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

在玉米的气孔复合体（Zea mays）中，光刺激保护细胞吸收K+和Cl-，但同时引发辅助细胞损失K+和Cl-。这种精巧的细胞分化是如何实现的，即来自环境的相同物理刺激在两种细胞类型中引起完全相反的离子运输过程，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。在保护细胞中，光激活H+ atp酶，使其挤出H+，从而通过K+选择性离子通道产生K+被动吸收的电梯度。对玉米保护细胞和辅助细胞原生质体进行膜片箝制，分析和测试其K+和H+ atp酶电流对光的调节作用。差异显示技术，以及在单个活细胞内通过膜片钳移液管在全细胞配置中引入的试剂合成cDNA的技术，将用于生产在两种细胞类型中差异表达的基因探针。探针将用于筛选文库以获得全长序列。在所有的实验中，保护细胞和辅助细胞的原生质体将使用最近开发的显微外科技术释放，该技术使用激光在细胞壁上烧一个洞，原生质体通常通过这个洞释放出来。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，这是以前的方法无法实现的。植物从大气中吸收二氧化碳，并利用太阳能将二氧化碳固定为碳水化合物。二氧化碳通过叶子表面被称为气孔的微小孔进入植物叶片。水分也通过气孔排出叶片。因此，严格控制气孔开度是很重要的，这样植物既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。气孔的开度是由一对保护细胞调节的，这些细胞定义和包围着每个气孔。保护细胞对气孔的调节需要从保护细胞中吸收和损失K+。在玉米中，当保卫细胞不需要K+时，它被储存在邻近的被称为附属细胞的细胞中。导致保护细胞吸收K+的环境信号也会触发辅助细胞失去这种离子，而保护细胞随后可以获得这种离子。如何达到这种精巧的细胞特化水平是本研究的目的。一种叫做“贴片夹紧”的电生理技术将用于研究光如何调节K+和其他离子穿过保护细胞和辅助细胞细胞膜的通量。分子生物学技术将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，哪些响应是提高植物生产力或降低植物对干旱的敏感性的目标。这些知识对于决定在太空中种植农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. ***