Role of Plastidic Dicarboxylate Translocators in Plant Ammonia Assimilation

氮同化对植物生命至关重要，因为它构成了无机氮转化为氨基酸并随后转化为其他含氮有机化合物的机制。氨是光呼吸的产物，它的同化对陆地植物是必不可少的，缺乏氨同化的突变体的致命表型证明了这一点。氨被2-氧-戊二酸的还原氨化同化，产生谷氨酸。该反应定位于叶绿体中，由谷氨酰胺合成酶(GS2)和谷氨酸合成酶(FD-GOGAT)催化。叶绿体不能合成2-氧戊二酸，因此需要从胞浆中导入2-氧戊二酸。氨同化的最终产物谷氨酸需要输出到胞浆中。两个不同的转运子具有重叠的底物专一性，催化这些转运步骤。在拟南芥中，相应的基因形成一个由三个成员(DiT1、DiT2.1、DiT2.2)组成的小家族。在这项研究中，将通过测试是否：1)DiT2.1和DiT2.2的基因功能是多余的，2)草乙酸酯/天冬氨酸穿梭或亚细胞代谢物库中的置换可以弥补DiT1的不足，以及3)改变二羧酸转运体的活性影响亚细胞代谢物水平，从而触发转录组的协调变化，来从功能上剖析可塑性的二羧酸转运体。该项目将提高对植物氨同化所涉及的运输活动的理解，以及对代谢物转运体对细胞间代谢通量的控制的理解。广泛的影响：本科生和研究生将接受跨学科的培训，这样他们就可以超越各自的专业领域，思考植物生物学的综合观点。初级科学家将接受处理膜转运蛋白的培训，研究生和博士后研究人员将参与植物膜转运蛋白领域的本科教学。通过与其他美国学术机构和国际合作伙伴的合作，将建立网络，并向广泛的科学家提供膜生物学领域的工具。该项目产生的植物材料将用于向K-12教师介绍植物基因工程，K-12教师将在暑期课程中接受现代分子技术培训。通过与统计和概率学院的合作，该项目将促进数学和生物科学研究生的跨学科培训。

Nitrogen assimilation is critical for plant life because it constitutes the mechanism by which inorganic nitrogen is converted into amino acids and subsequently other nitrogenous organic compounds. Ammonia is a product of photorespiration and its assimilation is essential for land plants, as demonstrated by the lethal phenotypes of mutants deficient in ammonia assimilation. Ammonia is assimilated by reductive amination of 2-oxoglutarate, giving rise to glutamate. This reaction is localized in plastids and catalyzed by the enzymes glutamine synthetase (GS2) and glutamate synthase (Fd-GOGAT). Plastids are unable to synthesize 2-oxoglutarate; hence 2-oxoglutarate needs to be imported from the cytosol. The end product of ammonia assimilation, glutamate, needs to be exported to the cytosol. Two distinct translocators with overlapping substrate specificity catalyze these transport steps. In Arabidopsis, the corresponding genes form a small family of three members (DiT1, DiT2.1, DiT2.2). In this study, plastidic dicarboxylate transporters will be functionally dissected by testing whether: 1) the gene functions of DiT2.1 and DiT2.2 are redundant, 2) an oxolacetate/aspartate shuttle or a displacement in sub-cellular metabolite pools can compensate for deficiency in DiT1, and 3) altered dicarboxylate transporter activity impacts sub-cellular metabolite levels that trigger coordinated changes in the transcriptome. This project will improve the understanding of transport activities involved in ammonia assimilation in plants, and of the control that metabolite transporters exert over metabolic fluxes between intracellular compartments. Broad Impact: Undergraduate and graduate students will receive cross-disciplinary training so they can think beyond their individual field of expertise towards an integrative view of plant biology. Junior scientists will be trained in handling membrane transporters, and graduate students and post-doctoral researchers will be involved in undergraduate teaching in the field of plant membrane transport. Through cooperation with other U.S. academic institutions and international partners, networks will be established and tools in the field of membrane biology will be made available to a broad range of scientists. Plant material generated in this project will be used to introduce K-12 teachers into genetic engineering of plants and K-12 teachers will be trained in modern molecular techniques in summer courses. Through the collaboration with faculty in Statistics and Probability, this project will foster the cross-disciplinary training of graduate students in mathematical and biological sciences.