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

小行星9416039 在玉米（Zea mays）的气孔复合体中，光刺激保卫细胞吸收K+和Cl-，但同时触发辅助细胞损失K+和Cl-。 如何实现这种精致的细胞分化水平，使得来自环境的相同物理刺激在两种细胞类型中激发完全相反的离子转运过程，是本提案的重点。 实验将利用激光辅助电生理学和分子生物学的微尺度（单细胞）技术的改进来分析细胞功能和基因表达。 在保卫细胞中，光激活H+ ATP酶，其挤出H+，从而通过K+选择性离子通道产生用于被动摄取K+的电梯度。 将玉米保卫细胞和附属细胞原生质体进行膜片钳，并分析和测试其K+和H+ ATP酶电流的光调节。 差异显示技术和通过在全细胞构型中经由膜片钳移液管引入的试剂在单个活细胞内合成cDNA的技术将用于产生在两种细胞类型中差异表达的基因的探针。 探针将用于筛选文库以获得全长序列。 对于所有实验，将使用最近开发的显微外科技术释放保卫细胞和辅助细胞原生质体，其中使用激光在细胞壁上烧一个洞，并通过洞释放原生质体。 激光显微手术方法避免了使用细胞壁消化酶来释放原生质体，这可能会诱导基因表达并改变细胞生理学和活力。 在这项研究中开发的方法应该允许膜片钳和单细胞的分子研究，已无法通过以前的方法。 植物从大气中吸收二氧化碳，并利用太阳的能量将二氧化碳固定为碳水化合物。 二氧化碳通过叶片表面的气孔进入植物叶片。 水也通过气孔离开叶片。 因此，严格控制气孔开度是很重要的，这样植物就不会因为二氧化碳吸收不足而挨饿，也不会因为水分流失过多而枯萎。 气孔的开度由成对的保卫细胞控制，保卫细胞界定气孔的边界。 保卫细胞调节孔孔径需要从保卫细胞摄取和损失K+。 在玉米中，当保卫细胞不需要K+时，K+就会储存在称为辅助细胞的邻近细胞中。 导致保卫细胞吸收K+的相同环境信号触发辅助细胞失去这种离子，然后保卫细胞可以访问。 细胞特化的这一精妙水平是如何达到的，这就是本研究的目的。 一种称为膜片钳的电生理技术将被用来研究光如何调节K+和其他离子穿过保卫细胞和附属细胞的细胞膜的通量。 分子生物学技术将被应用于研究这两种细胞类型在表达基因方面的差异。 了解气孔控制的机制是重要的，以便了解在育种或分子遗传操作中对目标的哪些反应，以提高植物生产力或降低植物对干旱的敏感性。 这些知识对于决定在太空中种植作物以支持载人空间站的最佳条件也很重要。 ***

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. ***