Cell-type resolution proteomics for functional annotation of the grain filling process in wheat endosperm

Grain development is a key life cycle stage of many plants. The development of seeds is the basis of agriculture and the primary source of calories consumed by humans. Here, we employ laser micro dissection (LMD) combined with shotgun proteomics to generate a cell-type proteome atlas of developing wheat endosperm at the early and late grain filling stages. We identified 1803 proteins from four different cell layers (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE), and endosperm transfer cells (ETCs)) of developing endosperm at 15 Days after anthesis (DAA) and 26 DAA. Sixty-seven differentially expressed proteins in the aleurone, 31 in the sub-aleurone, 27 in the starchy endosperm, and 50 in the endosperm transfer cells were detected between these two-time points. The results revealed highly distinguishable proteome dynamics in the different cell layers of endosperm over the time course. We observed high general metabolic activity of the grain with regard to carbohydrate metabolism, defence against oxidative stress, and signalling in the different cell layers during the grain filling process. Cell-specific identification of SUT and GLUT transporters suggest a grain filling model via nucellar projections and endosperm transfer cells (ETCs) initiating starch biosynthesis in the starchy endosperm (SE). The identification and regulation dynamics of proteins in the different cell layers demonstrate a functional switch of the proteome from the early to the late grain filling stage. Based on these data, we proposed a model for sugar loading and starch biosynthesis in wheat developing endosperm, including an abundance switch of cell-type-specific key proteins.

籽粒发育是多数植物关键的生命周期阶段。种子发育是农业生产的核心根基，亦是人类膳食热量的首要来源。本研究采用激光显微切割（laser micro dissection, LMD）结合鸟枪法蛋白质组学（shotgun proteomics），构建了小麦发育胚乳在籽粒灌浆早期与晚期的细胞类型特异性蛋白质组图谱。我们从开花后15天（Days After Anthesis, 15 DAA）与26 DAA的发育胚乳的四类细胞层——糊粉层（aleurone, AL）、亚糊粉层（sub-aleurone, SA）、淀粉胚乳（starchy endosperm, SE）以及胚乳转运细胞（endosperm transfer cells, ETCs）——中共鉴定出1803种蛋白质。在两个时间点间，我们分别检测到糊粉层中67种差异表达蛋白、亚糊粉层中31种、淀粉胚乳中27种以及胚乳转运细胞中50种。研究结果表明，在灌浆进程中，小麦胚乳不同细胞层的蛋白质组动态变化特征具有显著差异。我们观察到，在籽粒灌浆过程中，不同细胞层均表现出活跃的碳水化合物代谢、抗氧化应激防御以及信号转导相关的整体代谢活性。针对蔗糖转运蛋白（SUT）与葡萄糖转运蛋白（GLUT）的细胞特异性鉴定结果，提示了一条通过珠心突起与胚乳转运细胞（ETCs）启动淀粉胚乳（SE）淀粉生物合成的籽粒灌浆模型。不同细胞层中蛋白质的鉴定结果及其表达调控动态，证明了蛋白质组从灌浆早期至晚期的功能转换。基于上述研究数据，我们提出了小麦发育胚乳中糖分装载与淀粉生物合成的模型，该模型涵盖了细胞类型特异性关键蛋白的丰度转换。