Majeran Wojciech, Zybailov Boris, Ytterberg A Jimmy, Dunsmore Jason, Sun Qi, van Wijk Klaas J
Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
Mol Cell Proteomics. 2008 Sep;7(9):1609-38. doi: 10.1074/mcp.M800016-MCP200. Epub 2008 May 2.
Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.
玉米叶片的叶绿体分化为特定的维管束鞘（BS）和叶肉（M）类型，以适应C4光合作用。叶绿体含有类囊体膜和包膜，其中包含光合机制和转运蛋白，也包含参与例如蛋白质稳态的蛋白质。这些叶绿体膜必须在每种细胞类型中特化，以适应C4光合作用并调节代谢通量和活性。这项定量研究使用创新的基于凝胶和基于质谱的蛋白质定量方法，确定了BS和M叶绿体类囊体膜和包膜蛋白质组的分化状态及其寡聚状态。这包括天然凝胶、iTRAQ以及使用LTQ-Orbitrap的无标记定量。光系统I和II、细胞色素b6f以及ATP合酶复合物的亚基平均BS/M积累比分别为1.6、0.45、1.0和1.33，而光捕获复合物I和II家族的比例分别为1.72和0.68。观察到一种1000 kDa的BS特异性NAD(P)H脱氢酶复合物以及含有超过15种蛋白质的功能未知的相关蛋白质；我们推测，当核酮糖-1,5-二磷酸羧化酶/加氧酶的羧化速率低于苹果酸酶的脱羧速率时这个新复合物可能在无机碳浓缩中起作用。观察到类囊体蛋白酶（Egy和DegP）、状态转换激酶（STN7、8）以及光系统I和II组装因子的差异积累表明细胞特异性光合电子传递依赖于翻译后调控机制。确定了内膜转运蛋白磷酸烯醇丙酮酸/磷酸转运体、Dit1、Dit2和Mex1的BS/M比率，其反映碳代谢中的代谢通量。数百种其他蛋白质表现出不同的BS/M积累。质谱信息和功能注释可通过植物蛋白质组数据库获得。这些数据与先前的数据整合，形成了一个C4光合作用模型，从而为C4途径的代谢工程和协调C4分化遗传网络的靶向分析提供了新的理论依据。
