植物磷脂参与细胞自噬过程调控氮营养循环再利用的研究

Nitrogen (N) is the most-limiting nutrient for crop production. Knowledge concerning the regulation of plant sensing of and response to the macronutrient limitation will greatly facilitate the development of crops that yield more without increasing N inputs. Cell membranes are initial sites where nutrient cues are perceived and transduced into metabolic, cellular, and physiological responses. Our recent results indicate that phospholipase D (PLD) and associated membrane lipid changes play important, multifaceted roles in plant response to N availability, and that a better understanding of the lipid regulation has the potential to improve nutrient uptake and/or utilization in crop plants. The goal of the project is to understand the role of membrane lipids in plant response to N availability. We hypothesize lipids regulate plant responses to N through their roles in cell signaling and vesicular trafficking. Specifically, lipid mediators such as phosphatidic acid (PA) and phosphatydylethanolamine interact with specific autophagosomal proteins (ATGs) via covalent and noncovalent interactions and the interaction affect autophagosomal association and subcellular localization. Three interlaced objectives will be pursued: 1) determine the effect of altered PA and PE levels on autophagosomal components and processes; 2) probe the interaction of lipids with ATG proteins; and 3) explore the role of lipid-ATG interaction on autophagosomal process and N remobilization. Biochemical, genetic, cellular, and physiological approaches will be integrated in the proposed study. These will include genetic alterations of PLD expression by gene knockout and overexpression; metabolomic profiling of glycerolipidomes and hormones; identification and functional analysis of lipid-protein interactions; and cellular and physiological analyses of N remobilization and plant response to N. The proposed study will advance knowledge with potentials to improve crop production without increasing the use of N fertilizers. The lipid regulation-enhanced rice plants may be improved in nutrient acquisition and utilization for seed production. The knowledge may be applied to other crops. Decreases in fertilizer uses will have not only great economic values but also have far-reaching environmental benefits.

氮是限制作物产量最主要的营养因素，有关促进作物产量的研究表明氮营养对作物产量的影响最大。细胞膜是感知营养的初始点并引发植物体内代谢、细胞和生理反应过程。研究表明磷脂酶D及其导致的膜脂变化在植物响应氮营养过程中起着重要的、多元化的作用。前期研究发现脂质可能通过参与自噬及囊泡运输过程来调节植物对氮营养的再循环利用。本项目将通过生物化学、脂质代谢组、细胞学及生理学等方面进一步分析植物脂质调节者如磷脂酸（PA）及磷脂酰乙醇胺（PE）与相应的自噬体蛋白（ATGs）的互作而影响自噬体的组分、时空调控及其在氮循环利用中的生物学效应。研究内容包括：1. 确定磷脂PA和PE对自噬体形成和作用过程的影响；2.探索脂质和ATGs蛋白之间的互作关系；3.探索脂质-ATG互作通过自噬过程调控植物氮再循环利用的效应。本研究将推动我们认识脂质信号参与作物的氮营养高效循环利用的分子机制，以及在作物育种中的潜在应用价值。

自噬是真核细胞降解和回收再利用胞内生物大分子和受损细胞器的基本生理过程，对维持细胞生存及内稳态具有重要作用。目前有关酵母和动物细胞自噬过程已有大量的研究，并在自噬发生的过程及分子机理取得显著进展。然而，有关植物细胞自噬方面的研究相对滞后。本研究分析了水稻自噬体形成相关基因OsATG4b、OsATG7及OsATG8d在植物生长发育及逆境胁迫中的作用。首先观察GFP融合蛋白发现OsATG4b-GFP和OsATG7-GFP定位于细胞质，而GFP-OsATG8d定位于细胞核、细胞质且与微管蛋白共定位，盐胁迫及暗处理可使ATG8d显著积累。通过分离鉴定OsATG4b和OsATG7缺失突变体，结果表明OsATG4b或OsATG7的缺失突变均导致植株生长发育受阻，其突变体Osatg4b和Osatg7株高、结实率及生物产量均显著低于对照野生型植株，其中ATG7缺失导致植株生长严重受阻，Osatg7突变体高度不育，其结实率几乎为零。进一步分析发现OsATG7与磷脂酸（PA）存在特异性互作，表明PA介导自噬过程而调节植株生长发育。OsATG4b的缺失则导致幼苗生长受到一定程度的抑制，其种子萌芽速率和幼苗生长相对滞后于野生型，且Osatg4b突变体植株对盐胁迫更敏感。生化分析表明OsATG4b具有蛋白酶活性，可切除ATG8d的C-端，其酶活性受过氧化氢（H2O2）的抑制。同时，超表达OsATG8d也出现类似于Osatg4b突变体的表型，OsATG8d-OE种子千粒重显著低于对照野生型植株，种子萌芽力和幼苗生长滞后于对照株系。这些结果表明OsATG4b和OsATG8d共同参与水稻种子发育过程中的营养物质积累和种子萌芽过程中的营养释放和利用过程，从而正调控植物生长发育和逆境胁迫的响应过程，并且正常含量的OsATG8d对维持植物生长发育至关重要。