Molecular-Genetic Study of Aspartate Aminotransferase Genes/Isoenzymes in Arabidopsis thaliana

分子、遗传和生物化学方法被用于研究拟南芥中天冬氨酸氨基转移酶（AspAT）不同亚细胞同工酶的功能。由AspAT合成的天冬氨酸对植物生长和发育至关重要，因为天冬氨酸用于将同化的氮运输到种子，并且是必需氨基酸（包括甲硫氨酸和赖氨酸）的前体。因此，了解哪些亚细胞AspAT同工酶控制天冬氨酸合成可能有助于改变不太适合分子遗传学分析的作物中必需的天冬氨酸衍生氨基酸的合成。拟南芥含有五个编码AspAT同工酶的ASP基因，这些同工酶定位于细胞质、叶绿体、线粒体或过氧化物酶体。拟南芥突变体缺陷的主要AAT同工酶：胞质AAT 2（aat 2）或叶绿体AAT 3（aat 3）已被分离，并被用于确定功能的每个亚细胞同工酶在体内。aat 2突变体的分析表明，胞质AAT 2控制大量的N-同化成天冬氨酸在光和这池的天冬氨酸在胞质溶胶中转化为天冬酰胺的N-运输在黑暗中。对叶绿体aat 3缺陷突变体的平行分析表明，AAT 3控制叶绿体中天冬氨酸的合成，并且叶绿体中的天冬氨酸库用于甲硫氨酸生物合成，甲硫氨酸是一种必需的谷氨酸衍生氨基酸。因此，这项研究的aat 2和aat 3突变体的拟南芥提供了深入了解代谢通量从亚细胞池的天冬氨酸在体内，不能预测的基础上，在体外生化研究的分离的同工酶。其他研究包括分离AspAT的线粒体或过氧化物酶体同工酶缺陷的突变体。这些额外的研究应解决这些其他亚细胞AAT同工酶在天冬氨酸代谢方面的作用，包括植物特异性过程，如光呼吸。最重要的是，一个意想不到的发现是，ASP基因中的aat 2和aat 3突变中的某些突变导致天冬氨酸过度积累。相应ASP基因中的这些突变似乎会损害天冬氨酸降解，但不会损害突变酶的天冬氨酸合成。这些突变的ASP基因将在转基因植物中表达，以试图工程化过量生产天冬氨酸的植物。预计此类转基因植物可能会表现出更高的氮同化率和/或产生具有更高水平的天冬氨酸衍生必需氨基酸的种子。因此，这些关于模式遗传植物中天冬氨酸合成和催化的研究可能对改善作物植物中氮同化和/或必需天冬氨酸衍生氨基酸的合成具有显著影响。

Molecular, genetic and biochemical approaches are being used in Arabidopsis to study the function of distinct subcellular isoenzymes of aspartate aminotransferase (AspAT). Aspartate synthesis by AspAT is crucial to plant growth and development, as aspartate serves to transport assimilated nitrogen to seeds, and is the precursor to essential amino acids including methionine and lysine. Thus, understanding which subcellular AspAT isoenzymes control aspartate synthesis may have applications for modifiying the synthesis of essential aspartate-derived amino acids in crop plants less amenable to molecular-genetic analysis. Arabidopsis contains five ASP genes encoding AspAT isoenzymes localized to the cytosol, chloroplasts, mitochondria or peroxisomes. Arabidopsis mutants defective in the major AAT isoenzymes: cytosolic AAT2 (aat2) or chloroplastic AAT3 (aat3) have been isolated and are being used to determine the function of each subcellular isoenzyme in vivo. Analysis of aat2 mutants has shown that cytosolic AAT2 controls the bulk of N-assimilation into aspartate in the light and that this pool of aspartate in the cytosol is converted into asparagine for N-transport in the dark. A parallel analysis of mutants defective in chloroplastic aat3 shows that AAT3 controls the synthesis of aspartate in chloroplasts, and that this pool of aspartate in chloroplasts is used for methionine biosynthesis, an essential aspartate-derived amino acid. Thus, this research on the aat2 and aat3 mutants of Arabidopsis has provided insight into metabolic flux from subcellular pools of aspartate in vivo which could not have been predicted based on in vitro biochemical studies of the isolated isoenzymes. Additional studies include the isolation of mutants defective in mitochondrial or peroxisomal isoenzymes of AspAT. These additional studies should address roles of these other subcellular AAT isoenzymes in aspects of aspartate metabolism including plant-specific processes such as photorespiration. Most importantly, an unexpected finding is that certain mutations in the ASP genes of the aat2 and aat3 mutations lead to aspartate overaccumulation. These mutations in the corresponding ASP genes appear to impair aspartate degradation, but not aspartate synthesis by the mutant enzyme. These mutant ASP genes will be expressed in transgenic plants in an attempt to engineer plants that overproduce aspartate. Such transgenic plants may be expected to exhibit increased rates of nitrogen assimilation and/or produce seeds with higher levels of aspartate-derived essential amino acids.. Thus, these studies on aspartate synthesis and catabolism in a model genetic plant, may have significant impact on improving nitrogen assimilation and/or the synthesis of essential aspartate-derived amino acids in crop plants.