Comparative Proteomics Reveals a Role for Seed Storage Protein AmA1 in Cellular Growth, Development, and Nutrient Accumulation

Seed storage proteins are known to be utilized as carbon and nitrogen source for growing seedlings and thus are considered as potential candidates for nutritional improvement. However, their precise function remains unknown. We have earlier shown that ectopic expression of a seed storage protein, AmA1, leads to increase in protein besides high tuber yield in potato. To elucidate the AmA1-regulated molecular mechanism affecting increased protein synthesis, reserve accumulation, and enhanced growth, a comparative proteomics approach has been applied to tuber life-cycle between wild-type and AmA1 potato. The differential display of proteomes revealed 150 AmA1-responsive protein spots (ARPs) that change their intensities more than 2.5-fold. The LC–ESI-MS/MS analyses led to the identification of 80 ARPs presumably associated with cell differentiation, regulating diverse functions, viz., protein biogenesis and storage, bioenergy and metabolism, and cell signaling. Metabolome study indicated up-regulation of amino acids paralleling the proteomics analysis. To validate this, we focused our attention on anatomical study that showed differences in cell size in the cortex, premedullary zone and pith of the tuber, coinciding with AmA1 expression and localization. Further, we interrogated the proteome data using one-way analysis of variance, cluster, and partial correlation analysis that identified two significant protein modules and six small correlation groups centered around isoforms of cysteine protease inhibitor, actin, heat shock cognate protein 83 and 14-3-3, pointing toward AmA1-regulated overlapping processes of protein enhancement and cell growth perhaps through a common mechanism of function. A model network was constructed using the protein data sets, which aim to show how target proteins might work in coordinated fashion and attribute to increased protein synthesis and storage reserve accumulation in AmA1 tubers on one hand and organ development on the other.种子贮藏蛋白（seed storage proteins）可作为幼苗生长的碳源与氮源，因此被视为作物营养改良的潜在候选靶点。但其确切的生物学功能尚未明确。本团队此前的研究表明，在马铃薯中异位表达种子贮藏蛋白AmA1，不仅可提升块茎产量，还能增加块茎的蛋白质含量。为阐明AmA1调控的、影响蛋白质合成增强、贮藏物质积累与生长提速的分子机制，本研究针对野生型与转AmA1马铃薯的块茎生命周期开展了比较蛋白质组学分析。蛋白质组差异显示分析共鉴定出150个受AmA1调控的蛋白斑点（AmA1-responsive protein spots, ARPs），其表达量变化幅度超过2.5倍。通过液相色谱-电喷雾电离串联质谱（LC–ESI-MS/MS）分析，共鉴定出80个ARPs。这些蛋白大概率参与细胞分化过程，并调控多种生物学功能，包括蛋白质生物发生与贮藏、生物能量代谢与物质代谢，以及细胞信号转导。代谢组学研究显示，氨基酸的上调表达趋势与蛋白质组学分析结果相一致。为验证该结论，我们开展了解剖学研究，结果显示块茎皮层、髓前区与髓部的细胞大小存在显著差异，且该差异与AmA1的表达及定位模式相吻合。进一步地，我们通过单因素方差分析、聚类分析与偏相关分析对蛋白质组数据进行深度挖掘，鉴定出两个显著的蛋白模块，以及六个以半胱氨酸蛋白酶抑制剂、肌动蛋白、热休克同源蛋白83（heat shock cognate protein 83）和14-3-3蛋白亚型为核心的小型相关集群。这一结果提示，AmA1可能通过共同的功能机制，协同调控蛋白质积累与细胞生长这两个重叠的生物学过程。我们基于蛋白质组数据集构建了调控网络模型，该模型旨在阐明目标蛋白如何协同发挥作用：一方面提升AmA1转基因块茎的蛋白质合成与贮藏物质积累水平，另一方面调控器官发育。