RUI: Effects of Auxin and Abscisic Acid on Survival, Growth,and Differentiation of Cultured Guard Cell Protoplasts

该项目的长期目标是阐明调节植物叶片细胞和叶绿体感觉的分子机制。气孔两侧的保卫细胞不会经历与底层组织(叶肉)细胞一样的衰老过程。因此，建立了一套个体保卫细胞培养体系，以研究其对衰老的诱导和调控。保卫细胞原生质体(GCP，分离的去除细胞壁的保卫细胞)在36-40℃下成功培养了一周，但低温不能有效地保持保卫细胞作为保卫细胞，但允许再分化和愈伤组织的形成。提出了以下模型来解释温度敏感性：1)在较低温度(24-34oC)下，植物激素生长素诱导乙烯的合成。2)乙烯合成导致细胞衰老或导致愈伤组织形成的生长反应。3)在较高温度下，乙烯合成受到抑制。4)在较高温度下，脱落酸(ABA)中和生长素/细胞分裂素反应，从而阻止GCP的再分化。为了验证该模型，进行了以下实验：1)将GCP在ABA中进行较高温度的培养，以检测ABA本身是否能诱导去分化。2)检查GCP在较低温度下产生的乙烯水平。3)测试其他抑制乙烯作用的条件是否也允许GCP保持非衰老状态。这是一个由本科生研究机构(RUI)项目支持的项目。在之前三年的获奖期内，有26名本科生参与了研究。预计这一奖项也会有类似程度的参与。该项目的长期目标是阐明调控植物叶片细胞和叶绿体衰老(衰老)的分子机制。具有侧面气孔的保卫细胞不会经历像底层组织(叶肉)细胞那样的衰老过程。因此，建立了一套个体保卫细胞培养体系，以研究其对衰老的诱导和调控。在这种装置中，保卫细胞原生质体(GCP，去除细胞壁后分离的保卫细胞)在高温(36-40摄氏度)下成功培养相当长的时间(天)。较低的温度不能有效地保持保卫细胞作为保卫细胞，但允许再分化和一般细胞生长或愈伤组织的形成。假设是植物生长调节剂乙烯导致细胞在较低温度下衰老，但不是由保卫细胞在较高温度下产生。同样在较低的温度下，生长素和细胞分裂素等其他生长调节剂也是活跃的，导致更高的生长速度和细胞去分化。通过测量细胞在不同温度下产生的乙烯水平，以及检查外源添加的生长调节剂(如脱落酸)是否干扰衰老过程，来检验这些假设。这项研究有助于更好地理解保卫细胞为什么不衰老，以及诱导和调节植物细胞衰老的机制。***

MCB-9417761 Tallman The long-term objective of the project is to elucidate the molecular mechanisms that regulate senscence of cells and chloroplasts of plant leaves. Guard cells which flank stomata fail to undergo a senescence (aging) process like that which takes place in cells of the underlying tissue (mesophyll). Thus a system to culture individual guard cells has been developed to study the induction and regulation of senescence. Guard cells protoplasts (GCP, guard cells isolated with cell walls removed) have been successfully cultured for a week at 36-40o C, but lower temperatures were not effective at maintaining the guard cells as guard cells, but allowed redifferentiation and callus formation. The following model is suggested to account for the temperature sensitivity: 1) At lower temperatures (24-34oC) the plant hormone auxin induces synthesis of ethylene. 2) Ethylene synthesis results in cellular senescence or a growth response that results in callus formation. 3) Ethylene synthesis is inhibited at higher temperatures. 4) At higher temperatures, abscisic acid (ABA) counteracts the auxin/cytokinin response, thereby preventing redifferentiation of GCP. To test this model the following experiments are done: 1) The GCP are cultured at the higher temperature in ABA to test if ABA itself could induce dedifferentiation. 2) Examine the levels of ethylene produced by GCP at lower temperatures. 3) Test whether other conditions which which suppress ethylene action also allow the GCP to maintain a non-senescent state. This is a project supported through the Research at Undergraduate Institutions (RUI) program. Over the prior award period of three years there were 26 undergraduates involved in research. A similar level of involvement is expected in this award. %%% The long-term objective the project is to elucidate the molecular mechanisms that regulate the decline during aging (senescence) of cells and chloroplasts of plant leaves. Guard cells wh ich flank stomata fail to undergo an aging process like that which takes place in cells of the underlying tissue (mesophyll). Thus a system to culture individual guard cells has been developed to study the induction and regulation of senescence. In this set-up, guard cells protoplasts (GCP, guard cells isolated with cell walls removed) are successfully cultured for fairly long periods (days) at elevated temperatures (36-40o C). Lower temperatures are not effective at maintaining the guard cells as guard cells, but allow redifferentiation and general cell growth or callus formation. The hypothesis is that the plant growth regulator, ethylene, causes the cells to senesce at lower temperatures, but is not produced by the guard cells at higher tempertures. Also at lower temperatures, other growth regulators like auxin and cytokinin are active, resulting in higher growth rates and cellular dedifferentiation. These hypotheses are tested by measuring the levels of ethylene produced by the cells at different temperatures and by examining if exogeneously added growth regulators such as abscisic acid interfere with the aging process. The study leads to a better understanding of why guard cells fail to senesce and the mechnisms that induce and regulate senescence in plant cells. ***