植物SCAR/WAVE复合体与线粒体协同调节的自噬机制及其对柑橘果实品质的影响

Previously, we have revealed that the formation of autophagosomes in plants can be regulated by the SCAR/WAVE complex and cytoskeletons. Further study identified that the reduction of intracellular ATP level caused by abnormal mitochondrial activity could be one of the trigger factor; which is mainly through the alternation of autophagy regulating protein phosphorylating modification. Enhanced membrane/microtubules association of the SCAR or ATG protein is observed when their phosphorylation status has changed; as a consequence, the level of autophagy is elevated. In this project, we aim to further study the interaction among the SCAR complex，cytoskeletons and the membrane during the process of autophagy at different mitochondrial respiration condition. Moreover, it is known that the activity of mitochondria and intracellular ATP level changed significantly during fruit ripening and post-harvest senescence; and such change could be the trigger to initiate autophagy in fruit. Meanwhile, this also provides a good system to study the effect of mitochondria activity to autophagy. Therefore, this project plan to further study the mechanism of mitochondria and cytoskeleton regulated autophagosomes formation in fruits，especially the process, regulation and function of autophagy during fruit ripening and post-harvest senescence. Also,looking for mitochondrial protein candidates that regulate autophagy in citrus fruit, in order to provide new approach to improve citrus fruit quality during post-harvest.

SCAR/WAVE复合体通过与ARP2/3复合体调节肌动蛋白丝的聚合，这是大部分真核细胞共有的现象。最近我们发现SCAR/WAVE复合体也可与自噬膜相互作用，调节植物细胞自噬体的形成。后续实验证明，线粒体呼吸作用异常所导致的ATP浓度下降，是其主要诱导因素之一：通过改变相关自噬调控蛋白的磷酸化修饰，促进SCAR、ATG蛋白与自噬膜、微管结合，加速自噬。由于果实在成熟与采后衰老过程中线粒体活性变化显著，同时伴随着细胞内ATP浓度的改变，这为自噬的发生提供了可能的诱导因素，同时也为研究线粒体活性所导致的植物自噬提供了很好的研究对象。在此背景下，本项目拟以线粒体活性与细胞内ATP变化所导致的自噬为切入点，深入研究自噬通过细胞骨架与线粒体的调节机制，阐明呼吸作用异常所导致的自噬在柑橘果实成熟与采后衰老阶段的发生规律，同时找出调节柑橘果实自噬的线粒体蛋白，以期为果实品质调控提供新的依据。

柑橘采后衰老过程中线粒体活性改变，构成果实风味品质的有机酸含量随之减少，而低氧胁迫可诱导线粒体选择性自噬的发生，但其机制仍未知。在果实转色过程中自噬活性增加，因此自噬还可能参与柑橘果实色泽品质的建成。本项目聚焦于柑橘果实自噬的发生与调控、线粒体功能研究，旨在揭示自噬和线粒体活动对果实品质形成影响的新机制。由于柑橘果实的组织特殊性，细胞生物学与自噬的相关研究在其中很难有效开展。因此本项目优先进行柑橘愈伤组织稳定转化、柑橘果实瞬时转化、以及柑橘果实线粒体分离、原生质体分离线粒体染色观察试验；进而构建了自噬基因ATG7和ATG8在柑橘模式植物山金柑的稳定过表达和RNAi遗传转化材料。通过厌氧处理差异蛋白组数据分析，锁定了3个影响线粒体功能的蛋白；针对植物自噬的发生与调控机制，开展了转录水平、蛋白水平、细胞生物学观察等多角度的基础研究；与此同时，明确了红光处理可促使自噬活性增强，促进果实转色，并对其分子机制进行了深入的研究。总之，本项目初步明确线粒体活动和自噬对柑橘果实色泽品质和风味品质的形成具有重要作用，为深入的机制解析构建丰富的材料和数据基础。