Final Activity Report Summary - XYLODIMENS (Regulating plant quality by controlling xylem vessel dimensions during xylogenesis) This project was focussed on the role and timing of programmed cell death (PCD) in tracheary elements (TEs) formation during the trans-differentiation of mesophyll cells in a model system of Zinnia elegans cell culture. The aim is to obtain better understanding of the involvement of different endogenous and environmental factors in the process of xylogenesis, to elucidate the potential of pharmaceutical treatments for manipulation the dimensions of xylem vessels and to use the information obtained from the in vitro studies for in planta improvement of the post-harvest quality of cut flowers.Xylem vascular system serves the plant to establish soil-plant-atmosphere continuum for water transport and supports the mechanical strength of the stems. Hydraulic properties of xylem vessels and specifically the xylem dimensions are linked to crucial quality aspects of horticultural produce, such as the ability of cut flowers to recover from embolisms in the vascular system at the start of their postharvest vase life. Xylem vessel dimensions differ with genetic background and are influenced by environmental conditions during the growth. Appropriate watering strategy, temperature and light regime during growth and development can be used for manipulation of xylem conductance. Although post-harvest performance of flowers can be improved by vase-additives, bactericides and wetting agents, vascular patterns are robust once they are formed and therefore the actual control of xylem dimensions should take place during vessel formation (xylogenesis).Xylem vessels consist of a number of stacked tracheary elements (TEs) that originate through re-differentiation of root and shoot pro-cambium and cambium cells. TEs are dead hollow cells with a function supported by the neighbouring living cells. Re-differentiation process is influenced by endogenous and exogenous factors, including plant hormones (cytokinins and auxins) produced by leaves and roots and other signalling compounds (brassinosteroids, calcium, ROS, ethylene, nitric oxide, MAP-kinases, cell-to-cell communication, pH, osmotic values, etc.).Formation and fusion of TEs into a xylem vessel involves a cascade of processes, including cell division, cell elongation, cell wall synthesis, cell wall material deposition and lignification and, a final step of programmed cell death (PCD). The involvement of PCD suggests that xylogenesis could be manipulated by controlling the PCD. It is expected that modulations of the initiation and development of xylogenesis may alter the anatomy of the TEs, resulting in different properties of the xylem vessels and finally influencing the quality and vase life of the plants.Objectives of the project:Development of tools to study the timing and extent of cell death during TE formationDetermine the effect of hormonal and environmental factors on the timing and extent of cell death during TE formationDetermine the effect of treatments affecting cell death on the morphology and dimensions of TEs in vitro and in plantaFor achieving the goals we have used a model system of cell culture isolated from zinnia leaf mesophyll cells. Zinnia cell culture is a unique model system, which at the administration of certain ratio of cytokinins and auxins can be induced to trans-differentiate into vessel elements. The advantages also include production of one cell type isolated from complexity of leaf tissues, accessibility for chemical manipulations and microscope observations, high potential and synchrony of TE differentiation, preservation of vessel element patterning in similarity to zinnia hypocotyls in vivo. The cell culture was isolated from the first true pair of leaves from14 days old seedlings of Zinnia elegans, cv. Envy and Purple Prince, grown on peat-based commercial potting compost at conditions of photoperiod 16-h light/8-h darkness; 25/20o C day/night temperatures and 70% RH. 24-48 hours after isolation the cells were induced to trans-differentiate with a mix of plant hormones (1 mg/L benzyladenine (BA) and 0.1 mg/L naphthalene acetic acid (NAA)).For elucidation of the signalling involved in the process of re-differentiation of mesophyll cells into TEs and specifically the participation of caspase-like cascade, peptide caspase inhibitors, known to inhibit caspase proteases in animal and human cell lines, were administrated to hormone-induced culture. The timing of formation and the dimensions of produced TEs was scored in order to determine the effect of applied inhibitors on TE generation and to evaluate the apoptotic-like nature of cell death. Caspases are specific cysteine proteases that show a high degree of specificity with an absolute requirement for cleavage adjacent an aspartic acid residue and a recognition sequence of at least four amino acids N-terminal to this cleavage site. A sequence of caspase-activation events involving initiator caspases and down-stream activation of executioner caspases leads to the apoptotic phenotype. Morphological similarities have been found between animal cells undergoing apoptosis and dying plant cells, including condensation and shrinkage of the cytoplasm and nucleus, DNA and nuclear fragmentation and eventually formation of apoptotic-like bodies, but no engulfment of cellular debris by neighbouring living cells (phagocytosis) has yet been detected in plants. This suggests that no true apoptosis exists in the plant cells and plant PCD may primarily be of the non-lysosomal and autophagic type. These latter PCD types may, however, show biochemical and morphological features similar to animal apoptosis. Although no structural homologues of animal caspases have been identified in plants, there is accumulating evidence that cysteine proteases showing functional similarity to caspases, as in animal systems, participate in plant programmed cell death. No information has yet been available about participation of caspase-like proteases in xylogenesis.Impact of environmental factors during cultivation of zinnia plants on the dimensions and timing of TEs formation in isolated cell culture, the effect of extracellular osmolarity, the electrical conductivity in the root environment and light intensity during the cultivation of plants were studied. The results indicated that the increase of leaf osmolarity affected by either electrical conductivity or light intensity resulted in an increase of the percent of TEs formed in the culture after induction with the phytohormones.Experiments were performed to elucidate the involvement of caspase-like, cysteine and serine proteases and ethylene in the signalling during the process of differentiation in zinnia cell culture. The hormones were combined with treatments with irreversible broad-ranged human caspase-3 inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB), irreversible caspase-1 inhibitor Tyr-Val-ala-Asp-chloromethylketone (Ac-YVAD-CMK), and the reversible caspase-3/7 inhibitor Acyl-Asp-Glu-Val-l-aspartic acid aldehyde (Ac-DEVD-CHO), as well as cysteine protease inhibitors L- transepoxysuccinyl-leucylamido-[4-guanidino]butane) (E64), iodoacetamide (IA) and N-ethylmaleimide (NEM). Ethylene production after administration of the hormones was measured by laser-driven photoacoustic sensor. For microscope observations advanced light, fluorescent, confocal and differential interference contrast microscopy were used.In summary we have found that in the developed model system of zinnia cell culture:* The morphology of in vitro generated TEs show characteristic patterns of secondary wall thickening and formation of vessel-like structures in similarity to xylogenesis in planta;* Cultivation conditions during growing of zinnia plants (electric conductivity of the nutrient media and the light intensity) affect the leaf osmotic potential thus becoming critical factors that influence the potential of zinnia mesophyll cells for re-differentiation into TEs in hormone induced xylogenic culture and the dimensions of produced TEs. The ratio and type of the auxin and cytokinin might affect the process of re-differentiation by interfering with the cell death inhibiting chemicals.* Introduction of irreversible broad range caspase inhibitor Z-Asp-CH2-DCB and of the cysteine protease inhibitor E-64 delays the timing of TE formation and leads to larger dimensions of the TEs;* Administration of caspase 1 and caspase 3 inhibitors Ac-YVAD-CMK and Ac-DEVD-CHO, as well as the broad range caspase inhibitor Z-Asp-CH2-DCB significantly suppress the process of TE differentiation. Cysteine protease inhibitors IA and NEM completely abolish the formation of TEs;* Differentiating cells show typical programmed cell death hallmark of DNA laddering;* The plant hormone ethylene is involved in the process of re-differentiation.* The process of trans-differentiation of zinnia mesophyll cells into TEs involves three stages: I - de-differentiation of mesophyll cells and acquisition of competence for re-differentiation; II synthesis and deposition of secondary wall material and III - PCD associated with DNA laddering, disappearing of the nucleus, formation of large vacuole, rupture of the tonoplast, autolysis of cell content and formation of hollow dead TEs. Formation of vessel-like elements was also observed.* The tested caspase inhibitors are efficient for manipulation (delay of timing and increase of TE dimensions) of the PCD involved in phases II and III of xylogenesis in zinnia cell culture.Based on the performed experiments we assume that the re-differentiation in cultured mesophyll zinnia cells is a programmed cell death event that most probably involves caspase-like and cysteine proteases, and ethylene signalling and, the administration of the relative inhibitors may delay the TEs formation simultaneously leading to larger dimensions of the generated TEs. The application of tested PCD inhibitors can be used as a tool for controlling the dimensions of TEs in cell culture in vitro and for further manipulation of cell death signalling and vessel dimensions in planta.To our knowledge this study provides the first information indicating the involvement of caspase-like proteases in xylogenesis in zinnia cell culture. We believe that the undertaken research on in vitro manipulation of programmed cell death signalling during xylogenesis is significant contribution towards quality control in planta and provides a clue for elucidation of mechanisms underlying the control of xylem formation, for establishment of adequate markers of xylogenesis, for selection of treatments from developed model system in vitro with presumable beneficial effect on xylem architecture and, for further timely regulation of xylogenesis associated cell death genes.