Characterization of Aux/IAA Protein Degradation in Higher Plants

高等植物中 Aux/IAA 蛋白降解的表征

• 批准号: 0212659
• 负责人: Judy Callis
• 金额: $ 35万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212659&HistoricalAwards=false
• 关键词: Characterization; Aux; IAA; Protein; Degradation

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.

蛋白质水解的调节是细胞和生物体调节中的新兴范例。 已证明多种途径通过调节蛋白和下游蛋白的特异性和受控降解来调节。 在高等植物中，植物生长和发育的多个方面都需要生长素，其中吲哚-3-乙酸（IAA）是最丰富的天然存在的生物活性成员。 生长素调节细胞分裂、细胞伸长、细胞分化，并在侧根发生、顶端优势和向性反应等多个生物过程中发挥重要作用。分子和生物化学研究已经确定Aux/IAA蛋白家族是改变植物基因表达的生长素信号转导途径中的重要参与者。 除了Aux/IAA蛋白的活性（其特征仍然很差）之外，它们的快速降解已经成为其生物学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。 基于在其他系统中的工作，证明蛋白水解的顺式作用要求由可以靶向其他蛋白降解的小区域表示，Aux/IAA氨基酸与标记酶萤火虫荧光素酶的融合体的快速降解直接在转基因拟南芥植物中得到证明。 绝对需要的Aux/IAA氨基酸的性质被定义，然而，侧翼序列似乎以一种尚未完全理解的方式影响蛋白质水解。 这里提出的实验将定义这些序列的作用。 将进行Aux/IAA序列的点突变和缺失，与LUC融合，并在转基因植物中测定融合蛋白的半衰期。 为了确定所有Aux/IAA蛋白是否具有相同的半衰期，将确定另外的Aux/IAA蛋白靶向LUC降解的能力。将测试缺乏所需保守氨基酸的Aux/IAA蛋白作为LUC融合体的体内降解。 如果没有快速降解，则该蛋白质将用作确定Aux/IAA降解信号的充分性的起点。 外源生长素的应用改变了Aux/IAA蛋白水解的速率，为生长素调节基因表达的拟议机制提供了第一次实验证据。Aux/IAA蛋白质的损失增加与诱导转录的基因在其调控区含有生长素反应元件相关。 将进行实验以测试内源生长素的改变是否通过构建具有改变IAA稳态水平的酶的诱导表达的转基因植物来改变Aux/IAA蛋白水解。 上述研究将通过遗传方法来补充，以分离Aux/IAA：：LUC降解缺陷突变体。 将启动其他屏面，包括激活标记屏面。 对Aux/IAA：：LUC蛋白水解的影响，以前描述的突变体改变生长素的反应也将被确定，以确定基因的重要Aux/IAA蛋白水解。 鉴于Aux/IAA蛋白水解在生长素信号转导中的重要性以及生长素在调节植物生长和发育中的重要性，拟议工作的结果将是非常重要的。 Aux/IAA蛋白水解将作为一个模型，通过它可以调节其他细胞途径，提供在其他系统中有用的methodogy，试剂和实验方法。在这些研究中定义的序列可以用于其他情况下，以控制蛋白质的细胞内水平。