乙酸代谢在衣藻光合作用和黑暗异养中的作用机制研究

Photosynthesis (PS) is the most important chemical reaction in the world. Due to day/night diel cycle, half of the entire life of photosynthetic organism is in the dark. The reciprocal transition between dark and light is critical for photosynthetic organisms. Our previous study showed that the acetate metabolisms play important roles in the transition from dark heterotrophic growth to mixotrophic growth in Chlamydomonas. However, the occurring position and the regulatory mechanism of acetate metabolism are still undiscovered. Our laboratory acquired many acetate metabolisms related mutant strains. The preliminary results showed that acetate kinase (ACK) and acetyl-CoA synthetase function in converting acetate to acetyl-CoA under dark heterotrophic and mixotrophic conditions, respectively, and the acetyl-CoA synthetase may substitute the function of acetate kinase. In this proposal, we aim to analyze the detailed functional mechanisms of ACK and ACS in acetate metabolism; to detect the subcellular localization of the key enzymes in acetate metabolism; to demonstrate the expression patterns and activities of ACK and ACS of the various strains and the key metabolites in acetate metabolism of the various mutants under light and dark conditions; to identify the key downstream interacting partners in acetate metabolism. The implementation of this project will advance our understanding of acetate metabolism pathways, elucidate the functions of acetate in dark heterotrophic growth and photosynthesis, and provide new evidence and insights into the evolution of photosynthesis.

光合作用是地球上最重要的化学反应。昼夜交替使光合生物有一半时间是在黑暗中度过的，所以黑暗和光照顺利互转对光合作用至关重要。我们前期研究发现乙酸代谢在衣藻黑暗生长切换为光合兼养的过程中发挥重要作用。然而乙酸代谢发生的场所、调控机制及其在光合兼养与黑暗异养切换过程中的具体作用机制尚不清楚。本实验室已获得多个衣藻乙酸代谢突变藻株，初步分析表明乙酸激酶(ACK)和乙酰辅酶A合成酶(ACS)分别在黑暗异养和光合兼养过程中将乙酸转变为乙酰辅酶A，而且ACS可能替代ACK的功能。本项目拟分析 ACK和ACS具体作用机制；检测乙酸代谢关键酶的细胞定位，确定乙酸代谢发生的场所；分析ACS和ACK在光照和黑暗时的表达谱和酶活性，并检测乙酸代谢中的关键产物；通过遗传手段挖掘乙酸代谢下游的调控元件。本项目的实施有助于解析乙酸代谢通路、揭示乙酸在黑暗异养和光合作用中的作用，同时为光合作用进化研究提供新的理论依据。

光合作用是地球上最重要的化学反应。昼夜交替使光合生物有一半时间是在黑暗中度过的，所以黑暗和光照顺利互转对光合作用至关重要。我们前期研究发现乙酸代谢在莱茵衣藻黑暗生长切换为光合兼养的过程中发挥重要作用，然而乙酸代谢发生的场所、调控机制及其在光合兼养与黑暗异养切换过程中的具体作用机制尚不清楚。不仅如此，乙酸代谢过程所影响的其它生命过程以及响应机制仍不明晰，因此本实验室在已获得的乙酸相关代谢突变藻株中筛选了能够在黑暗异养和光合兼养过程中发挥作用的乙酸激酶（ACK）突变体以及乙酸代谢过程中Mn2+转运相关突变体作为研究对象，对ack及其相关突变体在自养兼养、光暗转换、乙酸浓度、乙酸同化效率、吸收光谱等条件下进行分析，证明了ACK1在黑暗异养条件下对维持衣藻生命活动的重要作用，而ACK2能够恢复ACK1在黑暗条件下的表型，说明ACK1和ACK2功能可能存在一定的互补性，同时发现ACK1突变体产生的色素含量和性质也发生了变化，该结果仍需通过实验进一步确定。在对乙酸代谢相关突变体mn050研究中发现，该突变体在乙酸代谢过程中对高光的响应发生变化，通过对高光响应蛋白LHCSR3以及NPQ水平的测定发现该突变体的Mn2+转运受限，ΔpH升高引起pmf升高，从而诱导了更高的NPQ，进一步通过自养异养、荧光参数、酵母异源互补实验、蛋白定位观察、跨膜质子梯度等实验的验证，证明其在乙酸代谢条件下Mn2+转运发生变化，阐明乙酸代谢过程中高光保护特性变化。