水稻内质网转运蛋白ER-ANT1调控光呼吸氨同化的分子机理

In higher plants, photorespiration involves a complex network of enzymatic reactions distributed chloroplast, peroxisome, mitochondrion. Ammonium released in the mitochondria by photorespiration must be reassimilated in the chloroplast by the glutamine synthetase/glutamate synthase (GS2/Fd-GOGAT) cycle. However, whether the other organelle or compartment modulates the assimilation of photorespiratory ammonia is largely unknown. Here we identified an endoplasmic reticulum (ER)-localized adenine nucleotide transporter1 (ER-ANT1), whose deficiency causes seedling lethality in air but viable under high CO2, exhibiting the typical photorespiratory phenotype. Metabolite analysis demonstrated that the absence of OsER-ANT1 obstructes the refixation of photorespiratory ammonia. These results imply the existence of the pathway of the refixation of photorespiratory ammonia. We will explore the relation between ER-ANT1 and GS/GOGAT by detecting the enzymatic activity, the amount of protein, gene expression and subcellular localization of protein and elucidate whether the GS2/Fd-GOGAT cycle is regulated by ER-ANT1, and if so, by what mechanism. And we will isolate a tagged protein along with its interacting partners from cell lysates by tandem afinity purification/mass spectrometry method (TAP/MS); we will also validate the protein interactions by bimolecular fluorescence complementation (BiFC) and investigate the phenotypic effects of specific gene encoding protein obtained from the BiFC by Reverse genetics approaches such as RNAi. This project will make a major contribution to our understanding of the underlying mechanisms of photorespiratory ammonia assimilation.

光呼吸是植物不可缺少的重要生理过程，其代谢产生的氨会对细胞造成毒害，必须被重新同化。叶绿体内的谷氨酰胺/谷氨酸（GS2/Fd-GOGAT）循环在该过程中具有重要作用。但是，是否有其它代谢通路或细胞器参与或调节了光呼吸氨的同化却并不清楚。本项目前期研究发现，内质网转运蛋白ER-ANT1参与了光呼吸氨同化过程，暗示着可能存在一条经由内质网的光呼吸氨同化途径。在此基础上，本研究拟从酶活性、基因转录、蛋白表达及其亚细胞定位等方面探索ER-ANT1与GS2/Fd-GOGAT循环的关系，阐明ER-ANT1调节氨同化是否依赖于GS2/Fd-GOGAT循环。采用串联亲和纯化/质谱联用技术、双分子荧光互补技术和RNAi等反向遗传学方法，筛选和鉴定ER-ANT1互作蛋白，验证ER-ANT1互作蛋白在光呼吸代谢中的生物学功能。其研究结果将为光呼吸氨同化理论提供新的参考，有助于进一步阐明植物氮代谢的分子调控网络。

光呼吸是植物不可缺少的重要生理过程，其代谢产生的氨会对细胞造成毒害，必须被重新同化。叶绿体内的谷氨酰胺/谷氨酸（GS2/Fd-GOGAT）循环在该过程中具有重要作用。但是，是否有其它代谢通路或细胞器参与或调节了光呼吸氨的同化却并不清楚。本项目前期研究发现，内质网ATP转运蛋白ER-ANT1参与了光呼吸氨同化过程，暗示着可能存在一条经由内质网的光呼吸氨同化途径。在此基础上，本研究从酶活性、基因转录以及正常条件和高CO2条件下的氨基酸代谢等方面探索了ER-ANT1与GS2/Fd-GOGAT循环的关系。结果表明，ER-ANT1调节氨同化不依赖于GS2/Fd-GOGAT循环。为进一步揭示内质网ER-ANT1调控光呼吸氨同化的机制，本项目采用串联亲和纯化/质谱联用技术进行了互作蛋白筛选。蛋白互作研究表明，ER-ANT1能够与线粒体电压依赖性阴离子通道蛋白(voltage- dependent anion- selective channel proteins，VDAC) 家族成员VDAC5发生互作。进一步研究表明，水稻VDAC5定位于线粒体-内质网形成的特殊物理连接处，与动物及酵母同源蛋白类似同属线粒体-内质网结构偶联蛋白（mitochondria-associated endoplasmic reticulum membrane, MAM），可能对线粒体Ca2+稳态及ATP生产和转运等一系列生理活动具有重要意义。本项目探索了内质网调节植物光呼吸氨同化的分子机理，提出了一条基于内质网的，独立于叶绿体光呼吸氨同化代谢途径的一条新途径，其研究结果将为光呼吸氨同化理论提供新的参考，有助于进一步阐明植物氮代谢的分子调控网络。