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

淀粉是植物重要的能量储存化合物，并构成人类饮食的重要部分。 在叶片中，淀粉在白天积累在叶绿体中，并在晚上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，但可能涉及a-淀粉酶、b-淀粉酶、脱支酶、a-葡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外，因此可能在正常的淀粉代谢中不起作用。 一些植物含有由生物和非生物胁迫诱导的分泌的α-淀粉酶。 拟南芥基因组含有三个α-淀粉酶基因，其中一个（AtAMY3）具有将其靶向至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型α-淀粉酶在拟南芥中的功能。植物病原体和非生物胁迫（例如盐、热、干旱、重金属和臭氧）引起植物组织的许多变化，包括韧皮部运输的抑制、碳水化合物（包括淀粉）的积累和呼吸增加。 当应激严重时，细胞产生活性氧，可诱导细胞程序性死亡。 在细胞死亡期间，细胞膜解体，允许来自不同隔室的分子混合。 在这些死细胞中，分泌的α-淀粉酶被认为作用于淀粉。 然后，产生的糖将可用于满足最终在压力下存活的相邻细胞增加的呼吸需求。本研究的目的是1）纯化和表征分泌的α-淀粉酶的性质和调节，2）鉴定该酶的功能。 从芥菜（Brassica juncea）和拟南芥中纯化该酶将使用环庚直链淀粉亲和层析。 将用各种形式的胁迫处理野生型植物以观察对分泌的淀粉酶的诱导、淀粉积累和随后的降解以及对细胞死亡的影响。 将使用在AtAMY3中具有T-DNA插入的拟南芥突变体来确认AtAMY3基因与质外体α-淀粉酶之间的联系，并测试该酶在严重胁迫的存活中起作用的假设。拟议工作的好处有三个。 首先，PI希望确定一种叶淀粉酶的作用，这种淀粉酶已经知道了一段时间，但由于其位于细胞壁中，其功能一直是个谜。 如果能够证明这种酶在应对严重胁迫中发挥作用，那么这一结果将增加对农业直接有益的知识。 第二，补助金将有助于培训一些本科生，其中许多人追求科学的职业生涯，和一个教学/研究博士后研究员选择在一个以本科为主的机构追求职业生涯。 第三，拨款将使首席研究员能够继续开展活动，通过本科生研究理事会和美国植物生物学家学会，为本科生课程发展研究实验室，并指导以本科生为主的院校的年轻教师。

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.