潮间带大型海藻孔石莼叶黄素循环机制研究

Coastal intertidal zone is a crucial place for living organisms landing from the marine to the terrestrial habitat. Most of the macro-algae inhabiting in intertidal zone frequently endure harsh abiotic stress during low tide. Xanthophyll cycle, which is used by these organisms to protect them against potential photodamages, is one of the focus among the field of studying the stress tolerance of algae. Xanthophyll cycle is composed of two key enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP), and the VDE has a pivotal role in the regulation of xanthophyll cycle. It is generally accepted that the illumination is required for the catalytic activities of VDE. However, our previous work with intertidal macro-alga, Ulva pertusa, shows that the VDE remains active under darkness. Therefore, Ulva pertusa would be used in this project to investigate the coordinating relationships between electron flows along the thylakoid membrane and the activities of the enzymes VDE and ZEP by applying a variety of specific metabolic inhibitors to inhibit photosynthetic linear electron flow, cyclic electron flow around PSI and chlororespiration, respectively, and then to inhibit the activities of VDE and ZEP, respectively. The results would be comprehensively analyzed by comparison with those obtained from other photosynthetic organisms of different degrees of evolution. Meanwhile, examine the content of VDE and ZEP combined with thylakoid membrane, especially LHCII complex, of the Ulva pertusa treated with various levels of illumination. The purpose of this project is to elucidate the regulation mechanisms of xanthophyll cycle in intertidal macro-alga Ulva pertusa, and to track the evolution process of xanthophyll cycle.

潮间带是海洋生物登陆进化为陆地生物的重要场所，大部分栖息在潮间带的大型海藻在退潮时处于严酷胁迫环境中，其中藻体利用叶黄素循环进行光保护是海藻抗逆研究的热点。叶黄素循环包括二种关键酶：脱环氧酶（VDE）和环氧酶（ZEP），其中VDE是调控叶黄素循环的关键。一般认为，VDE只在光照条件下才有活性。然而，我们的前期研究表明，潮间带大型海藻孔石莼VDE在黑暗时仍有较强活性。为此，本项目以孔石莼为对象，利用不同的专一性抑制剂，首先分别抑制光合线性电子传递、环式电子传递和叶绿体呼吸等，然后再分别抑制ZEP和VDE活性，以阐明电子传递与VDE和ZEP之间的协同关系；分离处于不同状态的孔石莼类囊体膜尤其是LHCII复合体，研究VDE和ZEP与类囊体膜的结合情况，解析叶黄素循环调控机制。同时，比较分析不同进化程度物种的叶黄素循环，阐释其演化过程。本项目的完成将可以明确孔石莼叶黄素循环机制及调控规律。

潮间带大型海藻周期性经历多种环境胁迫，由光能过剩所造成的氧化性胁迫是其中关键的一种。潮间带大型海藻能通过叶黄素循环耗散过剩的光能，然而其调控机制与高等植物明显不同。本项目中，我们以潮间带常见大型绿藻——石蒓为主要研究对象，首先通过特异性光合电子传递抑制剂研究了光合电子传递与叶黄素循环调控之间的关系，研究结果表明石蒓(Ulva sp.)叶黄素循环中的关键酶紫黄素脱环氧酶（VDE）在低光下也具有活性，抑制叶黄素循环的逆向反应酶玉米黄素环氧化酶（ZEP）后，即便是在低光下石蒓藻体内也能积累玉米黄素（Zx），Zx的积累与光强呈正相关，线性电子传递、环式电子传递、以及叶绿体呼吸等类囊体膜上电子传递介导的跨膜质子转运都能激活VDE。其次，研究结果还表明在低光下干出能激活孔石蒓(Ulva pertusa)的叶黄素循环，在严重脱水后细胞内积累玉米黄素，这种玉米黄素的积累与光合电子传递无关，我们认为类囊体腔在干出时收缩可能是诱导Zx积累的原因。通过测定Ulva prolifera在不同CO2浓度下光合作用的变化，我们发现无论是在正常条件下还是在适度失水或盐度胁迫条件下，U. prolifera的PSII活性都随着大气中CO2浓度的增加而显著增强。结果表明U. prolifera藻体即使经受适度的胁迫，依然可以直接利用空气中的CO2作为碳源进行固碳，形成生物量，这或许是漂浮U. prolifera在绿潮爆发时生物量大量积累的原因之一。最后，我们选择二次吞噬类海藻三角褐指藻（Phaeodactylum tricornutum）作为实验材料，研究了另一种叶黄素循环——硅甲藻黄素循环响应高光胁迫时的调控机制。本项目共发表SCI研究论文10篇，培养博士研究生6名，硕士研究生2名。