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

分子、遗传和生化方法正被用来研究不同的亚细胞天冬氨酸氨基转移酶(Aspat)同工酶的功能。天冬氨酸的合成对植物的生长发育至关重要，因为天冬氨酸是将同化的氮素输送到种子中的，也是蛋氨酸和赖氨酸等必需氨基酸的前体。因此，了解哪些亚细胞Aspat同工酶控制天冬氨酸的合成可能对修饰作物中不太适合分子遗传分析的必需天冬氨酸衍生氨基酸的合成具有应用价值。拟南芥含有5个编码Aspat同工酶的ASP基因，定位于细胞质、叶绿体、线粒体或过氧化物体。拟南芥的主要AAT同工酶缺陷突变体：胞质AAT2(Aat2)或叶绿体AAT3(Aat3)已被分离出来，并被用来确定体内各亚细胞同工酶的功能。对aat2突变体的分析表明，细胞质中的Aat2控制着大部分N-同化为天冬氨酸，而胞质中的这一池天冬氨酸在黑暗中被转化为天冬酰胺进行N-运输。对叶绿体aat3缺陷突变体的平行分析表明，aat3控制叶绿体中天冬氨酸的合成，叶绿体中的这一天冬氨酸池用于蛋氨酸的生物合成，蛋氨酸是天冬氨酸衍生的必需氨基酸。因此，对拟南芥aat2和aat3突变体的研究为了解体内天冬氨酸亚细胞池的代谢通量提供了洞察力，这是基于分离的同工酶的体外生化研究无法预测的。其他研究包括分离Aspat线粒体或过氧化物酶同工酶缺陷的突变株。这些额外的研究应该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，包括植物特有的过程，如光呼吸。最重要的是，一个意想不到的发现是，aat2和aat3突变的asp基因的某些突变会导致天冬氨酸过度积累。相应ASP基因的这些突变似乎会削弱天冬氨酸的降解，但不会损害突变酶合成天冬氨酸。这些突变的天冬氨酸基因将在转基因植物中表达，试图改造出过度生产天冬氨酸的植物。这样的转基因植物可能会表现出更高的氮同化率和/或产生更高水平的天冬氨酸衍生的必需氨基酸的种子。因此，这些关于模式遗传植物中天冬氨酸合成和分解代谢的研究，可能会对提高作物的氮同化和/或合成必需的天冬氨酸衍生氨基酸产生重大影响。

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.