NASA/NSF Collaborative Research: Light-Regulation of Z. maysStomata: Single Cell Electrical and Molecular Assays

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9416039 Assmann In the stomatal complex of maize (Zea mays), light stimulates uptake of K+ and Cl- into guard cells but simultaneously triggers loss of K+ and Cl- from subsidiary cells. How this exquisite level of cellular differentiation is achieved, such that the same physical stimulus from the environment elicits completely opposite ion transport processes in the two cell types, is the focus of this proposal. Experiments will utilize refinement of microscale (single cell) techniques of laser-assisted electrophysiology and molecular biology to analyze cellular function and gene expression. In guard cells, light activates a H+ ATPase which extrudes H+, thereby creating an electrical gradient for passive uptake of K+ through K+ selective ion channels. Guard cell and subsidiary cell protoplasts of maize will be patch-clamped, and their K+ and H+ ATPase currents will be analyzed and tested for light regulation. Techniques of differential display, and of cDNA synthesis within an individual living cell by reagents introduced via the patch- clamp pipette in whole cell configuration will be used to produce probes for genes that are differentially expressed in the two cell types. Probes will be used to screen libraries to obtain full length sequences. For all experiments guard cell and subsidiary cell protoplasts will be released using a recently developed microsurgery technique, in which a laser is used to burn a hole in the cell wall, and the protoplast is ten released through the hole. The laser microsurgery method obviates the use of cell-wall digesting enzymes to release protoplasts, which may induce gene expression and alter cell physiology and viability. Methods developed in this research should allow the patch-clamp and molecular study of single cells that have been inaccessible by previous approaches. %%% Plants take up CO2 from the atmosphere and use the sun's energy to fix CO2 into carbohydrates. CO2 enters plant leaves through tiny pores in the leaf surface called stomata. Water also exits the leaf through stomata. It is therefore important that stomatal apertures are tightly controlled, so that plants neither starve because of inadequate CO2 uptake, nor wilt because of excessive water loss. Stomatal apertures are regulated by pairs of guard cells which define and border each stomatal pore. Guard cell regulation of pore aperture requires uptake and loss of K+ from the guard cells. In corn, when K+ is not needed by the guard cells, it is stored in neighboring cells called subsidiary cells. The same environmental signals that cause guard cells to take up K+ trigger subsidiary cells to lose this ion, which the guard cells then have access to. How this exquisite level of cellular specialization is achieved is the purpose of t his study. An electrophysiological technique called patch-clamping will be used to study how light regulates the fluxes of K+ and other ions across the cell membranes of guard cells and subsidiary cells. Techniques of molecular biology will be applied o study how these two cell types differ in which genes they express. Understanding the mechanism of stomatal control is important, in order to learn which responses to target , in breeding or molecular genetic manipulations, to increase plant productivity or decrease plant sensitivity to draught. Such knowledge may also be important in deciding what the best conditions are for growing crop plants in space to support a manned space station. ***