Abstractbook ERO 13

2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Table of content

Welcome ...... II Organizing committee ...... III Local organizers ...... IV Sponsors ...... V General information about COST Action FA 1306 ...... VI General information about the meeting and maps ...... VII Program ...... XI Abstracts for oral presentations ...... 1 Abstracts for posters Theme 1 – WG 1: Phenotyping at plant and field level ...... 35 Theme 2 – WG 2: Phenotyping at cell level ...... 69 Theme 3 – WG 3: Integration of phenotyping on both levels and translation translation into good practices for applied use ...... 95 Theme 4 – Data processing and integration ...... 107 Theme 5 – Stakeholder interactions ...... 113 List of participants...... 115

I 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Welcome

Dear participants of the 2nd General Meeting of the COST Action FA 1306 – PhenomenAll – The quest for tolerant varieties – Phenotyping at the plant and cellular level

On behalf of the organizing committee of the COST Action and the local organizers we warmly welcome to Copenhagen. In Denmark we have a long tradition of collaboration between universities and this meeting is a joint arrangement between Department of Plant and Environmental Sciences (PLEN) at University of Copenhagen (UCPH) and Department of Food Science (FOOD) at Aarhus University (AU). We are looking forward to three days of new results, exchanging ideas and further developing our COST Action PhenomenAll by interesting discussions.

We have experienced an overwhelming interest in the meeting. The program contains six keynote speakers, 24 oral presentations and 70 posters. All together we have 136 registered participants. We are particularly happy to welcome 15 stakeholder representatives represent- ting both breeders and hardware manufacturers. Based on requests after last years meeting we have not only one session per workgroup but we have also invited one keynote speaker on the topic of Data processing and integration and two keynote speakers representing Stakeholder interactions. Furthermore we have set aside time for group discussions about how to streng- then and further develop our COST Action and general networking.

On Tuesday afternoon we will make an excursion to Campus Tåstrup, where Section of Crop Science of PLEN/UCPH resides and our phenotyping facilities are located. In the evening we will have the conference dinner in Tåstrup. We are particularly grateful to the six companies who are sponsoring this meeting. We would also like to thank three microbreweries on the island of Fyn (Funen), where the phenotyping facilities of FOOD/AU are located, for sponsoring a phenotyping session of beers. There would not have been a dinner without all of you.

We would also like to thank the COST Organization for financially supporting the majority of participants and taking care of the administration.

We will have three days with a tight and interesting schedule, looking forward to many interesting discussions!

On behalf on the Organizing Committee and the Local Organizers

Eva Rosenqvist Carl-Otto Ottosen PLEN/UCPH FOOD/AU

II 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Organizing committee

Eva Rosenqvist, University of Copenhagen, Denmark Carl-Otto Ottosen, Aarhus Universit, Denmark Sebastien Carpentier, KU Leuven, Belgium (Chair of COST Action FA 1306) Ulrich Schurr, Forschungszentrum Jülich, Germany (Co-chair of COST Action FA 1306) Andreas Voloudakis, Agricultural University of Athens, Greece Carla Pinheiro, ITQB, Portugal Diego Rubiales, Institute for Sustaniable Agriculture, CSIC, Spain Dyonisia Fasoula, Agricultural Research Institute, Cyprus Estelle Goulas, UMR 8567 CNRS/Université Lille 1, France Rick van de Zedde, Wageningen UR, The Netherlands

III 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Local organizing committee

University of Copenhagen Aarhus University Eva Rosenqvist Carl-Otto Ottosen Dominik Groβkinsky Benita Hyldgaard Dorthe Horn Larsen Jesper Svensgaard Jesper Cairo Westergaard

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Sponsors

V 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

General information about COST Action FA 1306 The quest for tolerant varieties – Phenotyping ant plant and cellular level – PhenomenAll

Mission Phenotyping is an emerging science that characterizes plant behaviour and quantifies features such as growth and stress tolerance in a precise and reproducible manner that allows linking to genetic control. As a highly interdisciplinary research area, phenotyping covers a wide range of methodological approaches from imaging based whole plant phenotyping (character- rization of morphological and physiological plant features), over molecular phenotyping approaches on a cellular level (transcriptomics, proteomics, metabolomics) up to computational methods for image analysis, plant growth modeling and statistical data analysis and data integration. The FA1306 work plan is divided into three work groups (WG).

WG 1: Phenotyping at plant level This WG will collect information and novel devselopments in: • Sensors and image analysis • Plant physiology/pathology • Modelling plant growth and real time monitoring • Data management & statistics • Screening the biodiversity of European gene bank collections • Screening the parents and the offspring of breeding programs and different genetically modified plants

WG 2: Phenotyping at cell level This WG will collect information and novel developments in: • Metabolomics and flux analysis • Proteomics • Transcriptomics • Genomics

WG3: Integration of phenotyping on both levels and translation into good practices for applied end use This WG will follow up the developments made in systems biology by integrating phenotyping on different levels and will collect the developments in translational research. • Phenotyping in practice (greenhouse/field) • Integration of different ‘omics’ technologies

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General information about the meeting and maps

The meeting will be held in auditorium A2-81.1 at Campus Frederiksberg, University of Co- penhagen, Thorvaldsens vej 40, 1871 Frederiksberg C (see map on page VIII)).

The excursion and conference dinner will be held at Campus Tåstrup, Højbakkegård Allé 17, 2630 Tåstrup (see map on page IX and X). The buses departs 12:50 (sharp!) from Grønne- gårdsvej (see map on page VIII)

Those who participate in the excursion but not in the dinner can take the free shuttle bus from Campus Tåstrup 16:35, arriving at the bus stop at Grønnegårdsvej 17:10.

Those who does not want to participate in the excursion but in the dinner can take a IC train from Copenhagen Central Station and bus 116 to bus stop Agrovej. It is 8 minutes walk from Campus Tåstrup. Consult Eva Rosenqvist about it.

Internet connection via WiFi Eduroam is available on campus. Employees from other scientific institutions that participate in Eduroam can use their home login name (username@homedomain) and password.

For participants who do not have Eduroam a Guest Login will be provided (see separate paper in the conference bag).

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Map Campus Frederiksberg

VIII 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Campus Frederiksberg and Campus Tåstrup

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Map Campus Tåstrup

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Program Monday April 18, 2016

08.00 Registration, mounting of posters, buying lunch tickets Welcome: Svend Christensen (UCPH), Sebastian Carpentier 09.00 (COST), Eva Rosenqvist (convener) Achim Walter, ETH 09.10 WG 1: Phenotyping at plant/field level - new methods Zürich, CH 10.00 Coffee, poster viewing, buying lunch tickets 10.30 Stefan Gerth, DE Maize root growth tracking using X-ray CT segmentation Gerd Patrick Bienert, Screening for and development of boron-efficient rapeseed 10.50 DE and Arabidopsis genotypes Pirjo Peltonen-Sainio, Phenotyping nutrient use efficacy and resistance to seed borne 11.10 FI diseases for conventional and organic breeding Theoharis Ouzounis, Phenotyping tomato genotypes under four LED light en- 11.30 NL vironments for energy efficient use of light Danijela Vukadinovic, Fully automated detection of western flower thrips Frankli- 11.50 NL niella occidentalis damage in chrysanthemum leaves 12.10 Lunch, poster viewing Björn Usadel, Data processing: Ontologies to link genomics and pheno- 13.10 RWTH, DE typing Christiaan Biemond, How to manage and explore large and diverse datasets in Di- 14.00 NL gital Phenotyping? Non-invasive phenotyping of postharvest quality traits in to- 14.20 Gerrit Polder, NL mato and strawberry Angelika Czedik- Non-invasive phenotyping of plant fungal infections during 14.40 Eysenberg, AT the asymptomatic stage 15.00 Coffee, poster viewing 15.40 Workshop – four groups (appointed conveners)

1. Development of collaboration in the COST Action

2. + 3. Standards and procedures WG1 + WG2

4. Stakeholders – future

17.00 Main conclusions presentation by group conveners 17.40 MC meeting Free evening for sightseeing

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Program Tuesday April 19, 2016

08.00 Registration, poster viewing WG 2: Phenotyping at the cell level and the need for a ho- Thomas Roitsch, 08.30 listic approach to implement deep physiological phenotyp- UCPH, DK ing Transcriptomic and metabolic responses of bread wheat 09.20 Guray Akdogan, TR against heat and drought stresses How do roots respond to osmotic stress? A transcriptomic ap- 09.40 Jassmine Zorrilla, BE proach to address this question in a non-model crop 10.00 Coffee, poster viewing Repeated drought stress reduces anti-oxidative capacity and 10.30 Monika Wimmer, DE alters primary metabolism of young sugar beets Natural variation in Arabidopsis Cvi-0 accession uncovers 10.50 Hannes Kollist, EE regulation of guard cell CO2 signaling by MPK12 Early detection and classification of Mla and mlo gene based 11.10 Matheus Kuska, DE resistance of barley using hyperspectral close-range imaging Christophe Djemiel, Phenotyping microbial diversity during flax dew-retting by 11.30 FR using targeted-metagenomics Reinhard Turetschek, Microbes Shape Defense Response of Pisum Sativum against 11.50 AT Didymella pinodes 12.10 Lunch, poster viewing 12.50 Departure to phenotyping facilities at Campus Tåstrup by bus Program specified 13.30 Group tours, presentations and company demonstrations. later • Multispectral + IR imaging and gravimetric phenotyping in greenhouse Group photo • Deep root phenotyping in the field – Radimax

Coffee • Demonstration of equipment for non invasive phenotyping by companies Snacks and drinks

18.00 Conference dinner at Campus Tåstrup 21.30 Departure from Campus Tåstrup by bus

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Program Wednesday April 20, 2016

08.00 Registration, poster viewing Carl-Otto Ottosen, WG 3: Phenotyping in greenhouses and the challenges of 08.30 AU, DK dynamic climate Integrated metabolome and whole plant phenotyping reveal 09.20 Thomas Altmann, DE genetic and physiological factors of vegetative growth and seed longevity Can specialized metabolites promote growth? Challenging Elizabeth Neilson, 09.40 traditional plant defense theories using dynamic imaging and DK metabolite analysis 10.00 Coffee, poster viewing Impact of treated wastewater on growth, respiration and hy- 10.30 Indira Paudel, IL draulic conductivity of citrus root systems in light and heavy soils Marta Vasconcelos, Can phenotyping at multiple levels shed light on the iron effi- 10.50 PT ciency trait in soybean plants? Benita Hyldgaard, 11.10 ModCarboStress – focus on photosynthesis DK Marta Malinowska, Phenomics analysis of drought responses in Miscanthus col- 11.30 UK lected from different geographical locations. The European landscape of plant phenotyping - novel deve- 11.50 Uli Schurr, DE lopments towards EMPHASIS - the ESFRI infrastructure for plant phenotyping 12.10 Lunch, removing posters Theo van Hintum, Stakeholder interaction: Phenotyping focus on stakehol- 13.00 WUR, NL ders Christian S. Jensen, 13.40 Stakeholder interaction: Phenotyping focus for breeders DLF, DK Can industry cooperation lead to more applicable phenotyping 14.20 Stefan Schwartz, NL systems? – A collaborative approach for plant phenotyping standards in industry Roland Pieruschka, International Plant Phenotyping Network: an association ai- 14.40 DE ming at integrating the plant phenotyping community 15.00 Final discussion, evaluation 15.40 Closure of conference 16.00 Goodbye

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Abstracts for oral presentations

In the order of the program

Theme 1: Phenotyping at plant and field level Theme 2: Phenotyping at cell level Theme 3: Integration of phenotyping on both levels and translation into good practices for applied end use Theme 4: Data processing and integration Theme 5: Stakeholder interactions

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Keynote speaker theme 1 Monday April 18, 2016, 9:10-10:00

Plant phenotyping in lab and field

Achim Walter Institute of Agricultural Sciences, ETH Zürich, Switzerland

For a lot of good reasons, plant phenotyping has recently become a hot topic. Basic science, crop breeding, the need to produce more food, feed and fuel in times of climate change and finally the possibilities of digitization with their impact on precision agriculture: All of these motivations have led to an increased effort to turn image-analysis-based methods often coming from the field of remote sensing into applicable tools in plant and crop research. In this talk, I will give a motivation and an overview of some of the methods and phenotyping platforms that are currently developed, refined and applied. In climate chambers, greenhouses and on field sites, imaging is performed at different spatial and temporal scales in the visual, near- infrared and thermal spectral range to quantify shoot and root architecture, dynamic and short- term growth processes as well as photosynthesis, gas exchange and compound composition of major and alternative crops alike. In plant biology and crop breeding, the intention behind phenotyping approaches is to quantify differences between genotypes based on certain plant traits. In ecophysiology and agronomy, the intention behind phenotyping is to adjust crop production systems ideally to regionally differing ecological niches or to an altering climate – often in the context of precision agriculture or smart farming. Thereby, phenotyping shall help elucidating basic rules of plant-environment-management interactions and facilitating knowledge transfer between lab and field, allowing for improved agricultural plants and practices in the future.

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Oral presentation theme 1 Monday April 18, 2016, 10:30-10:50

Maize root growth tracking using X-ray CT segmentation

Stefan Gerth, Joelle Claußen, Norbert Wörlein1 and Norman Uhlmann Fraunhofer Institute for Integrated Circuits, Development Centre X-ray Technology, Germany

Introduction During the last years, X-ray computed tomography (CT) has been applied to investigate be- lowground growth of plant organs such as potato tubers or root systems. Due to the non- destructive nature of this technique it is possible to observe time dependent the root growth in natural soil. With X-ray CT the 3D volume information of objects can be reconstructed using X-ray projections of the object from different points of view.

To analyze the root structure, a robust and automated data segmentation algorithm is needed. Using CT systems as a tool for plant phenotyping, the balance between measurement time and segmentation quality needs to be optimized. We present an algorithm for 3D CT data segmentation dealing to push CT plant phenotyping towards higher throughput, but still maintaining the segmentation of root systems in an automated way.

Materials and Method Over a period of two month B73 Maize was grown in a tube with 9 cm in diameter and 40 cm in height and scanned regularly. The resulting data are segmented using our algorithm witch is an adapted Frangi Vessel filter combined with local homogeneity analysis.

The scanning geometry used is the axial 3D-CT, where a conical X-ray beam projects the pot on a flat 2D image detector. The spatial sampling frequency of this setup is about 85 µm in the reconstructed volume allowing the extraction of roots down to a diameter of 0.2 mm.

Results and Discussion We will present the development of the root system of B73 Maize over two month analyzing the total segmented root length and the surface diameter distribution. Additionally we will show several different examples segmented and analyzed using this algorithm.

A detailed discussion about the influence of measurement time, soil and watering for the segmentation algorithm will be presented as well as the difference between automated and manual usage.

These are important aspects for the automated root biomass analysis in large scale plant phenotyping projects.

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Oral presentation theme 1 Monday April 18, 2016, 10:50-11:10

Screening for and development of boron-efficient rapeseed and Arabidopsis genotypes

Benjamin Pommerrenig1, Annett Bieber1, Jacqueline Fuge1, Isidro Abreu2, Till A. Diehn1, Astrid Junker1, Thomas Altmann1 and Gerd P. Bienert1 1 Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, 06466 Gatersleben, Germany 2 Universidad Autónoma de Madrid, c/ Darwin 2. 28049, Madrid, Spain

Boron (B) is an essential micronutrient for plants. Our group addresses the question of how B deficiency-sensitive Brassica crop plants process and regulate their B status during vegetative and reproductive growth. To work in defined B-deficient conditions with plants in early developmental stages is very difficult as B occurs as a trace contaminant essentially everywhere. Therefore the identification of B deficiency tolerant Brassica napus genotypes demanded the development of high-throughput compatible “B-free” growth conditions. In a pre-screen we identified a soil-substrate, which has B levels below the detection limit of 0.1 mg B (kg soil)- 1. Subsequently, we established two independent screening conditions which allow i) a phenotypic description of B-efficiency shoot traits in soil-substrate greenhouse pot conditions and ii) the quantitative assessment of B efficiency via the evaluation of parameters describing the rapid root growth inhibition under B deficiency in in vitro growth assays using a B chelating resin. We screened 357 winter-type and 242 summer-type rapeseed cultivars from the Gene- bank North of the IPK Gatersleben for B deficiency tolerance. These cultivars represent a set from all over the world including areas with B-sufficient and B-deficient soils. Both screens resulted in the identification of only three highly B deficiency tolerant summer-type cultivars. These three highly B deficiency tolerant and three representative B deficiency sensitive cultivars were selected for further comparative analyses. Using the high throughput phenotyping facilities at IPK growth and development of selected cultivars were compared under B deficient and sufficient conditions. Elemental analysis revealed significant differences in B con- tents of B deficiency tolerant and sensitive plants when grown under B-deficient conditions but not under standard conditions. These results indicate that the B deficiency tolerant cultivars can grow with a very limited amount of B. The genetic, morphological, physiological and molecular differences between B-efficient and -inefficient cultivars are currently analysed. An analogous high throughput phenotyping experiment is currently performed with a set of 192 Arabidopsis accessions in order to i) identify B deficiency tolerant Arabidopsis accessions and ii) to compare growth and development as well as element compositions between Arabidopsis and its closely related sister crop genus B. napus under B sufficient and B deficient conditions.

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Oral presentation theme 1 Monday April 18, 2016, 11:10-11:30

Phenotyping nutrient use efficacy and resistance to seed borne diseases for conventional and organic breeding

Marja Jalli, Ari Rajala, Satu Latvala and Pirjo Peltonen Sainio Natural Resources Institute Finland (Luke), Management and Production of Renewable Resources, FI-31600 Jokioinen, FINLAND

Improved resistance of barley to noxious diseases as well as improved nutrient use efficiency are of focal importance in both conventional and organic breeding programs. In the light of our findings shown below, both of these important traits can be incorporated by breeding into the novel cultivars.

A two-year field experiment was conducted to phenotype N and P use efficiency (NUE and PUE) traits of 195 spring barley genotypes (72 landraces and 123 cultivars from 1916‒2010). Low and high N and P regimes (35 and 70 kg N/ha, and 4.6 and 9.2 kg P/ha) with unfertilized control plots were used in the experiments. Results revealed wide variation in NUE and PUE among landraces and a positive breeding effect in cultivars and furthermore, NUE and PUE traits seemed to be coupled.

Resistance of 152 Nordic cultivars and landraces was phenotyped in field against Fusarium head blight (Fusarium culmorum), but without finding any resistance. The resistance of 985 barley cultivars and landraces with different geographical origin for net type (Pyrenophora teres f. teres) and spot type (Pyrenophora teres f. maculata) net blotch resistance was again phenotyped in a greenhouse. A significant improvement in the net blotch resistance was found in the European barley cultivars released within the last 40 years and the frequency of resistant genotypes was highest among European cultivars and Syrian landraces. For the spot type net blotch, the average resistance level was clearly weaker: the most promising material was found in the landraces originating from Jordania, while only one Swedish landrace showed resistance that might be useful in breeding programs. Leaf stripe (Pyrenophora gra- minea) and also loose smut (Ustilago nuda) resistance was phenotyped in a greenhouse with 125 genotypes and significant differences in resistance were found. Two of the leaf stripe resistant landraces were shown to carry Vada resistance. Importantly, four resistant cultivars and four resistant landraces were shown to have leaf stripe resistance, genetic background of which differed from Vada resistance. This is useful when searching for new resistance sources.

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Oral presentation theme 1 Monday April 18, 2016, 11:30-11:50

Phenotyping tomato genotypes under four LED light environments for energy efficient use of light

Theoharis Ouzounis1, Ep Heuvelink1, Henk J. Schouten1, Richard G.F. Visser2, and Leo F.M. Marcelis2 1Horticulture and Product Physiology group, Wageningen University, PO Box 16, 6700 AA Wageningen, The Netherlands 2 Wageningen UR Plant Breeding, Wageningen University & Research Center, PO Box 16, 6700 AA Wa- geningen, The Netherlands

The predictive quality of phenotyping is currently limited due to interactions between genotype and the environment. The already present knowledge on physiology and morphology in greenhouse tomato, as well as the recent elucidation of its genome sequence, make tomato one of the major model crops for physiological and genetic studies. Tomato cultivars vary in their responses to light. A core collection of 40 tomato genotypes based on their diversity, phylogeny, availability of re-sequencing information of their genomes, and agronomic traits was chosen. These 40 genotypes were screened for physiological, morphological and developmental parameters, such as plant height, plant fresh and dry weight, dry matter partitioning among plant organs, leaf area, leaf number, hypocotyl and epicotyl length, root length, stomatal conductance, chlorophyll, flavonol and anthocyanin content, as well as photosynthetic parameters (chlorophyll fluorescence, i.e. Fv/Fm, ΦPSII, ΦNPQ, NPQ, ETR).

This study examines the effect of a wider range of spectral distributions that include mono- chromatic, bichromatic, and full spectrum light treatments. Four different light environments all with the same light intensity (150±30 µmol m-2 s-1) were imposed in a climate room: 1) Control that consisted of UV, white, and far-red, 2) 88% red and 12% blue, 3) 100% red, and 4) 100% blue. The different LED light environments strongly affected the measured physiological, morphological and developmental parameters. We clearly show that genotypes can respond very differently to light environments. For example, the 88% red and 12% blue treatment demonstrated the highest total biomass on average, while the 100% blue showed the highest stomatal conductance and flavonol content. The 100% red induced morphological abnormalities in terms of downward and upward leaf curling, while the 88% red and 12% blue alleviated this effect, but this response is genotype dependent. Plant height was highest in the control treatment, hypocotyl and epicotyl length was favoured under the 100% blue, while leaf area was highest for the control and the 88% red and 12% blue light regimes. Principal component analysis helped to group genotypes with different types of responses. Conse- quences of this strong interaction between spectrum and plant response for phenotyping will be discussed.

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Oral presentation theme 1 Monday April 18, 2016, 11:50-12:10

Fully automated detection of western flower thrips Frankliniella occidentalis damage in chrysanthemum leaves

Danijela Vukadinović1, Gerrit Polder1, and Gerben Messelink1 1 Greenhouse Technology group, Wageningen University and Research Centre, The Netherlands

Western flower thrips Frankliniella occidentalis is the major pest in ornamental plants. This pest is the biggest pest problem in the ornamental greenhouse industry and causes serious crop damage by feeding from the plant and by transmitting plant-destroying viruses. Further- more, it is a big threat for the export of flowers to Russia (market value 80 million euro/year). There is an urgent need to develop alternative control measures as most available pesticides are not effective and have negative environmental impact and side effects on biological control agents. One of the new innovative control measures against this pest is the use of endophytes: fungi that grow into plants, without causing a plant disease and potentially making plants less at- tractive for pests. In order to investigate how successful this control method is, many isolates need to be screened on their effects on thrips. This results in hundreds of plants that need to be assessed on plant damage. The classical method is to assess this directly under a binocular microscope and score damage manually, which is very labour intensive. The development of an algorithm to automatically assess leaf damage with image analyses would be a major step forward, speed up the whole screening process and potentially automate the complete pest control process. An automated image analysis tool for these purposes would significantly increase the probability of successful pest control. Since the color of the thrips damage is almost equal to the color of the leaf veins, automating thrips damage quantification is a difficult task. We have developed an imaging system that consists of an 8-band multispectral camera. The spectral bands were selected based on the result of the preliminary experiments with the hyperspectral camera covering 192 bands of 400-1000nm. For the development of the automated thrips damage detection method, all leaves from 14 plants that were treated with 20 treatments were imaged during three days. This resulted in multispectral images of all leaves of 87 plants. Expert entomologist marked thrips damage on images of several leaves and this data was used to train our automated method. The fully automated method consists of two steps: separate leaves segmentation and detection and quantification of thrips damage on leaves. Separate leaves segmentation was performed using thresholding on color transformed images and connected component analysis. The success rate of separate leaves segmentation was 100%. In each segmented leaf areas of thrips damage were automatically segmented using machine learning method decision trees. The features of the pixel classification based decision trees algorithm were intensities of all 8 multispectral images. The accuracy of the automated thrips damage detection when compared to the manually labelled area was 95%.

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Keynote speaker theme 4 Monday April 18, 2016, 13:10-14:00

Data processing: Ontologies to link genomics and phenotyping

Bjørn Usadel RWTH Aachen and Forschungszentrum Jülich, Germany [email protected]

Phenotyping is becoming a mature technology and is producing significant amounts of data. However if there is anything “omics” sciences have taught us, it is that making data compara- ble not only paves the way for machine learning approaches but that it will open up new possibilities for data integration and analysis helping individual biologists and breeders.

This is because once curated, data sets can be used to unravel novel associations, and the data allow finding new biological principles, candidate genes or loci. Unlike in the disintegrate- omics disciplines there are however no clear and identical variable identifiers like gene names in typical phenotyping experiments. Therefore it is necessary to consistently name these traits using e.g. precise ontologies and to share the underlying data, including detailed and precise experimental descriptions. It is then and only then that the data can be linked with the disintegrate-omics and genetical genomic resources.

Some examples of “lost opportunities” will be presented alongside results on how unexpected associations can be mined to unravel novel biological hypotheses and/or to find candidate genes by combining with disintegrate-omics data.

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Oral presentation theme 4 Monday April 18, 2016, 14:00-14:20

How to manage and explore large and diverse datasets in Digital Phenotyp- ing?

Christiaan Biemond, Gert-Jan Speckmann, Koen Huvenaars, Timo Doeswijk, and Marco van Schriek.

KeyGene, Agro Business Park 90, 6708 PW Wageningen, The Netherlands

Phenotyping is a crucial aspect of plant breeding. However the applications of digital phenotyping technologies is rapidly changing the game. Digital cameras and sensors can phenotype large numbers of plants at great precision and reveal information about plant size, shoot color, and canopy architecture, while plant development can be followed over time from multiple angles. Where traditional phenotyping typically creates low resolution and often subjective data, digital phenotyping has the potential to make data-driven decisions based on high resolution, objective and reproducible data. However, the potential of the data and challenges in data management are two sides of the same coin. Digital phenotyping creates large and diverse data sets consisting of images, growth movies, data tables and figures, and all this data needs to be processed, analyzed, and stored. Where many researchers focus on camera technologies and image analysis, the data utilization by the end-user remains a challenge. Re- searchers would like to have the tools for quality control, access to all possible parameters measured, combine parameters to better understand the data, and further mine the data to zoom in into specific research questions. And all of that without droning in all the data that is available. Therefore, KeyGene developed an interactive data visualization tool. Phenoreport can handle large amounts of digital phenotype data and allows researchers to easily store and share the data within the company. It contains a dashboard interface with all phenotypic data collected in a single research project, ranging from raw to outlier corrected data, from growth curves to scatterplots, and from results to reporting. Phenoreport allows researchers to mine the results themselves at different integration levels. A unique feature is that researchers can apply filters and sorting to easily draft their own tables and graphs. This presentation will share the bottlenecks of data management in digital phenotyping, present a straightforward approach to overcome them, and underlines the benefits of accessibility and utilization of digital phenotyping data across your company or institute.

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Oral presentation theme 1 Monday April 18, 2016, 14:20-14:40

Non-invasive phenotyping of postharvest quality traits in tomato and strawberry

Gerrit Polder1, Orianne M. Gudenschwager Soruco2 , Zoraida Megias3, Yury Tikunov2 and Arnaud G. Bovy2 1 Wageningen UR, Greenhouse Horticulture, Wageningen, The Netherlands 2 Wageningen UR, Plant Breeding, Wageningen, The Netherlands 3Departamento de Biología y Geología, Universidad de Almería, Almería, Spain

A major challenge in ensuring global food security is to avoid that up to 50% of our produce is discarded. Postharvest shelf-life is an important determinant for the loss of fresh and processed fruit and vegetable products. Shelf-life is determined by quality traits such as firmness, mealiness, colour, flavour and antioxidant levels, but also by the resistance to fungal and bac- terial diseases. Postharvest quality traits are traditionally measured using invasive technologies, such as a potentiometer, a Brix meter, using taste panels, or by chemical measurements of extracts. In this case, every sample (a fruit) can be measured only once during its postharvest storage life and it is therefore very difficult to study the dynamics of the physiological processes on-going during storage and their effects on final product quality. Therefore there is a need to measure postharvest quality traits by non-invasive technologies, as for example VIS-NIR imaging. This greatly increases the throughput of postharvest measurements and allows studying the dynamics of various postharvest traits during storage.

In order to develop these non-invasive quality traits an experiment was started in which toma- to and strawberry fruits are used as model crops. Several tomato and strawberry varieties were phenotyped both invasive and non-invasive at different time steps after harvesting. Inva- sive techniques used were: Penetro hardness, Brix, Acidity, HPLC and PTR-MS. Non- invasive techniques were: Visable and Near InfraRed (VIS-NIR) transmission (400-900nm), NIR reflection (600-2400nm), acoustic firmness, and VIS-NIR hyperspectral – and multispectral imaging. Relations between the invasive and non-invasive techniques were modelled using Partial Least Square (PLS) regression. Results show good correlations for strawberry between NIR reflection and Brix (Q2 = 0.84) and a number of volatiles measured with PTR-MS (Q2 > 0.75). Preliminary results with tomato fruits indicate that both NIR reflection and VIS- NIR hyperspectral imaging are promising techniques for this crop as well.

In the next phase of the project an integrated HTP platform for automated, non-invasive diag- nostics of various postharvest quality parameters will be build, using the most promising techniques from the experiment. This system will be implemented as part of the instrumental flavour models developed for tomato and strawberry. The HTP platform allows us to screen a large collection of diverse tomato and strawberry varieties for variation in postharvest quality traits. The latter may form the basis of novel breeding programs geared towards the development of novel varieties with improved postharvest shelf-life.

10 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 1 Monday April 18, 2016, 14:40-15:00

Non-invasive phenotyping of plant fungal infections during the asymptomatic stage

Angelika Czedik-Eysenberg1, Michele Grieco2, Daniel Tholen3, Armin Schlereth4, Beatrice Encke4, Melanie Höhne4, Nichole Krohn4, Markus Teige2, Peter Hietz3, Mark Stitt4 and Ar- min Djamei1 1Gregor-Mendel-Insitute of Molecular Plant Biology, Dr-Bohr-Gasse 3, 1030 Vienna, Austria 2University of Vienna, Faculty of Life Sciences, Althanstraße 14, 1090 Vienna, Austria 3University of Natural Resources and Life sciences Vienna, Gregor-Mendel-Straße 33, 1180 Vienna, Austria 4Max-Planck-Initute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam, Germany

Plant fungal pathogens account for considerable crop yield losses worldwide and their management often requires large scale fungicide application. In order to become able to treat fungal infections in a more targeted manner, it is necessary to gain understanding of the early infection phases and to develop methods to detect fungal infections early on.

Early detection is particularly challenging in plant-fungal interactions in which the fungal infection is initially asymptomatic, i.e. doesn’t cause any specific recognizable symptoms and the plants appear healthy. This is the case in head smut infections, such as the crop infecting pathogens Ustilago hordei and Sporisorium reillianum. This class of fungal pathogens is asymptomatic up to the flowering stage of its host, when rapid fungal proliferation commen- ces and the plant’s seeds are replaced by fungal spores. We work with the head smut model pathosystem Ustilago bromivora - Brachypodium distachyon. This model system is closely related to cereal crop pathosystems, while Brachypodium distachyon as the grass host provides the major advantages of simple growth conditions, short life cycle and low space requirements as well as a growing molecular toolbox. The smut fungus Ustilago bromivora in- fects this specific host and is currently developed in our group to become an equivalent model biotroph as its relatives Ustilago hordei, Sporisorium reillianum or Ustilago maydis. In this pathosystem we compared 1) automatized phenotyping of morphological traits and 2) phenotyping of photosynthesis related traits by Pulse-Amplitude-Modulation (PAM) fluorimetry. Both of these methods proved successful in differentiating infected and uninfected plants during the asymptomatic infection stage. Moreover, these analyses as well as deepening analyses of photosynthetic carbon assimilation rate and carbohydrate levels in infected and uninfected plants reveal new insights into the physiological changes caused by the presences of the fungus during the early, asymptomatic infection phase.

11 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Keynote speaker theme 2 Tuesday April 19, 2016, 8:30-9:20

Phenotyping at the cell level and the need for a holistic approach to implement deep physiological phenotyping

Thomas Roitsch Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenha- gen, Højbakkegård Allé 13, 2630 Taastrup, Denmark Contact: [email protected]

Plant responses are affected by the complex interaction of genome x environment x management that determines phenotypic plasticity and explains the variability of genetic components. Whereas great advances have been made in the cost efficient and high through-put analyses of genetic information and non-invasive phenotyping, the large scale analyses of the underlying physiological mechanisms is lagging behind. The external phenotype is determined by the sum of the complex interaction metabolic pathways and intracellular regulatory networks that is reflected in an internal, physiological and biochemical phenotype. These various scales of dynamic physiological responses need to be considered and genotyping and external phenotyping must be linked to the physiology at the cellular and tissue level. A high-dimensional physiological phenotyping across scales is needed that integrates the precise characterization of the internal phenotype into high-throughput phenotyping of whole-plants and canopies. Thus complex traits can be broken down into individual components of physiological traits. Since the higher resolution of physiological phenotyping by wet chemistry is inherently limited in throughput, high-throughput non-invasive phenotyping needs to be validated and veri- fied across scales to be used as proxy for the underlying processes. Armed with this interdisciplinary and multi-dimensional phenomics approach, plant physiology, non-invasive phenotyping and functional genomics will complement each other, ultimately enabling the in-silico assessment of responses under defined environments with advanced crop physiology models. This will allow to generate robust physiological predictors also for complex traits to bridge the knowledge gap between genotypes and phenotypes for applications in breeding, precision farming and basic research.

D. K. Großkinsky, J. Svensgaard, S. Christensen and T. Roitsch (2015) Plant phenomics and the need for physiological phenotyping across scales to narrow the genotype-to-phenotype knowledge gap. J. Expt. Bot. 66: 5429-5440

12 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 9:20-9:40

Transcriptomic and metabolic responses of bread wheat against heat and drought stresses

Guray Akdogan1, Turgay Unver2, Mine Turktas2, Ebru D. Tufekci2, Tugba Gurkok2, Bay- ram Ozdemir3, Yakup Cikili4, Hussein A. Ahmed1, Deniz Kom1, Sancar F. Ozcan3, Gunseli Babaoglu2 and Arif Ipek2 1 Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara, Turkey 2 Department of Biology, Faculty of Science, Çankırı Karatekin University, Cankiri, Turkey 3 Ministry of Food, Agriculture and Livestock, Central Research Institute for Field Crops, Ankara, Turkey 4Cilimli Vocational School, Duzce University, Duzce, Turkey

Drought and high temperature (heat) stress are the two main abiotic stress factors influencing development, metabolic activities and yield potential of cultivated crops negatively. Bread wheat (Triticum aestivum L.) is one of the main staple crops for billions of people in the world. Drought and high temperature occurring especially at grain filling stage of plants at spring caused significant yield loses. The development of wheat varieties with increased tolerance to drought and/or high temperature stress is an important strategy for wheat breeders. Marker-assisted breeding is routinely used in wheat breeding programs for biotic and abiotic resistance. On the other hand, the numbers of molecular-marker that can be efficiently used for selecting wheat lines against drought and/or high temperature tolerance are very few, because drought and high temperature resistances are complex traits. The aim of this study to develop molecular (SNP) and metabolite markers to accelerate wheat marker-assisted breeding programs for drought and high temperature tolerance.

Here, we used 12 bread wheat cultivars grown in pots under natural conditions and then subjected to drought, high temperature and both drought and high temperature stress at grain filling stage. There were significant influences of drought and/or high temperature on measured physiological, growth and yield traits. It is also detected that significant interaction effects between bread wheat cultivar and stress treatments on most of measured traits. Utilizing these results, we selected two wheat cultivars, the most tolerant and the most sensitive against drought and/or high temperature stress for further analysis.

The tolerant and sensitive wheat cultivars subjected to the drought, high temperature, and both drought and high temperature stress for 14-days. Then, roots, leaves and grain tissue samples were collected for high-throughput RNAseq and metabolome profiling analyzes. RNAs isolated from different tissues of the wheat were sequenced via next-generation sequencing. Metabolome profiling was performed via GS-MS, LC/ToF-MS, and 2D-NMR tools. We identified several potential functional SNP markers that can be used for breeding of bread wheat with increased drought and/or high temperature tolerance. It was also found that there are significant differences between tolerant and sensitive wheat cultivars in terms of primary and secondary metabolites. This study is supported by TUBITAK with grant numbers 113O661 and 113O016.

13 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 9:40-10:00

How do roots respond to osmotic stress? A transcriptomic approach to address this question in a non-model crop

Jassmine Zorrilla1, Mathieu Rouard2, Alberto Cenci2, Ewaut Kissel1, Nicolas Roux2, Rony Swennen1,3,4, Sebastien Christian Carpentier1. 1Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, KU Leuven, B-3001 Leuven, Belgium. 2Bioversity International, Parc Scientifique Agropolis II, 34397 Montpellier Cedex 05, France. 3Bioversity International, Willem De Croylaan 42, B-3001 Leuven, Belgium. 4International Institute of Tropical Agriculture. c/o AVRDC - The World Vegetable Center. P.O. Box 10, Duluti, Arusha, Tanzania.

The non-model crop where our workflow has been tested on is banana (Musa spp.). It ranks among the top ten staple foods, with a total production over 145 million tons in 2013 (FAO- stat). Bananas need vast amounts of water and even mild-drought conditions are responsible for considerable yield losses. To characterize the reaction in roots to osmotic stress, we performed large-scale transcriptome sequencing on three triploid genotypes representing important subgroups of cultivated bananas and subjected them to mild osmotic stress (5% PEG treatment) for 3 days. Using Illumina technology, 18 cDNA libraries were sequenced producing around 568 million high quality reads, of which 70-84% were mapped to the diploid reference genome. Through uni-/multivariate statistics, 92 genes were commonly identified as differentially expressed in the three genotypes. Using our in house workflow to analyze GO enrichment and underlying biochemical pathways, we show that the applied stress leads to a drop in energy levels inducing a metabolic shift towards (i) a higher oxidative respiration, (ii) alternative respiration and (iii) fermentation. By validating a subset of genes in an independent experiment using RT-qPCR, we confirm the success of RNA-seq for evaluation of a non- model crop. Overall, the identification and annotation of this set of genes constitutes a step ahead to understand the complex network of root responses to osmotic stress. Our results also highlight the challenge to infer gene functions in a non-model crop and the additional complexity when dealing with polyploid genomes and gene families composed by paralogous copies.

14 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 10:30-10:50

Repeated drought stress reduces anti-oxidative capacity and alters primary metabolism of young sugar beets

Monika A. Wimmer, Heiner E. Goldbach Institute of Crop Science and Resource Conservation – Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115 Bonn, Germany; [email protected]

In most drought experiments, plants are exposed to single stress events. However, drought stress in Central Europe is often not terminal, but intermittent, which means that plants typically undergo non-lethal, but repeated drought stresses throughout their development. Little is known about the physiological and metabolic impacts of consecutive drought cycles, and how they may affect the plants’ ability to respond to subsequent stresses.

In the present experiment, sugar beets were grown in pots for 6 weeks, and subjected to no stress (control), or one (D1), two (D2) or three (D3) drought cycles preceding harvest. Each drought cycle consisted of 6 days of water cutoff, followed by 4 days of re-watering. At harvest, all plants were of the same age, and only differed in their drought history. Parameters of water relations, soluble sugars, starch and anti-oxidative metabolites were determined at two day intervals during drought and recovery.

In all drought treatments, visible wilting occurred between day 4 and 6, when relative water content and osmotic potential dropped to similar levels. However, both parameters did not recover to control levels in D2/D3 plants. Ascorbate pools were depleted more quickly in subsequent drought cycles, and only D1 plants recovered to control levels. We also observed highly dynamic changes in soluble sugars and starch, which were most prominent during the recovery period and clearly distinct between D1 and D2/D3 plants.

Collectively, our results indicate that repeated drought cycles can exhaust the anti-oxidative capacity and soluble sugar pools of young sugar beet plants, even though all plants visibly recovered during re-watering. Ongoing work aims to identify genotypic differences in the ability to fully and quickly recover from non-lethal drought stress. If sufficient genetic variability is present, this trait might be valuable for breeding of drought tolerant varieties in Cen- tral Europe.

15 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 10:50-11:10

Natural variation in Arabidopsis Cvi-0 accession uncovers regulation of

guard cell CO2 signaling by MPK12

Liina Jakobson1, Lauri Vaahtera2, Kadri Tõldsepp1, Maris Nuhkat1, Cun Wang3, Yuh-Shuh Wang1, Hanna Hõrak1, Ervin Valk1, Mart Loog1, Julian I. Schroeder1, Mikael Brosché2, and Hannes Kollist1 1Institute of Technology, University of Tartu, Estonia 2Division of Plant Biology, Viikki Plant Science Centre, University of Helsinki, Finland 3Cell and Developmental Biology Section, University of California San Diego, USA

Plant gas exchange is regulated by guard cells that form stomatal pores. Proper stomatal regulation is crucial for plant survival; stomata control uptake of CO2 for photosynthesis, loss of water and entrance of air pollutants such as ozone. Despite intense studies relatively few stomatal regulators specific for only one stimulus have been identified. The Arabidopsis thaliana accession Cvi-0 from Cape Verde Islands is hypersensitive to ozone, has more open stomata and impaired stomatal responses to changes in CO2 concentration. We mapped these traits to a single amino acid substitution in MAP kinase 12 (MPK12). In a parallel approach we showed that more open stomata and CO2-insensitivity phenotypes of a mutant cis (CO2- insensitive) are caused by a deletion of MPK12. Physiological experiments revealed that in guard cells the function of MPK12 is primarily related to CO2-induced stomatal movements and lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. MPK12 was found to interact with the protein kinase HT1, a negative regulator of CO2 signaling and double mutant analysis positioned MPK12 upstream of HT1. Phosphorylation assays showed that MPK12 functions as an inhibitor of HT1 kinase activity, whereas the substitution in Cvi-0 MPK12 impaired its ability to act as an inhibitor of HT1. Collectively these data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism how guard cell CO2 signaling controls plant water management.

16 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 11:10-11:30

Early detection and classification of Mla and mlo gene based resistance of barley using hyperspectral close-range imaging

Matheus Kuska, Mirwaes Wahabzada, Stefan Thomas, Sina Tüffers and Anne-Katrin Mahlein University of Bonn, Institute of Crop Science and Resource Conservation, Department of Plant Diseases and Plant Protection, Meckenheimer Allee 166a, D-53115 Bonn

To accelerate time and cost expensive visual rating in plant breeding procedures, sensor based phenotyping-platforms are necessary. Among different sensor technologies, hyperspectral imaging can assess different plant parameters non-invasively. In incompatible barley-powdery mildew interaction, plant resistance reactions are based on physiological and biochemical changes. Using a hyperspectral-imaging microscope it was possible to determine powdery mildew pathogenesis and to identify different resistance reactions of barley genotypes.

The mlo gene based resistance of barley is successful against infections by Blumeria graminis f.sp. hordei (Bgh), the causal agent of barley powdery mildew. Fast cell wall aggregations, so called papilla, inhibit the interaction. The modified epidermal cells remain intact. Contrary, barley Mla gene based resistance leads to a hypersensitive reaction in a single cell or in united cell structures and finally necrotizes the penetrated cells. The present study were conducted with a Bgh isolate K1 susceptible variety H. vulgare cv. Ingrid including a mlo gene based resistant near-isogenic line of cv. Ingrid and a Mla gene based resistant near-isogenic line of cv. Pallas. The hyperspectral reflectance of individual penetration spots was measured daily until 8 days after inoculation (dai). In parallel histological observation with coomassie bril- liant blue R-250 and 3,3´-diaminobenzidine were used to determine the Bgh development and barley resistance stage indicated by local H2O2 accumulations. Microscopic observation enabled a biological interpretation of spectral reflectance changes during pathogenesis and resistance reactions.

Further analysis of spectral signatures by data mining indicates specific reflectance patterns for pathogenesis and each resistance type. This information can be integrated in high- throughput phenotyping pipelines in order to evaluate the performance of multiple genotypes. Additional gene expression correlations of resistant genes may adjust and validate the resistance phenotyping by hyperspectral imaging.

17 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 11:30-11:50

Phenotyping microbial diversity during flax dew-retting by using targeted- metagenomics

Christophe Djemiel, Sébastien Grec, and Simon Hawkins Univ. Lille, CNRS, INRA, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France

Flax (Linum usitatissimum L.) is an economically important oil and fiber crop. Flax bast fibers, rich in cellulose, are traditionally used to produce textiles (linen) and can also be used for reinforcing composite polymers as an environmentally friendly alternative to glass fibers [1]. The ‘quality’ of both textiles and composite materials is influenced by the ‘quality’ of the plant fibers that arrive in the factory. Plant fiber quality itself is related to fiber cell wall structure determined during plant growth, and modified to a greater or lesser extent during fiber extraction. The first step in the industrial transformation of flax stems into fibers is achieved by dew-retting on the soil surface. At harvest, the flax stems are ‘pulled’ (up-rooted) and laid down directly on the soil in swaths (strips). Subsequent exposure to alternating periods of rain and heat combined with the development and the action of soil microflora favor the separation of cellulosic bast fibers from the stems via the hydrolysis of cell wall polymers (hemicellu- loses, pectins). Despite many studies of this process [2,3,4] relatively little is known about i) the composition of the microflora population, ii) the kinetics of microbial colonization of plant material and iii) the evolution of the microbial population in the soil. Soil retting remains a ‘black box’ (very few microbial species have so far been identified) and our current lack of knowledge of this process represents a major obstacle to a more efficient retting process. In the framework of a collaborative French ‘Future project’ ‘StructuratIoN de la filière Fibres techniques d’OrigiNe végétale pour usages matérIaux’ (SINFONI) we are conducting a targeted-metagenomics approach to phenotype for the first time the composition of the microbial communities (focusing on bacteria and fungi) involved in flax dew-retting. This approach has also allowed us to identify some potential major enzymatic functions related to cell wall degradation based on functional prediction using the bioinformatic software PICRUSt. In this communication we will briefly present the methodology used to explore microbial diversity using the Next Generation of Sequencing (Illumina MiSeq system) and explain how this data is helping us to understand the impact of plant cell wall degrading enzymes on flax stems. The range of identified biological markers should contribute to improving the extraction process and hence fiber quality. [1] Baley, C. (2002). Analysis of the flax fibres tensile behaviour and analysis of the tensile stiffness increase. Composites Part A: Applied Science and Manufacturing, 33(7), 939–948. http://doi.org/10.1016/S1359-835X(02)00040-4 [2] Akin, D. E. (2013). Linen most useful: Perspectives on structure, chemistry, and enzymes for retting flax. ISRN Biotechnology, 2013, 23. http://doi.org/http://dx.doi.org/10.5402/2013/186534 [3] Henriksson, G., & Akin, D. (1997). Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and europe. Applied and Environmental Microbiology. Retrieved from http://aem.asm.org/content/63/10/3950.short [4] Martin, N., Mouret, N., Davies, P., & Baley, C. (2013). Influence of the degree of retting of flax fibers on the tensile properties of single fibers and short fiber/polypropylene composites. Industrial Crops and Products, 49, 755–767. http://doi.org/10.1016/j.indcrop.2013.06.012

18 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 2 Tuesday April 19, 2016, 11:50-12:10

Microbes Shape Defense Response of Pisum Sativum against Didymella pinodes

Reinhard Turetschek1, Getinet Desalegn2, Hans-Peter Kaul2 and Stefanie Wienkoop1 1 University of Vienna, Department of Ecogenomics and Systemsbiology 2 University of Natural Resources, Department of Crop Sciences

Legumes, such as field pea, vitally benefit from the association with rhizobial bacteria and mycorrhizal fungi. Both symbionts affect the plants´ growth, metabolism and potentially prime the immune system for upcoming pathogen attacks. To date, little is known about the specific effects of microbes on Pisum´s above ground parts and their role during pathogenic infection. Through characterization of morphological traits and assessment of the leaf metabolism (primary metabolites and proteome – label free quantification) we aimed to elucidate the symbiotic effects on Pisum. Our results indicate that a varying composition of associated microbes substantially affects the leaf metabolism on different levels (hormone synthesis, secondary metabolism, ROS dampening). The infection with Didymella pinodes caused a common metabolic response among different symbiotic treatments. However, Rhizobia associated plants showed the most pronounced response. This suggests that the extent of Pisum´s defense response to D. pinodes depends on the prevailing symbiotic associations.

19 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Keynote speaker theme 3 Wednesday April 20, 2016, 8:30-9:20

Phenotyping in greenhouses and the challenges of dynamic climate Carl-Otto Ottosen

Department of Food Science, Aarhus University, Denmark [email protected]

One of the main differences between horticulture and agriculture is the immense number of cultivars or varieties used in horticulture both for food and non-food production. However phenotyping has been used systematically for centuries – although the naming has been different. The main difference in horticulture to agriculture especially in protected cultivation is the fact that most species are propagated by vegetative cuttings or by hybrid seeds so the genetic diversity is narrow. Thus the process of selecting genotypes based on single characters (flower color, yield) has been done widely, while new phenotyping methods have been developed for selecting genotypes with specific responses such as reduced stomatal conductance or improved tolerance to ethylene.

Horticultural growing experiences in commercial greenhouses are very useful for the explaining the plant responses during phenotyping as professional growers gain immense understanding of minute effects of different climate parameters. This is due to the fact that most abiotic and biotic climate factors can be controlled and monitored in controlled conditions. This allows for studies of the interaction between climate parameters in a controlled manner. With the right greenhouse equipment most parameters including the CO2 level can be manipulated and maintained relatively constant so limiting factors can be controlled and manipulated, as genotypes might be differently adapted to the actual growing conditions.

The challenges in phenotyping installations are often that climates are indeed kept as constant as possible with the exception of water and nutrient management. However, that is not how the natural climate is. In modern greenhouses, though, the climate can also be controlled in a dynamic manner e.g. following the light level as in nature, where the monitoring of the climate can benefit the explanation of the plant responses where e.g. the effect of climate change can be phenotyped.

We have focused on small-scale phenotyping of handful of cultivars using a variety of methods. They not only separate genotypes but also explain some of the mechanisms behind specific climate responses since the facilities allow for rapid changes the climate as well as monitoring of plant responses with high time resolution by e.g. PlantEye, DroughtSpotter and Moni-PAM for growth, water balance and chlorophyll fluorescence quenching.

20 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 3 Wednesday April 20, 2016, 9:20-9:40

Integrated metabolome and whole plant phenotyping reveal genetic and physiological factors of vegetative growth and seed longevity

David Riewe1, Manuela Nagel2, Jan Lisec3, Hea-Jung Jeon1, Marc Heuermann1, Janine Wie- bach1, Judith Schmeichel1, Monique Seyfarth1, Rhonda C. Meyer1, Andreas Börner2, Lothar Willmitzer3, and Thomas Altmann1 1Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Cor- rensstrasse 3, 06466 Stadt Seeland, OT Gatersleben, Germany; 2Department Genbank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Stadt Seeland, OT Gatersleben, Germany; 3Department of Molecular Physiology, Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam OT Golm, Germany

Biomass accumulation through vegetative plant growth can be regarded as the ultimate expression of the plants metabolic performance. While ample knowledge exists on natural variation of growth as well as on metabolic diversity, only rarely direct links have been established between a DNA polymorphism, the expression of the affected gene, activity of the encoded enzyme, substrate turnover and biomass accumulation. A cytosolic fumarase (FUM2) with function in carbon assimilation and nitrogen utilization occurs in A. thaliana and other Brassicaceae in addition to the mitochondrial fumarase enzyme involved in the TCA cycle. A major FUM2 promoter insertion/deletion (InDel) polymorphism coinciding with a fumarate QTL was found in the Arabi- dopsis Col-0 and C24 accessions, which also discriminates a large number of other accessions. By analyzing recombinant inbred lines, near isogenic lines and a selected accession panel with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate to malate ratio in the illuminated Arabidopsis leaf. The promoter InDel was also found to be signifycantly associated with the fumarate to malate ratio, with malate and fumarate levels, and with dry weight in 174 natural Arabidopsis accessions at 15 days after sowing (DAS). The association with biomass production was confirmed in another larger (251) accession population for dry weight at 22 DAS. These findings support a role of FUM2 in diurnal carbon storage and point to a growth advantage of accessions carrying the FUM2 Col-0 allele that occurs in preferentially in colder climates. Most plants are propagated via seeds, which are also the major storage form and agricultural product of crops. Seed viability is important for agriculture as well as for long term conservation of plant genetic resources. Germination phenotyping and metabolite profiling was performed using long term stored seeds of wheat and barley accessions. GC-MS analysis of the polar metabolome identified glycerol and related intermediates as highly correlated to germination rate. Fur- thermore, the lipidomic composition of the wheat panel was investigated using high-resolution LC-MS. From 26,000 chromatographic features (m/z), 650 (including membrane lipids like ga- lactosyl-lipids and phospholipids) were positively correlated to germination rate (max R = 0.77) and 3,600 (including degradation intermediates like phosphatidic acid, lysophosphatidic acid, diacylglycerols and fatty acids) were negatively correlated (max R = -0.89), linking seed viability to preservation of membrane integrity.

21 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 3 Wednesday April 20, 2016, 9:40-10:00

Can specialized metabolites promote growth? Challenging traditional plant defense theories using dynamic imaging and metabolite analysis

Elizabeth H Neilson1,2, Cecilia K Blomstedt, Bekzod Khakimov, Aaron Edwards, Birger Lindberg Møller1,2,4, Roslyn M Gleadow1,2 1)Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, Univer- sity of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2) VILLUM Research Center for ”Plant Plasticity”, Thorvaldsensvej 40, DK-1871 Frederiksberg C,Denmark 3) School of Biological Sciences, Monash University, Australia 4)Center for Synthetic Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Plants deploy a wide array of specialized metabolites that facilitate interactions with the biotic and abiotic environment, including the essential role of chemical defense against herbivores and pathogens. A trade-off is envisioned between growth and defense, such that resources allocated to the synthesis, transport and storage of specialized metabolites comes at the expense of resources allocated for growth and reproduction. We utilized the high-throughput phenomic imaging platform, The Plant Accelerator®, to accurately measure growth in sorghum lines varying in their ability to produce the specialized metabolite, dhurrin. Sorghum plants were grown under optimal and water-limiting conditions and imaged daily for four weeks. Contrary to traditional growth/defense hypotheses, we show that the sorghum line deficient in dhurrin, displayed slower initial growth rate compared to wild-type. Notably, high-throughput imaging enabled the detection of small, but significant differences in growth during the initial stages of abiotic stress. When grown under water-limiting conditions, however, dhurrin-deficient sorghum lines grew larger and faster compared to wild-type. GC-MS and LC-MS metabolite profiling analysis identified metabolites that may be involved in the turnover and recycling of dhurrin. These findings point to alternative in planta roles of dhurrin in plant growth, development and in response to abiotic stress, in addition to its action as a chemical defense compound.

22 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 3 Wednesday April 20, 2016, 10:30-10:50

Impact of treated wastewater on growth, respiration and hydraulic conductivity of citrus root systems in light and heavy soils

Indira Paudel1,2, Shabtai Cohen1, Avi Shaviv4, Asher Bar-Tal1, Nirit Bernstein1, Bruria Heu- er1and Jhonathan Ephrath3 1 Institute of Soil, Water and Environmental Sciences, ARO Volcani Center, Bet Dagan, Israel 50250, 2 The Robert H. Smith Faculty of Food Agriculture and Environment, The Hebrew University of Jerusalem, Re- hovot, Israel.3 Jhonathan E. Ephrath. Jacob Blaustein Institutes for Desert Research, French Associates Insti- tute for Agriculture and Biotechnology of Drylands, Ben-Gurion University of the Negev 4 Department of Environmental, Water and Agricultural Engineering, Faculty of Civil and Environmental Engi- neering, Technion-Israel Institute of Technology, Haifa, Israel

Roots interact with soil properties and irrigation water quality leading to changes in root growth, structure, and function. We studied these interactions in an orchard and in lysimeters with clay and sandy loam soils. Minirhizotron imaging and manual sampling showed that root growth was three times lower in the clay relative to sandy loam soil. Treated waste water (TWW) led to a large reduction in root growth with clay (45-55 %) but not sandy loam soil (<20%). TWW increased salt uptake, membrane leakage, and proline content, and decreased root viability, carbohydrate content, and osmotic potentials in the fine roots, especially in clay. These results are evidence that TWW challenges and damages the root system. Phenolo- gy and physiology of root orders were studied in lysimeters. Soil type influenced diameter, specific root area, tissue density and cortex area similarly in all root orders, while TWW influenced these only in clay soil. Respiration rates were similar in both soils and root hydraulic conductivity was severely reduced in clay soil. TWW increased respiration rate and reduced hydraulic conductivity of all root orders in clay but only of the lower root orders in sandy loam soil. Loss of hydraulic conductivity increased with root order in clay and clay irrigated with TWW. Respiration and hydraulic properties of all root orders were significantly affected by Na amended TWW in sandy loam soil. Further, aquaporin's 'PIPs gene' expressions were significantly reduced in roots of heavy soil and TWW irrigation. These changes in root order morphology, anatomy, physiology, hydraulic properties, and gene expression indicate rapid and major modifications of roots in response to differences in soil type and water quality.

23 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 3 Wednesday April 20, 2016, 10:50-11:10

Can phenotyping at multiple levels shed light on the iron efficiency trait in soybean plants?

Carla S. Santos1, Susana M.P. Carvalho123, Mohammad-Reza Hajirezaei4, Ricardo Giehl4, Ni- colaus von Wirén4, Marta W. Vasconcelos1 1Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Univer- sidade Católica Portuguesa, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto, Portugal, 2GreenUP/CITAB-UP & DGAOT, Faculty of Sciences, University of Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, 7. 4485-661 Vairão, Portugal, 3Department of Plant Sciences, Horticulture and Prod- uct Physiology Group, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands, 4Molecular Plant Nutrition, Department of Physiology & Cell Biology, Leibniz-Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

Iron (Fe) is an essential mineral nutrient for plant growth. Albeit present in the soil in high concentrations, it is often deficient for uptake in calcareous conditions, because it is mostly present in an oxidized state. Like most crops, soybean (Glycine max. L) is prone to developping Iron Defi- ciency Chlorosis (IDC), a condition that severely affects yield and plant growth. Phenotyping plants at a whole-plant, organ and cellular level is essential to understand the mechanisms underlying Fe uptake, trafficking and homeostasis and this integrated view is needed in order to prevent IDC. We conducted an in silico phenotyping approach in the Germplasm Resources Information Network (GRIN) looking at soybean plants that had been grown in multiple field locations, and we identified a set of novel Fe efficient and Fe inefficient soybean accessions. These were then brought to the lab, and a field-to-lab phenoltyping approach allowed confirming their efficiency and inefficiency traits. Subsequently, this plant material was phenotyped at multiple levels, looking at whole-plant, organ and cellular level by performing nutrient partitionning, physiological, biochemical, transcriptomic and metabolomic analyses. At an early growth stage, Fe efficiency was associated with higher chlorophyll content, and higher Fe reductase activity. The removal of the unifoliate leaves alleviated IDC symptoms, increased shoot-to-root ratio and trifoliate leaf area. Efficient plants were able to translocate Fe to the aboveground organs, whereas the inefficient plants accumulated more Fe in the roots. In a second experiment, a better understanding of the role of the Fe reductase was targeted. Soybean plants constitutively expressing the AtFRO2 iron reductase gene were analyzed for root and leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo), pod walls (Fe, K, P, Cu and Ni), leaves (Fe, P, Cu, Ca, Ni and Mg) and seeds (Fe, Zn, Cu and Ni). Recently, under the scope of an STSM, we grew a subset of the contrasting soybean lines under Fe limitation and Fe suffi- ciency, collected and concentrated their root exudates and subjected them to analysis for organic acids and Fe mobilization capacity. Altogether, results show that Fe efficiency results from a combination of individual traits expressed at multiple levels and that integrating such phenotyping data is a promising strategy to understand how tolerance to ID C in soybean can be improved.

24 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 1 Wednesday April 20, 2016, 11:10-11:30

MODCARBOSTRESS - Improving models and plant phenotyping pipelines for a smart agriculture under abiotic stress combination and elevated CO2

Benita Hyldgaard1, Thayna Mendanha1, and Carl-Otto Ottosen 1 1 Plants, Food and Climate, Dept. of Food Science, Aarhus University

Reliable crop models are of key importance in the innovation and breeding process towards creation of “climate efficient” or tolerant varieties of crops. In the EU project MODCARBO- STRESS, experiments are conducted in several phenotyping platforms focusing on acclimation of genotypes of wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) to changes in CO2, water availability and temperature. The experimental setup with multiple climatic factors enables us to create knowledge on the interacting effects of combined stresses

(elevated temperature and drought) plus elevated CO2 on plant responses as well as generating datasets to improve model parameterization in two different types of crop models (models included in the project: SiriusQuality and GECROS).

In our group in Årslev, DK, we have performed a CO2 x Drought x Temperature experiment with two wheat cultivars, including swapping of plants between the temperature treatments.

The talk will focus on the results from photosynthesis measurements (temperature and CO2 response curves) and include a comparison to results obtained in a similar experiment conducted by one of the project partners (Wageningen University and Research Centre, The Nether- lands).

In our experiment in Årslev, DK, one of the cultivars, Paragon, showed a shift in temperature optimum according to growth temperature when grown at ambient CO2, but not at elevated CO2. The other cultivar did not show a curve shift in response to growth temperature. Both cultivars shifted the curves upward in response to increased CO2 when grown at 18°C, but not at 28°C. The drought treatment resulted in a different response.

The project is an EU project funded by the ERA-net: FACCE JPI Agriculture, Food Security and Climate Change.

25 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 1 Wednesday April 20, 2016, 11:30-11:50

Phenomics analysis of drought responses in Miscanthus collected from different geographical locations.

Marta Malinowska, Iain Donnison, and Paul Robson Institute of Biological, Environmental and Rural Sciences, Aberystwyth University,UK

Miscanthus is a genus of C4 perennial grasses capable of high biomass potential even in tem- perate regions making it an ideal industrial crop for the renewable supply of energy and chemicals. Yield is strongly linked to water availability and many environments have limited water supply where otherwise irradiation and temperature are favourable.

A total of 47 Miscanthus genotypes, diverse regarding collection site and genotype, were screened in a high throughput phenomics facility under drought to generate high-quality time- course data for biomass accumulation and water use. Plants were subjected to three treatments: a watered control, mild drought (20% of field capacity), and a severe drought (water completely withdrawn). Daily visual spectrum images were calibrated to harvested biomass and used to assess biomass accumulation over the experiment.

Image analyses were used to determine growth and senescence as functions of time and treatment. An accurate prediction of plant biomass (R2=0.92***) was made by comparing actual harvested biomass and projected shoot area. Dynamic responses in senescence between the multiple genotypes under the three treatments demonstrated stay-green and senescence responses were not associated with species.

Phenomic analysis of drought responses was shown to have the potential to improve the selection of breeding candidates in Miscanthus and has identified interesting Miscanthus genotypes combining high biomass and high WUE for further characterisation.

26 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 4 Wednesday April 20, 2016, 11:50-12:10

EMPHASIS – a new European Research Infrastructure for Plant Phenotyping

Uli Schurr IBG-2, Research Centra Jülich, Vilhelm-Johnan-Strasse, 25428 Jülich, Germany

In March 2016 EMPHASIS was officially listed on the ESFRI-roadmap as one of only 5 new projects. The presentation will give a short update (relative to the presentation at the last COST-GM in Gatersleben) and will report about recent developments in the partnership and the propsed infrastructures of EMPHASIS. The further development of the project will be explained, options to participate as provider of infrastructure, technology developer and user (ACCESS modes) will be explained. EMPHASIS offers unique opportunities for the development of and access to plant phenotyping infrastructures in Europe.

27 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Keynote speaker 1 theme 5 Wednesday April 20, 2016, 13:00-13:40

The importance and challenges of phenotyping Plant Genetic Resources

Theo J.L. van Hintum Centre for Genetic Resources, The Netherlands (CGN), Wageningen UR, The Netherlands [email protected]

Plant Genetic Resources (PGR) serve as raw material for plant breeding and study material in most plant sciences. Genebank all over the world collaborate to make this PGR available to current users and to keep it available to future users.

These PGR, often seeds in freezers, hardly have any value without knowledge about their identity (taxonomic classification, variety name or collecting location, etc.) and even more importantly, their properties, both genotypic as phenotypic.

Collecting genotypic information has become relatively easy; increasingly PGR collections are being re-sequenced, the resulting data being made publicly available, and platforms for the analysis are being set up. This is in its infant stage but will happen; there are no fundamental technical obstacles. If it comes to phenotypic information, the situation is rather different.

Since the early stages in PGR conservation, it was clear that phenotypic data (so called characterization data) had to be collected to verify the identity of the accession during regeneration. Fixed descriptor lists were designed and used. The characters that were recorded had a high heritability and were easy to score. Soon it also became clear that phenotypic data (so called evaluation data) would be needed to allow potential users determine the value of the PGR for their purpose. Collections were screened for disease resistances and other, relatively easy to score traits. But there it stopped; results were rarely properly organized or made accessible on the Internet.

Currently we see that the genomics community asks for phenotypic data, to allow them to correlate their genetic data to actual phenotypes, for identifying genes and QTLs, for studying the effects of specific SNPs or chromosomal rearrangements. This community, with its bioinformatics expertise, wants to make the genebanks’ phenotypic data FAIR (findable, accessible, interoperable, and re-usable) only to find out that the data hardly exist, that making the existing data FAIR is more difficult than expected and that the phenotypic data often do not refer to the material that was genotyped.

In the presentation the different user groups of genebanks’ phenotypic data will be described, each with their specific needs. The problems of generating useful phenotypic data will be listed, as will be the complexities of linking the genomics community with the genebanks’ material and data. For all problems described, solutions will be proposed and illustrated with real life examples.

28 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Keynote speaker 2 theme 5 Wednesday April 20, 2016, 13:40-14:20

Phenotyping focus for breeders

Christian Sig Jensen, Morten Greve Pedersen, Niels Roulund, Ingo Lenk, Thomas Didion, and Dario Fè DLF Research Division, Højerupvej 31, 4660 Store Heddinge, Denmark

Modern breeding of most major crops is characterized by relatively big trial designs including hundreds or even thousands of plots distributed across several locations. Running such a breeding system is expensive not only due to extensive field management but also due to the substantial resources that are allocated to scoring of visual traits. It is all about breeding gains (BG). Accor- ding to the breeder’s equation, there are several ways to lift breeding gains: 1) Generation interval (time), which is the most potent driver of BG and sets the denominator for all other efforts; 2) Se- lection accuracy, which is determined by measurement precision and by the number of repeats; 3) Selection intensity, the effect of which is strongly correlated with the number of entries in the test. Although increasing the number of entries and repeats in field trials is an “easy” and effective way to increase BG, fixed breeding costs usually puts rather heavy constraints on the availability of this tool in practice. Shortening of the generation interval is being pursued to the maximum ex- tend wherever possible. Having breeding locations both on the southern and northern hemisphere makes it feasible in several species to have at least two generations each year. Thus, the only alternative left for spiking BG is improvement of measurement precision. During the past decade, a wide array of technological advancements has provided breeders and scientists with numerous phenotyping tools that potentially can assist in improving measurement precision. Many of these tools rely on non-destructive imaging that can be performed fast and consistently. While the first systems were designed for medium to large scale indoor screenings, new high throughput systems are being made also for outdoor screening, making them significantly more relevant for crop breeding. The big question is whether these technologies can compete with breeder’s scorings in terms of accuracy and time. If not, the new phenotyping tool must demonstrate its value in providing a new type of information, which the breeder was not able to achieve before, and which allows him to select for other traits that may be equally or more important than the traits he is selecting for today. Implementation of Genomic Selection in plant breeding, which is still ongoing, has further raised the request for improved phenotyping accuracy. Since GS allows for selection of harvest-related traits already at seedling stage, it is of outmost importance for prediction accuracy that phenotype data are precise, abundant, and reproducible. In my talk, I will present data on breeder’s accuracies and efficiencies in phenotyping forage ryegrass by visual scorings and by destructive measurements. I will discuss methods that may be tailored either to improve measurement accuracy or to complement breeding with new information. Such information may enable breeders to select for traits that will shape new crops for future climate challenges.

29 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 5 Wednesday April 20, 2016, 14:20-14:40

Can industry cooperation lead to more applicable phenotyping systems? – A collaborative approach for plant phenotyping standards in industry

Stefan Schwartz1, Klára Panzarova2, and Bas van Eerdt3 1 Phenospex B.V., Jan Campertstraat 11, 6416SG Heerlen, NL 2 Photon Systems Instruments, Starobrněnská 3, 602 00 Brno, CZ 3 PhenoKey, Zeelsterstraat 80, 5652 EL Eindhoven

With the increasing complexity of high throughput phenotyping technologies over the last years, it became obvious that solving the phenotyping bottleneck is a bigger challenge than expected. Major resources have been spent to develop and validate methods and too often those developments took place in parallel. Although this improved the method of plant phenotyping it did not help to make big leaps in the underlying biological applications. As a consequence, suppliers should follow a more open approach to guarantee comparability and exchangeability from one system to another to avoid double developments. To do that, we think it is now the time for suppliers of plant phenotyping hard- or software to open their systems and to take collaborative approaches. In this talk we want to introduce a first concept of industry collaboration that shall help to share competencies and optimize systems for certain applications by combining the best available technologies from different suppliers. This initiative is not only limited to the presenting companies, but should be a first step to organize an industry community, that should aim to improve plant phenotyping for suppliers and users in the long run. We want to achieve this by not only expressing intentions but by identifying and finding solutions for the most urgent needs that hinder industry collaboration. Therefore, we have set ourselves the goal to suggest ideas to the community within the next 12 months to tackle the following topics: 1) The book of traits: Finding a common language for methods in plant phenotyping that compares different methods for different applications. 2) Compatible Hardware: Developing simple protocols that allow the users to integrate sensors from different companies into the same automation systems. We will present a first case study for a collaborative phenotyping system that was developed. 3) Data Standard: Defining a common format for RAW- and computed-data from different phenotyping systems that allow the exchange and comparison of different datasets or the use of common databases independent of the supplier. Industry collaboration could evolve phenotyping into a better tool for science. Although, we have taken the initiative to start, this approach shall not be an exclusive partnership among the three (partly competitive) companies but an invitation to join this board in a first industry meeting we would like to host as a side meeting in Copenhagen. We would like to engage everybody in the market to give us their feedback and participate as an essential part for such an industry community, is a stronger communication between suppliers as well as between.

30 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Oral presentation theme 5 Wednesday April 20, 2016, 14:40-15:00

International Plant Phenotyping Network: an association aiming at integrating the plant phenotyping community

Roland Pieruschka1, April Agee Carroll2, Xavier Sirault3, Ulrich Schurr1 1IBG2: Plant Sciences Forschungszentrum Jülich, Jülich, Germany; 2College of Agriculture, Purdue University, West Lafayette, USA; 3Australian Plant Phenomics Facility, CSIRO Agriculture, Canberra, Australia

The quantitative analysis of structure and function of plants, the phenotyping of plants, has become a major bottleneck in research and applied use of plants. Innovative approaches targeting relevant traits are needed to quantitatively address key processes and understand the dynamic interactions between genetic constitution, molecular and biochemical processes with physiological responses leading to the development of phenotypes. To effectively address this bottleneck a close interaction within the plant phenotyping community is required. Therefore the International Plant Phenotyping Network (IPPN) has been initiated as an association with currently over 20 international organizations as members. The goal is to increase the visibility and impact of plant phenotyping and to use existing synergies by enabling communication and cooperation within the plant phenotyping community but also with different stakeholders from academia, industry, policy and general public. IPPN will enable and support events such as workshops and symposia, promote the establishment of different working groups, and as- semble and distribute all relevant information about plant phenotyping in a web-based platform. This presentation will introduce IPPN, summarize the recent activities and provide an outlook for future ones.

31 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

32 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Abstracts for posters

Alphabetic order of submitting author within sessions

33 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

34 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Theme 1

WG1: Phenotyping at plant and field level

35 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 1a Screening for rust resistance in cool season legumes: complementation of field and growth chamber studies with histology, genomics and proteomics

Diego Rubiales, María A. Castillejo, and Eleonora Barilli Institute for Sustainable Agriculture, CSIC, Córdoba, Spain [email protected]

Rusts are major biotic constraints of legumes worldwide. Breeding for resistance is regarded as the most cost efficient method for rust control. Preferred resistance for breeders for decades has been complete resistance, which is usually controlled by single genes. The draw back is the easy breakdrown by development and spread of new races of the pathogen. This has reaised an increasing concern on durability and in the search of more durable types of resistance.

The situation is a bit different in cool season legumes. In contrast with the better studied common bean in which complete monogenic resistance has been identified and is efficiently used in breeding, only incomplete resistance of complex inheritance has been described in faba bean, pea, chickpea and lentil and several of their associated QTLs have been mapped. However, the relatively large distance between the QTLs and their associated molecular markers hampers their efficient use for MAS. However rust resistance breeding is not only slow due to the difficulty and the relatively low investment on genomics of the legume crops, but also, and mainly because of the little knowledge on the mechanisms of resistance opera- tive and on the biology of the rust pathogens. Comprehensive studies on host status and virulence of the various rust species are often missing, and in most of the examples listed above, there is little agreement on the existence of races and on their distribution. Also, available information on levels of resistance and on the responsible mechanisms is often incomplete. On- ly after significant input to improve existing knowledge on biology of the causal agents and in phenotyping plant responses, will resistance breeding be efficiently accelerated. Phenotyping on rust resistance performed at Cordoba will be presented and critically discussed.

Keywords: resistance, rust, legume

36 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 1b Selecting maize genotypes for drought resistance and low input agriculture

Maria C. Bicho1, Joaquim M. Costa1,2, Miqueias Permanhani1,3, Maria C. Vaz Patto1 1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal 2 LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Portugal 3 Fitotecnia-Universidade Federal Rural do RJ, Seropédica, Rio de Janeiro, Brazil

Maize (Zea mays L.) is one of the most important agricultural crops worldwide. Selection for drought resistance is a major issue for both maize breeders and agronomists. This applies to conventional, but especially, to the more sustainable, low input agriculture, that requires better adapted genotypes to stressful environmental conditions and limited inputs. Older varieties, landraces, and synthetic populations can provide interesting materials for use is such selection. In this context, phenotyping and selection of individuals more resistant to drought, within known and/or new populations, are major strategies to achieve a more sustainable and still productive farming. Fast, simple and remote non-destructive methods to phenotype and select maize for drought resistance are required in modern plant breeding. Our aim was to set up and optimize screening / phenotyping procedures for selecting outstanding maize genotypes within populations grown under controlled conditions. We studied two maize open pollinated varieties (Fandango and Pigarro) grown in pots. Control plants were continuously well irrigated. Water deficit was imposed to the remaining seedlings by water withholding until soil water content has reached 35 % of field capacity, which has occurred 17 days after germination, at the stage of four fully expanded leaves. Phenotypic characterization was based on the following parameters: Relative water content, photosynthetic pigments content, leaf temperature (Tleaf) and individual leaf gas exchange. Tleaf was assessed by thermography and leaf gas exchange was measured using an infrared gas analyzer. No differences were found for these parameters between the two maize populations. Leaf gas exchange was negatively influenced by drought stress. Physiological parameters allowed to detected significant variability among individuals of the same maize population for drought response. The variability found for drought responses may indicates the existence of individual maize plants better adapted to low input agriculture and with potential for future breeding programs. Strategies to optimize growing conditions and screening procedures are discussed.

Key-Words: drought, phenotyping, maize, populations, resistance.

37 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 2 Photosynthetic traits in phenotyping of wheat genotypes for tolerance to drought and high temperature

Marian Brestic, Marek Zivcak and Katarina Olsovska Department of Plant Physiology, Slovak University of Agriculture, Nitra, Slovakia

Identification of traits contributing to drought and high temperature tolerance represent the major goals of phenotyping of genetic resources in cereal crops. The presented study was aimed at the evaluation of several photosynthetic methods and technical approaches in assessment of tolerance to drought and high temperature in wheat genotypes of different origin. The post-anthesis drought stress was induced by withholding of irrigation under a rainout shelter. The set of physiological parameters was monitored regularly, including the leaf water content and osmotic adjustment. Using the values of CO2 assimilation and transpiration, the leaf transpiration efficiency was calculated; moreover, the stomatal conductance was measured by porometer.

The numerous measurements of rapid chlorophyll a fluorescence kinetics in wheat leaves were recorded and the drought stress-associated changes at the chloroplast level were analysed. This technique was used also for the assessment of photosynthetic thermostability. The drought tolerance of genotypes was classified according to harvested grain yield in non- stressed and stressed conditions. The correlations of physiological traits with yield-based drought tolerance of genotypes were assessed. Our results indicated that the drought-tolerant genotypes had a higher transpiration efficiency due to lower stomatal conductance in non- stressed conditions, but the higher stomatal conductance associated with higher osmotic adjustment in drought. The analysis of chlorophyll fluorescence kinetics based on numerous measurements enabled to identify parameters, which well reflected the drought stress level and hence, they correlated well with genotypes’ drought tolerance. In contrary, the commonly used fluorescence parameters were fully insensitive until the sub-lethal drought level. The simple methodical approach enabled to identify also the significant genotypic differences in heat resistance. The results confirmed the link between the leaf transpiration efficiency and yield in conditions of post-anthesis drought stress in wheat. Moreover, the rapid, non- invasive, easy-to-do measurements of chlorophyll fluorescence kinetics in wheat leaves were shown to be useful in assessment of drought stress severity and the drought stress tolerance.

38 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 3 Monitoring canopy temperature patterns in grapevine: a tool to compare genotypes and optimize deficit irrigation?

Joaquim M. Costa1,2*, Ricardo Egipto2, Ivan Garcia-Tejero1,3, Margarida Vaz4, Carlos M. Lo- pes2, Manuela Chaves1 1 LEM-ITQB, Universidade Nova de Lisboa, Apartado 127, 2780-901 Oeiras, Portugal 2 LEAF, ISA,Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal 3 Centro IFAPA, LasTorres–Tomejil Ctra. Sevilla-Cazalla Km12, 41200. Alcalá del Río, Sevilla, Spain 4 ICAAM, Universidade de Évora, 7002-554 Évora, Portugal

Mediterranean wine production must address the increasing concerns of stakeholders on more environmentally sustainable wine production. Deficit irrigation can help viticulturists to save water but more precise irrigation must account for variation of plant needs in time (day and phenology) as well as the variation due to the genotype. A better comprehension of grapevine stress physiology in response to drought and heat is important to optimize crop/soil management, save water and improve berry composition. Canopy temperature (Tc) is a proxy of stomatal conductance to water vapor (gs) and an indicator of vine’s water status. Thermography permits to visualize and quantify the range and spatial distribution Tc in row crops such as grapevines. Our hypothesis is that diurnal and seasonal Tc patterns may differ between genotypes due to different stomatal control and heat loss strategies. To that extent, we monitored the diurnal and seasonal course of Tc in two V. vinifera cvs, Aragonez (syn. Tempranillo) and Touriga Nacional subjected to deficit irrigation under typical Mediterranean climate conditions in Alentejo (South Portugal). The diurnal and seasonal course of Tc, leaf water potential

(ψleaf) and leaf gas exchange and vines’s agronomical performance were assessed. Measure- ments were done along three years (2013- 2015) and at different phenological stages of the crop: i) green berry stage (end May/mid June), ii) veraison (mid-july), iii) early ripening (mid August) and iv) late ripening (end August). Deficit irrigation saved on average 46 mm (460 m3 /ha) of water per year as compared to conventional farm’s schedule and had no negative effect on neither yield nor berry traits. The relationship between Tc14-17h, gs and ψ was more robust under drier and warmer years. Correlations between Tc and the parameters ψleaf, gs and An were tested for the two genotypes but no differences were observed. Strategies to optimize the use of thermal imaging in field conditions are proposed.

Key-words: evaporative cooling, diurnal and seasonal water stress, grapevine phenotyping, remote sensing, sustainable water use.

39 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 4 Elucidating specific root phenotypes that arise during metal stress using phytohormones

Stefanie De Smet, Jaco Vangronsveld, and Ann Cuypers Centre for Environmental Sciences, Environmental Biology, Hasselt University, Belgium

In order to survive in a changing environment, plants possess a high level of plasticity, especially at the root system. The root system is the first contact point of the plant with contami- nants, such as metals, in the soil. Therefore, the positioning of these roots is the first factor determining metal uptake and subsequent plant growth. Metal contamination is a very common and widespread type of pollution that forms a major threat to the general public health. However, the effects of metal stress on plant growth and the underlying mechanisms remain poorly understood. We found that roots of Arabidopsis thaliana plants exposed to cadmium (Cd), copper (Cu) or zinc (Zn) showed different morphological responses. Both exposure to Cd or excess Cu stimulated the outgrowth of lateral roots, but Cd inhibited their elongation more severely than Cu. On the other hand, Zn exposure inhibited both lateral root outgrowth and elongation. Micro- scopic analysis of lateral root formation using a cyclin B-GUS reporter line showed that Zn exposure affects the emergence of lateral root primordia as well as the activation of meri- stems. Our results indicate the existence of metal-specific interference with the intrinsic pathways of lateral root development, outgrowth and elongation. The metal-specific root phenotypes were further investigated using heterogeneous metal exposure. Using a split-root system we investigated the ability of the root system to colonise control medium when part of the root system was exposed to metal stress. During Cd and Cu stress the root growth was diminished in the metal-containing zones and stimulated in the control zone, indicating a redistribution respons. For Cd this redistribution respons was only found in lateral roots, while excess Cu induced growth of laterals and primary root. Exposure to excess Zn showed a systemic inhibition of both lateral and primary root growth.

We postulate that phytohormones are involved in the metal-specific phenotypes, since lateral root development is strongly connected with the action of phytohormones. Furthermore, several reports indicate altered phytohormone concentrations or gradients during stress including metal exposure. Analysis of root architecture after homogenous Cd, Cu or Zn exposure of the abscisic acid insensitive abi4-1 mutant indicated that the ABI4 transcription factor may be involved in the inhibition of lateral root emergence after Zn exposure. In addition, we also observed that the Cu-induced increase of lateral root number described for wild-type plants is absent in the abi4-1 mutant. Exposing the ethylene insensitive ein2-1 mutant to Zn showed that EIN2 plays a role in the Zn-induced inhibition of lateral elongation. Finally, using the d14-1 mutant showing dysfunctional strigolactone signalling, we concluded that the D14 receptor is potentially involved in the effects of Cd stress on lateral root elongation.

40 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 5 Budget Remote Phenotyping – Potential and limitations of consumer-grade NIRGB and Red Edge cameras for early detection of plant leaf diseases

Johannes Fahrentrapp1, Martin Geilhausen2, and Patrick Laube2 1 Zurich University of Applied Sciences, Life Sciences and Facility Management, Research Group for Viticulture 2 Zurich University of Applied Sciences, LifeSciences and Facility Management, GIScience research group

Plant leaf diseases can be very destructive and for many crops they lead to lower fruit quality and yield. To safe our harvests and income many preventive pesticides treatments are applied to manage diseases. Early detection could help to substantially reduce pesticide application to healthy crops allowing a site-specific treatment. Diseases in leaves can be detected and specified using hyperspectral images. Some approaches even allow the detection before eye-visible symptoms appear. These imagers are expensive, usually not very handy, and typically not constructed for field use.

We analyzed series of images of Phytophthora infestans and Botrytis cinerea artificially inoculated tomato leaves within their first 48 hours of incubation. Images were taken with two different handheld, low cost Canon Power Shot cameras producing three band JPEGs (near- infra-red, green, blue, and red-edge, green, blue). At 24 hours past inoculation (hpi) first differences could be detected in single band reflectance between healthy (mock-inoculated) and pathogen-inoculated leaf disks. Significant differences could be found in reflectance range, mean and median values as well as in the index pigment specific simple ratio (PSSRb). Cur- rent findings will be presented.

41 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 6 On the value of the individual plant as the unit of evaluation and selection for accurate field phenotyping: three case studies with barley segregating and uniform genetic material

Dionysia A. Fasoula1, Michalis D. Omirou1 1Agricultural Research Institute, P.O. Box 22016, 1516 Nicosia, Cyprus

The concept of field phenotyping of individual plants grown at ultra-wide distances excluding interplant competition is apparently at odds with conventional wisdom which dictates that field phenotyping should be performed at the actual conditions of the farmers’ fields, i.e., at the usual crop densities, considered to be well approximated by the conventional plots. How- ever, there are a number of drawbacks associated with the latter, seemingly reasonable hypothesis, and the breeding community needs innovative solutions to address challenges like decelerating response to selection and reduced stability of yield under the current erratic environmental and climatic changes. The drawbacks are associated with the masking effects of the soil and the associated rhizosphere heterogeneity, the hindering of the full phenotypic expression, the negative correlation between yielding and competitive ability, the limited seed quantities which prevent conventional replications during the early, but critical, segregating generations, and others.

Plants are sessile organisms, so the masking effects of soil heterogeneity and competition are crucially important parameters during evaluation and selection in plant breeding. As a unique compensatory mechanism, plants can offer an extensive and valuable range of phenotypic differentiation, when grown under appropriate conditions. The range of phenotypic differentiation, that remains unexpressed in conventional plots, can be captured in detail through the use of a novel, whole-plant field phenotyping equation with prognostic capabilities. In this presentation we demonstrate the use of the equation, discussing phenotyping data for yield and stability of yield in barley under real breeding situations. The cases presented concern whole-plant phenotyping in segregating generations, as well as in uniform, homozygous materials.

42 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 7 Effect of bHLH gene regulators of anthocyanin pathway of wild strawber- ries (Fragaria vesca L) in the polyphenol content on commercial strawber- ries fruit (Fragaria x ananassa)

Andrea Lorena Herrera V 1 , Katja Schulenburg 2 , Antje Feller 1, Stefan Martens 1 1 Fondazione Edmund Mach, DQAN. Via E. Mach, 1 38010 S. Michele all'Adige, Italy, 2 Technische Universität München

The cultivated strawberry (Fragaria x ananassa) is one of the most important berry of the market not only during the summer period of the northern hemisphere, but all year around, world wide; being Italy one of the top producer countries in the EU. Red coloration on berry fruits is due to the presence of anthocyanin pigments mainly produced during the late stages of the fruit maturation process and is an important phenotypical feature. In the past years the interest on anthocyanins and other phenylpropanoids compounds has increased due to their antioxidant properties, for which beneficial health effects were found in both in vitro and in vivo systems, making plant breeders around the world consider these plant metabolites as an important trait to follow up.

As part of the MYB-bHLH-WD40 complex bHLH transcription factors play an important role in phenylpropanoid biosynthesis by their effect on the regulation of genes involved in the pathway of these compounds, This transcription factor complex has been characterized in several crops from diverse families, including some Rosaceae species, and is also involved in the biosynthesis of polyphenols.

The aim of this study is to evaluate the role of bHLH transcription factors in the biosynthesis of the broad group of polyphenols including anthocyanins and proanthocyanidins and ellag- itanins. We achieve this goal by performing agro-infiltrations on immature fruits of the cultivated strawberry Fragaria x ananassa with constructs that are designed to silence bHLH candidate genes of the wild species Fragaria vesca. Obtained fruits were further investigated on their effect on polyphenols biosynthesis by methanolic extraction, and detection by targeted UHPLC-MS/MS.

43 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 8 The Finnish Plant Phenotyping Infrastructure – the road to high throughput and high precision

Kristiina I.H. Himanen1, Katriina Mouhu1, Tarja Kariola2 and E. Tapio Palva2 1Department of Agricultural Sciences, University of Helsinki, Finland 2 Department of Biosciences, University of Helsinki, Finland

Current bio economy strategies strongly depend on sustainable plant production for food, feed, fiber and biofuel. In this process fundamental and applied plant research approaches should form a single continuum aiming at meeting the demands of the growing world population. To facilitate fundamental plant research and to translate the vast amount of genomic knowledge to crop plant performance we have recently established a National Plant Phenotyping Infrastructure (NaPPI) at the University of Helsinki Viikki campus as a Finn- ish Research Infrastructure (FIRI) platform. The aim of NaPPI is to provide access for the plant research community in Finland and the Nordic region to up-to-date phenotyping facilities. These high throughput facilities accommodate analysis tools for both small model plants such as Arabidopsis, large crops (legumes, cereals, potatoes, berries) and trees (spruce, pine, birch, poplar). Multi-analysis of the plant phenotypes and phenomes will supplement the existing research infrastructures on different molecular omics on campus (transcriptomics, genomics, proteomics, and metabolomics). The system is based on automated plant management, imaging and data processing provided by Photon Systems In- struments (www.psi.cz). The imaging tools allow automated detection of morphological and growth parameters, analysis of photosynthetic capacity as well as many physiological responses. Automation of these research aspects greatly enhances data collection and analysis in addition to making the data more accurate since the imaging tools exceed human capacities for detecting and modelling morphological changes. These high throughput facilities are part of a distributed infrastructure together with the University of Eastern Fin- land high precision unit (www.spectromics.org) that focuses on hyperspectral imaging and multimodal approaches, including chemical imaging. At the University of Helsinki our flagship research projects focus on high throughput screens such as Arabidopsis mutant collections and breeding populations of crops.

44 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 9 Non-destructive root phenotyping of ten alpine plant species in a natural soil system

Csilla Hudek1,2, Craig J. Sturrock3, Brian S. Atkinson3, Silvia Stanchi1and Michele Freppaz1 1 University of Torino, Department of Agricultural, Forestry and Food Sciences, Grugliasco, Italy 2 T2M, Marie Curie Cofund Fellow 3 Hounsfield Facility, School of Biosciences, The University of Nottingham, Leicestershire, England

Mountains reach from the Artic to the Tropics, have an essential role in the World’s water cycle and provide greater biodiversity than lowland areas. Mountain ecosystems are particularly sensitive to land degradation and any recovery is extremely slow or even impossible. Twenty percent of Europe’s native vascular plant species are found in alpine areas above the treeline, covering only 3 % of the entire European continent.

Moraine sites above the treeline are particularly prone to land degradation due to the low level of soil aggregation and scarce vegetation cover. Soil erosion processes, landslides and debris flows are frequently occurring processes on fresh moraine sites. In the process of soil stabilization, vegetation colonization plays an essential role. As the length of the growing season of alpine plants above the treeline is limited to a few months (1-4 month), the major role on soil stabilization is played by the root system of the plants. Increased soil stability of moraine sites above the treeline could potentially reduce the risk of natural hazards occurring at lowland areas where civilians and their properties are at risk.

The objective of this study is to visualize and compare the root architecture of the ten, most dominant moraine species of the Lys valley (NW Italian Alps) in natural soil system by using a non-destructive phenotyping technique to determine plant efficiency in soil stabilization.

Ten alpine plant species with six replicates of each were cored and collected from the moraine of the Lys glacier, in the Monte Rosa massif. One sample of each species (ten in total) was placed in a PVC cylinder and scanned with X-ray MicroCT to non-destructively investigate plant root structure. After the completion of scanning, roots were segmented and quantified using VG Studio Max 2.2 (Volume Graphics GmbH, Germany) image analysis software.

With the MicroCT scanner we were able to visualize the 3D root architecture and gain data on the exact position, orientation and elongation of the root system of the studied alpine species under natural soil conditions. This allowed us to visualize root-soil contact and to obtain data on intact root systems without damaging the roots. We have identified the differences in root architecture between the studied species and found different strategies in plant root architecture to overcome the unstable soil conditions.

45 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 10 High-throughput plant phenotyping at the Vienna Biocenter: VBCF Plant Sciences Facility (VBCF GmbH)

Jakub Jez, Anneliese Auer & Jolanta Ambroz-Kumorowski Vienna Biocenter Core Facilities GmbH, Dr. Bohr Gasse 3, 1030 Vienna

The Vienna Biocenter Core Facilities GmbH (VBCF; formerly called CSF) is a public funded non-profit research institute, situated at the Vienna Biocenter (VBC). We offer access to state of the art research infrastructure and scientific services. VBCF is organized in ten scientific core facilities equipped with cutting-edge instruments and highly skilled technical and scientific personnel.

The VBCF Plant Sciences Facility (PlantS) operates 22 high quality, highly specialized and state-of-the-art plant growth chambers and provides professional support to the outstanding “green research” at the VBC. Several chambers are capable of providing exceptional environmental conditions i.e. low temperature (frost), high temperature, different light intensities, different light spectra (LED: UV375, blue405, blue450, white and red660,730) and different gas conditions (e.g. CO2) allowing precise environmental simulation across different climate zones and the simulation of various environmental stress conditions.

For the objective, reproducible and high-throughput assessment of plant phenotypic traits we operate an automated, sensor-to-plant screening system linked to LemnaTec image analysis software. The phenotyping system is fully integrated into one of the chambers allowing highly precise control of environmental conditions throughout the experiment.

Beside high-throughput screening of Arabidopsis we provide also custom phenotyping services: i.a. low-throughput (side-view) phenotyping of crop plants, screening of seedlings (agar-plates), root phenotyping (agar-plates), phenotyping of seeds and confocal microscopy image analysis.

46 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 11 Integrated analysis of plant growth and development using high throughput multi-sensor platforms at IPK

Astrid Junker, Jean-Michel Pape, Henning Tschiersch and Thomas Altmann Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstr. 3, 06466 Seeland, OT Gatersleben, Germany

Plant phenomics describes automated and high throughput amenable techniques that allow for quantitative monitoring of large plant populations with respect to breeding-relevant traits such as higher yield or biomass accumulation, plant resistance to biotic and abiotic stresses as well as plant nutrient use efficiency. Currently, the IPK runs three plant phenotyping facilities for high-throughput imaging of whole plants of small size (such as Arabidopsis thaliana), medium size (such as Hordeum vulgare) and large size (such as Zea mays, Junker et al. FIPS 2015). In each of these phenotyping systems plant images are acquired in near-infrared (NIR) and visible spectra (for RGB and fluorescence imaging) from top and side views. Imaging is performed repeatedly and thus enables to follow plant growth dynamics over development. The Image Analysis Platform (IAP, Klukas et al. Plant Phys 2014) automatically extracts plant architectural traits (such as plant height and width, projected leaf area, estimated volume), color-related and physiological traits (such as static fluorescence, moisture content- related parameters). The existing high throughput plant phenotyping facilities have been ex- tended for the analysis functional chlorophyll fluorescence using pulsed amplitude-modulated chlorophyll fluorescence imaging systems and for the acquisition of 3D height profiles of plants and plant stands. These systems are integrated into the plant phenotyping procedures and thereby allow for the simultaneous acquisition of multiple complementary plant traits. Co-registration of feature data derived from all different sensors (sensor fusion) will be used to deduce novel and refined information on plant architectural and physiological traits. Op- portunities and challenges of integrated analyses will be discussed using the example of the phenotypic screening of maize natural diversity as represented by the maize Genebank collection at IPK.

47 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 12 Analysis of drought tolerance traits in Solanum tuberosum

Manuela Haas1, Heike Sprenger1, Katharina Rudack2, Jürgen Gremmels1, Sylvia Seddig2, Rolf Peters3, Dirk Hincha1, Craig Sturrock4, Awais Khan5, Renate Horn6and Karin Köhl1

1 Max Planck Institute of Molecular Plant Physiology, Germany 2 Julius Kühn Institute for Resistance Research and Stress Tolerance, Germany 3 Agricultural Research Station Dethlingen, Germany 4The Hounsfield Facility,University of Nottingham, UK 5International Potato Center, Lima, Peru 6 Department of Plant Genetics, University of Rostock, Germany

Climate models predict an increased likelihood of seasonal droughts for many areas of the world. Potato (Solanum tuberosum L.) produces high food energy per volume water invested, but is drought-sensitive. The low drought tolerance results from the shallow root system of potato and its low ability to recover from drought. However, a significant variation in drought tolerance was revealed within the group of European Solanum tuberosum ssp. tuberosum cultivars, when drought effects on tuber starch yield was measured in controlled environments and multisite-multiyear field trials. Based on these results, we selected cultivars of contrasting drought tolerance for in-depth analysis and as parents to generate a population that segregates for drought tolerance. Within the project VALDIS TROST, this population and selected sub- populations are genotyped and phenotyped for metabolite and transcript levels, shoot growth, yield, yield quality under optimal and drought conditions in pot and field trials on three sites for three years. The aim is to identify genomic and phenotypic markers for drought tolerance and to gain further insight into the drought tolerance mechanisms employed by the species.

In a collaborative project with the CIP, the short-term response of shoot growth and turgor to a drying – re-watering cycle is investigated by time resolved measurements with the Fieldscan laser scanning device. By comparing cultivars of contrasting drought tolerance and genotypes from the segregating population we will elucidate, how short-term shoot response contributes to drought tolerance. Detailed analysis of shoot response pattern will identify new phenotypic markers for drought tolerance. These new markers will subsequently be tested in independent population assayed for drought tolerance in pot and field trials in semi-arid areas of the sub- tropics.

The contribution of root reaction to drought under field conditions to tolerance is presently under-investigated. We used X-ray computer tomography to analyze root patterns of four potato genotypes of contrasting tolerance. By time resolved measurement in a drying re- watering regime, we monitor root response during drought recovery. Thus, we will gain insight into genotype, environment and genotype × environment interaction on root development and their contribution to drought tolerance in potato.

Taken together, these methods will enhance our understanding drought tolerance mechanisms and help us to develop strategies for efficient drought tolerance breeding.

48 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 13 Effect of Ppd alleles on phenological characteristics of wheat

Ankica Kondić-Špika1, Dragana Trkulja1, Nikola Hristov1, Ljiljana Brbaklić2, Simon Griffits3

1 Institute of Field and Vegetable Crops, Novi Sad, Serbia 2 Biogranum, Novi Sad, Serbia 3 John Innes Centre, Norwich, UK

The study was conducted at the experimental field of the Institute of Field and Vegetable Crops in Novi Sad (IFVCNS), Serbia in order to determine the effect of different Ppd alleles on phenology and performance of winter wheat genotypes. The material consisted 10 modern Serbian wheat varieties and 54 NILs of cv. Paragon with single, double and triple changes in Ppd alleles. The genotypes were sown in the plot size of 2 m2 (2x1) with 6 rows per plot at the location of Rimski sancevi (45°20`N, 19°51`E). The experiment was carried out in randomized complete block design with 3 replications. Phenology data (heading, flowering and maturity) were collected during the two growing seasons (2014 and 2015).

The results indicated that Serbian varieties (set 1) were significantly earlier regarding the heading, flowering and maturity than original cv. Paragon, from which NILs were produced. Also, the most of the NILs with introgressed single (set 2) and double (set 3) insensitivity and early Ppd-1 alleles were earlier then original cv. Paragon. The differences were higher in heading and flowering time then in maturity. Most of these NILs were still later but more similar to our Serbian cultivars. The NILs with single, double or triple introgressed Ppd-1 null alleles or knock-outs and late alleles (set 4) were significantly later then original cv. Paragon, especially regarding the maturity.

The growing season 2014 had more favourable temperature and moisture conditions from March to July then the season 2015. All indicated differences among the groups of genotypes were more pronounced in the less favourable season 2015. It means that genetic differences in Ppd alleles among the genotypes were activated and expressed to a higher extent in stress conditions. Only the difference between the set 4 and other genotypes regarding the maturity was clearly seen in both growing seasons. PCA analyses have confirmed clear separation of the fourth set of genotypes from others in both growing seasons, while other sets were not completely separated from each other.

In the fourth set of genotypes some lines had heading time as the original cv. Paragon, but they had later maturity. The combination of alleles in these lines could be interesting for our agroecological conditions. One of the goals of breeding programs is to keep varieties as early as possible in heading and flowering but at the same time to have later maturity in order to prolong grain filling and increase the yield. The results have shown that this approach provides the tools for creation of genotypes with specific allelic combinations leading to more appropriate constructions of earliness.

ACNOWLEDGEMENT: The study was conducted as a part of project FP7-KBBE-2011-5: "Genetics and physiology of wheat development to flowering: Tools to breed for improved adaptation and yield potential " (ADAPTAWHEAT, Project Number: 289842).

49 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 14 Field phenotyping of maize growth parameters using UAV acquired high- resolution hyperspectral images

Lammert Kooistra1, Sander Mücher2, Darell van der Voort1, Henk Kramer2, Jappe Franke2, Juha Suomalainen1, Eva Ampe3 and Piet Reyns3 1 Laboratory of Geo-Information Science and Remote Sensing, Wageningen University, The Netherlands; lam- [email protected] 2 Alterra, Wageningen University and Research Centre, The Netherlands 3 Limagrain Nederland BV

Quantitative analysis of plant phenotypes in field trials has become a major bottleneck due to the large amount of breeding trials that need to be scored and analyzed. In controlled circum- stances like greenhouses significant progress has been made using image based techniques in high-throughput phenotyping facilities. The next step would be to adopt camera based technology in field experiments, however, a multitude of factors are influencing these observations: light conditions, wind, moisture on leaves, etc. Both ground based vehicles and aerial platforms have been considered for field-based phenomics. To develop these platforms further, additional progress is required on experimental protocols (spatial, temporal, spectral set- tings) which: 1) optimize the required signal to quantify the relevant trait(s); and 2) which reduces the effect of disturbing factors resulting in robust and reproducible retrieval of trait values including the associated uncertainty.

In this paper we evaluate the usability and accuracy of high-resolution hyperspectral images acquired from an Unmanned Aerial Vehicle (UAV) to quantify the growth parameters biomass and plant height for a large-scale maize breeding trail. Furthermore, this research aims to test which spectral range and growing stage gives the best relation with biomass and plant height in order to find out which sensors are most suited and what flying periods are essential for phenotyping these traits. The field under research consists of a phenotyping experiment with 3838 plots with a size of 10.20 m2 planted with different maize (Zea Mays) cultivars. Bi- omass and height measurements of each cultivar were done at harvest. At four different growth stages, a multi-rotor system equipped with a combined RGB and hyperspectral camera, resulting in three geo-rectified products: RGB-orthomosaic, digital surface model and a hyperspectral dataset with 100 bands ranging from 450-950 nm. In the subsequent method development, both individual spatial images and also the fusion (including time) was evaluated using vegetation indices and multivariate statistical approaches.

For the spatial estimation of final height, a good accuracy (R2 = 0.70) could be achieved when compared to the field observations. For the estimation of final biomass, the prediction accuracy is R2 = 0.74 when the spatial layer of the canopy plant model is combined with spectral information from specific parts of the growing season in a multivariate approach. We will also discuss the uncertainties introduced due to limitations in geo-rectification and effects of changing illumination conditions.

50 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 15 Leaves color and morphometric analysis of plants in responses to stress: the high-throughput phenotyping approach

Marek Kovar, Katarina Olsovska, Marian Brestic, Marek Zivcak, Kristyna Kunderlikova and Oksana Sytar Slovak University of Agriculture, Slovak Republic

Drought is one of the most important abiotic stress situation decreasing the crop productivity worldwide. Using PlantScreenTM phenotyping platform in AgroBioTech Research Center in Nitra, Slovakia, have been studied growth dynamics of different genotypes sorghum and soybean in response to drought. Calculated morphometric parameters from RGB imaging in top and side view (with different angles) positions (such as plant area, growth height, relative growth rate, plant perimeter, compactness), as well colour segmentation and greening index were used to discriminate between genotypes with different drought tolerance. The study was completing by measurements of leaf RWC and osmotic potential, concentration of chlorophylls (spectrophotometrically and SPAD-index) and calculation of water use efficiency. De- tail morphometric analysis showed that sensitive genotypes both crops respond to drought by rapid decline of growth which was accompanied by stress-induced senescence and leaf chlorophyll breakdown. Colour segmentation on leaf area have been used on calculation of greening index via the user defined algorithm clusters hues of green and chlorotic areas on leaves. This technique allowed to show the dynamics of greening during plants ontogeny in well- hydrated conditions as well as the effect of drought on the start of senescence of assimilation organs. Calibration of the selected scale of greens with spectrophotometrically determined concentration of chlorophylls (and SPAD-index) allows the indirect evaluation of assimilatory pigments from RGB images. Therefore, the RGB imaging became an important tool in high- throughput plant phenotyping and the final morphometric analysis can be reliably used to assess growth dynamics and plant fitness under stress situation.

This work was co-funded by European Community under project No. 26220220180: Building Research Centre, AgroBioTech".

Keywords High-throughput phenotyping, RGB imaging, greening index, sorghum, soybean, drought

51 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 16 Crop phenotyping of forage grasses using a Remotely Piloted Aircraft Sys- tem: assessment of sward density in a breeding context

Peter Lootens1, Jonas Aper1, Tom De Swaef1, An Ghesquiere1, Koen Mertens2, Joost Baert1, Jürgen Vangeyte2, Isabel Roldán-Ruiz1 1 ILVO, Plant Sciences Unit, Caritasstraat 39, B-9090 Melle, Belgium 2 ILVO, Technology and Food Sciences Unit, Burg. Van Gansberghelaan 115 Bus 1, B-9820 Merelbeke, Belgium Corresponding author: [email protected]

Close remote sensing using Remotely Piloted Aircraft Systems (RPAS) equipped with a camera offers a huge number of possibilities for phenotyping crops in the field. Hereby, individual plants, plots, but also entire crops can be phenotyped in a frequent, objective and non- destructive manner. Compared to remote sensing it has several advantages, of which a higher temporal and spatial resolution is the most important.

Therefore ILVO has established a Precision Crop Farming platform (PCF-ISense) to explore the practical applicability of RPAS technology with the appropriate sensors for the farmer (related to crop management) and breeder (related to plant and crop phenotyping).

Within the ILVO perennial ryegrass breeding program, the evaluation of sward density after 2-3 years of mowing is an important breeding target because it is a major component of sward quality. A quick deterioration of the sward quality is economically undesirable because it forces the farmer to renovate his grassland. In general, tetraploid perennial ryegrass has a lower sward density than diploid perennial ryegrass.

In this framework, we established a field experiment in April 2013 that consists of 294 plots of four diploid perennial ryegrass trials and 360 plots of four tetraploid perennial ryegrass trials. The trials were composed of varieties and breeding material such as half-sib families in two or three replications. In October 2015, we monitored sward density of the experiment using a visual camera attached to a RPAS.

From these images an orthophoto was constructed. Dedicated image analysis allowed to determine several coefficients related to ground cover per plot. Correlations up to r = 0.78 were found between the scores given by the breeder and the image analysis derived coefficients. The borders within the trial also allowed to evaluate field variability, which again can be used to optimize the statistical analysis of the whole field trial.

52 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 17 Selection of wavelengths for quantification of nitrogen concentration in winter wheat by multispectral vision

Marlier Guillaume, Mercatoris Benoit, Leemans Vincent, Destain Marie-France Department of Biosystem Engineering, Gembloux Agro-Bio Tech, University of Liège, 2 Passage des Déportés, 5030 Gembloux (Belgium)

Hand-held sensors (SPAD meter, N-Tester, …) are used for detecting the leaves nitrogen concentration (Nc) on basis of an optical detection of the chlorophyll concentration. These devices are active sensors: an internal radiation source emits light and transmission through a leaf is measured in the red (650 nm) and in the near-infrared (920 nm) spectral regions. These devices present several drawbacks. The nitrogen concentration is gained by an indirect way through the chlorophyll concentration and the leaves have to be fixed in a defined position for the measurements. These drawbacks could be overcome by an imaging device that measures the canopy reflectance. Hence, the objective of the paper is to analyse the potential of multispectral imaging for detecting nitrogen concentration.

The tests were carried out on parcels submitted to nitrogen inputs varying from 0 to 180 kg N.ha-1. Reference Nc measurements were obtained by the Kjeldahl method and a Hydro N- Tester (Yara). The developed imaging system comprised a CMos camera and a set of 22 interference filters ranging from 450 to 950 nm mounted on a wheel steered by a stepper motor. The image acquisition and the motor rotation were controlled by a program written in C++. The crop was imaged vertically at one meter height. The raw images presented 1280*1024 pixels covering an area approximately 0.5*0.4 m and were recorded with a 12 bits luminance resolution. To deal with the natural irradiance variability of the scene, a white reference was used and the integration time was automatically adjusted for each image. The image treatment included the segmentation of Photosynthetically Active Leaves (PAL) by using Bayes theo- rem and the computation of the mean PAL reflectance after correction of background and illumination fluctuations. Nc was estimated on basis of the 22 filters by Partial Least Square (PLS) method and by four filters selected by Best Subset Selection (BSS).

In comparison with the Kjeldahl method, the estimation of Nc by the Hydro N-Tester, the PLS and the BSS (filters 600-80, 950-100, 650-40 and 450-80 nm) gave determination coeffi- cient and standard error respectively equal to of 0.53, 0.29 %; 0.67, 0.21%; 0.56 and 0.25%. This indicated that the full multi-spectral approach gave significantly better Nc estimation than a portable device and suggested that a camera equipped with four filters would give similar results.

53 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 18 Modelling the effects of climate change on crops: phenotyping the com-

bined effect of CO2, temperature and drought on wheat

Thayna Mendanha, Benita Hyldgaard, Carl-Otto Ottosen

Plant, Food, Climate Department of Food Science Aarhus University.

The ERAnet project ‘MODCARBOSTRESS’ started in 2015 and aims to revisit key modelling hypotheses as well as parametrizing models under stress combinations. The project is being conducted by an international consortium of seven partners working in either manipulat- ing stress combinations under phenotyping platforms or in modelling and uses wheat and rapeseed as core species. During 2015 we analyzed the morphological responses of combined effect of [CO2], temperature and drought on two wheat cultivars (‘Paragon’ and ‘Gladius’).

In nature, several stress factors act simultaneously. Drought and heat often occur con- comitantly, but can have very different effects on various physiological, growth, developmental, and yield processes. The combination of multiple stresses can have a significantly higher detrimental effect when compared to each of the stresses applied individually, but when combined with elevated [CO2], counteracting factors might potentially alleviate stress.

Single seeds per pot from the two cultivars were sown in 11cm plastic pots filled with a peat based potting mix and grown in a greenhouse at ambient CO2 for 2.5 weeks prior to the experiment. Plants were moved to controlled climate chambers in a complete randomized design and pre-acclimated for one week before the swapping between temperatures. The plants were divided into six subsets: Subset 1: cold control (18/14°C); 2: warm swapped to cold; 3: cold + drought, 4: warm control (28/24°C), 5: cold swapped to warm, 6: warm + drought. The [CO2] treatments were separated in time and performed during two rounds of the experimental setup: 400 ppm and 800 ppm.

Activities of this task included the investigation over the following parameters: Plant Height (PH), Fresh weight (FW), Dry weight (DW), leaf area (LA). The biomass and growth parameters were found to be significantly cultivar dependent. For both cultivars, the elevated temperature subset exhibited a negative effect on all studied growth parameters (PH, FW, DW and

LA). Elevated [CO2] decreased the vegetative biomass significantly (FW, DW) but had no effect in PH and LA. Drought stress increased PH for plants of Gladius cultivar but shown no significant effect in Paragon plants. Biomass parameters suffered a negative effect under all drought stress treatments. The plants submitted to higher temperature treatment suffered a more severe reduction in biomass parameters then the ones under the combination of drought + heat. Plants swapped from cold to warm presented the most significant reduction in biomass for both CO2 concentration studied.

54 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 19 The application of agriculture remote aerial sensing in Serbia

Sanja Mikić1, Dušan Stanisavljević1, Rastko Čarapić2, Nebojša Pavičić2, Bojan Mitrović1, Miroslav Zorić1, Milan Dobrota2 1 Institute of Field and Vegetable Crops, Maskima Gorkog 30, Novi Sad, Serbia 2 Logit d.o., Bulevar Milutina Milankovića 25b, 11070 Belgrade, Serbia

The modern agriculture is facing many challenges such as farmland degradation, soil erosion, nutrient excess and insufficiency, drought, flooding, weed, disease and pest outbreaks, poor seed quality and emergence, etc. To address these problems in a cost-effective and environmentally-sound way, new information-driven technologies are being increasingly implemented in the last several years. One of them is remote sensing, an integrated part of precision agriculture, that facilitates surveying large farm areas, gathering data in near real- time manner, and processing and analysing data using statistical methods and machine learning algorithms. Despite its great possibilities, the application of remote sensing is still largely exclusive to developed countries and dependent on the technology providers. Moreover, it requires certain skills for its utilisation, which farmers often lack. To make this technology more accessible to the end users in the agricultural sector on Balkans and South East Europe and less dependent on foreign developers, a one-year European Commission funded FRACTALS (Future Internet Enabled Agricultural Applications) FP7 project for ICT SMEs was granted to the AREAS (Agriculture Remote Aerial Sensing) project. The granted SME Logit from Serbia developed technology platform for gathering, processing and analysing large amounts of data, improving remote sensing processes, giving the output that addresses specific agriculture problem solving, thus increasing the productivity and yields on farms and significantly reducing the costs and risks. After obtaining aerial images with the right spatial resolution and wavelength, in the right time, they have to be pre-processed, using specific algorithms and advanced statistical methods, with initial expert corrections using suitable vegetation indices. For the AREAS platform to be complete and comprehensive, the project involves UAVs acquired images. The outputs of the platform include crop counting for sowing quality assessment and yield estimation, weed identification and early detection for optimization of herbicides application and crop stress monitoring for disease and pest occurrence or water shortage based on leaf and canopies changes. The evaluation and application of the product started at the pilot scale with assistance of the Institute of Field and Vegetable Crops, one of the two largest agriculture research institutes and state-owned seed companies. The institute also provides a network of extension service throughout Serbia that serves as a knowledge transfer and a link between scientists and farmers. The technology proved to be efficient in detecting poor emergence rate, plant counting and weeds. Since the relatively late start of the project in 2015, the more detailed trials were planned for 2016 that include soil type and fertility evaluation, nutrient uptake and biotic stress identification and assessment. The aim of this paper was to present the results of the endeavours to develop and apply cheap and self-sufficient technology that would accommodate needs of farmers, seed companies and agriculture researchers in developing countries.

55 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 20 Do specialized metabolites improve drought tolerance in crops?

Lucia Montini1, Elizabeth H Neilson2, Birger Lindberg Møller1,2 and Nanna Bhjarnholt1

1 Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, Univer- sity of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2 VILLUM Research Center for ”Plant Plasticity”, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Sorghum bicolor is the fifth most important cereal crop in the world and a key model plant to study environmental stress due its natural tolerance to drought and high temperatures. This tolerance is due to factors such as C4 anatomy, root structure and stay-green, however, it may also be due the presence of specialized metabolites belonging to the cyanogenic glucosides family. It has been long suggested that the most abundant cyanogenic glucoside found in Sor- ghum, dhurrin, serves a dual purpose in the plant as defence compounds to adapt to the biotic damages or abiotic stresses of changing environment as well as nitrogen and carbon source produced during its turnover. Under certain conductions, such as drought, the content of dhurrin increases. This may suggest that dhurrin is used as a nitrogen source, which could be remobilized when the plant is re-watered. Considering that many crop plants are cyanogenic, sorghum can be used as a model plant to clarify how cyanogenic glucosides and their derivatives are regulated and metabolized in plant and identify their role in plant drought stress response. For this project, various lines sorghum – differing in cyanogenic glucoside content – were grown at The Plant Accelerator ® under drought stress conditions for a period of 4 weeks and then were re-watered for a further week. The Australian Plant phenomics facilities were used to monitor plant growth and performance, and identify specific traits associated with improved drought tolerance and recovery. In addition, the phenomic platform enabled the identification of key time points to harvest material for metabolomic analysis to gain an in-depth understanding of the role of dhurrin- and its derivatives under environmental stress.

56 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 21 Using image analysis to study the day-length effect on anatomical stem traits of flax genotypes (L. usitatissimum L.)

Janka Nozkova1, Andrey Pavlov2, Miroslava Savrtkova1, Vladimir Koshkin2, and Nina Brutch2 1 Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Genetics and Plant Breeding, Tr. A. Hlinku 2, 949 76 Nitra, [email protected] 2 The Federal Research Center the N.I. Vavilov All-Russian Institute of Plant Genetic Resources, B.Morskaya str., 44, St. Petersburg, Russia

Fiber flax is known as one of the oldest crops with dual purpose for natural fibers and oil seeds production. This crop is widespread throughout the world, so it is necessary to identify differences in fiber content between genotypes growing under different environmental conditions. This experiment was designed to identify basis of variation in fiber content and to understand the stem anatomical differences between 13 selected genotypes of fiber flax (L. usitatissimum L.) grown under various conditions of day-length, with use of image analysis.

The genotypes were grown under conditions of short (12 hours) and long photoperiod natural for 60o N (17.5 to 19 hours) in vegetative pots (10 plants per 5 liter pot) where other environmental factors were equal. For each genotype stem cross-sections of 10 plants were taken from the middle part of stem. From stem cross-sections were taken images by fully automatic macroscope Zeiss Discovery V20 with digital Camera AxioCam MRC5. Images were analyzed using software for image analysis AxioVision 4.8 with module for automatic measuring. The images were adjusted using various tools such as thresholdings, dynamic, events to get the most accurate results. To identify differences between selected genotypes, in total 6 anatomical parameters were analyzed namely, number of bast fiber bundles (n), number of elementary fibers (n), total area of bast fiber bundles (µm2), total area of stem cross-section (mm2), total area of xylem (µm2), and total area of pith (µm2). To get more quantitative parameters we used automatic measurement software tools and on mentioned analyzed parts of stem cross-sections were evaluated these parameters – perimeter, area, bound width and bound height. For statistical analysis was used software SAS 9.3. Basic statistics of the obtained data showed wide variation in the values for all the genotypes and for all the traits in this study. All data were subjected to ANOVA and significant differences (p < 0.05) were found among genotypes and different photoperiod conditions.

The genotypes grown under long photoperiod conditions got higher average values in traits: number of bast fiber bundles, number of elementary fibers, and total area of bast fiber bundles. Significant differences between short- and long- photoperiod conditions were confirmed on the evaluated traits only in some genotypes. But in our experimental collection were also genotypes where significant differences between various photoperiod conditions on the anatomical stem traits have not been confirmed. Even so the flax is the long-day species, these genotypes are interesting as the base material for further breeding of fiber varieties suitable for regions with short photoperiod conditions.

57 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 22 Ecotype-level differences in the electron flow under desiccation and rehydration of the resurrection plant Haberlea rhodopensis

Kenny Paul 1, Imre Vass1, and Ádám Solti2 1Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62. Szeged, 6726 Hungary 2Department of Plant Physiology and Molecular Plant Biology, Institute of Biology, Eötvös Loránd University, Pázmány Péter stny. 1/C, Budapest, 1117 Hungary.

Resurrection plants possess vegetative tissues that are able to tolerate severe dehydration and resume normal physiological function upon rehydration. Haberlea rhodopensis is a homoio- chlorophyllous poikilohydric plant which preserves most of its chlorophyll content and photosynthetic apparatus during desiccation. Besides the more studied shade-acclimated ecotype, ecotypes growing on habitats of high irradiance were also discovered. Although recent investigation indicated a better physiological and morphological adaptation to desiccation, the photosynthetic functions have not been analysed in details yet. During desiccation and rehydration, high and low irradiance-acclimated ecotypes were subjected to fluorescence relaxation kinetics and chlorophyll a fluorescence transient measurements by a double-modulation fluo- rometer, together with simultaneous measurements of chlorophyll a fluorescence and P700 oxidation by a Dual-PAM-100 system. Under drought stress cyclic electron flow is enhanced, whereas under severe desiccation, the Photosystem II becomes inactive and acceptor-side limitation factors start to inhibit the Photosystem I function in the high irradiance acclimated ecotype. Under rehydration, the photosynthetic function recovers rapidly together with an increase in cyclic electron flow. In contrast, the shade ecotype only showed an increased cyclic electron flow under severe desiccation and upon rewatering, all the recovery processes proved to be slower. Therefore, growth conditions and acclimation processes in the natural habitat strongly influence the desiccation tolerance.

Keywords cyclic electron flow; Haberlea rhodopensis; photochemistry; resurrection plant; desiccation tolerance

58 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 23 Physiological phenotyping of plant responses to post-anthesis drought and

elevated CO2

Dorthe Horn Larsen, Xiangnan Li, Fulai Liu, Eva Rosenqvist University of Copenhagen, Faculty of Science, Department of Plant and Environmental Sciences, Højbakkegaard Allé 9, DK-2630 Taastrup, Denmark

The concentration of atmospheric CO2 has been steadily increasing for decades while crop productivity is increasingly threatened by extreme climate events. If drought occurs during grain filling it is known to have a detrimental effect on grain yield, whereas an increase in

CO2 may result in a higher yield and biomass especially in C3 crops. Current methods of in- house plant phenotyping are providing a new tool to detect the early responses to abiotic stress and predict the potential stress effect on yield. A pot experiment with four elite spring wheat (Triticum aestivum L.) cultivars (‘Paragon’, ‘Cadenza’, ‘Gladius’ and ‘Yecora Rojo’) was conducted in a climate-controlled greenhouse, in which drought stress and well-watered treatments were given at ambient (400 ppm) and elevated (800 ppm) CO2. The plants were watered with nutrient solution. The drought treatment was applied during grain filling, where water was withheld for 14 days reaching 25-30% of pot holding capacity at day 7. Non- invasive or rapidly measured parameters, such as imaging of plant phenotypes, stomatal conductance (gs), chlorophyll content, gas exchange for Vcmax and Jmax, and water status parameters were investigated. The leaf water potential and relative water content were significantly reduced by drought, while no significant effect of elevated CO2 on plant water status was found. However, drought significantly decreased the grain yield of all cultivars, mainly due to a lower spike number and grain number per spike. Drought stressed plants of ‘Paragon’, ‘Ca- denza’ and ‘Yecora Rojo’ increased the grain yield by 30.3, 24.9 and 30.8%, respectively in elevated CO2, while ‘Gladius’ showed a slight decrease compared to ambient CO2. Collec- tively, the results indicate that elevated CO2 can alleviate the effect of drought on yield in wheat.

During the drought treatment periods all plants were watered with water to avoid differences in nutrient availability between the treatments. This resulted in nitrogen deficiency symptoms in some cultivars, which prevented them to fully respond to the elevated CO2. This illustrated the problems involved in phenotyping for stress tolerance in future climate scenarios where other abiotic resources than the studied may become limiting.

59 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 24 Exploring the rhizosphere: Imaging root-soil interactions using X-ray Computed Tomography

Saoirse Tracy*1, Malcolm Bennett2, Tony Pridmore2, Craig Sturrock2, Sacha Mooney2 1School of Agriculture, University College Dublin, Ireland 2University of Nottingham, United Kingdom *Contact: [email protected]

Although roots play a crucial role in plant growth and development through their acquisition and delivery of water and nutrients to the above-ground organs, our understanding of how they interact with their immediate soil environment largely remains a mystery as the opaque nature of soil has prevented undisturbed in situ root visualisation. The spatial arrangement of roots and the soil structure are extrinsically linked to the overall productivity of a plant, as they control the ability of a plant root to extract essential resources for growth. In a world with a rapidly increasing population and the threat of climate change maximising plant productivity is vital. Therefore the visualisation and quantification of root growth in soil is needed to understand plant root growth dynamics. The use of non-invasive techniques such as X-ray Computed Tomography (CT) means that it is now possible to visualise a growing root within an undisturbed soil core. X-ray CT is a technique that enables non-destructive 3-D investigations into root:soil interactions at the micro-scale. By imaging the actual 3D geome- tries of the soil structure and visualising the interfaces between roots, soil, water and air filled pores an accurate representation of water movement and root growth in soil is achieved. Pre- vious commonly employed destructive methods for root studies, such as root washing could not provide detailed information on root architecture, including branching characteristics and extension rate, which are inherently linked to conditions within the soil matrix. Therefore this technique can enable root phenotyping of different crop species and varieties in soil. This information is crucial if laboratory research is to be translated to an understanding of responses under field conditions.

60 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 25 Photosynthesis-related features of willow (Salix spp.) plants in response to waste water sludge treatments

Ivaylo N. Tsvetkov1, Hristina Gr. Hristova1, Svetla Marinova2, Vera Z. Petrova2 and Nikolina P. Tzvetkova3 1 Forestry Research Institute, 132, Kl. Ohridski Blvd., Sofia 1756, Bulgaria 2 Institute of Soil Science Agrotechnology and Plant Protection”N. Poushkarov”, 7, Shosse Bankia Str, Sofia 1080, Bulgaria 3 University of Forestry, 10, Kl. Ohridski Blvd, Sofia 1756, Bulgaria

Some basic photosynthesis-related parameters of young willow plants subjected to waste water sludge treatments were studied in the context of following general biomass productivity. Two willow clones (S. alba. L and S. viminalis rubra) were included in a field trial (on Ver- tisoil) and only S. alba was used in a pot experiment with different doses of sludge from waste water treatment plants (WWTPs). Photosynthetic characteristics (net CO2 assimilation, transpiration, stomatal conductance) were measured together with studying important bio- metric indexes (height, diameter, number of shoots, fresh and dry weight). The physiological measurements in the pot experiment were carried out in two subsequent vegetation seasons and during the second vegetation season of the field trial, with all the aboveground biomass being cut at the end of the first season. While sludge treatment dose significantly (p<0.01) affected the photosynthetic activity of willow plants during the first year, no such effect was noticed during the second vegetation season. Both stomatal conductance and transpiration of pot plants were not influenced by the sludge treatment. Physiological performance of the white willow (S. alba L.) plants was strongly influenced by the waste water sludge additive applied in field conditions. All studied photosynthesis-related parameters followed a trend of decreasing values with raising the treatment dose. The hybrid clone (S. viminalis rubra) displayed similar values of net CO2 assimilation in all variants, while both stomatal conductance and transpiration at the maximal treatment dose (6t/dka) were significantly (p<0.05) higher than those of the control variant. Based on the biometrics data obtained, the hybrid willow clone was nearly twice more productive than the white willow.

61 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 26 Prioritizing root system architecture QTLs for marker-assisted selection in durum wheat grown across a broad range of water regimes

Marco Maccaferri1, Walid El-Feki1 , Nazemi Gashemali1,2, Silvio Salvi1, Maria Angela Canè1, Chiara Colalongo1, Sandra Stefanelli1, Roberto Tuberosa1 1Department of Agricultural Sciences, University of Bologna, 40127 Bologna, Italy 2Department of Agriculture, Haji Abad Branch, Islamic Azad University, 21100 Haji Abad, Iran

Optimization of root system architecture (RSA) traits for capturing soil moisture and nutrients is an important objective for modern wheat breeding. Linkage and association mapping for RSA evaluated in seedlings of two recombinant inbred line populations and one association mapping panel of 183 elite durum wheat (Triticum turgidum L. var. durum Desf.) evidenced 20 clusters of QTLs for root length and/or number as well as 30 QTLs for root growth angle (RGA). QTLs were mapped on a high-density tetraploid consensus map based on a transcript- associated Illumina 90K SNP assay developed for bread and durum wheat, thus allowing for an accurate cross-referencing of RSA QTLs between tetraploid and hexaploid wheat. Among the main QTL clusters for root length and number highlighted in this study, 15 overlapped with QTLs for multiple RSA traits reported in bread wheat while out of 30 QTLs for RGA, only six showed colocation with previously reported QTLs in wheat. Based on their relative additive effects, allelic distribution in the AM panel and co-location with QTLs for yield and kernel weight across 15 environments with a broad range in soil moisture, the RSA QTLs have been prioritized in terms of breeding value. Three major QTL clusters for root length and number and five QTLs for RGA appear particularly valuable for further characterization towards their positional cloning and possible deployment in marker-assisted selection to enhance yield and yield stability.

62 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 27 Phenotyping of a maize introgression library reveals ample genetic variability for root architecture, water-use efficiency and grain yield under different water regimes

Silvia Giuliani1, Silvio Salvi2, Giuseppe Sciara1, Riccardo Bovina1, Mariangela Cané1, Claude Welcker2, Lorenç Barera-Bosquet2, Antonin Grau2, François Tardieu2, Pierluigi Meriggi3 and Roberto Tuberosa1 1 Department of Agricultural Sciences, University of Bologna, Italy 2 Laboratory of Plant Ecophysiological Responses to Environmental Stresses, INRA, Montpellier, France 3 Horta srl, Piacenza, Italy

The genetic dissection of root system architecture (RSA) provides valuable opportunities towards a better understanding of its role in determining yield under different water regimes. To this end, a maize introgression library comprised of 75 BC5 lines derived from the cross between Gaspé Flint (an early line; donor parent) and B73 (an elite line; recurrent parent) were evaluated in two experiments conducted under well-watered and water-deficit conditions (WW and WD, respectively) in order to identify QTLs for RSA, biomass accumulation (BA), water use efficiency (WUE), flowering time (FT), grain yield (GY) and yield components (YC). RSA features at maturity (shovelomics), FT, GY and YC were investigated in the field while BA and WUE were measured in plants grown in the high-throughput phenotyping platform PhenoArch (INRA, Montpellier). QTLs were identified for all investigated traits. Sever- al QTLs for FT affected also RSA, BA, WUE, YC and/or GY, thus underlying the pivotal role of phenology in controlling morpho-physiological traits and yield components. For several QTLs, a sizeable QTL x water regime interaction was evidenced in both experiments. A particularly interesting QTL for WUE in both WW and WD conditions was mapped on bin 9.01, with Gaspé Flint contributing the beneficial allele. Strong concurrent QTL effects on RSA and YC were identified on bin 1.03, 2.04 and 3.03. The most interesting QTLs are being considered for fine mapping and positional cloning.

63 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 28

Investigation of the Interaction Between Gibberellin (GA3), Hydrogen Per- + oxide (H2O2), K and Antioxidant Defense Mechanism in the First Inter- node Elongation of Triticum aestivum cv. Tir

Tolga Yalçınkaya1, Ismail Turkan1, and Askim Hediye Sekmen1 1Department of Biology, Faculty of Science, Ege University, Bornova, 35100, Izmir [email protected]

Plants develop several mechanisms to cope with limited water conditions. Deep sowing tolerance is the one of the most efficient way to avoid drought, which can be described as the ability of the elevation of the shoot apical meristem above the soil surface by elongation of meso- cotyl or first internode. In the literature, there are only a few reports concerning the first internode elongation. Recent studies have showed that reactive oxygen species (ROS) also have emerged as key factors of plant growth and development. However, there is no study investi- gating the relationship between ROS, gibberellin (GA3), H2O2 and first internode elongation. + With this aim, we tried to characterize the interaction between GA3, ROS, H2O2 K and antioxidant machinery in the internode elongation of a deep sowing tolerant wheat variety cultivated in Turkey, namely Triticum aestivum cv. Tir. Therefore, wheat seeds were sown depths to 2 cm and 10 cm of soil. For GA3 and Uniconazole treatments (GA3 biosynthesis inhibitor), they dissolved in dimethyl sulfoxide was added to the 1 % agar medium at a concentration of -6 -7 2.89 × 10 M and 2 × 10 M. To determine increment or inhibition effect of H2O2 on the first internode elongation, H2O2 treatment was achieved by soaking the seeds for 24 hours in sev- + eral H2O2 solutions at different concentrations (0.05, 1, 10 µM, and 80 mM). K (1.5 mM) applied via added to %1 agar medium. At 10 days, both first internode and coleoptile length of Tir wheats were measured. H2O2 content and the activities of antioxidant enzymes in the first internodes of Tir were determined. On the other hand, expression levels of the GA3 biosynthesis genes (Ta20ox1 and Ta30ox2), semidwarfing genes (RHT-A1, RHT-B1 and RHT- D1) and GAMyB, transcription factor were determined. epidermal cell lengths in the first internode of all groups were measured. To our knowledge, this is the first study that concerns + the relationship between GA3, H2O2, K and antioxidant defence system in the first internode elongation under deep sowing.

64 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 29 Plant architecture measurement using a 3D reconstruction method suitable for high throughput plant phenotyping

Rick van de Zedde1, Gert Kootstra1, Gerwoud Otten1 and Franck Golbach1 1 Wageningen University & Research Centre, The Netherlands

In plant phenotyping, there is a demand for high-throughput, non-destructive systems that can accurately analyze various plant traits by measuring features such as plant volume, leaf area, and stem length. Existing vision-based systems either focus on speed using 2D imaging, which is consequently inaccurate, or on accuracy using time-consuming 3D methods. Within the EPPN project – WP3 Novel sensors - we developed a 3D setup and computer vision software. The objectives were to: • A new system combining both accuracy and fast three-dimensional (3D) reconstruction, suitable for high throughput plant phenotyping. • Development of image processing methods for the identification and segmentation of plant organs (stem and leaf) from the 3D plant model. Various measurements of plant features were obtained from the 3D model such as plant volume, leaf area, and stem length. • Evaluation of the accuracy of our system by comparing the measurements of our methods with ground-truth measurements obtained destructively.

To establish the ground truth, we manually cut individual leaves and the remaining stem and positioned them on a flatbed scanner. The resulting scans were used to calculate the ground- truth measurements. The results indicate that the proposed system is very promising.

65 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 30 Screening for drought tolerance in a crossing population of garden roses.

Marie-Christine Van Labeke1, Katrien De Dauw1, Hannes De Deurwaerder1, Leen Leus2 1 Department of Plant Production, Ghent University, Coupure links 653, 9000 Gent, Belgium 2 Plant Sciences Unit, Institute for Agricultural and Fisheries Research, Caritasstraat 39, 9090 Melle, Belgium

Water availability and plant resilience to water deficit is also an issue in ornamental plant production. Rose is the economically most important ornamental plant and differences in drought tolerance between garden roses are reported. The aims in breeding are often morphological and anatomical changes as frequently observed in numerous crops after chromosome doubling. However, polyploids can also exhibit a number of novel physiological characteristics as e.g drought tolerance. We studied drought tolerance in a crossing population of Rosa wichurana x Rosa ‘Yesterday’ (garden rose).

In a preliminary study 69 genotypes of this population were screened for drought sensitivity. Based on the relative water content (RWC) of detached leaves and the predawn leaf water potential (ψ) in a greenhouse experiment, the genotypes were assigned to a drought tolerant, a drought sensitive or an inconclusive category.

Based on these results a distinct set of representatives of each category (10 genotypes in total) were polyploidized and both diploid and tetraploids were used for a further drought experiments For each genotype, ten replicates were selected for uniformity and allocated to a treatment (control or drought stress). After an acclimatisation period of two weeks, a complete irrigation stop was imposed on the plants contained in the drought treatment and this for a period of four weeks. It was not possible to maintain the volumetric water content of the sub- strates in the drought treatment uniform as genotypes had a different vigour. The above ground dry biomass (shoots and leaves) was determined after four weeks. Using DW, various stress tolerance indices were determined and differences in drought tolerance could be evaluated using these indices.

For genotypes with a similar vigour stomatal conductance was an efficient tool to screen for drought tolerance. Comparing mild (deficit irrigation) and severe drought stress (irrigation stop) we found that osmotic adjustment (proline, soluble sugars) only occurred under severe drought (volumetric water content of the substrate below 15 vol%).

66 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 31 Mining natural variations of photosynthesis to optimize ’biomass and grain yield’ in contrasting wheat cultivars under severe drought

Imre Vass1, Kenny Paul1, János Pauk2, Zsuzsanna Deák1, László Sass1, 1Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, H-6726 Szeged, Tem- esvári krt. 62, Hungary 2Department of Biotechnology, Cereal Research Non-Profit Ltd., H-6726 Szeged, Alsó kikötő sor 9, Hungary

We report a case study of ntural variations and correlations of some photosynthetic parameters, green biomass and grain yield in Cappelle Desprez and Plainsman V winter wheat (Triti- cum aestivum L.) cultivars, which are classified as being drought sensitive and tolerant, respectively. We monitored biomass accumulation in the vegetative phase and grain yield in the grain filling period. Interestingly, we observed higher biomass production, but lower grain yield stability in the sensitive Cappelle cultivar, as compared to the tolerant Plainsman cv. High biomass production in the sensitive variety was correlated with enhanced water use efficiency. Increased cyclic electron flow around Photosystem I was also observed in the Cap- pelle cv. under drought stress as shown by light intensity dependence of the ratio of maximal quantum yields of Photosystem I and Photosystem II, as well by the plot of the Photosystem I electron transport rate as a function of Photosystem II electron transport rate. Higher CO2 uptake rate in flag leaves of the drought stressed Plainsman cv. during grain filling period corre- lates well with its higher grain yield and prolonged transpiration rate. The increase in drought factor (DFI) and performance (PI) indices calculated from variable chlorophyll fluorescence parameters of secondary leaves also showed correlation with higher biomass in the Cappelle cultivar during the biomass accumulation period. However, during the grain filling period, DFI and PI parameters of the flag leaves were higher in the tolerant Plainsman V. cultivar and showed correlation with grain yield stability. Our results suggest that overall biomass and grain yield may respond differentially to drought stress in different wheat cultivars and therefore phenotyping for green biomass cannot be used as a general approach to predict grain yield.

67 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 32 Phenotyping for drought tolerance in lettuce: the limits and possibilities of chlorophyll fluorescence imaging

Marek Zivcak1, Marian Brestic1 and Klaudia Bruckova1 1 Department of Plant Physiology, Slovak University of Agriculture, Nitra, Slovakia

Chlorophyll fluorescence imaging (CFI) represents an important technique involved in numerous phenotyping platforms. CFI is expected to serve as a measure of photosynthetic functions, especially in studies of genotype × environment interactions. In our study, we tested opportunity to apply CFI to recognize drought sensitivity in lettuce genotypes. Eight genetically distinct parental lines of cultivated lettuce (Lactuca sativa L.) and one drought resistant wild lettuce (Lactuca serriola L.) were cultivated in a growth chamber under limited/non- limited water supply (20% / 70% of available water capacity). At the end of the experiment, plants were exposed to severe drought stress by withholding of irrigation for 3 more days. In parallel with other phenotyping methods applied in the experiment, CFI was recorded regularly in light exposed plants at the actinic light intensity set on the ambient level.

Depending on genotypes, total dry mass in drought stressed plants decreased by 20-50% compared to control; the relative plant dry mass decrease (DMD) was used as a measure of drought sensitivity of genotypes. CFI analyses have shown a significant decrease in the apparent electron transport rate, ETR, in all genotypes (having the same trend as the efficient quantum yield, ΦPSII). However, contrary to expectations, the moderate drought stress led to negligible decrease or even a slight increase of ETR, which did not correspond to the observed decrease of photosynthetic performance. It indicates that the electron transport was efficiently re-directed to alternative energy-consuming pathways, such as photorespiration and others. On the other hand, we observed a significant decrease of steady-state fluorescence intensity (Fs), both in moderate and severe drought. Fs decrease can be explained by activation of energy-dissipating processes (non-photochemical quenching) and/or structural changes in the leaves. The correlation analyses indicated a very low correlation between the drought sensitivity indicator DMD and ETR in moderate drought stress (p=-0.05), a moderate correlation between DMD and ETR in severe drought stress (p=0.45), but the relatively good correlation between DMD and Fs observed in moderate drought (p=0.65) and a very high correlation between DMD and Fs observed in severe drought (p=0.94). The better correlation in severe drought can be well explained by the fact that all plants were functionally in a very similar status (stomata closed, photosynthesis stopped), and the differences in Fs were dominantly due to structural changes.

Thus, our results suggest that the steady-state fluorescence signal (Fs) reflected well the effects of water deficit on photosynthetic apparatus and hence it can be used in phenotyping for drought tolerance in lettuce. In contrary, parameter ETR, which is often referred as the most useful parameter for phenotyping, was not sensitive enough. This is an important information emphasizing the need of testing the techniques in individual crops and different stress scenarios.

68 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

69 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Theme 2

WG2: Phenotyping at cell level

70 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 33 Lipidomic fingerprinting of olive seeds as a basis for identifying new markers of geographical traceability for olives and olive oil

Eliana Alves1, Pedro Domingues1, Luísa Pato2, Luís Pato2, Maria R. M. Domingues 1,* 1 Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal 2 M&LPato, Lda, Rua Homem Cristo, 20, 2R, 3810-064 Aveiro, Portugal *Corresponding author: [email protected]

Plant lipidomics has been used mostly to identify changes in the composition of lipids, associated with plant development and metabolic pathways involved in stress responses. It may also be useful to identify potential biomarkers of traceability.

In olives, major lipids as fatty acids and triacylglycerols, known as compositional markers, are frequently used in traceability studies. However, they cannot confirm their botanical origin, a factor that determines their identity and traceability. Concerning olive seed lipids, there is no information on their lipid molecular composition. Lipidomics is nowadays recognized as a valuable tool for tracking the plant lipidome at the molecular level. So, this work aims at developing a lipidomic-based methodology to unequivocally establish the molecular fingerprint of olive seeds from the Portuguese autochthonous olive (Olea europaea L. cv. Galega vulgar), for the isolation of minor key plant biomarkers.

Samples of olive fruits were collected in Bairrada (littoral centre region of Portugal), in Sep- tember 2015, one month before the picking season. Bairrada is a protected designation of origin (PDO) wine region, known for having vineyards with peculiar and excellent features (and also olive orchards), resulting from the diversity of soils, mainly clayey and sandy soils.

The lipidomic approach used herein relied on total lipid extraction of olive seeds with organic solvents, followed by lipid fractionation by solid-phase chromatography with further identify- cation of the different molecular species of lipid classes by electrospray ionization tandem mass spectrometry.

The lipidome of olive seeds comprises triacylglycerols (major components) and a minor por- tion that includes diacylglycerols, glycosides (acylated sterol glycosides) and phosphorlipids (phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines and lyso- phosphatidylethanolamines).

By obtaining the lipidome of olive seeds fully identified by mass spectrometry fingerprinting and a set of typical lipid markers, it will be possible to screen how this profile can shift in different conditions (edaphological and orographic), and further suggest a lipid identity to Galega vulgar.

Keywords: mass spectrometry, olive, seeds, triacylglycerides, acylated sterol glycosides, glycerophospholipids, traceability

71 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 34 Small RNAs transcriptome differences between low- and high-temperature stress in Arabidopsis

Vesselin Baev, Ivan Milev, Elena Apostolova, Georgi Minkov, Mariyana Gozmanva, Galina Yahubyan Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tsar Assen St, 4000 Plovdiv, Bulgaria

Small RNAs (sRNAs) include two major groups distinguished by their different type of bio- genesis – microRNAs (miRNAs) and small-interfering RNAs (siRNAs). Despite of numerous studies on the involvement of particular sRNA in plant stress response, there are only few reports on the genome-wide sRNA profiles generated under different stress treatments. To understand the involvement of the small RNAs and miRNAs in plant response to different stress factors, we characterized sRNA-seq datasets produced by high-throughput sequencing (HTS) from Arabidopsis plants suffering from low-temperature (LT) and high-temperature (HT) stress for 24 hour.

Our study for the first time presents the genome-wide sRNA profiles of LT and HT treated plants and reveals that they greatly differed in the first 24 hours of stress onset. Under the NT and LT conditions, the sRNA populations were dominated by 21-nt sRNAs. Another important finding is that there are many protein-coding genes that give rise to differentially expressed sRNAs following temperature shifts. Amongst the temperature induced small RNAs and miRNAs, several can be selected whose profiles can be used to distinguish NGS libraries generated under low-temperature stress from those generated under high-temperature treatment.

72 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 35 Novel tool to dissect apoplastic pH homeostasis important for cell growth regulation in Arabidopsis thaliana roots

Elke Barbez, Angelika Gaidora, Thomas Lendl, Wolfgang Busch Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

Unlike animals, plant cells are surrounded by a rigid cell wall, which needs to undergo loosening if plant cells expand. Cell wall loosening in areal tissues of the plant is described to be induced by the plant hormone auxin, which has been postulated in the 70ies as ‘The acid growth theory’ (Rayle and Cleeland, 1971; Krauss, 1971; reviewed in Hager, 2003). Interes- tingly, this theory only holds true for the plants areal parts but is not plausible for roots since auxin inhibits instead of stimulates cell and thus root growth (Chadwick et al., 1967, Löfke et al., 2015). Recent work has shed significant light on the role of auxin in apoplastic pH homeostasis in roots (Fasano et al., 2001; Monshausen et al., 2011; Gjetting et al., 2012; Shih et al., 2015). However, the available methods to assess apoplastic pH are quite laborious or do not offer the required cellular resolution. Here we present a novel tool to address apoplastic pH in Arabidopsis thaliana roots. We therefore exploit the pH dependent spectral dynamics of a non-toxic fluorescent dye that easily penetrates the Arabidopsis root apoplast enabling the analysis of its pH using confocal microscopy. This method allowed us to study the apoplastic pH dynamics during A. thaliana root growth confirming that root cell expansion requires apoplast acidification. Our results additionally suggest a highly complex role for endogenous auxin in apoplastic pH homeostasis important for cell growth regulation in roots. We are currently using this fluorescent dye to perform genome wide association studies in order to identify novel regulators of apoplastic pH homeostasis in A. thaliana roots.

73 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 36 Unravelling the aspects of sodium chloride stress in maize roots: identification of the differential proteomic response specific to the chloride (Cl-)- stress component!

Nádia Campos1, Christoph-Martin Geilfus1, Sebastien Carpentier1,2 1 Department of Biosystems KULeuven, Leuven, Belgium 3 SYBIOMA: Facility for SYstems BIOlogy based MAss spectrometry, Leuven, Belgium

Salinity is a common and aggressive kind of stress, that impacts on global agricultural production. When fertile soils become salinized, vegetative growth and yield of most conventional crops decreases. Zea mays L. (maize) is one of the most important crops around the world and is considered as sensitive to salinity, with big losses in the field. Chloride is the predominant anion in salinized soils. It is known to be taken up by the plant in large amounts. Due to its high mobility it reaches, also via the apoplastic path, aboveground tissues where it accumulates to toxic concentrations. Despite this relevance, research on chloride-mediated aspects of salinity is neglected. The presented study has been carried out to tackle this problem on the level of the root proteome. Our work aimed to identify differential proteomic response that ensue specific to the chloride (Cl-)-stress component of NaCl-induced salt stress. For this, maize plants (cv. Susann, Nordsaat Saatzucht GmbH, Langenstein, Germany) were grown in hydroponic culture in a controlled environment chamber. The separate effects of Cl- and sodium (Na+) ions on the root proteome were examined using the macro-ion gluconate− as a sodium-accompanying counter-anion; glucosamine+ was used as the chloride- accompanying counter-cation. 50 mM Na-gluconate or glucosamine-chloride was added to the nutrient solution and short-term effects were studied, doing so by stressing the plants for 3 hours. The separate effects of Na+ and Cl− were compared with the combined effect of NaCl. Proteins from root samples were extracted by phenol extraction and ammonium acetate precipitation, followed by trypsin enzymatic digestion and loaded on the Q-exactive for mass spectrometry analysis. In total 2277 root proteins were confidently identified, with FDR of 0.3%. A preliminary analysis showed that 73 were identified only on sodium-stressed roots and 98 only under chloride stress. Most of the proteins were identified on the 3 conditions (1416) and 126 were common to both stress conditions. The identification of chloride-specific stress effects may potentially arm the breeders with tools to increase crop tolerance by molecular means.

74 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 37 Molecular networks involved in Brassica napus microspore embryogenesis

Ewa Dubas1, Jérôme Duclercq2, Iwona Żur1, Monika Krzewska1, Anna Nowicka1 1The F. Górski Institute of Plant Physiology Polish Academy of Sciences, Krakow, Poland 2 Unité EDYSAN CNRS FRE 3498, Université de Picardie Jules Verne, Amiens, France Presenting author’s email: [email protected]

Molecular analysis of genes involved in embryogenesis is an important approach for modern plant embryologists and breeders. Induction of embryogenesis in isolated microspores provide a unique model to analyse the embryo development starting from its earliest stages. Moreo- ver, the use of a liquid medium enables the targeted impact on the embryo formation and its phenotype e.g. axis formation. The auxin transport inhibitors are known to interfere with embryogenesis induction and microspore-derived embryo (MDE) quality. Therefore, the analysis of the effect of 1-naphthoxyacetic acid (1-NOA), the IAA influx and efflux inhibitor, could help in the understanding of auxin function in microspore embryogenesis. Identification of expressed genes, more likely to be involved in MDE development, would help in the discovery of novel molecular markers for embryogenesis studies.

Highly embryogenic ‘DH4079’ of B. napus line was used in the study. MDEs were induced and in vitro cultured under various experimental conditions (two heat shock treatments) in the presence of 1-NOA or exogenous auxins. Gene expression quantification in MDEs was performed using the RT-qPCR technique.

Our results confirmed that 1-NOA affected B. napus microspore embryogenesis. The efficiency of the process induction and phenotypic abnormalities were associated with changes in the expression levels of genes encoding an auxin response protein BDL (BODENLOS), auxin transporters AUX1 (AUXIN TRANSPORTER PROTEIN 1), LAX3 (LIKE AUX1), PIN1, PIN7 (AUXIN EFFLUX CARRIERS) and cell fate regulators WOX2, WOX8 (WUSCHEL RELATED homeobox) as well as a controller of embryo development LEC1 (LEAFY cotyledon).

In conclusion, the gene expression analysis in the presented study for B. napus MDEs provides new insights into understanding of the molecular mechanisms that control microspore embryogenesis.

ACKNOWLEDGMENTS: The research was supported by institutional funding of the F. Górski Institute of Plant Physiology Polish Academy of Sciences, project no. T1Zb1/2016.

75 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 38 Blue light stress-dependent ROS formation by Arabidopsis cryptochrome may have a novel conserved signaling role

El-Esawi M.1,2, D’Harlingue A.1, and Ahmad M.1 1 Pierre and Marie Curie University, 75252 Paris Cedex 05, France 2 Botany Department, Faculty of Science, Tanta University, Egypt

Cryptochromes are wide-spread blue-light absorbing flavoproteins with important signaling roles. In plants, they mediate de-etiolation, developmental, and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cryptochrome 1 (cry1) activation by blue light stress also results in direct enzymatic conversion of molecular oxygen

(O2) to ROS (reactive oxygen species) and H2O2 in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 could play a physiological role. ROS formation resulting from cry1 expression was measured by fluorescence assay in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death is determined by colorimetric assay. We found that cryptochrome activation resulted in a rapid increase in ROS formation within cellular compartments that express the cryptochrome protein. This ROS formation resulted in significant cell death over long term illumination, and therefore has physiological consequences. Another important discovery in the course of preforming quantitative real-time PCR analysis of ROS marker and antioxidant genes in plant seedlings has been made. Our very new results showed that economically important genes that are regulated by ROS can also be significantly up- or down-regulated by cryptochromes. This new finding will form the basis for a novel strategy using timed high intensity blue light pulses as a means to optimize crop stress resistance. We conclude that ROS formation by cryptochrome is indeed of physiological relevance under certain defined environmental conditions and could represent a novel evolutionarily conserved paradigm for cryptochrome signaling that can be exploited for biotechnology.

76 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 39 Loss-of-function mutations of Polyamine Oxidase 4 in Arabidopsis thaliana results in increased tolerance to nitro-oxidative stress via modifications in root system architecture and RFO-related metabolites

Miren Sequera-Mutiozabal1, Stefanie Schmidt2, Joachim Kopka2, Chrystalla Antoniou3, Ru- ben Alcazar1, Antonio Fernandez-Tiburcio1 and Vasileios Fotopoulos3* 1 Departamento de Productos Naturales, Biología Vegetal y Edafología, Facultad de Farmacia, Universidad de Bar- celona, Barcelona, Spain 2 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany 3 Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, P.O. Box 50329 Limassol, Cyprus * Corresponding author: [email protected]

Polyamines (PAs) are amine compounds that are essential components of signaling mechanisms under challenging environments, leading to tolerance or survival. However, their metabolic or signaling targets are not well characterized. One of the essential elements regarding PA homeostasis is AtPAO4, a major isoform in Arabidopsis roots, which was shown to be actively involved in spermine regulation in roots and leaves of mature plants. In continuation of recent findings showing that T-DNA insertional mutants of AtPAO4 accumulate 10-fold more spermine and result in delayed dark-induced senescence, the current study shows that these mutants presented increased root length and lateral root density compared with WT Col-0 plants under normal growth conditions, suggesting a role for this enzyme on root architecture. An established cross-talk exists between PAs and reactive oxygen/nitrogen species (RONS) under abiotic stress conditions, therefore exogenous addition of H2O2 or NO donor SNP generates changes on pao4 root architecture by increasing the number of lateral roots and last lateral root site of insertion, both important elements to increase root surface of absorption, indicating this as a possible mechanism to enhance tolerance to stressful environments. 15 day-old Arabidopsis seedlings demonstrated increased tolerance to nitro-oxidative stress. After both stresses, pao4 mutants showed increase in spermine content suggesting a link between its accumulation and the plant’s overall tolerance capacity. In order to identify down-stream signaling targets, metabolomic profiling by GC-TOF-MS after H2O2 and SNP treatment revealed that PA signaling pathway under nitro-oxidative stress conditions is related to glutamate-5-oxoproline regulation which influences glutathione homeostasis, as well as to galactinol-myo-inositol which is a key metabolite on Raffinose Family Oligosaccharides (RFO) biosynthesis pathway. RFOs are currently emerging as powerful antioxidant molecules in addition to their role as carbon re- allocation molecules in abiotic stress response. Current results suggest that PA down-stream signaling under nitro-oxidative stress conditions greatly relies on antioxidant capacity regulation, establishing a novel connection between PAs and RFO homeostasis.

77 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 40 Flax transcriptomics and proteomics for deciphering the influence(s) of genotype and environment on fiber development

Chabi Malika1, Lucau Anca2, Arribat Sandrine1, Goulas Estelle1, Blervacq Anne-Sophie1, Re- naud Jenny3, Hawkins Simon1 1 Univ. Lille, CNRS, UMR 8576 – UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France2 CIIL - UMR 8204-INSERM U1019 CNRS-Pasteur Institute of Lille, University North of France, Trans- criptomics & Applied Genomics, 1 rue du Professeur Calmette, BP 245, F 59019 Lille, France3 Department Environmental Research and Innovation (ERIN), Luxembourg Institute of Science and Technology (LIST), L- 4422 Belvaux, Luxembourg

Flax (Linum usitatissimum L.) is an ancient crop cultivated for many hundreds of years for its cellulose-rich bast fibers that are used in the textile industry. Fibers are also nowadays more and more used in composite and bio-based materials. The quality of fibers is tightly linked to the architecture and structural composition of the fiber cell walls, which consist of a middle lamella, primary cell wall and a thick secondary cell wall characterized by high amounts of crystalline cellulose and low amounts of lignin [1, 2, 3]. It is of great interest to better understand the different factors that can influence the structure of flax cell walls, such as genetic variability and the influence of the environment. To this end, we conducted comparative whole genome transcriptomic and proteomic analyses on the fiber-bearing tissues from different varieties grown under field conditions for two consecutive years. The seven analysed fiber and oil varieties, spring (planted in the spring and are harvested at the end of the summer in the same year) and winter varieties (planted in the winter and harvested the following year) were previously selected by breeding programmes in France and other European countries (FIBRAGEN consortium). Our results indicated significant inter-year and inter-variety differences at i) the fiber morphology level (differences in bundle area and fiber number), ii) the protein level (278 protein spots showing significant differential abundance between varieties and years) and iii) the transcriptome level (659 differentially-expressed genes at the variety level, and 1,571 genes at the environmental level of which a substantial number are involved in cell wall metabolism). Interestingly, our results also suggest that xyloglucan endotransglu- cosylases/hydrolases could be associated with variety- and stress-related differences in flax cell wall metabolism.

[1] Morvan C, Andème-Onzighi C, Girault R, Himmelsbach DS, Driouich A, Akin DE (2003) Building flax fibres: More than one brick in the walls. Plant Physiol Biochem 41: 935–944

[2] Day A, Addi M, Kim W, David H, Bert F, Mesnage P, Rolando C, Chabbert B, Neutelings G, Hawkins S (2005) ESTs from the Fibre-Bearing Stem Tissues of Flax (Linum usitatissimum L.): Expression Analyses of Sequences Related to Cell Wall Development. Plant Biol 7: 23–32

[3] Bourmaud A, Ausias G, Lebrun G, Tachon M-L, Baley C (2013) Observation of the structure of a composite polypropylene/flax and damage mechanisms under stress. Ind Crops Prod 43: 225–236

78 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 41 Physiological phenotyping by determination of phytohormone and enzyme activity signatures

Dominik K. Großkinsky 1,3, Eric van der Graaff1, Rebecca Dölker1 and Thomas Roitsch1,2

1 Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenha- gen, Højbakkegård Allé 13, 2630 Taastrup, Denmark 2 Global Change Research Centre, Czech Globe AS CR, v.v.i.., Drásov 470, Cz-664 24 Drásov, Czech Republic 3Contact: [email protected]

The availability of novel germplasm and cost-efficient molecular techniques for whole genome sequencing, mapping and genomic selection as well as complementary techniques to image the whole plant phenome non-destructively, give access to information unimaginable just a few decades ago. However, to be able to generate cost-efficient, reliable and robust non- invasive predictors for yield and quality it will be essential to link the current high-throughput genotyping and phenotyping of whole plants to the underlying physiological processes. Only the physiology of a plant integrates various levels of regulation of the expression of the genetic information as well as the impact of the environment and agricultural management.

The activities for enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance and crop yield, as well as abiotic and biotic stress responses. Since enzyme activities integrate several modes of regulation at the mRNA and protein level, the analysis of enzyme activities is very suited to analyze the physiological state of plants. This work assessed the establishment of a simple, fast and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism, mainly using coupled spectrophotometric kinetic assays. These assays proved to be robust and highly suitable for the characterization and the diagnosis of the physiological state for various plant species, including several crop species. Plant growth and development depend on a strong regulatory network of phytohormones. Only in the recent years it became evident that the central stress signalling backbone in response to pathogen infection, comprising ethylene, jasmonic and salicylic acid, is also modulated by other phytohormones. Due to extensive interactions of abscisic acid, auxin and newly also cytokinins with the stress signalling compounds, it is required to quantify spatial and temporal dynamics of the whole phytohormone spectrum, but typically established method only determine individual, single phytohormones. Based on published procedures, we developed an easy, rapid extraction and analysis method enabling determination of abscisic, indole-3-acetic, jasmonic and salicylic acid as well as eight cytokinin derivatives within the same extract and analytical run on a UHPLC-MS/MS device. Furthermore, certain phytoalexins, especially important as additional defense compounds within generalized pathogen responses and also cytokinin induced resistance, can be determined in the very same extract. Parallel determination of these phytohormones is also essential for physiological phenotyping to assess the function of various regulatory processes in plants. We successfully applied the established method to determine complex phytohormone profile in five model and crop plant species and also different cell suspension cultures.

79 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 42 Phenotyping of Tolerant/Resistant Varieties with Proteomic Tools

Leonor Guerra-Guimarães1,2, Rita Tenente1, Ana Vieira1, Carla Pinheiro3,4, Inês Chaves3,5, Sebastien Planchon6, Danielle R. Barros1,7, Vagner T. Queiroz1,8, Jenny Renaut6, Cândido P. Ricardo3, Maria Céu Silva1,2 1Centro de Investigação das Ferrugens do Cafeeiro (CIFC), Instituto Superior de Agronomia (ISA), Univer- sidade de Lisboa, Quinta do Marquês, 2784-505 Oeiras, Portugal 2Linking Landscape, Environment, Agriculture and Food (LEAF), ISA, Universidade de Lisboa, Tapada da ajuda, 1349-017 Lisboa, Portugal; 3Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa (UNL), Quinta do Marquês, 2780-157 Oeiras, Portugal; 4Instituto de Biologia Experimental e Tecnológica (IBET), Quinta do Marquês, 2780-157 Oeiras, Portugal 5Faculdade de Ciências e Tecnologia, UNL, 2829-516 Caparica, Portugal 6Luxembourg Institute of Science and Technology (LIST); 41, rue du Brill, L-4422 Belvaux, Luxembourg 7Dep. De Fitossanidade, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, 96010-000, Brazil 8 Departamento de Química e Física, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo (UFES), 29500-000 Alegre, ES, Brazil

We have conducted a 2-DE proteomic analysis of the apoplastic proteins of coffee leaves subject to different temperature ranges (abiotic stress) or infected with H. vastatrix (biotic stress). The main objective was to understand the dynamic nature of 29 proteins superfamilies identified in the coffee leaf apoplast, when subject to the different stress conditions. The main biological processes that differentiate the two types of stress are: i) stress/defense proteins, whose number and diversity is much higher in the biotic stress situation (23% vs. 6%); ii) proteins of the secondary and redox metabolism that only change in abundance during the biotic stress; iii) proteins involved in protein glycosylation metabolism identified only in the abiotic stress. More than 50% of the affected proteins participate in proteolysis and degradation of the cell wall. However, distinct proteins of similar metabolic function and/or the same proteins with minor structural modifications (PTMs, isoforms, etc.) modulate the responses in the two stress conditions. Only a deep knowledge on the type, function and quantity of proteins expressed at a given time, condition and cellular compartment, integrated with information from other areas, will in the future, generate knowledge that can be used in the phenotyping of tolerant/resistant varieties.

Acknowledgements: Work supported by Portuguese Funds through FCT (Fundação para a Ciência e a Tecno- logia) under the projects PTDC/AGR-GPL/109990/2009 and PEst-OE/EQB/LA0004/2011 and by a Brazil- Portuguese collaborative project funded by FCT and CAPES (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and a Pos-PhD grant of CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico – Brazil).

80 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 43 Hormonal regulations and genome stability in the plant stress response

Jan Hejatko, Helene S. Robert, Vanesa Tognetti, Jiří Fajkus CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Hormonal regulation represents one of the key mechanisms in the regulation of plant adaptive response to various environmental stress conditions. Changes in the endogenous levels of plant hormones and/or signaling were shown to be associated with changes in the stress response. Environmental stress and genotoxic treatments can threaten genetic stability. Telo- mere shortening, either due to their incomplete terminal replication, disturbed nucleoprotein structure or due to DNA lesions emerging in telomeres due to e.g., oxidative damage, ultimately results in telomere dysfunction and chromosome instability. Thus, stable telomere maintenance can be used as a factor underlying and promoting genome stability. However, our knowledge on the molecular mechanism underlying both genetic stability and involvement of plant hormonal regulations in the plant stress response is scarce.

Our recent unpublished results suggest that cytokinins control development of the shoot vascular tissue via control of the tracheary element developmental pathway. We found that ma- nipulating the endogenous cytokinin levels and/or cytokinin signaling pathway could be used in the targeted modification of the onset of the secondary cell wall formation and functional properties of tracheary elements, mainly in terms of their ability to conduct water, affecting thus the water distribution within the plant and potentially influencing draught resistance.

Using Arabidopsis cytokinin receptor as a molecular probe, we have identified novel cytokinin receptors in the oilseed rape, implying the existence of similar mechanisms in crops. Based on these findings, we propose the hormonal pathways as a suitable target in the development of novel marker-assisted strategies. We will test our transgenic and hormonal signaling defective lines in order to determine their stress (draught and cold) resistance. Based on our results, the suitable targets involved in the hormonal biosynthesis, signaling and/or the downstream signaling targets associated e.g. with the secondary cell wall formation will be selected.

In addition, we are currently testing auxin modules and gain- or loss-of-function lines for high impact for stress tolerance by imaging of chlorophyll fluorescence. Our preliminary data show that stress-induced changes in auxin synthesis and transport affect chloroplast photopro- tection mechanisms. Variants of the Arabidopsis orthologues in the molecular regulators involved in hormonal signaling and/or downstream effector genes will be determined in crops with stress contrasting phenotypes to assess the transferability our knowledge from the model plant to the real crops. The newly established core facility Plant Science will be involved in reaching the project objectives. Supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601).

81 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 44 Glutathione as a modulator of endoreduplication in leaves of Arabidopsis thaliana plants grown under control and cadmium exposure conditions.

Sophie Hendrix, Els Keunen, Jaco Vangronsveld, and Ann Cuypers Environmental Biology, Centre for Environmental Sciences, Hasselt University, Diepenbeek

Endoreduplication is an alternative form of the cell cycle in which DNA replication is repeated without intervening mitosis, resulting in endopolyploidy. It plays an important role in the growth and development of many plant species, but can also be affected by environmental stress factors, such as UV-B irradiation, drought and cadmium (Cd) exposure. In this study, the extent of endoreduplication was kinetically monitored in leaves of Ara- bidopsis thaliana seedlings hydroponically grown under control conditions. Using flow cytometry, nuclear ploidy levels of different leaves were determined at regular intervals from the day they appeared to day 21 after sowing. Results showed a clear day-to-day progression of nuclei to higher ploidy levels, consistent with increasing leaf area.

In addition, the effects of Cd on endoreduplication were determined in leaves of A. thaliana plants exposed to a range of environmentally realistic Cd concentrations (0, 1, 2.5, 5 and 10 µM) starting from the day the first “true leaves” appeared. Nuclear ploidy levels in leaves of Cd-exposed plants were significantly decreased as compared to the control, suggesting a role for endoreduplication in plant responses to Cd.

Cadmium is known to affect the level and redox state of the antioxidative metabolite glutathione (GSH), which is also involved in cell cycle regulation. Therefore, the extent of endoreduplication was compared between leaves of wild-type (WT) A. thaliana plants and the GSH- deficient cadmium-sensitive 2-1 (cad2-1) mutant grown under control and Cd exposure conditions. Even under control conditions, nuclear ploidy levels significantly differed between leaves of both genotypes. These results were supported by differences in epidermal cell area and number, determined by a combination of differential interference contrast microscopy and phenotypic analysis of leaf area. Furthermore, quantitative real-time PCR analysis revealed significant differences in transcription levels of cell cycle-related genes consistent with the different extent of endoreduplication between leaves of WT plants and cad2-1 mutants.

Taken together, these data suggest a role for GSH in regulating endoreduplication in A. thaliana plants, both under control and Cd exposure conditions. In the long run, the knowledge obtained in this study can contribute to the development of strategies to increase plant growth on Cd-polluted soils for phytoremediation purposes.

82 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 45 Characterization and pollen phenotyping of AtbZIP transcription regulators acting in Arabidopsis male germline

Antónia Gibalová1, Lenka Steinbachová1, Veronika Bláhová1, Zuzana Gadiou1, Roman Pleskot2, Said Hafidh1 and David Honys1,3 1Laboratory of Pollen Biology, Institute of Experimental Botany AS CR, v. v. i., Rozvojová 263, 165 02 Praha 6, Czech Republic 2Laboratory of Cell Biology, Institute of Experimental Botany AS CR, v. v. i., Rozvojová 263, 165 02 Praha 6, Czech Republic 3Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44, Praha 2, Czech Republic

We have aimed to identify transcriptional regulators acting in male germline to better understand many important processes required for successful gametogenesis. The understanding of gamete production in the male gametophyte represents crucial aspect how to improve plants vigour and fertility. Candidate genes were chosen exploiting pollen developmental transcriptomic data and based on a wide screen of T-DNA mutant lines showing phenotype de- fects in pollen development. We selected bZIP family transcription factors (TFs) counting 75 members in Arabidopsis thaliana. Out of these, few genes exhibited pollen enriched expression profile and shared properties for possible interaction analyzed in amino acid sequences alignment. Here we report the identification of putative bZIP TFs regulatory network potentially active in Arabidopsis thaliana pollen and highlight the important component of this regulatory complex – AtbZIP34 and AtbZIP18. Both genes are highly expressed in pollen, but also possess lower promoter activities in several sporophytic tissues. This observation overlapped with the localization of AtbZIP34 and AtbZIP18 proteins throughout plant development. In subcellular context, the localization of AtbZIP34 was restricted to the nucleus whereas AtbZIP18 was found in the nucleus and cytoplasm/ER. To address the role of At- bZIP TFs in the male gametophyte, we performed phenotypic screen of the T-DNA knockout alleles together with their genetic analysis, which showed reduced transmission of the mutant alleles through the male gametophyte. Two other bZIP proteins expressed in pollen – At- bZIP52 and AtbZIP61 interacted with AtbZIP34 and AtbZIP18 in Y2H analysis, suggesting that co-operation of these bZIP TFs might be important to govern certain regulatory processes in pollen. To uncover the pathways controlled by this network, we evaluated atbzip34 and at- bzip18 microarray data and identified number of downstream genes.

Acknowledgement: The authors gratefully acknowledge the financial support from ESF- COST (Action FA1306), Czech Science Foundation (15-16050S, 14-32292S and 13-06943S) and Czech Ministry of Education, Youth and Sports (LD14109).

83 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 46 Pointing for evaluation of proteome of Portuguese cultivars Castanea sativa Mill.

Michael Freitas1,2, Catarina Marinho1,2, Tiago Santos1,2, José L. Capelo3,4, Hugo M. Santos3,4, J Ferreira-Cardoso5, Gilberto Igrejas1,2,3,* 1 Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal 2 Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal 3 BIOSCOPE Group, UCIBIO-REQUIMTE, Chemistry Department, Faculty of Science and Technology, Univer- sity NOVA of Lisbon, Lisbon, Portugal 4 ProteoMass Scientific Society. Faculty of Sciences and Technology. Campus de Caparica, Caparica. Portugal 5 Centre for Research and Technology of Agro-Environment and Biological Sciences,University of Trás-os- Montes and Alto Douro, 5000-801 Vila Real, Portugal. *[email protected]

The European chestnut (Castanea sativa Mill.) is an agro-forestry specie with great economic importance for Central and Northern regions of Portugal. For many centuries chestnuts provided the staple food in rural populations, because of their good adaptation to soil-climatic conditions, until the appearance of maize and potato.

Portuguese cultivars present several characteristics recognized in the international market, thus it is important to create programs that are capable of monitor its biodiversity considering also the preservation of the national genetic heritage.

Storage proteins characterization in chestnut is an important tool in the evaluation of genetic variability. In this sense, we proposed to evaluate the applicability of storage protein of chestnut as markers of genetic diversity in Castanea sativa Mill. It was performed one-dimensional electrophoresis (SDS-PAGE) of 21 Portuguese cultivars to define their protein profiles, which revealed to be highly polymorphic for albumins and globulins.

At the second stage, it was performed two-dimensional gel electrophoresis (2-DE) enabling the separation of protein, firstly by their pI and then further separated by molecular weight through SDS-PAGE, in order to obtain the complete proteome of two of the most important Portuguese cultivars.

Subsequent analysis by matrix-assisted laser desorption/ionization mass spectrometry (MAL- DI-TOF-MS) and bioinformatics it was possible to identify protein sequences present in chestnut and infer their function. Overall, 259 reproducible protein spots were detected by two-dimensional electrophoresis on pH 3-10 gels. The majority of the proteins identified were related to binding, catalytic, cellular or metabolic processes.

These results correlated to protein content expand our knowledge of the Portuguese cultivars of chestnut and improve our understanding of the cellular biology of chestnut seeds. The analysis of storage protein proved to be a useful tool to give complementary information to explain the genetic and adaptive diversity within different cultivars.

Supported by Project INNOFOOD - NORTE-07-0124-FEDER-0000029

84 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 47 Signaling towards abiotic and biotic stress resistance

Hansjörg Stampfl1, 2 and Claudia Jonak 1,2 1 Austrian Institute of Technology, Department of Health & Environment, Konrad Lorenz Strasse 24, 3430 Tulln an der Donau, Austria 2 Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr Gasse 3, Vienna, Austria [email protected]

Unfavorable environmental conditions and pathogen infections limit plant growth and development and thus reduce agronomic yield. Plants have evolved complex cellular and physiological mechanisms to prevent damage and enable growth under stress conditions. These responses are controlled by different stress-type specific but also common and interacting signaling pathways, which may inhibit each other explaining the trade-off between biotic and abiotic stress responses.

Studies of the regulatory circuits induced by stress revealed the importance of protein kinases for directing cellular responses to abiotic and biotic stresses. We discovered the Arabidopsis protein kinase ASKα as a positive regulator of both abiotic and biotic stress responses. During salt stress ASKα is activated and it positively regulates stress tolerance and redox homeostasis. Combining molecular, biochemical, and physiological analyses we showed that ASKα phosphorylates cytosolic glucose-6-phosphate dehydrogenase on an evolutionary conserved residue, thereby stimulating its activity. Glucose-6-phosphate dehydrogenase is the entry point into the oxidative pentose phosphate pathway and is essential for maintaining the cellular redox status. Interestingly, ASKα is also a positive regulator of innate immunity. Pathogen infection and the perception of several, unrelated pathogen-associated molecular patterns (PAMPs) induce ASKα kinase activity. Loss of ASKα attenuates, whereas its overexpression enhances, pattern-triggered immunity responses, ultimately affecting pathogen resistance. Remarkably, while under salt stress conditions, ASKα-induced stimulation of G6PD activity contributes to the removal of excess levels of reactive oxygen species (ROS) via the ascorbate/glutathione cycle and to stress tolerance, upon pathogen infection G6PD activity is necessary for ROS production by NADPH oxidases and a successful defense response. Overall, our data provide evidence that ASKα and G6PD constitute an evolutionary conserved signaling module that links protein phosphorylation cascades to metabolic adjustment under both abiotic and biotic stress conditions.

In our current work, we seek to analyze the role of the ASKα-G6PD module in plants exposed to combinatorial stresses. Furthermore, we investigate its potential for the development of multi-stress tolerant crop plants.

85 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 48 Effect of meteorological factors on metabolomics of geraniol and its “satellites” in geraniol chemotype of Thymus pulegioides

Kristina Ložienė, Vaida Vaičiulytė Nature Research Centre, Institute of Botany, Lithuania

The commercially important acyclic monoterpene alcohol geraniol with flowery-rose-like odour is a fragrance ingredient usable in multiple cosmetic products, and it is characterized by antibacterial activity against food borne pathogens (Burt, 2004; Lapczynski et al., 2008; Chen and Viljoen, 2010). Geraniol, the main compound of essential oil of geraniol chemotypes of species of genus Thymus, always goes along with “satellites” – geranial, nerol and neral. The- se four compounds are interdependent: nerol is the Z-enantiomer of geraniol and occurs in small quantities in many essentialoils, where it is being found together with geraniol; the ter- pene aldehydes geranial (or citral A) and neral (or citral B) are isomers E and Z, respecttively (Demyttenaere, 2001). Some commercial species of genus Thymus are cultured, therefore, is actually as same meteorological factors influence on composition of essential oils. The aim of this work was to establish the effect of meteorological factors on percentage of geraniol, nerol, geranial and neral in essential oil of Thymus pulegioides growing under the same environmental conditions.

The parent plant of T. pulegioides, belonged to geraniol chemotype, was vegetatively propagated and grown in the field collection of the Nature Research Centre (Vilnius, Lithuania) six year in an open ground under the same micro-edaphoclimatic environmental conditions. The aerial parts were annually collected at the full flowering stage and dried at room temperature. The essential oils of leaves-inflorescences (free from stems) were isolated by hydrodistillation in the European Pharmacopoeia apparatus during two hours. The metabolomic analysis carried out by gas chromatography and mass spectrometry. The meteorological data (temperature, precipitation, photosynthetically active solar radiation and sunshine duration) were obtained from the meteorological bulletins of the closest station of meteorology of Lithuanian Hydrometeorological Service under the Ministry of Environment.

Geraniol negatively correlated with geranial, nerol and neral (r = –0.60, p = 0.2, r = –0.49, p = 0.3 and r = –0.43, p = 0.4, respectively), i. e. the increasing of geraniol was accompanied by the reduction of other three compounds. The amount of geraniol was positively influenced by temperature and negatively – by photosynthetically active solar radiation (r = 0.89, p < 0.05 and r = – 0.83, p < 0.05, respectively).

86 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 49 Halophyte plants as a natural source of antioxidant compounds

Elisabete Maciel1,2, Tânia Melo2, Bruna Marques1, Rosário Domingues2, Ricardo Calado1 and Ana Lillebø1 1 Department of Biology & CESAM, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal 2 Mass Spectrometry Centre, Department of Chemistry & QOPNA, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal

The unprecedented rate of salinization of soils formerly used for agriculture is a world-wide issue, with salt stress being one of the most serious environmental factors limiting the productivity of conventional crop plants. Halophytic species have evolved several physiological traits that enable them to thrive under high salinity conditions. These traits include the biosynthesis of several bioactive molecules, including a number of lipid species, which might dis- play antioxidant, anti-inflammatory, antimicrobial and antitumoral activities. This chemical diversity has fostered a growing interest for possible multitude applications, with halophytes still being considered an untapped reservoir of novel bioactive compounds. In this study we used a lipidomic approach based on liquid chromatography coupled to mass spectrometry (LC-MS) to characterize the lipidome of Salicornia ramosissima and Halimione portulacoides and explore the antioxidant potential of their lipids. The lipidome signatures retrieved unraveled a plethora of polar lipids (> hundred species) which included: phosphorlipids (5 classes) and glycolipids (3 classes). High content of omega-3 fatty acids were found in both halophyte species. The lipophilic extract from both species also exhibited a promising antioxidant potential (S. ramosissima IC50 = 0.459 ± 0.022 mg/mL; H. portulacoides IC50 = 0.201 ± 0.008 mg/mL). It has been recognized that halophytes have the potential to replace common crop plants in salinized soils. The overall results of this study suggest that these halophytes may contribute to the development of value added products for future

87 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 50 Subcellular phenotyping to characterize the molecular framework regulating auxin efflux carrier proteins (PINs) expression and localization.

Ivan A. Paponov1 and Klaus Palme2 1 NIBIO, Norwegian Institute of Bioeconomy Research, NO-1431 Ås 2 Molecular Plant Physiology, Albert-Ludwigs-University of Freiburg, D-79104Freiburg, Germany

The specific expression and localization of PIN proteins, which involved in auxin polar transport, is responsible for the establishment of auxin gradients in the root, which is critical for root apical meristem activity and root growth. However, the molecular mechanisms which determine the expression of PINs remain elusive. Critical factors for the regulation of PIN gene expression pattern are the PLETHORA (PLT) transcription factors, which are major de- terminants for root stem cell specification and serve to stabilize maximum auxin levels at the distal root tip. For further investigations into the molecular framework which regulates PIN expression, mutants with altered root growth were selected, assuming that this phenotype could be due to perturbation in PIN expression and localization. Analysis of Arabidopsis mutants defective in root meristem activity and examination of PIN expression and localization using ummunolocalization technique in these mutants helped to identify genes, which regulate the expression and localization of PINs. We investigated PIN expression and localization in the auxin receptor mutant tir2, triple and quadruple mutants of the F-box proteins tir1afb1afb2afb3, the mutant of HALTED ROOT, a subunit of the 26S proteasome hlr, and the root pattern defective mutants short-root (shr) and scarecrow (scr) which a short root phenotype. In summary, the main regulators of PINs at the transcriptional level are the PLETO- RA genes, which are required for quiescent centre identity and stem cell specification. At a post-transcriptional level, PINs are regulated by SHORT-ROOT.

88 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 51 Pinus pinaster provenance assay: climatic scenarios impact in the needle proteome

Carla Pinheiro1,2, Adelaide Machado1, Alicia Horta3, Rita Tenente1, Sébastien Planchon4, Jenny Renaut4, Teresa David5,6 and Isabel Correia5,6 1Instituto de Tecnologia Química e Biológica, Universidade NOVA de Lisboa, Portugal 2Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Portugal 3Instituto Superior de Agronomia, Universidade de Lisboa, Portugal 4Luxembourg Institute of Science and Technology, Luxembourg 5Instituto Nacional de Investigação Agrária e Veterinária, I.P., Portugal 6 Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Portugal

Pinus pinaster Ait. (maritime pine) is a highly relevant resource in Mediterranean areas, where summer droughts frequently occur. Predicted climatic scenarios point out for an increasing severity of summer droughts with impact on P. pinaster stands. Therefore, selection of populations adapted to drought-prone environments is under course in Portugal. Here we present a P. pinaster provenance assay considering two modelled water regimes (dry and very dry). We used populations from distinct climatic groups: Atlantic (Leiria), Mediterranean (Cuenca) and Maghrebian (Algeria). Growth and survival were evaluated in a field trial (Companhia das Lezírias, Portugal) under a Randomized Complete Block design. Needle proteome (2DE-MALDI/TOF-TOF) was characterized on 1.5 year-old seedlings. Analysis of variance showed significant differences (Pr≤0.05) in growth traits (total height, diameter and shoot diameter) between populations. Leiria and Cuenca exhibited opposite trends: Leiria showed higher growth and lower shoot diameter while an inverse ranking was observed for Cuenca. We also found significant differences in survival between P regimes, but not between provenances. Regarding the needle proteome, significant differences were found between the three populations and the two regimes, but significant Genotype x Environment interactions were only found for Cuenca (Pr≤0.01) and Algeria (Pr≤0.07). Under a very dry scenario, nee- dles up-regulate several heat-shock proteins and stress-inducible proteins. RuBisCO was also found to show provenance specific and stress responsive isoforms (under stress, RuBisCO isoforms of lower molecular weight were found less abundant). Our data showed that the biochemical responses to stress were provenance specific. Changes at protein level reflect the growth performance ranking, Leiria being the most stable population and Cuenca the most affected under the very dry climate scenario. The distinct strategies displayed by these provenances illustrate genetic differences that will be explored to potentiate P. pinaster resilience in the face of climate change.

89 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 52 Phenotyping at cellular level: stress induced changes in somatic embryo structure during cryopreservation procedure

Terezia Salaj1, Bart Panis2, Rony Swennen2,3,4, Radoslava Matusova1, Jan Salaj1 1Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, 950 07 Nitra, Slovak Republic 2Bioversity International, Willem de Croylaan 42, 3001 Leuven, Belgium 3 KU Leuven, Laboratory of Tropical Crop Improvement, Willem de Croylaan 42, 3001 Leuven, Belgium 4 International Institute of Tropical Agriculture, POB 10, Duluti, Arusha, Tanzania

Cryopreservation is a valuable method allowing safe and efficient storage of plant genetic resources. In recent years several cryopreservation techniques have been developed and applied to a wide variety of plant species, including in vitro propagated plant material, meri- stems, buds, seeds, embryos, pollen. The method is also very convenient for the storage of conifer embryogenic tissues. Somatic embryos can be stored at a very early developmental stage and after cryopreservation they can regenerate into whole somatic seedlings. Embryo- genic tissues of hybrid firs Abies alba x A. cephalonica and Abies alba x A. numidica have been cryopreserved using a slow-freezing method and showed high recovery frequencies (90- 100% for individual cell lines). During the cryopreservation procedure that comprises several steps, the somatic embryo structure underwent different changes as a response to the stresses to which they are exposed. The aim of the study presented here is to describe these changes. Immediately after pretreatment (sorbitol 0.5M) the bipolar structure of somatic embryos was disintegrated as a result of partial damage or complete disruption of the vacuolised supensor cells. Fluorescence microscopy showed good survival of meristematic embryonal cells. In some cases part of the meristematic embryonal cells were also damaged and the fluorescence was concentrated in the embryo „head“ periphery. Storage in liquid nitrogen did not cause profound changes in comparison with the pretreatment. During the post-thaw recovery, somatic embryos regained their original bipolar structure. Mitotic activity some of survived meristematic cells led to cell cluster formation and their polarisation resulted in bipolarity and finally somatic embryo regeneration.

The work was supported by grant MVTS COST FA1306.

90 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 53

Searching for novel proteins involved in CO2-induced stomatal movements

Yana Sindarovska, Mikael Brosche, Liina Jakobson, Hanna Hõrak, Yuh-Shuh Wang, and Hannes Kollist Plant Signal Research Group, Institute of Technology, University of Tartu, Estonia

Stomata are pores in epidermal plant tissues, which are formed by surrounding guard cells and control gas exchange in plants. Stomatal regulation is under intense research; however there are still gaps in understanding molecular details of guard cell signalling. Previous research showed that the Arabidopsis protein kinase HT1 regulates CO2-induced stomatal movements. HT1 is classified as a mitogen-activated protein kinase kinase kinase (MKKK). Our recent studies have identified MPK12 as an interactor of HT1 that lies upstream of HT1 in the signalling cascade. This is an unexpected result, since typically MPKKK should be at the top of the signal transduction chain.

The objective of this research is to study signalling cascade(s) that involve MPK12 and HT1. Both MPK12 and MPK9 are preferentially expressed in guard cells and have functional re- dundancy. Our previous studies showed that mpk9 single mutant behaved like A. thaliana

Col-0 wild type in response to CO2 changes, while mpk12 single mutant demonstrated constitutively more open stomata and impairments in stomatal responses. Thus, mpk9mpk12 double mutant was generated and characterized for stomatal CO2 responses. The mpk9mpk12 mutant showed even more open stomata than mpk12 single mutant, but stomatal responses to CO2 were similar to mpk12. So together these two MPKs possibly play an essential role in CO2- induced stomatal regulation.

As MPK12 is a protein kinase, it likely interacts with MKKs. Thus some MKKs may play an essential role in CO2-induced stomatal movements. Three Arabidopsis MKK knockout mutants, namely mkk1, mkk2, and mkk3 have already been analyzed for CO2 responses, darkness and ABA treatment. However, all of these demonstrated wild type responses. Other Ara- bidopsis MKK knockout mutants will be analyzed as well. MKKs having impaired response will be used for pair-wise interaction tests with MPK12 and/or HT1. As protein kinases may interact with phosphatases, knockout mutants of phosphatases related to MPK signaling will also be analyzed for their stomatal CO2 response. Additionally, new MPK12 interactors will be isolated and characterized.

91 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 54 Analysis of cereal response to abiotic stress using 2D-DIGE approach

Pavel Vítámvás1, Klára Kosová1, Jenny Renaut2, and Ilja T Prášil1 1 Division of Crop Genetics, Breeding and Product Quality, Crop Research Institute, Drnovská 507, 161 06 Pra- gue 6 - Ruzyně, Czech Republic 2 Luxembourg Institute of Science and Technology, Rue du Brill, 4422 Belvaux, Luxembourg

Cold, drought and salinity represent the major abiotic stress factors with a dehydrative component. Plant exposure to stress induces a dynamic proteome response aimed at an establishment of a new homeostasis. Recovery after stress is also important since it affects further plant growth and development. The two-dimensional differential gel electrophoresis (2D- DIGE) analysis enables protein relative quantification leading to an identification of the protein spots revealing an enhanced abundance in stress-treated or stress-tolerant varieties which could be further tested as potential markers of stress tolerance. Proteomic experiments aimed at wheat and barley proteome response to cold, drought and/or salinity were analyzed. The aim of these analyses was to identify protein spots revealing differential abundance between different stress treatments or differently tolerant genotypes that could be potentially used for abiotic stress phenotyping. Unsurprisingly, the majority of potential proteins for phenotyping belong into stress- and defence-related proteins. The results of proteomic analyses were inter- preted with respect to other physiological data such as parameters related to stress tolerance (membrane stability, LT50), water regime-related characteristics (water saturation deficit, osmotic potential), and others. The role of gel-based proteomic analysis in understanding plant stress response and acquisition of plant stress tolerance is discussed.

Acknowledgement: The work was supported by the Ministry of Agriculture of the Czech Re- public (QJ1310055 and MZe RO0415), EU COST action FA1306 and by the Ministry of Ed- ucation, Youth and Sports (LD15167 and LD14087).

92 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 55a Molecular plasticity during drought recovery is characterized by metabolite and protein turnover dynamics in Medicago truncatula

Stefanie Wienkoop1, David Lyon1,2, and Ma. Angeles Castillejo1,3 1 Dept. of Ecogenomics and Systems Biology, University of Vienna, Austria 2 Center for Protein Research, The Novo Norsk Foundation, Copenhagen, Denmark 3 Institute for Sustainable Agriculture, CSIC, Córdoba, Spain

Plants are continuously exposed to extreme environmental changes. Especially water availability is subjected to a considerable fluctuation; several days of drought are often followed by periods of sufficient rain. This requires a molecular plasticity that enables plants to regulate drought acclimation and deacclimation processes for recovery and continuous growth.

A complex network, including proteomic and metabolomic turnover dynamics, is part of this regulatory process.

To study this, a partial 15N-metabolic labelling strategy in planta was introduced. Nitrogen incorporation was analyzed over a drought-recovery experiment determining the relative iso- tope abundances (RIA). Severely drought stressed plants (10 days of water withhold) were re- watered over a period of 4 days until full physiological recovery. The RIA of metabolites and proteins was monitored using mass spectrometry from samples taken 2, 24, 48, 72 and 96 hours after re-watering.

The data reveal independent regulatory mechanisms for stress recovery with different dynamic phases that, during the course of recovery, define the plants` deacclimation from stress. An early transition phase that seems key for recovery initiation through water re-supply was observed. Furthermore, the data indicate that plasticity may also be related to the nutritional status of the plant prior to stress initiation.

93 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 55b Comparative proteome analysis highlights metabolic adjustments under drought stress in Pisum sativum

M. Ángeles Castillejo1,2, Rebeca Iglesias-García2, Stefanie Wienkoop1, and Diego Rubiales2 1 Department of Molecular Systems Biology, University of Vienna, Austria 2 Institute for Sustainable Agriculture, CSIC, Córdoba, Spain

Abiotic stresses caused by adverse environmental conditions are responsible for heavy economic losses on pea crop, being drought one of the most important abiotic constraints. Devel- opment of pea cultivars well adapted to dry conditions has been one of the major tasks in breeding programs. A better understanding of molecular mechanisms for drought tolerance is a priority to cope with the increasing food requirements. We used a shotgun proteomic approach (nLC-MSMS) with the aim of understanding the complexity of pea crop responses to drought stress. Three pea genotypes showing different level of tolerance to water deficit were used. Multivariate analysis of data and functional cluster unraveled 395 significant differences of 700 identified when genotypes and/or treatment were compared. We propose different mechanisms to cope drought in the genotypes studied. The susceptible genotype activates a series of regulatory processes to maintain homeostatic equilibrium, especially those of the primary metabolism. The tolerant genotypes used a strategy for providing the energy required for activating stress defenses. We describe a specific mechanism against drought for the most tolerant genotype in which proteins drought-stress related and other involved in strengthening of cell wall seem to have an active role.

94 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 56 Activity of low molecular weight antioxidants affects the effectiveness of embryogenesis initiation in isolated microspore cultures of triticale (× Triticosecale Wittm.)

Iwona Żur, Ewa Dubas, Monika Krzewska, Franciszek Janowiak The F. Górski Institute of Plant Physiology Polish Academy of Sciences, Krakow, Poland Presenting author’s email: [email protected]

Isolated and in vitro-cultured immature cells of male gametophyte (microspores) have two developmental options: they could continue the standard developmental pathway forming mature pollen grains or enter the process of microspore embryogenesis (ME), which ends in the formation of haploid/doubled haploid plants. The latter option requires a precisely balanced stress treatment. Its intensity should meet the threshold required to redirect microspore development, but not exceed stress tolerance and diminish cell viability. It has been suggested that enhanced cell defence ability should improve the effectiveness of ME.

Our previous investigation proved that in anther cultures of triticale, high activity of antioxidative enzymes was required for the effective induction of ME, whereas low molecular weight (LMW) antioxidants were of minor importance. To establish the role of LMW antioxidants in isolated microspore cultures, their activity was determined in triticale microspores by DPPH (2,2-Diphenyl-1-picrylhydrazyl) method.

The analysis was done first in non-embryogenic microspores (isolated from freshly cut tillers) and then in microspores redirected towards embryogenesis by low temperature tillers pretreatment (3 weeks at 4°C). Additionally, the effect of the short or long-term treatment with reduced glutathione (0.3 M GSH) – one of the most important LMW antioxidants – was estimated.

The five studied DH lines of triticale differed significantly both in their embryogenic potential and in microspore LMW antioxidant activity. The most distinctive feature differentiating genotypes with various embryogenic potential was the activity of LMW antioxidants in microspores isolated from freshly cut tillers, significantly higher for recalcitrant ones. GSH increased antioxidative activity of microspores only when supplemented shortly before (4 days) microspore isolation. It also had a positive effect on the effectiveness of microspore embryogenesis. These results suggest that the activity of endogenous LMW antioxidants is not sufficient to cope with oxidative stress connected with microspore isolation and in vitro culture. It should be supported by supplementation of exogenous antioxidants e.g. GSH.

ACKNOWLEDGMENTS: The research was supported by institutional funding of the F. Górski Institute of Plant Physiology Polish Academy of Sciences, project no. T1Zb2/2016.

95 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

96 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Theme 3

WG3: Integration of phenotyping on both levels and translation into good practices for applied use

97 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 57 A novel GC/MS method for rapid differentiation of cereal and pseudocereal species

Marijana M. Ačanski, Radojka N. Razmovski, and Kristian A. Pastor University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

In this paper hexane extracts of flour samples, made of different cereal and pseudocereal species: buckwheat, amaranth, spelt, corn and small grains (wheat, barley, oats, rye and triticale), were analyzed. Extracted lipid components were derivatized using transesterification reagent, 0.2 M trimethylsulfonium hydroxide (TMSH) solution in methanol. Hexane extracts of cereal and pseudocereal species were analyzed using gas chromatography - mass spectrometry system. Methyl esters of dominant and minor fatty acids and compounds comprising unsaponifi- able matter, such as phytosterols, tocopherols and squalene, were identified. The possibility of cereal and pseudocereal flour differentiation was examined by applying binary system in multivariate analysis of automatically integrated surface areas of extracted components, after the elimination of dominant fatty acid methyl esters, present in the hexane extract of every investigated variety. The results show that buckwheat, amaranth, corn and spelt flour samples are clearly and unambiguously classified into different groups according to plant species. Given the high degree of botanical similarity, flour samples of all cultivars belonging to small grain species (wheat, barley, oats, rye and triticale) constitute a separate group.

98 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 58 Leaf development of maize inbred lines under different water and nitrogen supply during mid-growth vegetative stage

Sofija Božinović1, Ana Nikolić1, Jelena Vančetović1, Dragana Ignjatović Micić1, Astrid Jun- ker2, Kathleen Weigelt-Fischer2, Dejan Dodig1, and Thomas Altmann2 1PlantBreeding Department, Maize Research Institute ZemunPolje, Belgrade, Serbia 2Research group Heterosis, Dept. of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany

A set of 20 maize (Zea mays L.) inbred lines, including 15 tolerant and five susceptible to drought stress, were chosen for the experiment. The genotypes were defined either as drought tolerant or drought susceptible according to their response to water deficiency during early reproductive stage in field conditions, with the grain yield as the main parameter. Out of 15 tolerant inbreds 12 are introduced from different countries and belong to the drought tolerant mini core collection of Maize Research Institute Zemun Polje gene bank. In this study genotypes were grown in a greenhouse under optimal conditions until most of them reached V6 growth stage (six fully developed leaves). From V6 stage plants were subjected to the limited water and/or nitrogen supply and imaged for four weeks at the automated phenotyping and imaging platform (LemnaTec Scanalyzer) at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). The experimental design was a randomized block design with eight replications, two treatments (water and nitrogen) and two levels of stress in each treatment. Optimum and stress levels for water were 75% and 30% of field capacity, respecttively. For the nitrogen stress treatment no fertilizer was added during the experiment. Besides visible, near-infrared and fluorescence images being taken every day, leaf number and chlorophyll content in the last fully developed leaf were manually measured two times a week. At the end of the experiment fresh and dry biomass weight, water and nitrogen use efficiency and relative water content were measured. The main goals were to collect a complete set of phenotypic data related to maize morphological and physiological response to water and nitrogen availability and to explore the relationship between drought tolerance in maize at vegetative and reproductive stages. Image data have currently being extracted and analyzed using IAP (Integrated Analysis Platform) software, developed at IPK. Preliminary data analysis of leaf number collected manually during the experiment has been done. The timing and the magni- tude of plant’s leaf number response to limited water and nitrogen supply differed among the genotypes. In 14 genotypes (five susceptible and nine tolerant to drought) significant effect of water and water + nitrogen stress on leaf number could be detected ten days after the treatment start, when the plants had 9.3 leaves on average (from 8.4 to 10.5). Remaining six (tolerant) inbreds responded to water and water + nitrogen stress somewhat later, indicating possible higher tolerance. The biggest difference in leaf number was one to two leaves, depending on the genotype, and was detected 15 to 20 days after start of the stress treatment between control and water/water + nitrogen treatments. In most of the inbreds, the effect of nitrogen treatment could not be observed until the very end of the experiment. Although limited water supply was the main restrictive factor in leaf development, combined water + nitrogen stress has been slightly higher than just water stress.

99 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 59 Development of integrated phenotyping

Simon Fraas, Sabine Lüthje, and Hartwig Lüthen Biozentrum Klein Flottbek, Universität Hamburg [email protected]

Our journey into phenotyping started with the initial challenge to analyze an increasing number of images showing roots and hypocotyls of wild type Arabidopsis and auxin-response- mutants. Initially it was done manually in a time-consuming process utilizing ImageJ, but with an increasing number of lines studied measuring the images became a bottleneck of our research.

This lead to the development of HansaTrace, an ImageJ Plugin developed to automatically measure root length by extracting the longest shortest path of a binary skeleton of Arabidopsis plants. As auxin inhibits root growth while it stimulates shoot elongation, we also developed a protocol for measuring elongation of excised hypocotyl segments. The imaging aspect of hypocotyl growth measurement turned out to be much more challenging than it was in root systems. Segments float freely in auxin-solution filled modified multi-well plates, while roots grow on agar plates and are treated by spraying them with auxin solutions.

Our collaborators focus on cell based phenotyping, measuring auxin induced swelling of wild type and mutants defective in the possible auxin receptor ABP1 and found that the mutant lines did not show the response. We looked if ABP1 is a growth relevant auxin receptor. As both responses to the hormone are very rapid, the aim had to be to study the auxin growth responses at a high temporal resolution, resulting in a large number of images to be analysed.

Iterating through several setup generations for each experiment we managed to establish growth setups providing optimal combinations of growth conditions and imaging techniques. We adopted our image analysis techniques to mature Arabidopsis plants at different stages of development, as well as to young maize plants and successfully analysed stress responses in for a proteomic study targeting the abundance of plasma membrane bound peroxidases and other redox proteins.

Both phenotyping tracks involve plants of very different stages of development, and very different technical problems of image acquisition were encountered. We will discuss how we optimized our imaging train in order to avoid problems ranging from reflections, irregular illumination and how to make our analysis software cope with them. We will also demonstrate of imaging based phenotyping approaches are able to efficiently characterize the complex physiology of stress adaptation and hormone signaling.

100 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 60 Apoplastic pH that is triggered by chloride stress has a pivotal role in the modulation of the proteome.

Christoph-Martin Geilfus1* and Sebastien Christian Carpentier1,2 1SYBIOMA, Proteomics Core Facility, KU Leuven, O&N II Herestraat 49 - bus 901, B-3000 Leuven, Belgium. 2Division of Crop Biotechnics, Department of Biosystems, KU Leuven, Willem de Croylaan 42 - box 2455, B- 3001 Leuven, Belgium. * [email protected]

A chloride or drought-stress induced increase of leaf apoplastic pH has been linked with the regulation of stomatal aperture via effects exerted through abscisic acid. Apart from this function, little is known about other physiological implications arising from the transitions in the pH of the leaf apoplast. It is subject of debate whether stress-induced changes in leaf apoplast pH have further remote functions to adjust the physiological state. During the establishment of chloride stress the pH of the leaf apoplast undergoes a transition and we show here that this pH event links chloride stress with proteomic changes associated to different cellular compartments and biological processes in leaves of maize (Zea mays L.). Proteomic footprints being acquired by ultra performance liquid chromatography in combination with mass spectrometry were analyzed in dependency to the leaf apoplastic pH relations. Leaf apoplastic pH was non-invasively phenotyped in real-time via microscopy-based ratiometric imaging analysis. 44 proteins were identified to specifically increase in abundance in response to chloride stress via effects exerted through the leaf apoplastic proton relations. Identified proteins are implicated in responses to stress. Among them was calmodulin, a further group of abscisic acid-responsiveness proteins and proteins that play a role in the synthesis of phenylpropanoids or compatible osmolytes. Further enzymes participate in carbohydrate metabolism, protein biosynthesis or in the production of compounds from the cell wall or cytoskeleton. Results highlight a new and unexpected role of the apoplastic pH pivotal in modulating global proteomic responses. For the first time the importance of integrating leaf apoplastic pH relations into models of proteomic adjustments that ensue upon the initiation of chloride stress is reported. This indicates that apoplastic pH is a factor with the potential to provide physiological context through the integration of protein abundances.

101 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 61 Integrated phenotyping of model and crop plants: Development of experimental setups

Simon Fraas1, Anne Hofmann1, Anna Tahmasian1, Pietro Magnabosco2, Zaneta Michalec- Warzecha3, Momme Opitz1, Marta Libik-Koieczny3, Antonio Masi2 and Sabine Lüthje1 1 Biozentrum Klein Flottbek, Universität Hamburg 2 University of Padua, Italy 3 Polish academy of Science, Krakow, Poland [email protected]

Habitus of plants is different and due to this phenotyping approach has to be adapted to the species under investigation. Besides thale cress (Arabidopsis thaliana HILL.), we investigate barrelclover (Medicago truncatula GAERTN.), ice plant (Mesembryanthemum crystallinum L.) and maize (Zea mays L.). Except for the latter they all have a flat rosette-like phenotype during the time of experiment, which enables us to deploy consumer grade 2D digital cameras above the specimen. For analysis of digital images Arabidopsis and plants of similar habitus we utilize a custom ImageJ plugin. This plugin creates binary masks of the plants utilizing HSB colorspace based thresholding and uses the ImageJ particle analyzer to extract a set of parameters including but not limited to area, circularity and perimeter.

In the present work we compared phenotypes of Arabidopsis wildtype and a mutant. The data showed onsetting difference in leaf habitus 14 days after germination. The same set-up was used for barrelclover to investigate alterations by osmotic stress in comparison to control experiments.

For ice plant, as a well-known model plant for salt stress, a first run with phenotyping led us to the conclusion to choose a different setup stabilizing the leaves and a different timeframe for salt stress induction.

Finally we studied maize under different stress conditions (flooding, osmotic stress etc.) on plant organ and cellular level (aerenchyma formation, peroxidase profiling, etc.). Without the convenient rosette habitus maize poses a challenge in registering phenotypic traits, thus we concentrated on roots and leaves in different setups.

102 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 62 Phenotyping Phaseolus sp. Germplasm for Chilling Tolerance at Seedling Stage

Faik Kantar1, Bilgehan Turan Certel1, Volkan Gözen2, Akın Tepe2, Vahdettin Çiftçi3, Hati- ce İkten1, Cengiz Toker4 1Akdeniz University, Faculty of Agriculture, Department of Agricultural Biotechnology, Antalya Turkey /corresponding author [email protected] 2Western Mediterranean Akdeniz Agricultural Research Institute, Muratpaşa Antalya Turkey. 3Abant İzzet Baysal University, Faculty of Natural and Agricultural Sciences, Gölköy Bolu Turkey. 4Akdeniz University, Faculty of Agriculture, Department of Field Crops, Antalya Turkey.

Phaseolus beans are one of the most important food legume species in the world. Cold stress (temperatures below 10 oC) at seedling stage in the spring is primary stress factor limiting the growth and production of beans. A project was initiated to screen Phaseolus germplasm collection of 826 genotypes from national and international sources and gene banks. The germplasm was tested at seedling stage at 5°C in comparison with control plants at 28/22°C (16°C h day/8°C h night) at 300-700 µmol m-2 S-1 light intensity in controlled climatic conditions. Phenotyping was made on the bases of visual assessment of chilling damage, measurements of membrane leakage, Chlorophyll (SPAD) and leaf colour parameters and dry matter production.

Correlations and regression analysis were made of chilling damage scale with other phenotyping parameters investigated of cell membrane leakage conductivity, Chlorophyll (SPAD) values, leaf colour parameters (L value = Lightness, a value = redness – greenness, b value = yel- lowness-blueness, Chroma (C value) = colour saturation or intensity and hue angle = h = arc tan a/b) and seedling dry matter production in order to find out whether they can be used as reliable screening criteria for cold tolerance assessment.

Considerable level of variation in chilling tolerance existed within the germplasm screened. Of 826 genotypes that was grouped by Chilling Damage Assessment Scale (CDAS = 1 (highly tolerant = no visible damage) to 9 (highly sensitive = no plants survived), 10 accessions showed high levels of chilling tolerance (CDAS 1), 29 accessions tolerance (CDAS 2) and 66 genotypes slight tolerance (CDAS 3) while 102 accessions sensitive (CDAS 8) and 53 accessions highly sensitive (CDAS 9) with the rest being in between.

Fractional SPAD values of the difference between control and cold treatment were positively correlated with cold damage index values. Correlations between the parameters of variables investigates were mostly statistically insignificant whereas significant correlations were recorded between the parameters within the variables investigated. The results were discussed in order to find significant relations between phenotyping parameters measured. This paper highlights the results of correlation and regression analysis.

103 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 63 Exploiting the forgotten common bean landrace phenotypes: Disease resistance and genomic data integration

Susana T. Leitão1, Mara L. Alves1, Diego Rubiales2, and Maria C. Vaz Patto1 1Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal 2Institute for Sustainable Agriculture, CSIC, Cordoba, Spain

Common bean (Phaseolus vulgaris L.) is the worldwide most important grain legume for human consumption. In addition to its nutritional and health benefits, it is an environmentally friendly resource because of its ability to fix atmospheric nitrogen as a result of the endosym- biotic association with the soil bacteria Rhizobium. However, due to yield instability caused by biotic and abiotic stresses, this crop is underused in Europe. It is therefore essential to genetically improve common bean varieties stress resistance in order to attain more stable, reliable and sustainable local production. Fusarium wilt (caused by the soil borne fungus Fusari- um oxysporum f. sp. phaseoli) is among the most important constraints to bean production, being intimately related to yield loss.

Portugal holds a very promising national common bean germplasm collection, resulting from more than five centuries of natural adaptation and farmer’s mass selection, not yet fully explored in breeding. In this work we have used an integrated phenotyping approach to search for the genomic regions controlling fusarium wilt resistance in a collection of Portuguese landraces. This will pave the way for the future improvement of resistance in common bean, based on this valuable, highly diverse and unexplored genetic resource.

To do so, a collection of 150 Portuguese common bean landraces was phenotyped for resistance to fusarium wilt under growth chamber controlled conditions. Disease symptoms were assessed, using a 1-5 visual scale, every 3 days, from the 7th to 30th day after inoculation. Two weeks after infection, observed disease severity varied greatly among landraces, ranging from landraces completely dead till landraces with no symptoms or only low levels of yellow- ing or discoloration on the leaves margins.

The same collection was screened with more than 12.000 SNPs (Single Nucleotide Poly- morphism) uniformly distributed throughout the genome. A genome-wide association study, joining the disease resistance phenotypic information with the genomic information, is currently ongoing using mixed linear models to account for population structure and familiar re- latedness.

This integration of phenotyping at different levels will allow the identification of molecular tools to assist future common bean resistance precision breeding.

104 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 64 High-throughput screening tools for identification of traits contributing to salinity tolerance in Arabidopsis thaliana

Klára Panzarová1, Mariam Awlia2, Ariana Nigro3, Jiří Fajkus1, Zuzana Benedikty1, Mark Tester2 and Martin Trtílek1. 1PSI (Photon Systems Instruments), Drásov, Czech Republic. 2Center for Desert Agriculture, KAUST, Saudi Arabia. 3Institute of Plant Biology, University of Zürich, Switzerland.

Non-invasive capturing and interpreting of plant structural and functional phenotypes in controlled or dynamically changing environment is long-standing and necessary requirement for genetic and physiological research by crop breeders, agricultural industry, and academia. To sustain global food security the major challenge global agriculture and plant biology field has been facing is the identification of new high-yielding genotypes of agricultural crops that are adapted to our future climate. Soil salinity is one of the main stress factors that are severely affecting the agriculture land in global scale and results in significant reduction of plant growth and yield. It was shown that plants suffer a rapid growth reduction upon the first exposure of their roots to salt stress, which is occurring prior to the accumulation of ions to toxic concentrations in the shoots. During this early phase, symptoms of growth reduction include slower leaf emergence and a small growth size. The phenotypic traits associated with this type of tolerance can be quantified in the days immediately after imposition of stress using non-destructive image-based phenotyping.

To enhance our understanding of the early responses to salinity, we designed an experimental protocol based on using high-throughput and non-invasive imaging technologies developed at Photon Systems Instruments (PSI, Czech Republic). The methodology presented is based on automated integrative analysis of photosynthetic performance, growth analysis and color index analysis at the onset and early phase of salinity stress response of Arabidopsis thaliana ecotypes grown in soil. Here we show that stress imposition significantly and rapidly affected photosystem II operating efficiency, subsequently impacted growth dynamics and greening index of Arabidopsis plants at different stages of stress response.

Our work provides quantitative insights into early phase of salinity response and provides robust protocol for high-throughput image-based analysis of phenotypic traits associated with this early phase of salinity response. We show that the integrative concept of PlantScreenTM high-throughput phenotyping platform provides a powerful tool for acquisition and selection of morphological, physiological and biochemical parameters, which can be used for identification of various components underlying early plant responses to various environmental conditions.

105 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 65 High-throughput screening tools for identification of traits contributing to salinity tolerance in Arabidopsis thaliana

Non-invasive capturing and interpreting of plant structural and functional phenotypes in controlled or dynamically changing environment is long-standing and necessary requirement for genetic and physiological research by crop breeders, agricultural industry, and academia. To sustain global food security the major challenge global agriculture and plant biology field has been facing is the identification of new high-yielding genotypes of agricultural crops that are adapted to our future climate. Soil salinity is one of the main stress factors that are severely affecting the agriculture land in global scale and results in significant reduction of plant growth and yield. It was shown that plants suffer a rapid growth reduction upon the first exposure of their roots to salt stress, which is occurring prior to the accumulation of ions to toxic concentrations in the shoots. During this early phase, symptoms of growth reduction include slower leaf emergence and a small growth size. The phenotypic traits associated with this type of tolerance can be quantified in the days immediately after imposition of stress using non- destructive image-based phenotyping.

106 2nd General Meeting of COST FA1306, PhenomenAll, Copenhagen April 18-20, 2016

Poster 66a IDENTIFICATION OF SELF-COMPATIBLE VINE CACTI HYBRIDS BY USING FLOW CYTOMETRIC ANALYSIS

Noemi Tel-Zur French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research, Sede Boqer Campus, Ben-Gurion University of the Negev, Israel

Self-incompatibility (SI) is a widespread genetic mechanism by which plants recognize and reject their own pollen grains. The ability to select self-compatible (SC) hybrids would greatly benefit growers of vine cacti by eliminating the need to grow complementary cultivars for cross pollination. With this breeding objective in mind, reciprocal interspecific-interploidy crosses and a first back-cross were performed between the SC tetraploid Hylocereus megalanthus and a SI diploid, either Hylocereus undatus or H. monacanthus, at Ben-Gurion Universi- ty of the Negev (Israel). Viable hybrids were successfully obtained, and preliminary data showed that the SC hybrids were tetraploid and fertile. Thus, there is most probably a relationship (yet to be determined) between the tetraploid level and SC.

Determining the ploidy level by chromosome count is technically challenging. A reliable and rapid alternative technique to estimate DNA content and ploidy is flow cytometry analysis. In the current work, nuclear DNA content (2C-DNA) was used to estimate ploidy level in comparison with an external standard, which was either of the diploids H. monacanthus or H. undatus, each alone or in combination with the tetraploid H. megalanthus. Ge- nome size was calculated using P. sativum as the internal standard reference. Flow cytometric analysis was carried out according to Dolezêl et al. (1989, Biol Plant 31:113–120) with minor changes (Cisneros and Tel-Zur 2010, Euphytica 174:73–82) due to the high polysaccharide content in the tissues of these species. Flow cytometric analysis showed nuclear DNA content values ranging from 8.35 to 9.59 pg/2C for tetraploid hybrids. These findings indicate that flow cytometry analysis could indeed be used as a first selection criterion to identify promising hybrids; thereafter additional rounds of selection could be used for other traits, such as improved fruit quality. Thus, the new selection methodology proposed here would facilitate early screening of a large number of hybrids. Although all our vine cacti crosses all yielded SC tetraploid hybrids, the correlation between ploidy level and SC still needs to be demonstrated conclusively. For this purpose, flow cytometric analyses of a large number of SI and SC hybrids are required.

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Poster 66b Saving banana from thirst: -omics integration for the characterization towards drought tolerance

Jelle van Wesemael, Ewaut Kissel, Jassmine Zorilla and Sebastien Carpentier Laboratory for Tropical Crop Improvement, KU Leuven, Willem De Croylaan 42, 3001 Leuven.

Water deficit is one of world’s major constraints in agriculture and will aggravate in the future. An important crop that needs vast amounts of water for optimal production is banana (Musa spp.). Millions of people depend on banana as staple food, while it is mostly grown in rain fed systems and hence very prone to fluctuating water availability. Worldwide a huge (uncharacterized) biodiversity is available, and we believe optimal exploration and exploitation of this diversity will alleviate issues with drought and also with pests and diseases. The International Transit Center of Bioversity International holds the world’s biggest collection of banana biodiversity (>1500 accessions) which is hosted by the Laboratory for Tropical Crop Improvement (KU Leuven).

Our first aim is to perform a long term phenotyping model to evaluate drought tolerance within a diverse set of edible sterile bananas. Acquired experimental phenotypic data will also be included in accession information sheets available to the end users of the Bioversity world banana collection. Our second aim is to identify potential tolerance related markers. Those markers are sought by measuring variables on three levels: plant phenotype, transcriptome, and proteome. By integrating the data from the high throughput phenotyping system to the molecular analysis we are able to correlate the presence of specific alleles to tolerant phenotypes. In this perspective, we have already established proof of concept experiments to find molecular markers at transcript and protein level. We seek to further establish the phenotyping effort to guide more detailed marker selection, and get a more detailed picture of the phenotypic response to drought within the banana biodiversity.

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Theme 4

Data processing and integration

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Poster 67 A data-driven model to explain the emergence of tan spot in the UK

Anyela Camargo, Odín Morón The National Plant Phenomics Centre, IBERS, Aberystwyth University, Aberystwyth, SY23 3EB

The future of global agriculture and its impact on food security is one of the most urgent issues in today’s world. Farmers must prepare for changes in the climate that is likely to feature more erratic weather patterns that will necessarily have an effect in the emergence and re- emergence of plant diseases. One such disease is Tan Spot, caused by Pyrenophora tritici- repentis, which affects wheat crops worldwide. Although Tan Spot is not yet a disease critical for UK’s wheat production, global warming may change this. Historical data on disease incidence showed a steady increase in Tan Spot outbreaks since 2005. Current strategies to control Tan Spot are mainly through the use of agrochemicals, the preferred method for integrated pest management (IPM). However, relying solely on pesticides for IPM has numerous problems and limitations. In order to prepare farmers for the risks posed by pests/diseases, scientists must work closely with farmers to ensure that agriculture can stand up to the impact of climate change. Emerging ‘omics’ technologies combined with data modelling can help scientists to accurately identify the disease’s ideal conditions to grow, estimate its severity, predict its effect on yield, and discriminate possible virulence mechanisms. In this research, we have used High-throughput plant phenotyping technologies as well as historical weather and disease incidence data to try and explain the sudden raise of tan spot in the UK. We analysed images of MAGIC wheat lines infected with tan spot which were screened at the Na- tional Plant Phenomics Centre. Phenotypic data was integrated with climate and field data to create a machine-learning based model to explain the incidence of tan spot in the UK and its correlation with other European countries were the disease is economically important.

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Poster 68 ELIXIR Portugal - Integrating Genomic and Phenotypic Data for Woody Plants

Inês Chaves1,2, ELIXIR.PT-NODE3 1 IBET, Apartado 12, 2781-901 Oeiras, Portugal 2 ITBQ-UNL, Avenida da República, 2780-157 Oeiras, Portugal 3elixir-portugal.org

ELIXIR Portugal is a consortium of Portuguese research institutions (INESC-ID, iBET, IGC, ITQB-UNL) which is part of the European Research Infrastructure for Life-Science Infor- mation (ELIXIR, http://www.elixir-europe.org/) and of the national biological information network BioData.pt (http://www.biodata.pt). Like ELIXIR itself, the Portuguese node is organized as a decentralized network of specialized centers, under a common computing infrastructure, and with shared training and industry/entrepreneurship programmes with a Europe- an dimension.

Woody plants are a major natural resource in Europe, with a huge ecological and economical impact, supporting millions of jobs across diverse industries (e.g. wine, fruit, olive oil, coffee, paper, timber and cork) and strongly contributing to the European GDP. In Portugal, woody plants represent 10% of the GDP, and are a central research domain in both academia and industry.

Massive sequencing and genotyping of woody plants (and their pathogens and pests) is generating large quantities of molecular data. Phenotypic data collected for each plant genotype, often in multiple locations and field conditions, has also been generated within different breeding programmes. Moreover, novel approaches are being implemented to identify and collect large sets of quantitative phenotypes and to explain the genetic basis of important traits.

Elixir Portugal aims to secure and deliver the core data resources underpinning woody plant research, provide tools and services to drive data access and exploitation, develop and maintain controlled vocabularies and standardized APIs for data interoperability, reuse and integration, and provide training programmes in this domain.

By focusing on genotype-phenotype analysis based on the widest available public datasets, enabling more powerful association analysis and opening the way to understanding of function, candidate gene prioritisation, and improved plant breeding, this initiative will contribute to build a framework that is of added-value to the woody plant user communities including industry and academia.

Acknowledgements: FCT for financial support through project EXCL/EEI-ESS/0257/2012, and EU for support through H2020 project ELIXIR-EXCELERATE, nº 676559.

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Poster 69 Fast Processing of Hyperspectral Data for Phenotyping of Diseased Plants

Mirwaes Wahabzada1, Kristian Kersting2, Matheus Kuska1, Anne-Katrin Mahlein1 1INRES-Phytomedicine, University of Bonn, Bonn, Germany, 2 CS Department, Technical University of Dortmund, Dortmund, Germany

Understanding the adaptation process of plants to biotic stress is essential for improving management practices and breeding strategies of crops for a sustainable agriculture in the coming decades. In this context, non-invasive plant phenotyping by means of sensor technologies, such as hyperspectral imaging, has been shown to be successful in monitoring plant physiological traits and genotype specific responses to biotic stresses. Furthermore, changes in cellular leaf structures or biochemical characteristics are detectable via hyperspectral imaging. However, the highly informative spectral data sets are usually very large and need new appropriate analysing methods in terms of scalability and interpretability.

In this work we consider a fast processing of hyperspectral images with the aim of automated segmentation of spectral information at pixel level. To achieve this, we consider to reduce the complexity of data using a hierarchical decomposition of spectral signatures within the image. In a second step we apply standard clustering algorithms on the reduced data to find a clear separation of different areas of diseased leaf. The benefits of this proposed method is that it is fast and does not require any prior information such as labels or training data. For instance, recorded signatures for pixels are seldom annotated, as even stressed plants show only local signs of stress. On the other hand, the efficiency is of great importance often due to the ab- sence of prior information and the inability to adapt previous efforts. That is, as the data is often acquired under changing conditions, it requires a large number of repeated experiments with different parameters to get optimal results.

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Poster 70 PlotCut: A high-throughput data extraction tool for UAS (drone) imaging used in phenotyping agricultural research plots and research fields

Jesper Cairo Westergaard University of Copenhagen, Department of Plant and Environmental Sciences.

Agricultural research plots in-situ can be imaged not only by stationary or tractor mounted imaging sensors, but also by unmanned aerial systems (UAS). Deploying the UAS and subsequently having the many hundreds of individual images made into one orthomosaic image is not the big hurdle anymore. Extracting data from the several hundred research plots (some- times thousands when we’re talking plant breeders), is now the most time consuming part of the process outlined below:

Fly drone + take pictures è Create orthomosaic è Extract pixel data from individual plots è Calculate relevant indices (e.g. Excess Green Index) è Deliver phenotyping research data to scientist and/or data to aid the plant breeder in decision making.

It used to be that the data extraction accuracy relied solely on a human, visually based, mark- ing off and extracting of the image snippet matching each research plot. This left much room for naming errors, and the repeatability would often suffer as well.

Now, perform these tasks upwards of fifteen times a season for just one research area, and it becomes apparent that a templating tool is needed. We are now building such a tool at the University of Copenhagen.

The tool we are developing is a high-throughput data extraction platform, named PlotCut. We do this together with several Nordic plant breeders. Their continuing input in the process – from one software version to the next – is essential for the long term success of the platform.

In its current status the platform calculates and outputs (to CSV files) for each research plot in the images the following:

RGB values, excess green index, normalized excess green index, crop coverage (only relevant in certain instances, depending on growth stage and the image resolution).

Future versions are planned to include options for CIR/NIR cameras (NDVI), user annotation of plots (while quality checking phenotyping rectangles), automated statistical operations on the phenotyping results and report generation.

Acknowledgements: PlotCut is developed in the Public Private Partnership – Plant Pheno- typing Project (6p). Funded by the Nordic genetic resource center NordGen and coordinated by the University of Copenhagen, Dept. of Plant and Environmental Sciences.

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Theme 5

Stakeholder interactions

No poster contributions

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List of participants

Themes: 1. Phenotyping at plant and field level Contribution: E. Exhibitions 2. Phenotyping at cell level K. Keynote speaker 3. Integrated phenotyping O. Oral presentation 4. Data processing and integration P. Poster 5. Stakeholder interactions

Surname E-mail University/Company Theme University of Novi Sad, Marijana Ačanski [email protected] Faculty of Technology, 3 P Serbia Guray Akdogan [email protected] Ankara University, Turkey 2 O Thomas Altmann [email protected] IPK Gatersleben, Germany 3 O University of Aveiro, Por- Eliana Alves [email protected] 2 P tugal University of Plovdiv, Vesselin Baev [email protected] 2 P Bulgaria Gregor Mendel Institute of Elke Barbez [email protected] molecular plant biology, 2 P Vienna, Austria Eleonora Barilli [email protected] CSIC, Spain 1 P Rene Benjamins [email protected] Syngenta, The Netherlands 1 Universidade NOVA de Maria Catarina Bicho [email protected] 1 P Lisboa, Portugal Christiaan Biemond [email protected] KeyGene, The Netherlands 4 O Leibniz Institute of Plant Gerd Patrick Bienert [email protected] Genetics and Crop Plant 1 O Research, Germany University of Bologna, Federico Bosetto [email protected] Italy Maize Research Institute Sofija Bozinovic [email protected] 3 P Zemun Polje, Serbia Slovak University of Agri- Marian Brestic [email protected] 1 P culture, Slovakia Aberystwyth University, Anyela Camargo [email protected] 4 P UK Nadia Campos [email protected] KU Leuven, Belgium 2 P Sebastien Carpentier [email protected] KU Leuven, Belgium Jens Michael Carstensen [email protected] Videometer A/S, Denmark 1 E ITQB-UNL/IBET, Portu- Inês Chaves [email protected] 4 P gal University of Copenhagen, Svend Christensen [email protected] 2 P Denmark ITQB-UNL and ISA-UL, Joaquim Miguel Costa [email protected] 1 P Portugal

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Gregor-Mendel Institute of Angelika Czedik- [email protected] Molecular Plant Biology, 1 O Eysenberg Austria Universiteit Hasselt, Bel- Stefanie De Smet [email protected] 1 P gium Klaus J. Dehmer [email protected] IPK Gatersleben, Germany Getinet Desalegn [email protected] BOKU, Vienna, Austria 3 Université de Lille Christophe Djemiel [email protected] 2 O 1/UGSF, France

The Franciszek Górski Ewa Dubas [email protected] Institute of Plant Physio- 2 P logy, Krakow, Poland

Mohamed El-Esawi [email protected] Tanta University, Egypt 2 P Zurich University of Ap- Johannes Fahrentrapp [email protected] plied Sciences ZHAW, 1 P Switzerland Agricultural Research In- Dionysia Fasoula [email protected] 1 P stitute, Cyprus Carlsberg Research Labo- Christine Finnie [email protected] 2 ratory, Denmark Cyprus University of Vasileios Fotopoulos [email protected] 2 P Technology, Cyprus University of Hamburg, Simon Fraas [email protected] 3 P Germany Christoph-Martin Geilfus [email protected] Kiel University, Germany 3P Fraunhofer Development Stefan Gerth [email protected] Center X-ray Technology, 1 O Germany Estelle Goulas [email protected] University of Lille, France 2 P University of Copenhagen, Dominik Grosskinsky [email protected] 2 P Denmark Leonor Universidade de Lisboa, [email protected] 2 P Guerra-Guimarães Portugal University of Copenhagen, Thure Pavlo Hauser [email protected] Denmark Central European Institute of Technology, Masaryk Jan Hejatko [email protected] 2 P University, Czech Repub- lic Hasselt University, Belgi- Sophie Hendrix [email protected] 2 P um Andrea Lorena Fondazione Edmund [email protected] 1 P Herrera Valderrama Mach, Italy University of Helsinki, Kristiina Himanen [email protected] 1 P Finland University Hamburg, Anne Hofmann [email protected] 3 P Germany Institute of Experimental David Honys [email protected] Botany ASCR, Czech Re- 2 P public

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Csilla Hudek [email protected] University of Torino, Italy 1 P Worldwide Phenomics Mark Humble [email protected] 1 Services, Netherlands Phenospex, The Nether- Grégoire Hummel [email protected] 1 E lands Aarhus University, Den- Benita Hyldgaard [email protected] 1 O mark University of Trás-os- Gilberto Igrejas [email protected] Montes and Alto Douro, 2 P Portugal Christian Sig Jensen [email protected] DLF, Denmark 5 K Vienna Biocenter Core Jakub Jez [email protected] 1 P Facilities GmbH, Austria Austrian Institute of Tech- Claudia Jonak [email protected] 2 P nology, Austria Leibniz-Institute of Plant Astrid Junker [email protected] Genetics and Crop Plant 1 P Research (IPK), Germany Johannes Ravn Aarhus University, Den- [email protected] Jørgensen mark Akdeniz University, Facul- Faik Kantar [email protected] 3 P ty of Agriculture, Turkey Wageningen University Atiyeh Kashaninia [email protected] and Research Centers, The 3 Netherlands University of Tartu, Fin- Hannes Kollist [email protected] 2 O land Institute of Field and Ve- Ankica Kondic-Spika [email protected] getable Crops, Novi Sad, 1 P Serbia Wageningen University, Lammert Kooistra [email protected] 1 P The Netherlands Slovak University of Agri- Marek Kovar [email protected] 1 P culture, Slovakia University of Bonn, Ger- Matheus Kuska [email protected] 2 O many MPI of Molecular Plant Karin Köhl [email protected] 1 P Physiology, Germany University of Copenhagen, Dorthe Horn Larsen [email protected] 1 Denmark Clas- Lantmännen Lantbruk, Clas-Tomas Larsson [email protected] Sweden ITQB-NOVA, Universida- Susana Leitao [email protected] de Nova de Lisboa, Portu- 3 P gal University of Copenhagen, Xiangnan Li [email protected] 1 Denmark University of Copenhagen, Fulai Liu [email protected] 1 Denmark Peter Lootens [email protected] ILVO, Belgium 1 P

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Nature Research Centre, Kristina Loziene [email protected] 2 P Lithuania University of Hamburg, Hartwig Lüthen [email protected] 3 Germany University of Hamburg, Sabine Lüthje [email protected] Germany University of Aveiro, Elsabete Maciel [email protected] 2 P Germah Aberystwyth University, Marta Malinowska [email protected] 1 O UK Ulg, Gembloux Agro-Bio Guillaume Marlier [email protected] 1 P Tech, Belgium Antonio Masi [email protected] University of Padova, Italy 1 Aarhus University, Den- Thayna Mendanha [email protected] 1 P mark University of Liège, Belgi- Benoit Mercatoris [email protected] um Heinz Walz GmbH, Ger- Oliver Meyerhoff [email protected] 1 E many Institute of Field and Ve- Sanja Mikić [email protected] getable Crops, Novi Sad, 1 P Serbia University of Copenhagen, Lucia Montini [email protected] 1 P Denmark University of Copenhagen, Elizabeth Neilson [email protected] 3 O Denmark

Slovak University of Agri- Janka Nôžková [email protected] 1 P culture in Nitra, Slovakia

Andreas Olsen [email protected] Knud Jepsen a/s, Denmark 5 Aarhus University, Den- Carl-Otto Ottosen [email protected] 1 K mark Wageningen University, Theoharis Ouzounis [email protected] 1 O The Netherlands PSI (Photon Systems In- Klara Panzarova [email protected] struments, s. r o.), Czech 3 PE Republic Ivan Paponov [email protected] NIBIO, Norway 2 P Biological Research Cen- Kenny Paul [email protected] 1 P tre, HAS, Hungary LemnaTec GmbH, Germa- Stefan Paulus [email protected] 1 E ny Natural Resources Institute Pirjo Peltonen-Sainio [email protected] 1 O Finland (Luke), Finland University of Copenhagen, Thi Thuy Hong Phan [email protected] 1 Denmark Forschungszentrum Jülich, Roland Pieruschka [email protected] 5 O Germany Universidade NOVA de Carla Pinheiro [email protected] 2 P Lisboa, Protugal

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Wageningen UR, The Net- Gerrit Polder [email protected] 1 O herlands The Hebrew University of Indira Pudel [email protected] 3 O Jerusalem, Israel University of Copenhagen, Jesper Rasmussen [email protected] 1 Denmark University of Copenhagen, Thomas Roitsch [email protected] 2 K Denmark University of Copenhagen, Eva Rosenqvist [email protected] 1 P Denmark Filippo Rossi [email protected] GMR Strumenti SAS, Italy 1 E Slovak Academy of Sci- Terezia Salaj [email protected] 2 P ences, Slovakia Forsachungszentrum Jü- Ulrich Schurr [email protected] 4 O lich, Germany Phenospex B.V., The Net- Stefan Schwartz [email protected] 5 O herlands University of Tartu, Esto- Yana (Iana) Sindarovska [email protected] 2 P nia University of Copenhagen, Simon Fiil Svane [email protected] 1 Denmark University of Copenhagen, Jesper Svensgaard [email protected] 1 Denmark Aarhus University, Den- Chris Sørensen [email protected] mark Ben-Gurion University of Noemi Tel Zur [email protected] 3 P the Negev, Israel Saoirse Tracy [email protected] UCD, Ireland 1 P Forest Research Instiute, Ivaylo Tsvetkov [email protected] 1 P Bulgaria University of Bologna, Roberto Tuberosa [email protected] 1 P Italy University of Vienna, Au- Reinhard Turetschek [email protected] 2 O stria Ismail Turkan [email protected] Ege, Turkey 1 P RWTH Aachen, For- Björn Usadel [email protected] schungszentrum Jülich, 4 K Germany Wageningen UR, The Net- Rick van de Zedde [email protected] 1 P herlands Agri Information Partners, Berno van der Geest [email protected] The Netherlands PhenoKey B.V., The Net- Bas van Eerdt [email protected] 4 herlands Wageningen UR, The Net- Theo van Hintum [email protected] 5 K herlands Marie-Christine van La- [email protected] Ghent University, Belgium 1 P beke Katholieke Universiteit Jelle van Wesemael [email protected] 3 P Leuven, Belgium

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Catholic University of Por- Marta Vasconcelos [email protected] 3 O tugal Biological Research Cen- Imre Vass [email protected] 1 P tre, Szeged, Hungary Crop Research Institute, Pavel Vítámvás [email protected] 2 P Czech Republic Phenospex B.V., The Net- Philipp von Gillhaußen [email protected] 1 S herlands Wageningen UR, The Net- Danijela Vukadinovic [email protected] 1 O herlands University of Bonn, Ger- Mirwaes Wahabzada [email protected] 4 P many Achim Walter [email protected] ETH Zürich, Switzerland 1 K Jesper Cairo Wester- University of Copenhagen, [email protected] 4 P gaard Denmark University of Vienna, Au- Stefanie Wienkoop [email protected] 2 P stria Donald Danforth Plant Melinda (Mindy) Wilson [email protected] Science Center, Missouri, USA University of Bonn, Ger- Monika Wimmer [email protected] 2 O many Slovak University of Agri- Marek Zivcak [email protected] culture, Dept. of Plant 1 P Physiology, Slovakia Jassmine Zorrilla [email protected] KU Leuven, Belgium 2 O The F. Górski Institute of Plant Physiology Polish Iwona Zur [email protected] 2 P Academy of Sciences, Po- land

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