DIFFERENTIAL EXPRESSION OF THE DIVERSE PLANT ACTINS

我们一直在探索假设(1)，即古老的和不同种类的植物基因在维管植物的进化过程中被保存下来，因为它们具有独特的调节模式和/或编码具有不同功能的蛋白质。我们利用拟南芥基因组紧凑，生命周期快，体积小，易于转化和再生，以及简单的遗传学来剖析这一假说的不同部分。通过对数百种转基因植物中肌动蛋白启动子/报告基因融合体的分析，利用等变异体特异性单克隆抗体检测RNA水平和蛋白质水平，我们确定了三种营养肌动蛋白和五种生殖肌动蛋白各自具有强烈而独特的时空表达模式。从大多数营养组织中ACT2的组成表达，到植物激素诱导的ACT7，再到胚珠和花粉中ACT1的表达。首次开发了基于序列的植物插入突变体分离方法，并用于分离8个表达肌动蛋白基因中的4个突变体。当植物在特定条件下生长时，这些突变体具有令人兴奋和微妙的细胞和发育表型。对三个肌动蛋白基因突变的多代研究表明，每个基因都受到强烈的选择约束，是拟南芥存活所必需的。植物肌动蛋白同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，多个肌动蛋白同变异体的共同表达导致了同变动力学，从而允许生物系统的时间和生化扩展和缓冲反应。需要广泛的分子、细胞、遗传和生化方法来解决以下具体目标：1)分离每个肌动蛋白和profilin基因的突变体；2)表征这些突变体的表型；3)确定肌动蛋白同型变异体的时空表达模式；4)确定共表达actis的亚细胞分布；5)利用酵母中产生的功能性肌动蛋白，剖析区分肌动蛋白同变异体的物理/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.