Regulation of Proteolysis During Legume Germination

在萌发的种子中，蛋白质储备的动员需要在胚胎生长的适当时间内许多蛋白酶的作用。在绿豆（Vigna radiata）中已经鉴定出四种这样的蛋白酶：蛋白酶F、芦笋肽水解酶、羧肽酶I和II。绿豆子叶中这些蛋白的水平将在种子发育和萌发的不同阶段使用特异性抗体进行量化。每个蛋白酶的lambda-gt11 cDNA克隆将被分离并用作特异性探针来表征和定量蛋白酶mRNA水平。这些结果将与蛋白酶基因转录物的体外可翻译性和蛋白酶活性的直接测定相结合。这些研究将确定合成对转录、mRNA加工、翻译、翻译后加工或其组合的控制水平。在萌发后不久与胚轴分离的子叶中，蛋白酶及其mrna的水平也将被检测。子叶的分离破坏了蛋白酶的正常变化模式，并可能为植物激素在这些酶的调节中所起的作用提供见解。种子萌发和种子活力的一个重要组成部分是动员储存的食物储备，如碳水化合物，蛋白质和脂质。关于高等植物种子中蛋白质储备的动员，我们所知甚少。目前尚不清楚为什么不同的蛋白质降解酶在发芽的不同时间以不同的数量出现，也不知道这个过程是如何控制的。本提案所述的工作将开发分子工具来研究绿豆种子中参与蛋白质降解的四种不同酶的合成调控。这些工具的发展将在未来提供控制蛋白质降解过程的潜力，这将影响农业，特别是麦芽和酿造工业。//

The mobilization of protein reserves in the germinating seed requires the action of a number of proteases at appropriate times during growth of the embryo. Four such proteases have been identified in the mung bean (Vigna radiata): proteinase F, vicilin peptidohydrolase, and carboxypeptidase I and II. The levels of these proteins in mung bean cotyledons will be quantified at different stages of seed development and germination using specific antibodies. cDNA clones in lambda-gt11 for each of the proteases will be isolated and used as specific probes to characterize and quantitate protease mRNA levels. These results will be combined with those for the in vitro translatability of protease gene transcripts, and the direct determination of protease activities. These studies will pinpoint the level of control of synthesis to transcription, mRNA processing, translation, post-translational processing, or a combination thereof. The levels of proteases and their mRNAs in cotyledons detached from the embryonic axis shortly after germination will also be examined. Detachment of the cotyledons disrupts the normal pattern of changes in the proteases, and may offer insight into the role of phytohormones in the regulation of these enzymes. An important part of seed germination and seed viability is the mobilization of stored food reserves such as carbohydrate, protein and lipids. Little is known about the mobilization of protein reserves in the seeds of higher plants. It is not known why different protein-degrading enzymes appear in different amounts at different times during germination or how this process is controlled. The work described in this proposal will develop the molecular tools to study the regulation of the synthesis of four different enzymes involved in protein degradation in mung bean seeds. The development of these tools will offer in the future the potential for controlling the process of protein degradation which would impact agriculture, particularly the malting and brewing industries.//