Hydraulic properties of Zinnia elegans from cellular development in vitro to performance in planta Peter Twumasi Promotoren: Prof. Dr. A. M. C. Emons Hoogleraar Plantencelbiologie Wageningen Universiteit Prof. Dr. O. van Kooten Hoogleraar Tuinbouwproductieketens Wageningen Universiteit Co-promotoren: Dr. Ir. W. van Ieperen Universitair docent bij de leerstoelgroep Tuinbouwproductieketens Wageningen Universiteit Dr. J. H. N. Schel Universitair hoofddocent bij de leerstoelgroep Plantencelbiologie Wageningen Universiteit Promotiecommissie: Prof. Dr. C. Mariani (Radboud Universiteit, Nijmegen) Prof. Dr. L. H. W. van der Plas (Wageningen Universiteit) Dr. Ir. E.J. Woltering (Wageningen Universiteit en Research Centrum) Prof. Dr. M. De Proft (Katholieke Universiteit Leuven, België) Dit onderzoek is uitgevoerd binnen de C.T. de Wit Onderzoekschool ‘Production Ecology and Resource Conservation’. Hydraulic properties of Zinnia elegans from cellular development in vitro to performance in planta Peter Twumasi Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff in het openbaar te verdedigen op dinsdag 8 mei 2007 des namiddags te 16:00 uur in de Aula Peter Twumasi, 2007 Hydraulic properties of Zinnia elegans: from cellular development in vitro to performance in planta PhD Dissertation, Wageningen University, The Netherlands With summaries in English and Dutch Keywords: Apoptosis, average daily temperature, caspase, cell death, cysteine, cytochemistry, differential day and night temperature, differentiation, DNA fragmentation, electrical conductivity, in vitro culture, leaf osmolarity, light intensity, mesophyll cells, osmolarity, osmotic potential, plant quality, programmed cell death, protease, suspension culture, tracheary element, vase life, vessel element, water stress, xylem hydraulic conductance, xylogenesis, Zinnia elegans ISBN 90-8504-658-5 Contents Chapter 1 General introduction 9 Chapter 2 Modulation of xylem conduit dimensions by soil water availability and the effect on the vase life of Zinnia elegans cut flowers 27 Chapter 3 Effect of temperature on xylem vessel and vessel element lengths in Zinnia elegans stems 45 Chapter 4 Establishing in vitro xylogenic Zinnia elegans suspension culture with improved efficiency in tracheary element differentiation 61 Chapter 5 Osmotic potential of Zinnia elegans plant material affects the yield and morphology of tracheary elements produced in vitro 81 Chapter 6 Effects of osmotic stress and timed hormone application on tracheary element differentiation in xylogenic cultures from two Zinnia elegans cultivars 99 Chapter 7 Manipulation of programmed cell death in xylogenic Zinnia elegans suspension cell cultures influences the kinetics and dimensions of tracheary elements produced 117 Chapter 8 General discussion 137 Summary in English 147 Summary in Dutch 150 Acknowledgements 153 About the author 155 Appendix 156 Education certificate of PE&RC 158 To my dear parents, Mr Kwame Ofori Atta, Sr (late) and Madam Adwoa Addai Pomaah My wife, Agnes Twumasi My sons, Jim O. Twumasi and Rodney Twumasi My daughter, Miranda A. Twumasi Selection of symbols and abbreviations used in this thesis Variable Description Units %TE Percentage of tracheary element differentiation ABA Abscisic acid ADT Average daily temperature oC BA Benzylaminopurine DAPI 4, 6-Diamidino-2-phenylindole DIC Differential interference contrast DIF Difference in day and night temperature oC DMSO Dimethyl sulfoxide DT Daytime temperature oC E-64 N-[N-(L-3-trans-carboxirane-2-carbonyl)- L-leucyl]-agmatine EC Electrical conductivity dSm-1 FA Formaldehyde FDA Fluorescein-diacetate FTL First true leaves GA Glutaraldehyde Ht Harvest time for first true leaves (FTL) days -1 -1 Kh Stem hydraulic conductivity mgs kPa cm Lmax Maximum vessel length cm LO Leaf osmolarity mOsm NAA α-Naphthalene acetic acid NT Nighttime temperature oC OP Osmotic potential MPa PCD Programmed cell death TE Tracheary element TUNEL Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling VE Vessel element VPE Vacuolar processing enzyme YVAD-AMC YVAD-7- amino-4-methylcoumarin Zasp Z-Asp-CH2-DCB (a general caspase inhibitor) τ Vessel half-length or median length cm Chapter 1 General introduction Chapter 1 Background Plants, like other organisms, require a combination of essential genetic and environmental factors for proper growth and development. Any adjustment in the correct balance of interactions between these factors may seriously affect the development of a particular phenotype with subsequent compromise of quality. Thus, for any set of genes, the phenotype is strongly dependent on the prevailing environmental conditions. While many studies have been devoted to the improvement of cut flower quality, such as increased flower density and stem length, less have been focused on the structure and function of the xylem vessels in the stem. Most especially, the hydraulic properties of the fundamental units of the xylem, the tracheary elements (TEs), have been insufficiently studied. In order to gain insight into the effect of xylem vessel dimensions on cut flower quality, this thesis deals with the regulation of the xylem vessel structure and function in the cut flower Zinnia elegans. It focuses on mechanisms involved in the development and regulation of hydraulic properties of TEs at the cellular level, and of vessels and elements at the whole plant level. A further consideration is given to the interaction of genetic and environmental factors with the xylogenesis process. Determinants of plant quality A range of physiological and morphological traits are commonly used as a measure of plant quality. In general, plant quality characteristics differ between species. In most plant species, the shoot and internode lengths, or specific length and weight measurements, are sufficient for quality determination. Quality determination requires further measurements, such as the root growth potential, root and shoot electrolyte leakage, leaf properties, flower density, vase life of cut flowers, and form, size and taste of the plant products. One parameter as an important index for plant quality, especially in cut-flowers and many ornamentals, is the stem length (Sysoyeva and Markovskaya, 2004). Occasionally, certain internal tissue characteristics, such as the anatomy of different cell files in the stem, can be used to determine quality. In cut flowers, for example, the xylem conduit dimensions (length and diameter) are good indicators of vase life properties and for that matter the quality. Thus, each set of xylem conduit dimensions dictates a specific hydraulic functioning of the xylem system that in effect regulates the vase life properties of the cut flower. For instance, the restoration of water uptake and turgidity at the start of the vase life of cut flowers is strongly affected by vascular 10 General introduction hydraulic conductance and its recovery from embolism (Zimmermann and Jeje, 1981; Comstock and Sperry, 2000; Nijsse et al., 2001; Twumasi et al., 2005). The interaction between the genetic makeup of the plant and the conditions within which the plant develops strongly affects any particular phenotype under study, for example, in our case the xylem conduit dimensions (Gregorius, 1977; Cavalli-Sforza and Feldman, 1978). Thus, the environmental (E) and genotype (G) interaction can be mathematically simplified as: P = f(E, G). In a more complex set of genetic and environmental factors, the phenotype for the kth observation on the ith genotype in the jth environment (Pijk) can be approached by the linear equation: Pijk = Ej + Gi + (GE)ij + εijk where, Ej is an environmental effect, Gi is a genotypic main-effect, (GE)ij is a genotype-by- environment interaction effect, and εijk is a residual effect (Cooper et al., 2004). These environmental and genetic interactions have over the years been harnessed to improve traits in a particular organism. Environmental conditions, well established for plant quality improvement, include the application of day and night temperature differences (DIF) during plant growth and development (Erwin et al., 1994; Carvalho et al., 2002; Bachman and McMahon, 2006), application of different photoperiods (Boyle and Stimart, 1983; Runkle et al., 2001; Runkle and Heins, 2002), use of different light intensities and qualities (Runkle and Heins, 2001), humidity (Mortensen and Gislerod, 1997), adjusted soil water (Nonami, 1998) and plant nutrition (Rodriguez-Perez et al., 2001; Williamson et al., 2001). The structure and function of the vascular system in general, and the xylem conduits in particular, like the other plant characteristics mentioned above, are also susceptible to changes in environmental conditions during plant growth and development (Lovisolo and Schubert, 1998; Nijsse, 2001). Therefore, a careful regulation of xylem structure and function by environmental factors during growth could enhance plant quality. Evolution of xylem conduit Higher plants make use of a vascular system to achieve long distance transport of water and mineral components from the soil to the leaves for transpiration, photosynthesis and redistribution of photosynthetically manufactured foods and signaling compounds throughout the plant (Sieburth and Deyholos, 2006). As a common phenomenon, all large and multicellular organisms require this sort of system to ensure transport of water, food and 11 Chapter 1 signal molecules to and from tissue targets. Unlike animals