Proteome Analysis of Plastids from Developing Seeds of <i>Jatropha curcas</i> L.

In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level.本研究针对处于蛋白质与脂质储备初始沉积阶段的发育中麻疯树（Jatropha curcas）种子胚乳分离得到的质体，开展了蛋白质组学分析。从质体中提取的蛋白质经胰蛋白酶消化后，将所得肽段接入与电喷雾电离（ESI）-LTQ-Orbitrap Velos质谱仪在线联用的EASY-nano液相色谱系统进行分析，最终鉴定得到1103种蛋白质，归属于804个蛋白质群组；其中923种蛋白质被认定为可靠鉴定结果，这极大扩充了当前已报道的麻疯树鉴定蛋白质集合。在所有鉴定得到的蛋白质中，仅有5种由质体基因组编码，且无一参与光合作用过程，这证实了所分离的质体不具备光合活性。923种可靠鉴定蛋白质中，有824种的同源蛋白存在于PPDB、SUBA或PlProt数据库中；剩余13种蛋白质未在上述三个质体蛋白质数据库中检索到同源物，但被至少一种所用的预测程序标注为质体定位蛋白。功能分类结果显示，参与氨基酸代谢的蛋白质为最主要的功能类别，其次依次为碳水化合物代谢、能量代谢与脂质代谢。本研究鉴定得到了核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）的大小两个亚基，该酶在该质体中的存在被认为是一种适应性特征，可抵消糖酵解途径中将碳水化合物转化为油脂时，三分之一碳以CO₂形式流失所带来的影响。尽管鉴定得到了多种参与二萜类前体生物合成的酶类，但未检测到任何萜烯合酶/环化酶，这提示从发育中种子胚乳分离得到的质体无法合成佛波酯（phorbol esters）。综上，本研究在全蛋白质组层面上，为解析发育中种子胚乳质体的主要生物合成通路与部分独特特征提供了新的认知。