RUI: Differentiation of Cultured Guard Cell Protoplasts

气孔是叶片表面上的微小气孔，不透气。 每个气孔（气孔）两侧是两个相对的保卫细胞，其形状会响应环境信号（例如光质量和强度，细胞间叶二氧化碳水平和脱落酸[阿坝]的质外体浓度）而变化。 保卫细胞的形状变化允许每个气孔的尺寸作为对主要环境条件的综合响应而变化，从而调节叶片和空气之间的气体交换速率，并且因此调节蒸腾和光合作用的速率。 气孔保卫细胞信号转导机制是气孔变化的重要机制。 由于保卫细胞高度特化以传递这些信号，因此历史上它们被认为是最高度分化的植物细胞类型之一。 然而，最近的实验表明，在响应激素信号，在32 ℃下培养的保卫细胞原生质体（GCP）可以重新进入细胞周期，去分化，并分裂产生愈伤组织，从中可以再生植物;因此保卫细胞是全能的。 这些数据表明，保卫细胞并不像以前认为的那样远离分生组织状态，并且像其他植物细胞一样，它们必须积极维持在分化状态。 关于维持保卫细胞原位分化状态的信号转导途径或基因知之甚少。 升高的温度和阿坝是维持培养的GCP在体外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通过限制由激素诱导的转录本的翻译来抑制培养的GCP的细胞周期依赖性再进入。 更具体地说，假设在38 ℃下热休克转录因子HSF的活化导致HSP的产生，HSP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 ℃下乙烯合成的抑制剂也阻止培养的GCP完成细胞周期，所以进一步假设在32 ℃下植物激素生长素刺激氨基环丙烷-1-羧酸（ACC）合酶和/或ACC氧化酶的产生，导致乙烯的产生增加。 设想乙烯然后通过双组分调节系统起作用以激活细胞重新进入和/或完成细胞周期所需的MAPK途径。 根据这一基本假设，可以预期ACC合酶和ACC氧化酶的转录物在38 ℃不产生或不翻译，因此MAPK在38 ℃不被激活。 初步实验旨在阐明保卫细胞对高温反应的主要特征。 研究的特征包括：1）暴露于32 C和38 C的时间长度对GCP重新进入细胞周期的能力的影响; 2）分析细胞命运的可逆性/相互转化性; 3）其他条件如营养饥饿是否可以模拟38 C培养的效果;和4）细胞周期中在38 ℃下培养的GCP能够进行的点。 进一步的实验旨在探索所描述的模型的可行性，包括研究：1）细胞周期蛋白依赖性激酶同源物、细胞周期蛋白D3、ACC合酶、ACC氧化酶和MAPK的基因是否在32 ℃、38 ℃或两者都转录; 2）已知的HSP是否在38 ℃产生，而不是在32 ℃; 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的动力学相关;和4）MAPK是否被乙烯依赖性机制激活。 研究维持植物细胞分化状态所需的蛋白质可以深入了解涉及少数表达蛋白质的普遍机制问题，这些蛋白质在多细胞生物中维持分化状态。这些研究还可以告诉我们，这些蛋白质的表达是否会使某些植物细胞对培养产生排斥作用。 如果能够鉴定出保持在分化状态的GCP的标记蛋白，那么最终将有可能克隆这些蛋白的基因，从而使它们能够在植物组织中在所选启动子的控制下表达，以控制植物细胞响应于各种环境条件的分化。

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.