A Developmental Study of Essential Amino Acid Biosynthesis in Chloroplasts

本课题主要研究高等植物叶绿体必需天冬氨酸家族氨基酸代谢的发育调控。正在测试的假设是，将测试以下参数以确定它们在叶片发育过程中的变化：1)氨基酸的水平和细胞内分布，2)叶绿体被膜对氨基酸的通透性，以及3)必需氨基酸产生的关键酶的活性。从大麦植株不同发育阶段的叶片中制备的纯化叶绿体被用来测定整个发育过程中叶绿体和其他细胞室中的氨基酸浓度，测量不同年龄叶片中天冬氨酸和代谢相关的氨基酸赖氨酸、苏氨酸、蛋氨酸和异亮氨酸进入叶绿体的运输，并分离天冬氨酸激酶，不同年龄组织叶绿体的高丝氨酸脱氢酶和二氢二氢吡啶甲酸合成酶及其相对催化活性。抗体也将被提高到酶，酶水平的变化将通过免疫学方法得到确认。在过去的十年中，很明显，叶绿体是植物叶片中营养必需的天冬氨酸家族氨基酸生物合成的主要部位，如果不是唯一的部位的话。在植物叶片的发育过程中，产生这些氨基酸和其他氨基酸的需求会发生变化。本研究探索叶绿体合成必需的天冬氨酸衍生氨基酸的能力在叶片发育过程中是否会发生变化，以便更好地了解叶绿体的发育及其调控。这些研究将进一步提供有关必需氨基酸生物合成的性质和调控的信息。鉴于两种天冬氨酸衍生氨基酸的缺乏是导致人类营养不良的主要因素，有关其合成调控的新信息特别令人感兴趣。

This project focuses on the developmental regulation of essential aspartate-family amino acid metabolism in chloroplasts of higher plants. The hypothesis being tested is that the following parameters will be tested to determine how they vary during leaf development: 1) the level and intracellular distributions of amino acids, 2) the permeability of the chloroplast envelope to amino acids and 3) the activities of key enzymes of essential amino acid production. Purified chloroplasts prepared from leaves of barley plants at various stages of development are used to determine concentrations of amino acids in chloroplasts and other cellular compartments throughout development, to measure the transport of aspartic acid and the metabolically related amino acids, lysine, threonine, methionine and isoleucine, into chloroplasts from leaves of varying ages, and to isolate the enzymes aspartate kinase, homoserine dehydrogenase and dihydrodipicolinate synthetase from plastids of different age tissues and relative catalytic activities. Antibodies also will be raised to the enzymes and variations in enzyme levels will be confirmed by immunological methods. In the last decade it has become clear that the chloroplast is the main site, if not the sole site, for the biosynthesis of the nutritionally essential aspartate-family amino acids in plant leaves. During the development of plant leaves, the requirement for the production of these and other amino acids changes. This research explores whether the capacity of chloroplasts to synthesize the essential aspartate-derived amino acids varies during leaf development, so that chloroplast development and its regulation can be better understood. These studies will further provide information regarding the nature and regulation of essential amino acid biosynthesis. Given that deficiency of two of the aspartate-derived amino acids is a main factor contributing to human malnutrition, new information regarding the regulation of their synthesis is of particular interest.