RUI: Secreted alpha-amylase and stress-related starch metabolism

淀粉是一种重要的植物储能化合物，在人类饮食中占有相当大的比例。在叶片中，淀粉在白天积累在叶绿体中，晚上被分解，为植物提供能量。淀粉降解的酶途径的全部细节尚不清楚，但它可能涉及a-淀粉酶、b-淀粉酶、去支化酶、a-葡萄糖苷酶和淀粉磷酸化酶。有趣的是，这些酶的大部分活性位于叶绿体之外，因此可能在正常的淀粉代谢中没有作用。一些植物含有一种分泌的a-淀粉酶，这种酶是由生物和非生物胁迫诱导的。拟南芥基因组包含三个α-淀粉酶基因，其中一个(AtAMY3)具有针对其分泌途径的预测信号序列。本研究的主要目的是了解这种分泌的α-淀粉酶在拟南芥中的功能。植物病原体和非生物胁迫(如盐、热、干旱、重金属和臭氧)会对植物组织造成许多变化，包括抑制韧皮部运输、碳水化合物(包括淀粉)的积累和呼吸作用的增加。当压力很大时，细胞会产生能诱导细胞程序性死亡的活性氧。在细胞死亡过程中，细胞膜解体，允许来自不同隔室的分子混合。正是在这些死亡细胞中，分泌的a-淀粉酶被认为作用于淀粉。然后，产生的糖分将可用于满足邻近细胞增加的呼吸需求，这些细胞最终将在压力下生存下来。本研究的目的是：1)纯化和鉴定分泌的α-淀粉酶的性质和调控；2)鉴定该酶的功能。利用环七直链淀粉亲和层析法从芥菜和拟南芥中提纯该酶。野生型植物将受到不同形式的胁迫处理，以观察对分泌淀粉酶的诱导、淀粉积累和随后的降解以及对细胞死亡的影响。一个在AtAMY3中插入T-DNA的拟南芥突变体将被用来证实AtAMY3基因和质外体α-淀粉酶之间的联系，并检验这种酶在严重胁迫中发挥作用的假设。拟议中的工作有三个好处。首先，PI希望确定一种叶淀粉酶的作用，这种酶已经知道一段时间了，但由于它位于细胞壁中，其功能一直是个谜。如果能够证明这种酶在对严重压力的反应中发挥作用，结果将增加对农业直接有益的不断增长的知识体系。其次，这笔拨款将有助于培养一批本科生，其中许多人追求科学职业，以及一名选择在以本科生为主的机构从事职业生涯的教学/研究博士后研究员。第三，这笔拨款将使私人助理继续为本科课程开发调查实验室，并通过本科生研究理事会和美国植物生物学家协会在以本科生为主的机构指导年轻教师。

Starch is an important energy-storage compound for plants and makes up a significant portion of the human diet. In leaves, starch accumulates in chloroplasts during the day and is broken down at night to provide energy for the plant. The full details of the enzymatic pathway for starch degradation are not understood but it probably involves a-amylase, b-amylase, debranching enzyme, a-glucosidase, and starch phosphorylase. Interestingly, much of the activity of these enzymes is located outside the chloroplasts and, as such, probably plays no role in normal starch metabolism. Several plants contain a secreted a-amylase that is induced by biotic and abiotic stress. The Arabidopsis thaliana genome contains three a-amylase genes, one of which (AtAMY3) has a predicted signal sequence targeting it to the secretion pathway. The primary goal of this research is to understand the function of this secreted a-amylase in Arabidopsis. Plant pathogens and abiotic stress (e.g. salt, heat, drought, heavy metals, and ozone) cause a number of changes to plant tissues including inhibition of phloem transport, accumulation of carbohydrates (including starch), and increased respiration. When the stress is severe, cells generate reactive oxygen species that can induce programmed cell death. During cell death membranes disintegrate, allowing the mixing of molecules from different compartments. It is in these dead cells that the secreted a-amylase is thought to act on starch. The resulting sugars would then be available to meet the increased respiratory demand of the neighboring cells that will ultimately survive the stress. The objectives of this research are 1) to purify and characterize the properties and regulation of the secreted a-amylase, and 2) to identify the function of the enzyme. Purification of the enzyme from mustard (Brassica juncea) and Arabidopsis will make use of cycloheptaamylose affinity chromatography. Wild type plants will be treated with various forms of stress to observe the effects on induction of the secreted amylase, starch accumulation and subsequent degradation, and on cell death. An Arabidopsis mutant with a T-DNA insertion in AtAMY3 will be used to confirm the link between the AtAMY3 gene and the apoplastic a-amylase, and to test the hypothesis that the enzyme plays a role in survival of a severe stress. The benefits of the proposed work are three. First, the PI hopes to define the role of a leaf amylase that has been known for some time but whose function has been a mystery due to its location in cell walls. If it can be demonstrated that the enzyme plays a role in the response to severe stress, the results will add to a growing body of knowledge that is directly beneficial to agriculture. Second, the grant will help to train a number of undergraduates, many of which pursue careers in science, and a teaching/research postdoctoral fellow choosing to pursue a career at a predominantly undergraduate institution. Third, the grant will enable the PI to continue his activities of developing investigative laboratories for undergraduate courses and mentoring younger faculty at predominantly undergraduate institutions through the Council on Undergraduate Research and the American Society of Plant Biologists.