Characterization of Aux/IAA Protein Degradation in Higher Plants

蛋白质水解的调节是细胞和有机体调节的一个新兴范例。多种途径已被证明是通过调控蛋白和下游蛋白的特异性和受控降解来调节的。在高等植物中，植物生长发育的多个方面都需要生长素，其中吲哚-3-乙酸（IAA）是最丰富的天然生物活性成分。生长素调节细胞分裂、细胞伸长和细胞分化，并在侧根形成、顶端优势和热带反应等多种生物过程中发挥重要作用。分子和生物化学研究已经确定了Aux/IAA蛋白家族在植物生长素信号转导途径中是改变基因表达的重要参与者。除了Aux/IAA蛋白的活性（其特征仍然很差）外，它们的快速降解已经成为其生物功能的一个组成部分。这种快速蛋白水解的机制和作用是本研究的重点。基于其他系统的研究表明，蛋白质水解的顺式作用需求是由一个可以靶向其他蛋白质降解的小区域代表的，在转基因拟南芥植物中直接证明了Aux/IAA氨基酸与标记酶萤火虫荧光素酶融合的快速降解。绝对必需的Aux/IAA氨基酸的性质已经确定，然而，侧翼序列似乎以一种尚未完全了解的方式影响蛋白质水解。这里提出的实验将确定这些序列的作用。Aux/IAA序列将发生点突变和缺失，融合到LUC中，并在转基因植物中确定融合蛋白的半衰期。为了确定是否所有的Aux/IAA蛋白都具有相同的半衰期，将确定额外的Aux/IAA蛋白靶向LUC降解的能力。缺乏所需的保守氨基酸的Aux/IAA蛋白将作为LUC融合进行体内降解测试。如果不能快速降解，该蛋白将作为确定Aux/IAA降解信号充分性的起点。外源生长素的应用改变了Aux/IAA蛋白水解的速率，首次为生长素调控基因表达的机制提供了实验证据。更快的蛋白质水解增加了Aux/IAA蛋白的损失，这与在其调控区域诱导含有生长素反应元件的基因的转录有关。实验将通过构建转基因植株，通过诱导表达改变IAA稳态水平的酶来测试内源生长素的改变是否会改变Aux/IAA蛋白水解。上述研究将辅以遗传方法分离Aux/IAA::LUC降解缺陷突变体。将启动其他屏幕，包括激活标记屏幕。之前描述的突变体在生长素反应中改变对Aux/IAA::LUC蛋白水解的影响也将被确定，以确定Aux/IAA蛋白水解的重要基因。鉴于Aux/IAA蛋白水解在生长素信号传导中的调节作用以及生长素在调节植物生长发育中的重要性，本研究的结果将具有重要意义。Aux/IAA蛋白水解将作为一种模型，通过它可以调节其他细胞途径，为其他系统提供有用的方法、试剂和实验方法。在这些研究中定义的序列可以在其他情况下用于控制蛋白质的细胞内水平。

The regulation of proteolysis is an emerging paradigm in cellular and organismal regulation. Multiple pathways have been demonstrated to be regulated through the specific and controlled degradation of both regulatory and downstream proteins. In higher plants, multiple aspects of plant growth and development require auxin, of which indole-3-acetic acid (IAA) is the most abundant naturally occurring biologically active member. Auxin regulates cell division, cell elongation, cell differentiation, and it plays an important role in multiple organismal processes such as lateral root initiation, apical dominance, and tropic responses. Molecular and biochemical studies have identified the family of Aux/IAA proteins as essential players in the auxin signal transduction pathway that alters gene expression in plants. In addition to the activity of the Aux/IAA proteins (which remains poorly characterized), their rapid degradation has emerged as an integral part of their biological function. The mechanism and role of this rapid proteolysis is the focus of this study. Based on work in other systems demonstrating that the cis-acting requirement for proteolysis is represented by a small region that can target other proteins for degradation, the rapid degradation of a fusion of Aux/IAA amino acids to the marker enzyme firefly luciferase was demonstrated directly in transgenic Arabidopsis plants. The nature of absolutely required Aux/IAA amino acids were defined, however, flanking sequences appear to affect proteolysis in a way that was not completely understood. Experiments proposed here will define the role of these sequences. Point mutations and deletions of Aux/IAA sequences will be made, fused to LUC and the fusion protein half-life determined in transgenic plants. To determine whether all Aux/IAA proteins have the same half-life, the ability of additional Aux/IAA proteins to target LUC for degradation will be determined. An Aux/IAA protein lacking the required conserved amino acids will tested for in vivo degradation as a LUC fusion. If not rapidly degraded, this protein will serve as the starting point to determine the sufficiency of the Aux/IAA degradation signal. Application of exogenous auxin alters the rate of Aux/IAA proteolysis, providing for the first time experimental evidence for a proposed mechanism for auxin-regulated gene expression. Increased loss of Aux/IAA proteins by faster proteolysis correlated with induction of transcription of genes containing auxin-response elements in their regulatory regions. Experiments will be performed to test whether alterations in endogenous auxin alter Aux/IAA proteolysis through the construction of transgenic plants with inducible expression of enzymes that alter IAA steady state levels. The above studies will be complemented by a genetic approach to isolate mutants defective in Aux/IAA::LUC degradation. Additional screens, including an activation tagging screen, will be initiated. The effect on Aux/IAA::LUC proteolysis of previously described mutants altered in auxin responses will also be determined to identify genes important for Aux/IAA proteolysis. The results from the proposed work will be highly significant given the importance of the regulation of Aux/IAA proteolysis in auxin signaling and the importance of auxin in regulating plant growth and development. Aux/IAA proteolysis will serve as a model by which other cellular pathways may be regulated, providing methodogy, reagents, and experimental approaches useful in other systems. Sequences defined in these studies may be used in other contexts to control the intracellular level of a protein.