RUI: Differentiation of Cultured Guard Cell Protoplasts

RUI：培养的保卫细胞原生质体的分化

• 批准号: 9900525
• 负责人: John Tallman
• 金额: $ 24.65万
• 依托单位: Willamette University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-10-01 至 2002-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9900525&HistoricalAwards=false
• 关键词: RUI; Differentiation; Cultured; Guard; Cell

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.

气孔是叶片表面不透气的微小气孔。每个气孔两侧都有两个相对的保护细胞，它们根据环境信号（如光的质量和强度、细胞间叶片二氧化碳水平和脱落酸[ABA]的外胞浓度）改变形状。保护细胞的形状变化允许每个气孔的尺寸变化，作为对当前环境条件的综合反应，从而调节叶片与空气之间的气体交换速率，从而调节蒸腾和光合作用的速率。关于导致气孔变化的保护细胞信号转导机制，我们已经了解得很多。由于保护细胞是高度特化的转导这些信号，历史上它们被认为是高度分化的植物细胞类型之一。然而，最近的实验表明，在32℃下培养的保护细胞原生质体（GCP）响应激素信号，可以重新进入细胞周期，去分化并分裂产生愈伤组织，从而可以再生植株；因此，保护细胞是全能性的。这些数据表明，保护细胞并不像以前认为的那样远离分生组织状态，而且像其他植物细胞一样，它们必须主动维持在分化状态。我们对保护细胞维持原位分化状态的信号转导途径或基因知之甚少。升高的温度和ABA是维持体外培养GCP分化状态所需的唯一信号。一些证据表明，热休克蛋白（HSP）可能参与维持分化状态的模型。工作假设是HSP通过限制激素诱导的转录本的翻译来抑制激素依赖性的培养GCP重新进入细胞周期。更具体地说，假设在38℃时，热休克转录因子HSF的激活会导致热休克蛋白的产生，从而减少细胞周期蛋白依赖性激酶和细胞周期蛋白D3的产生。因为在32℃时，乙烯合成抑制剂也会阻止培养的GCP完成细胞周期，因此进一步假设在32℃时，植物激素生长素刺激氨基环丙烷-1-羧酸（ACC）合成酶和/或ACC氧化酶的产生，导致乙烯的产生增加。根据设想，乙烯随后通过双组分调控系统激活细胞重新进入和/或完成细胞周期所需的MAPK通路。根据这一基本假设，可以预期ACC合成酶和ACC氧化酶的转录本在38C时不产生或不翻译，因此MAPK在38C时不被激活。初步实验的目的是阐明保护细胞对高温反应的主要特征。待研究的特性包括：1)暴露于32C和38C的时间长短对GCP重新进入细胞周期能力的影响；2)细胞命运的可逆性/互转性分析；3)营养饥饿等其他条件是否能模拟38℃培养的效果；以及4)在38℃下培养的GCP能够进入细胞周期的哪一点。进一步的实验旨在探索所描述的模型的可行性，将包括调查：1)周期蛋白依赖性激酶同源物、周期蛋白D3、ACC合成酶、ACC氧化酶和MAPK的基因是否在32℃、38℃或两者同时转录；2)已知热热蛋白是否在38C下产生，而在32C下没有产生；3) HSP产生的动力学是否与培养的GCP对生长素和/或细胞分裂素的反应性丧失动力学相关；4) MAPK是否被乙烯依赖的机制激活。对维持植物细胞分化状态所需的蛋白质的研究，可以深入了解多细胞生物中维持分化状态的少数表达蛋白的普遍机制。这些研究也可以告诉我们这些蛋白质的表达是否使一些植物细胞对培养产生抗性。如果能够鉴定出维持在分化状态的GCP标记蛋白，那么最终将有可能克隆出这些蛋白的基因，使其能够在选定的启动子控制下在植物组织中表达，从而控制植物细胞在各种环境条件下的分化。