2 State Institute of Viticulture and Enology, Freiburg i. Br., Germany, University of Hohenheim, Institute of Crop Science, Stuttgart, Germany. Email: [email protected]

Introduction Berry shrivel is becoming an increasing concern for winegrowers all over the world. Affected berries stop accumulating sugars at the onset of ripening, the must weights stay at around 10-14°Brix (Knoll et al., 2010) and they lose their turgescence. Due to the low sugar contents, these berries cannot be used in the wine-making process and must be removed before harvest. Until today, no single factor causing this physiological disorder has been determined. Research conducted in Austria has shown that an unbalanced ratio of K and Mg in the soil may cause berry shrivel (Redl, 2005, Reisenzein and Berger, 2001). To prove this theory and understand better the causes of berry shrivel, two fertilization trials were established in Southern Germany.

Material and Methods A three-year soil fertilization trial with different amounts of K- and Mg-fertilizers was performed on two sites in Southern Germany with the varieties Pinot Blanc and Zweigelt. In addition, foliar fertilization trials with application of K-, Mg- and Ca- fertilizer were conducted. Amounts of K, Mg and Ca in affected and healthy berries were analyzed with AAS at harvest.

Results and Discussion In the first two years, no significant differences in the soil fertilization treatments could be observed. In the variety Pinot Blanc in the third year, however, the plots that were given 300% of the recommended amount of K (102 kg/ha K 2O) and no Mg showed significantly less incidences of berry shrivel than the untreated control (Fig. 1). At the Pinot Blanc site in 2011, vines treated with Mg foliar fertilizer showed significantly less incidence of berry shrivel compared to the untreated control. In plots where Ca fertilizer was used, significantly higher incidences were observed compared to plots treated with K or Mg fertilizers. Analysis of K, Mg and Ca in affected berries showed a significantly increased concentration of Ca in affected berries of both varieties. If this is a cause for berry shrivel or it is caused by berry shrivel remains uncertain. Due to the annual variability of this disease, climatic conditions seem to have an

221 DGP Meeting September 5-9, 2012 influence on the occurrence of berry shrivel as well. Extreme weather conditions such as high precipitation at important phenological stages as flowering and ripening seem likely to increase the incidence of berry shrivel. However, research on berry shrivel must be continued and should focus more on physiological aspects such as xylem/phloem flow or leaf/fruit ratio (Kührer and Gabler, 2012).

a 50%

40% a a

30%

a 20%

a b a b b

Incidence of berry shrivel berry of Incidence a b 10%

0% 1) 0 K2O, 2) 49 3) 147 1) 0 K2O, 2) 34 3) 102 4) 0 K2O, 5) 0 MgO, 0 MgO kg/ha kg/ha 0 MgO kg/ha kg/ha 117 kg/ha 102 kg/ha K2O, 26 K2O, 26 K2O, 39 K2O, 117 MgO K2O kg/ha kg/ha kg/ha kg/ha MgO MgO MgO MgO

Treatments Zweigelt Pinot Blanc

Figure 1: Incidences of berry shrivel at both trial sites with different soil fertilization treatments in 2011. Means followed by the same letter do not differ significantly at p < 0.05.

Literature Knoll, M., D. Achleitner and H. Redl. (2010). Sugar accumulation in 'Zweigelt' grapes as affected by "Traubenwelke" . Vitis 49: 101-106. Kührer, E. and C. Gabler. (2012). Indikator für das Risiko eines Traubenwelkebefalls. Der Winzer 5: 20-23. Redl, H. (2005). Der Traubenwelke auf der Spur . Deutsches Weinbaujahrbuch 56: 83-90. Reisenzein, H. and N. Berger. (2001). Untersuchungen zur Zweigeltkrankheit im österreichischen Weinbau . Pflanzenschutzberichte 59: 67-78.

222 DGP Meeting September 5-9, 2012 Nitrogen and carbohydrate fractions provide potential quality markers in young plant supply chains for ornamental flowers

Siegfried Zerche 1, Dieter Lohr 2, Elke Meinken 2 and Uwe Druege 1

1Leibniz-Institute of Vegetable- and Ornamental Crops (IGZ), Großbeeren and Erfurt; 2Forschungsanstalt für Gartenbau Weihenstephan (FGW), Freising. E-mail: [email protected]

Introduction Young plant supply chains for serving ornamental flower markets comprise global production and logistics. According to experimental data, levels of nitrogen (NF) and carbohydrate (KH) fractions (as amide, nitrate, amino, protein N; glucose, fructose, sucrose, starch) determine survival, vitality and rooting capacity of cuttings. Their allocation depends on e.g. genus, light and N-supply to stock plants, and duration and temperature of dark exposure in logistics. Involving global supply chains, genotype-environment responses of NF and KH were studied 1 in relation to rooting capacity with Chrysanthemum (C) and Pelargonium (P).

Materials and Methods Cuttings were yielded of stock plants in companies in Middle East (P), East Africa (P) and Central America (C) or in Germany (C & P) at Forschungsantstalt für Gartenbau (FGW) while nitrogen was fertilized (ND) at a high level according to commercial practice or graduated at FGW (low, high, excess). Sampling of cuttings for NF & KH analyses occurred at planting day of cuttings, either at day of harvest (P, n=85 & C, n=82) at FGW in Germany or after delivery from Middle East (P, n=79), East Africa (P, n=79) and Central America (C, n=77) to Germany. Analyses of NF and KH were carried out by Kjehldahl method and enzymatic assays, respectively.

Results and Discussion Total levels and size allocation to NF&KH confirm genera differences between C&P and vary markedly with season, fertilization and logistics (Zerche et al. 2011). At FGW, low nitrogen fertilization resulted in reduced total nitrogen which corresponded to increased carbohydrate allocation especially of starch in leaf and stem for both P and C (Figures 1 A and B). Cuttings which endured dark cold stress when delivered by companies showed low leaf KHs

(especially starch) while no strong depletion was observed in stem. Total nitrogen (N t) was at similar levels in cuttings from Companies and FGW, except the low ND fertilization at FGW.

However, cuttings from companies occasionally showed lower ratios of protein-N to N t in favour of mobile organic N (amide N + amino N) (Figures 1 A and C). Nitrate N seemed unaffected by transport. Generally, negative correlations were noticed between NF and KH in leaf and stem while degrees of correlation differed with P and C, depending on cultivar, origin of cuttings and range of N t accumulation. Negative correlations among N t and the ratios of -1 -1 protein-N to N t ceased at high N t levels (P > 35 mg N g DM; C > 47 mg N g DM), indicating nitrogen limitation below these values. At the high N t levels, positive correlations were found between KH levels and ratios of protein-N to Nt for each specific company, while negative

223 DGP Meeting September 5-9, 2012 50 A Figure 1: Pelargonium zonale cuttings 45 Total Nitrogen (N t) in cuttings A) total nitrogen - Nt (sum of amide, 40 nitrate, amino and protein N); 35 B) total carbohydrates in leaf (sum of DM) -1 30 glucose, fructose, succhrose, starch); C) protein-NF (% ratio of N t) 25 when supplied with different rates of 20 Nitrogen Fertilization (ND): ND high – 15

Sum of NF (mg g (mg NF of Sum FGW; ND low – FGW; ND excess 10 – FGW; ND commercial Co. Dümmen 5 ND commercial Co. Endisch

0 55 correlations appeared between KH levels and mobile organic N. This may be an 50 B Carbohydrates in leaf indication that during certain delivery 45 dates cuttings endured particular 40 acuteness of carbohydrate depletion. This DM)

-1 35 might have triggered decrease in protein 30 N in favour of mobile organic N, while 25 constant Nt levels are retained (Figure 1 A 20 and C).

Sum of KH (mg g (mg KH of Sum 15 Positive correlations persisted among NF 10 and rooting capacity with P and C though 5 cultivar specific variation occurred. 0 Without N limitation, weak positive 90 correlations emerged among leaf KH and Protein-N ratio of N t C rooting capacity of Pelargonium while 85 Chrysanthemum showed no correlations. ) t 80 Whether observed negative correlations between stem KH levels and rooting 75 capacity reflect causal relationships or are 70 the result of intercorrelations with other 65 factors such as nitrogen requires further Protein-NFN of (%

investigations. 60 1The research project under attendance of the 55 young plant grower companies Dümmen, Dec 09+10 Feb 10 Feb 11 May 11 Jan 10+11 Mrch 10+11 Mrch 11 Endisch and Kientzler is funded by the Delivery (Month Year) German Federal Ministry of Food, Agriculture and Consumer Protection (BMELV) Literature Siegfried Zerche, Dieter Lohr, Elke Meinken und Uwe Druege (2011). Präzisierte N-Düngung bei globalisierter Jungpflanzenproduktion von Zierpflanzen. Mitt. Ges. Pflanzenbauwiss. 23: 122–123

224 DGP Meeting September 5-9, 2012 In-vivo staining of reduced iron by 2,2’ bipyridine in rice exposed to iron toxicity

Mathias Becker and Katrin Engel

Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; E-mail: [email protected]

Screening of rice for tolerance to conditions of iron toxicity is hampered by the fact that traditional methods cannot differentiate between the potentially toxic reduced FeII and the oxidized FeIII forms in plants. We propose a protocol to visualize FeII in rice tissues. The method is based on the selective formation of a purple-red colour complex between 2,2’ bipyridine and FeII. Rice genotypes with known responses to iron toxicity conditions were exposed to 0, 0.9, 1.8, 39.0 and 18 mM FeII in nutrient solution for two days. Root systems of intact plants were subsequently placed in 2,2’ bipyridine solutions of 2.5, 5.0 and 10 mM for 3, 6, 12 and 24 hours. Roots, leaf sheaths and leaf blades were assessed for the formation of the [Fe(bipy)32+] colour complex using bifocal microscopy. The best visual differentiation of resistant excluder, tolerant includer and sensitive genotypes was obtained when root systems of plants stressed for five days at 9-18 mM FeII were incubated in 5 mM bipyridine for 6 hours. The exclusion of iron in the rhizosphere resulted in a weak colour expression in the xylem vessels of the resistant genotype Pokkali. The sensitive genotype Nipponbare exhibited a dark purple staining in all xylem vessels, which was associated with the expression of intense leaf bronzing symptoms. The method could improve the selection of candidate genotypes in view of accelerating the breeding for iron toxicity tolerance in rice.

225 DGP Meeting September 5-9, 2012 Spectral determination of field emergence – a high-throughput phenotyping tool for plant breeding and field experimentation

Sebastian Kipp, Bodo Mistele, Jörg-Peter Baresel and Urs Schmidhalter

Chair of Plant Nutrition/Technische Universität München, [email protected]

Introduction Yield formation of cereals starts already in the early growth period when the seedlings´ vigor and several environmental factors permit germination rate and field emergence to a greater or lesser extent (Rajala et al. 2011). Thus it is obvious that a high germination rate and field emergence is a highly relevant trait for cereal breeders. As this trait is a substantial factor affecting the final yield it has to be seen as an early selection criteria in the entire breeding process. Considering that the amount of field trials and plots is increasing rapidly it becomes clear that plant breeders need a high throughput tool for phenotyping such traits. Scoring field emergence visually and with several persons is laborious, biased and expensive. Even experienced breeders determine field emergence mostly individually. However scoring of field emergence is still conventional because of a lack of alternative methods (Lukina et al. 1999).

Materials and Methods The 2-year field experiments were conducted at the Dürnast research station of the Technische Universität München in southern Germany (11°41´60´´ E, 48°23´60´´ N) in 2011 and 2012. The experimental design, a randomised block design with four replicates, was identical in both years. Fifty-one winter wheat ( Triticum aestivum L. ) varieties were grown conventionally with uniform nitrogen fertilization. 320 kernels per m² were sown in each plot having a size of 12 m x 1.50 m. Photos of each plot were generally taken using a Nikon D500 reflex camera under cloudy conditions to avoid shadows and to provide a constant operation mode. For all photos, the percentage of green pixels was calculated using R statistics 2.15 (Fig. 1) and averaged for each plot and variety. Spectral measurements were carried out at the beginning of the growing season using a multispectral active flash sensor, containing a xenon flash as light source, which emits light in a range between 370 and 1026 nm. Sensor readings were recorded (40 measurements per second) and directly co-recorded along with GPS coordinates from the TRIMBLE RTK-GPS (real-time kinematic global positioning system) (Trimble, Sunnyvale, CA, USA). Approximately 70 sensor readings were recorded per plot and averaged to one value.

226 DGP Meeting September 5-9, 2012 Subsequently a spectral index, the field emergence index (FEI) was calculated, using three single wavelength values in the visible (VIS) and near infrared (NIR) spectrum. To assess the ability of spectral reflectance data for evaluating field emergence, averages of each variety for FEI and photo analysis of green pixels were correlated. Coefficients of determination were calculated to evaluate the relationship between both components.

Fig. 1: Analysis of the amount of green pixels using R statistics 2.15

Results and Discussion A high correlation between FEI and digital image analysis could be shown with R² = 0.86 for 2012 and R² = 0.80 for 2011 (Fig. 2). Thus spectral reflectance measurements and the FEI can be adopted as a high-throughput phenotyping tool for scoring field emergence. Photo imaging is suitable as a precise reference method to validate the ability of spectral measurements for that trait. Using this method for scoring field emergence will be advantageous in plant breeding and field experimentation, considering the fact that it is time-saving, unbiased and less expensive than manual field scoring.

Fig. 2: Correlation between FEI and photo imaging for 2011 (red) and 2012 (blue)

Literature Rajala, A., Niskanen, M., Isolahti, M. and Peltonen-Sainio, P. (2011): Seed quality effects on seedling emergence, plant stand establishment and grain yield in two-row barley. Agricultural and Food Science , 20:228-234. Lukina, E.V., Stone, M.L. and Raun, W.R. (1999): Estimating vegetation coverage in wheat using digital images. Journal of Plant Nutrition . 22:341-350.

227 DGP Meeting September 5-9, 2012 Determination of Plant Water Status with THz and Sub-THz waves

Ralf Gente 1 , Wiebke Sannemann 2 , Agim Ballvora 2 , Nino Voß 1 , Konstantin Hobuß 1 , Arno Rehn 1 , Michael Schwerdtfeger 1 , Stefan Busch 1 , Kristian Altmann 1 , Merle Noschinski 2 , Jens Léon 2 and Martin Koch 1

1Fachbereich Physik, Philipps-Universität Marburg, 2INRES, Rheinische Friedrich-Wilhelms-Universität Bonn, E-mail: [email protected]

Introduction Research in plant breeding and plant nutrition has to face the challenge of handling shrinking resources and increasing demands for yield and nutritional qualities. In this context the non-destructive investigation of the water status of agricultural crops becomes more and more important. THz and Sub-THz measurement systems are promising candidates to accomplish this task. Such systems for the determination of the water status of plants are currently not far away from the border between basic research and practical applications. The key parts of the two CROPSENSe.net subprojects S4 and S5 are the construction of such measurement systems and the development of algorithms for data analysis in a close cooperation with the subproject GS2. The algorithms are based on a physical model of the barley plant.

Materials and Methods The underlying idea for the measurements is the simple concept that the absorption of radiation in a plant is connected to its water content. This means, the more water is inside a plant, the less electromagnetic radiation will reach the receiver after passing the plant or parts of it. Yet, closer investigation shows that getting decent measurement results is not as easy as it might seem at first sight. In the following we will describe, how we propose to solve the inevitable problems of this process. In the subproject S4 a fiber coupled THz time domain spectrometer is used to investigate the water status of single leaves with pulses of electromagnetic radiation in a broad frequency range. The dielectric properties of the leaf are obtained by comparing the spectrum, which results from the transmission of a THz-pulse through the leaf, and the reference spectrum, which is measured without any sample in the measurement system. An effective medium theory is used to build a physical model of the leaf. In this model the leaf is described as a mixture of pure plant material, water and air. An optimization algorithm is used to ensure a good fit of the theoretical model and the measured data. This way, the ratio of the involved components is determined for each measurement. A different approach is the investigation of a whole plant at once. In the subproject S5 this is realized by using continuous microwave radiation at 35 GHz and 60 GHz. The plant is placed on a rotation mount in the intersection point of the two microwave beams. Thus, measurements at both frequencies can be performed at the same time, while the plant is being rotated. Additionally, a camera records images of the geometrical shape of the plant. The recorded information is used to obtain the 228 DGP Meeting September 5-9, 2012 transmission characteristics of the plant, which are correlated to its water content.

Results and Discussion To assess the accuracy of the measurements, gravimetric measurements are performed in parallel to the THz and Sub-THz measurements. In the case of the THz measurements, leaves from a barley plant are cut off and their fresh weight is measured. Afterwards an oven is used to dry them. The leaves are taken out of the oven in regular intervals to repeat the measurements. Figure 1 shows the comparison of the gravimetric measurements and the THz measurements. Because of the gravimetric measurements this is still an invasive experiment. But when solely THz measurements are used, this is a non-destructive method and the leaves can stay connected to the plant.

100 90 EMT-model gravimetric measurement 80 70 60 50

rel. water contentrel. water (grav. %) 0 20 40 60 80 100 120 drying time (min) 10 5 0 0 20 40 60 80 100 120 drying time (min) deviation deviation (%) Figure 1: Comparison of gravimetric and THz measurements. The graph shows the average relative water content of 10 leaves.

In the case of the Sub-THz measurements whole barley plants are exposed to drought stress. Also here, gravimetric measurements of water content are performed for comparison. Thus, we have to rely on a homogenous behavior of the plants. But when solely Sub-THz measurements are used, also this is a non-destructive method. 30 Control 25 Stress Null Control (grav.) 20 Stress (grav.) Null (grav.) 15

10 absolute water content absolute water (g)

5 0 2 4 6 8 10 12 14 16 time (days) Figure 2: Comparison of gravimetric and Sub-THz measurements. The graph shows the absolute water content of three groups of plants: A control group, which was watered appropriately, a draught stress group and a completely unwatered group (“Null”).

Literature Evaluation of leaf water status by means of permittivity at terahertz frequencies, C. Jördens, M. Scheller, B. Breitenstein, D. Selmar and M. Koch, Journal of Biological Physics, (2009), 35(3), pp. 255-264

229 DGP Meeting September 5-9, 2012 Autorenverzeichnis

Abd El Lateef ...... 103 Böhlendorf ...... 219 Abreu ...... 39, 167 Bolaños ...... 39, 167 Adam ...... 129 Bollig ...... 56, 146 Adolphs ...... 126 Bonierbale...... 104 Ahmed ...... 176 Bonilla ...... 39, 167 Altmann ...... 228 Borchard ...... 126 Amâncio ...... 188 Braun ...... 74, 197 Amany ...... 103 Bremer ...... 151, 184 Amelung ...... 83 Brodersen ...... 18 Amoros ...... 104 Brown ...... 3 Ander ...... 140 Brunn ...... 208 Apelt ...... 120 Burkhardt ...... 13, 22, 179, 205, 206, 209 Arias ...... 123 Busch ...... 228 Arnold ...... 121, 123 Buschmann ...... 83 Ashraf ...... 154 Bustamante ...... 179, 205, 206 Avsaroglu ...... 173, 194 Cabeza ...... 95, 158, 162, 186 Bach ...... 146 Carvalho ...... 188 Bachteler ...... 221 Černý ...... 101, 111 Baginsky...... 216 Changwony ...... 28 Bakry ...... 103 Chaumont ...... 24 Balazadeh ...... 132 Chebil ...... 176 Balík ...... 101, 111 Chen ...... 199 Ballvora ...... 228 Choat ...... 18, 20 Baresel ...... 226 Clemens ...... 118, 207, 210 Bareth ...... 83 Cremer ...... 83 Basi ...... 13 Cuhls ...... 210 Baudisch ...... 216 Czauderna ...... 9 Baumgarten ...... 158, 162, 186 Dahlin ...... 202 Becker ...... 28, 76, 142, 204, 225 Daum ...... 9 Bengough ...... 84 De Bona ...... 93, 190 Benito ...... 108 Delhaize ...... 144 Beschow...... 178, 180 Demiral ...... 173, 194, 195 Beßler ...... 127, 129 Deras ...... 206 Beuters ...... 156 Dietrich ...... 84 Bienert ...... 24 Dittert ...... 158, 162, 211, 213 Binner ...... 115 Dold ...... 28

230 DGP Meeting September 5-9, 2012 Domínguez ...... 108 Giesemann ...... 211 Downie ...... 51 Giller ...... 27 Dreiseidler ...... 205 Goldbach ...... 22, 83, 113, 117, ...... 118, 123, 124, 181 Druege ...... 223 Gong ...... 217 Dursun ...... 137 Graner ...... 83 Duschyk ...... 72, 197 Gregorio ...... 142 Eckhardt ...... 104 Hacker ...... 181 Egle ...... 180 Hajirezaei ...... 64 Eichert ...... 8, 22 Hakki ...... 99, 173, 194, 195 Eitenmüller ...... 139 Hamurcu ...... 173, 194, 195 Elewa ...... 103 Hanstein ...... 174 Engel ...... 76, 225 Happeck ...... 171 Engels ...... 104, 127, 129, 192 Harmankaya ...... 99, 107 Erban ...... 60 Hartmann ...... 201 Erdle ...... 87 Hegenberg ...... 126 Eschemann ...... 126 Herbst ...... 120 Falk ...... 208 Herrmann ...... 211 Faust ...... 174 Heyer ...... 219 Fernández ...... 7 Hinrichs ...... 151 Fiorani ...... 91 Hobuß ...... 228 Fleck ...... 68, 164 Hogekamp ...... 56 Förstner ...... 83 Höller ...... 64 Frank ...... 216 Hong ...... 123, 207 Franke ...... 27 Horst ...... 29, 56, 60, 72, 132, Frei ...... 64, 76, 142 ...... 134, 144, 146, 197 Freitag L...... 171, 178 Hund ...... 86 Freitag N...... 141 Hunsche ...... 13, 22, 209 Führs ...... 72 Hütsch ...... 37 Gabriel ...... 106 Jansen ...... 18 Gans ...... 120 Janssen ...... 170, 217 Gárate ...... 108 Jones ...... 84 Geilfus ...... 98, 172, 175 Jung C...... 83 Gente ...... 228 Jung S...... 140 George ...... 106 Kabish ...... 103 Georgii ...... 208 Kage ...... 213 Gezgin ...... 99, 137, 173, 195 Kaiser ...... 209 Gezgýn ...... 107 Kasal ...... 181 Gibon ...... 81 Kiep ...... 217 231 DGP Meeting September 5-9, 2012 Kipp ...... 226 Maurer ...... 144 Kirchgessner ...... 86 McElrone ...... 18 Kirchner ...... 60 Meier ...... 199 Kleist ...... 181 Meinken ...... 223 Klug ...... 60 Merbach ...... 180 Koch H.J...... 149 Merkt ...... 221 Koch M...... 228 Meyer ...... 134 Köhler ...... 171 Mistele ...... 87, 226 Kopka ...... 60 Mohammed ...... 192 Köslin-Findeklee ...... 132, 134 Monteiro ...... 93, 188, 190 Köster B...... 158, 162, 186 Morgan ...... 175 Köster J.R...... 211 Morieri ...... 51 Kremer ...... 158 Möseler ...... 126 Kreye ...... 204 Mühling ...... 98, 136, 144, 172, 175, ...... 211, 213, 218, 219 Krippner ...... 208 Müller-Röber ...... 132 Kuhlmann ...... 83 Mustroph ...... 199 Kulhánek ...... 101, 111 Myint ...... 56 Küster ...... 56 Nadeau ...... 202 Lawson ...... 9 Neuhaus ...... 98 Le ...... 123 Neuhoff ...... 179 Lee ...... 18 Ngwene ...... 106 Lemme ...... 149 Noga ...... 83 Léon ...... 83, 228 Noschinski ...... 228 Liebisch ...... 86, 141 Nowak ...... 178 Liese ...... 158, 162, 186 Öborn ...... 202 Lindberg ...... 175 Oerke ...... 83 Linse ...... 202 Oldgee ...... 142 Löhnertz ...... 97 Oldroyd ...... 51 Lohr ...... 223 Osthushenrich ...... 37 Lucena ...... 108 Ozdemir ...... 173, 194, 195 Lynch ...... 47 Pacholski ...... 213 Maaß ...... 183 Pariyar ...... 13, 22, 209 Mähl ...... 210 Peiter ...... 54, 170, 171, 178, 180, Manaa ...... 176 ...... 183, 199, 216, 217 Martín ...... 108 Peiter-Volk ...... 183, 199, 216 Matthews ...... 18 Peter ...... 54, 199 Mattusch...... 68 Pfliehinger ...... 97 Matute ...... 206 Philips ...... 18 232 DGP Meeting September 5-9, 2012 Phong ...... 210 Senbayram ...... 211 Pilz ...... 179 Shackel ...... 18 Plümer ...... 83 Shejbalová ...... 101, 111 Pohlmeier ...... 83 Siles ...... 179, 205, 206 Post ...... 83 Smit ...... 97 Postma ...... 47 Spoth ...... 83 Pourmoyyed ...... 160 Stahl ...... 208 Pullinen ...... 181 Stamm ...... 118 Rascher ...... 83 Staver ...... 179, 205, 206 Reguera ...... 39 Steffens ...... 153, 154 Rehn ...... 228 Steingrobe ...... 95 Reichelt ...... 219 Strochalska ...... 153 Reichert ...... 104 Subedi ...... 204 Riedel ...... 221 Sulieman ...... 186 Riederer ...... 6 Tamkoc ...... 173, 194, 195 Rissel ...... 54 Tawfik ...... 103 Rohwer ...... 213 Techow ...... 211 Rumpf ...... 83 Thor ...... 54, 170, 183, 217 Ryan ...... 144 Töpfer ...... 83 Salas ...... 104 Trimborn ...... 124 Sannemann ...... 228 Ulas ...... 29 Saqib ...... 37 Uschkerat ...... 83 Schenk ...... 68, 115, 151, 164, 184 Valdivia ...... 205 Scherer ...... 33, 113, 117, 156, 160 Van Do ...... 117 Schmidhalter ...... 83, 87, 226 van Dusshoten ...... 83 Schmidt ...... 83, 121, 188, 190 Vanlauwe ...... 27 Schneider ...... 28, 86 Vašák ...... 101, 111 Schreiber ...... 164 Vetterlein ...... 156 Schubert ...... 37, 140, 153, 154, 174, 208 von Arx ...... 141 Schulte auf’m Erley ...... 29, 136, 219 von Wirén ...... 46, 64 Schultz ...... 160 Voß ...... 228 Schulze J...... 158, 162,186 Walter ...... 86, 141 Schulze S...... 164 Wang ...... 136 Schulze-Lammers ...... 83 Waßmann ...... 164 Schumann ...... 83 Wedde ...... 33 Schurr ...... 83, 91 Weiermüller ...... 83 Schwerdtfeger ...... 228 Weiß ...... 197 Seehuber...... 113 Well...... 211

233 DGP Meeting September 5-9, 2012 Welp ...... 33 Yilmaz ...... 99, 107, 137 Wendler ...... 144 York ...... 47 Wening ...... 153 Yu ...... 166 Wester ...... 124 Yunta ...... 108 Weyer ...... 83 Zahn ...... 56 White ...... 4, 84 Zeng ...... 158 Wiesler ...... 29 Zerche ...... 223 Wimmer ...... 43 Zhang ...... 47 Windt ...... 85 Zingore ...... 27 Wu...... 76, 142 Zörb ...... 98, 172, 218 Wünsche ...... 221 Zwieniecki ...... 15 Xue ...... 218

234 DGP Meeting September 5-9, 2012