RUI: Differentiation of Cultured Guard Cell Protoplasts

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Stomata are microscopic pores in the otherwise gas-impermeable surface of leaves. Each stomatal pore (stoma) is flanked by two opposing guard cells that change in shape in response to environmental signals (e.g. light quality and intensity, intercellular leaf carbon dioxide levels, and apoplastic concentrations of abscisic acid [ABA]). The shape changes of guard cells allow the dimensions of each stoma to be varied as an integrated response to prevailing environmental conditions, thus regulating the rate of gas exchange between leaves and the air, and, therefore, rates of transpiration and photosynthesis. A great deal is known about the guard cell signal transduction mechanisms that result in stomatal changes. Because guard cells are highly specialized to transduce such signals, historically they have been considered among the most highly differentiated plant cell types. Recent experiments have shown, however, that in response to hormonal signals, guard cell protoplasts (GCP) cultured at 32C can reenter the cell cycle, de-differentiate, and divide to produce callus from which plants can be regenerated; thus guard cells are totipotent. These data suggest that guard cells are not as far from a meristematic state as was once thought, and that like other plant cells, they must be actively maintained in their differentiated state. Little is known about the signal transduction pathways or genes that might be involved in maintaining guard cells in their differentiated state in situ. Elevated temperature and ABA are the only signals required for maintenance of cultured GCP in their differentiated state in vitro. Several lines of evidence suggest a model where heat shock proteins (HSP) might be involved in maintaining the differentiated state. The working hypothesis is that HSP inhibit hormone-dependent re-entry of cultured GCP into the cell cycle by limiting translation of the transcripts induced by hormones. More specifically, it is hypothesized that at 38C activation of the heat shock transcription factor HSF results in production of HSP that reduce production of cyclin-dependent kinases and cyclin D3. Because at 32C inhibitors of ethylene synthesis also prevent cultured GCP from completing the cell cycle, it is further hypothesized that at 32C the plant hormone auxin stimulates the production of aminocyclopropane-1-carboxylic acid (ACC) synthase and/or ACC oxidase, resulting in increased production of ethylene. It is envisaged that ethylene then acts through a two-component regulatory system to activate a MAPK pathway that is required for cells to re-enter and/or complete the cell cycle. Following this basic hypothesis, it would be expected that transcripts for ACC synthase and ACC oxidase are either not produced or not translated at 38C and that MAPK is, therefore, not activated at 38C. The initial experiments to be conducted are aimed at elucidating the primary characterisctics of the responses of guard cells to elevated temperature. Among the characteristics to be studied are: 1) the effects of the length of exposure to 32C and 38C on the capacity of GCP to re-enter the cell cycle; 2) an analysis of the reversibility/inter-convertiblility of cell fates; 3) whether other conditions such as nutrient starvation can mimic the effects of cuture at 38C; and 4) the point in the cell cycle to which GCP cultured at 38C are able to proceed. Further experiments aimed at exploring the plausibility of the model described will include investigation of: 1) whether genes for cyclin-dependent kinase homologs, cyclin D3, ACC synthase, ACC oxidase, and MAPK are transcribed at 32C, at 38C, or both; 2) whether known HSP are produced at 38C, but not at 32C; 3) whether kinetics of HSP production are correlated with kinetics of loss of responsiveness of cultured GCP to auxin and/or cytokinin; and 4) whether MAPK is activated by an ethylene-dependent mechanism. Investigation of the proteins required to maintain plant cells in a differentiated state could provide insight into the question of a universal mechanism involving a small number of expressed proteins that maintain differentiated states in multicellular organisms. Such studies could also tell us whether the expression of these proteins makes some plant cells recalcitrant to culture. If marker proteins for GCP maintained in the differentiated state can be identified, it should eventually make it possible to clone the genes for these proteins such that they can be expressed in plant tissues under the control of selected promoters for control of plant cell differentiation in response to various environmental conditions.