穿孔蛋白MACPs调控植物低氧响应的分子机理

Hypoxia caused by flooding affect serious injury to terrestrial plants, which is one of the common/important abiotic stresses. However, the molecular basis of plant responses to hypoxia remains to be further investigated. Our preliminary data show the transcripts of plant Membrane Attack Complex/Perforin (MACP) proteins were significantly induced by hypoxic stress in Arabidopsis thaliana. MACP2-overexpressors (MACP2-OEs) display enhanced plant tolerance to hypoxia, while suppression of MACP2 in MACP2-KO showed hypersensitivity to hypoxia. Interestingly, the MACP2-GFP fusion protein is located in the plasma membrane under normal oxygen conditions, but it could be translocated in the vesicle structures under low oxygen treatment. Our results further showed that MACP2-GFP and EPSINR2-mCherry co-located in the MACP2+ vesicles, suggesting that MACP2 might be involved in plant response to hypoxic stress by regulating vesicular transport pathway. By reverse genetic, cell biology and biochemical approaches, we intend to further elucidate the mechanism of how MACP2 protein participates in the vesicular transport pathway as well as their potential roles in the regulation of plant response to hypoxia in Arabidopsis. The expected results of this proposal have important theoretical values for understanding of plant hypoxic signaling pathway and application potentials for genetic engineering of plant flooding tolerance.

洪涝导致的低氧胁迫对植物造成严重伤害，是最常见的非生物胁迫之一，但目前对植物低氧响应的分子网络了解有限。申请人前期研究发现，植物穿孔蛋白MACPs在低氧处理下表达量显著升高，过表达MACP2可以明显增强植物对低氧的耐受性，而其缺失突变体则异常敏感。有趣的是，MACP2-GFP融合蛋白在常氧条件下定位在细胞质膜上，而在低氧下则会形成囊泡结构。共定位实验表明，低氧胁迫显著诱导MACP2-GFP和衔接蛋白EPSINR2-mCherry共定位于MACP2+囊泡，因此推测MACP2可能通过调控囊泡运输途径，参与植物响应低氧胁迫。本项目拟在研究MACP2在低氧下精细表达谱的基础上，结合反向遗传学、细胞生物学和生物化学手段，深入探究MACP2蛋白介导的囊泡运输途径调控植物低氧响应的生理功能和分子机制。预期研究成果对于深入理解植物低氧信号通路和植物抗涝遗传改良具有重要的理论价值和实践应用前景。

近年来洪涝灾害在全球气候变暖和生态环境恶化的影响下，发生频率升高，影响范围扩大，严重威胁全球农业生态系统，是最常见的非生物胁迫之一。植物有一套响应低氧胁迫的调控机制，但是因为植物响应低氧调控信号复杂，模拟条件难控制，但目前对植物低氧响应的分子网络了解有限。植物穿孔蛋白与革兰氏阳性细菌的成孔毒素同源，在响应外界逆境、免疫应答过程中起重要作用。本研究在基因组水平上鉴定了15种植物的MACP基因，解析了其进化规律。我们发现拟南芥MACP2被低氧胁迫诱导。利用反向遗传学方法构建并获得过表达OE、突变体KO等纯合植株；表型分析发现过表达MACP2可以显著增强植物对低氧和水淹的抗性，而突变体则更加敏感，提示MACP2能提高植物对低氧和水淹胁迫下的耐受性。营养缺陷分析发现MACP2能增强植物对营养缺陷的敏感性，提示MACP2可能与自噬途径有关。病害分析发现MACP2主要通过SA信号参与响应Pst. DC3000和病原菌侵染Botrytis cinerea，并呈现出相反的耐受性。蛋白定位分析发现，MACP2定位于细胞膜，同时又与EPSINR2具有共定位；低氧和病虫害等逆境胁迫下，MACP2除定位于细胞膜外，还呈现囊泡状结构。本项目研究成果有助于深入探究植物响应低氧胁迫的分子调控机制，对深入理解植物低氧信号以及植物抗涝及病虫害遗传改良具有重要的理论价值和实践应用前景。