Light Induced Changes in Protein Expression and Uniform Regulation of Transcription in the Thylakoid Lumen of <em>Arabidopsis thaliana</em>

In plants oxygenic photosynthesis is performed by large protein complexes found in the thylakoid membranes of chloroplasts. The soluble thylakoid lumen space is a narrow and compressed region within the thylakoid membrane which contains 80–200 proteins. Because the thylakoid lumen proteins are in close proximity to the protein complexes of photosynthesis, it is reasonable to assume that the lumen proteins are highly influenced by the presence of light. To identify light regulated proteins in the thylakoid lumen of Arabidopsis thaliana we developed a faster thylakoid preparation and combined this with difference gel electrophoresis (DIGE) of dark-adapted and light-adapted lumen proteomes. The DIGE experiments revealed that 19 lumen proteins exhibit increased relative protein levels after eight hour light exposure. Among the proteins showing increased abundance were the PsbP and PsbQ subunits of Photosystem II, major plastocyanin and several other proteins of known or unknown function. In addition, co-expression analysis of publicly available transcriptomic data showed that the co-regulation of lumen protein expression is not limited to light but rather that lumen protein genes exhibit a high uniformity of expression. The large proportion of thylakoid lumen proteins displaying increased abundance in light-adapted plants, taken together with the observed uniform regulation of transcription, implies that the majority of thylakoid lumen proteins have functions that are related to photosynthetic activity. This is the first time that an analysis of the differences in protein level during a normal day/night cycle has been performed and it shows that even a normal cycle of light significantly influences the thylakoid lumen proteome. In this study we also show for the first time, using co-expression analysis, that the prevalent lumenal chloroplast proteins are very similarly regulated at the level of transcription.在植物中，产氧光合作用由叶绿体类囊体膜（thylakoid membrane）中的大型蛋白质复合物完成。类囊体腔（thylakoid lumen）是类囊体膜内一处狭窄且受压的可溶性空间，内含80至200种蛋白质。由于类囊体腔蛋白与光合作用相关蛋白质复合物距离极近，因此可合理推测腔内蛋白会受到光照的显著调控。 为鉴定拟南芥（Arabidopsis thaliana）类囊体腔中的光调控蛋白，我们开发了一种快速类囊体制备方法，并将其与暗适应、光适应状态下的腔蛋白质组的差异凝胶电泳（difference gel electrophoresis，DIGE）技术相结合。DIGE实验结果显示，经8小时光照后，共有19种腔内蛋白的相对表达水平出现上调。其中表达量上调的蛋白包括光系统II（Photosystem II）的PsbP与PsbQ亚基、主要的质体蓝素（plastocyanin），以及其他若干功能已知或未知的蛋白。此外，对公开转录组数据的共表达分析表明，腔内蛋白的表达共调控并非仅局限于光照，而是腔蛋白基因整体呈现出高度一致的表达模式。 光适应植株中大量类囊体腔蛋白表达量上调，结合观测到的转录水平统一调控模式，可推断绝大多数类囊体腔蛋白的功能与光合活性相关。本研究首次针对正常昼夜周期内的蛋白质水平差异开展分析，结果表明即使是常规光照周期，也会对类囊体腔蛋白质组产生显著影响。此外，本研究还首次通过共表达分析证实，叶绿体类囊体腔中的丰度较高的蛋白在转录水平上的调控模式高度相似。