文献来源：A comprehensive analysis of chromoplast differentiation reveals complex protein changes associated with plastoglobule biogenesis and remodelling of protein systems in orange flesh. Plant Physiol. 2015 Jun 8. pii: pp. 00645. 2015. [Epub ahead of print]
文章摘要：
球形晶体状的成色素母细胞在柑橘（(Citrus sinensis Osbeck)）果肉中的分布具有明显的区域特异性，并且在果肉成熟过程中由造粉体转化而来。而造粉体与某些特殊的类胡萝卜素组成以及相关基因的表达有关。通过对分化以及衰老期间的成色素母细胞采用iTRAQ技术进行定量蛋白质组学的检测一共检测到1386个蛋白质，其中1016个蛋白质具有定量信息。与此同时，其中三个蛋白质的表达量还通过免疫印迹的方法得到了验证。
iTRAQ结果显示成色素母细胞的生成主要与三大类蛋白质的表达相关：
（1）与核糖体组装相关而参与转录的蛋白质丰度显著降低；
（2）与萜类化合物（含类胡萝卜素）合成、胁迫应答（含氧化还原，抗坏血酸以及谷胱甘肽等途径）以及发育相关的蛋白质丰度显著上调；
（3）信号转导相关以及DNA&RNA 相关的蛋白质则丰度比较恒定。
与此同时，另外一个非常有趣的发现是当有色体中的质体小球形成时有一些定位在质体小球中的蛋白质也随之丰度显著增加。
蛋白质组学数据还显示蛋白质转运的稳定、核糖体组装的抑制以及成色素母细胞蛋白酶的累积都与造粉体向成色素母细胞转化相关，因此推测这些过程在色素母细胞的形成和分化中共同发挥作用。
与此相反，成色素母细胞的衰老过程则可能与压力应激以及能量供给密切相关。总体来说，此次蛋白质组学分析结果新发现了很多定位在质体中的蛋白质，也揭示了柑橘果肉中成色素母细胞形成、分化以及衰老背后的可能的分子机制。
实验过程：
Figure 7. Schematic of experimental design for iTRAQ proteomic analysis. Plastids were purified from 40 fruits at each ripening stage for each independent replicate (for details, see ‘Materials and Methods’). A total of three biological replicates plus two technological replicates at MG and Br stages (MG 3+1, Br 3+1, Ma 3, and OMa 3) were performed. A total of 1,905 unique proteins were identified (see Supplemental Table S3A), of which 1,386 proteins were predicted to be plastid-localized, and 1,016 of which were further annotated with iTRAQ values as shown in Supplemental Table S3B.
实验结果：
成色素母细胞具有极强的合成和储存类胡萝卜素的能力，能使植物呈现鲜艳的橙色、红色、黄色等色彩。成色素母细胞通常由发育成熟的有色体或者其他一些与光合作用无关的质体演变而来，具有晶体状、球状、管状、膜状等多种形态。到目前为止，成色素母细胞的发育机制仍然十分神秘。该实验以发生发育过程中不同阶段的成色素母细胞及其前体为研究对象，采用iTRAQ技术对这些对象进行了蛋白质组学的定性定量分析。此次分析一共鉴定到一千多个蛋白质，其中将近两百个定位在质体中。并通过Western blot对其中三个定位在质体中的蛋白质进行了表达量的验证。
Figure 8. Comparison of protein abundance determined by proteomic analysis and immunoblotting. The abundance of proteins determined by the iTRAQ analysis is expressed as the value of individual/internal standard (IS). 30 μg of total proteins extracted from purified plastids at the indicated stages was separated using SDS-PAGE, blotted and subjected to immunoassays using specific antibodies that recognize the following three proteins: TIC40, a component of the translocon complexes at the inner envelope membrane; FBN1, Fibrillin 1 targeted to the plastoglobules in plastids; RbcL, the large subunit of Rubisco targeted to the plastid stroma.
经过对此次iTRAQ结果进行生物信息学分析发现鉴定到的蛋白质在不同的成色素母细胞发生和发育阶段中呈现七种不同的分布模式。经过进一步的总结发现：（1）与核糖体组装相关而参与转录的蛋白质丰度显著降低；（2）与萜类化合物（含类胡萝卜素）合成相关，胁迫应答（含氧化还原，抗坏血酸以及谷胱甘肽等途径）以及发育相关的蛋白质丰度显著上调；（3）信号转导相关以及DNA&RNA 相关的蛋白质则丰度比较恒定。
Figure 9. Abundance model of plastid-localized proteins during chromoplast differentiation. A, Abundance model of plastid-localized proteins being classified into seven categories during fruit ripening (MG-Br-Ma). These seven categories are stable, early increase, late increase, continuous increase, early decrease, late decrease, and continuous decrease. Excluding the ‘not assigned’ class and 91 proteins that lack a consistent and logical pattern of abundance, we established an abundance model for 828 proteins. B, Pattern of protein abundance summarized according to functional classes. Note that the number (right-hand side) and percentage (bottom) of proteins in the coloured-coded MapMan functional classes were shown in (A).
进一步的生信分析发现在成色素母细胞衰老过程中表达量发生变化的蛋白质还与电子转运/ATP合成、压力应激、氧化还原、激素代谢、CHO代谢等相关。
Figure 10. A heatmap showing the proportion of proteins of each functional class in each of the three abundance (stable, increase, and decrease) patterns during chromoplast differentiation. The magnitude of the percentage is represented by a color scale (top left) going from low (white) to high (red).
此次研究揭示了成色素母细胞发生发育过程中的蛋白质组变化，揭示了成色素母细胞发生发育可能的分子机制，也为类似的研究提供了实验思路和数据积累。
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