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.
