DIFFERENTIAL EXPRESSION OF THE DIVERSE PLANT ACTINS

我们一直在探索假说（1），即古老而不同的植物基因类在维管植物进化过程中得以保留，因为它们具有独特的调控模式和/或编码具有不同功能的蛋白质。 我们利用拟南芥紧凑的基因组，快速的生活周期，小尺寸，易于转化和再生，以及简单的遗传学来剖析这一假设的不同部分。 基于肌动蛋白启动子/报告基因融合在数百个转基因植物，RNA水平和蛋白质水平的分析与isovariant-specific单克隆抗体，我们确定了三个营养肌动蛋白和五个生殖肌动蛋白每个具有强大的和独特的空间和时间的表达模式。 这些范围从组成型表达的ACT 2在大多数营养组织，植物激素诱导ACT 7，表达的ACT 1在胚珠和花粉。 开发了第一种基于序列的分离植物插入突变体的方法，并用于分离八个表达的肌动蛋白基因中的四个中的突变体。 这些突变体具有令人兴奋的和微妙的细胞和发育表型，当植物在特定条件下生长时就会显示出来。 对三个肌动蛋白基因突变体的多代研究表明，每个基因都受到强烈的选择约束，是拟南芥生存所必需的。 植物肌动蛋白同功变异体的极端序列多样性及其复杂的表达模式导致了第二种假设。 假设2指出，在同一细胞中的多个肌动蛋白异构体的共表达导致异构体动力学，允许生物系统的响应的时间和生化扩展和缓冲。 需要各种各样的分子、细胞、遗传和生物化学方法来解决以下特定目标：1）分离每个肌动蛋白和profilin基因中的突变体; 2）表征这些突变体的表型; 3）确定肌动蛋白同功变异体的空间或时间表达模式; 4）确定共表达肌动蛋白的亚细胞分布; 5）使用在酵母中产生的功能性肌动蛋白来剖析区分肌动蛋白同种变体的物理/分子参数。

We have been exploring the Hypothesis (number 1) that the ancient and divergent classes of plant genes have been preserved throughout vascular plant evolution because they have unique patterns of regulation and/or encode proteins with distinct functions. We made use of the compact genome, rapid life cycle, small size, ease of transformation and regeneration, and simple genetics of Arabidopsis to dissect different parts of this hypothesis. Based on the analysis of actin promoter/reporter fusions in hundreds of transgenic plants, RNA levels, and protein levels assayed with isovariant-specific monoclonal antibodies we determined that the three vegetative actins and five reproductive actins each have strong and distinct spatial and temporal expression patterns. These range from constitutive expression of ACT2 in most vegetative tissues, to the phytohormonal induction of ACT7, to the expression of ACT1 in ovules and pollen. The first sequence-based method of isolating plant insertion mutants was developed and used to isolate mutants in four of the eight- expressed actin genes. These mutants have exciting and subtle cell and developmental phenotypes that are revealed when plants are grown under specialized conditions. Multigenerational studies on mutants in three actin genes demonstrated that each gene is under strong selective constraint and is required for the survival of Arabidopsis. The extreme sequence diversity of the plant actin isovariants and their complex expression patterns lead to a second hypothesis. Hypothesis number 2 states that the coexpression of multiple actin isovariants in the same cell results in isovariant dynamics that allow for the temporal and biochemical expansion and buffering of responses of a biological system. A wide variety of molecular, cellular, genetic, and biochemical approaches are needed to address the following Specific Aims: 1) to isolate mutants in each actin and profilin gene; 2) to characterize the phenotypes of these mutants; 3) to determine the spatial or temporal expression patterns of the actin isovariants; 4) to determine the subcellular distribution of coexpressed actins; 5) to dissect the physical/molecular parameters that distinguish the actin isovariants using the functional actin proteins being produced in yeast.