Elucidating the spatial and temporal control of granule initiation in wheat

本项目旨在开发淀粉颗粒如何在小麦籽粒发育中启动的完整机制理解。淀粉是我们主要谷类作物的主要热量成分。它是在胚乳的质体（淀粉体）中合成的，由葡萄糖聚合物、支链淀粉和直链淀粉组成的半结晶不溶颗粒。虽然对这些聚合物的合成已经相当了解，但我们才刚刚开始了解淀粉颗粒是如何在质体中形成的，以及决定颗粒形状和大小的因素。这些性状是淀粉功能的重要决定因素，从而影响作物品质。在不同的谷类作物中观察到不同的淀粉颗粒形状和大小，这至少部分是由谷物发育过程中颗粒形成模式的差异决定的。特别是小麦科（小麦、大麦、黑麦）的谷物在胚乳中产生两种不同的淀粉颗粒：大的a型颗粒和小的b型颗粒。这是由两个在空间和时间上分离的颗粒形成波引起的：a型颗粒在淀粉体的早期形成，b型颗粒在淀粉体基质的后期形成。我们最近对拟南芥叶片的研究发现了启动每个质体正确数量颗粒所需的保守“起始蛋白”。有趣的是，我们还发现这些蛋白质参与小麦颗粒形成的不同方面，其中一些是正确的a型颗粒形成所必需的，另一些是正确的b型颗粒形成的数量和时间所必需的。在颗粒发育过程中，这些单独的蛋白质如何在一个整体机制中协同作用以协调颗粒的形成尚不清楚。我们的目标是建立一个完整的机制模型的空间和时间控制的颗粒形成在小麦籽粒发育。在遗传方法中，我们将使用在单个起始蛋白中有缺陷的四倍体小麦突变体进行杂交。淀粉颗粒表型将在缺乏多个起始蛋白的突变体中进行分析，以揭示功能依赖性。在一种互补的生化方法中，我们将描述起始蛋白的丰度和相互作用如何通过谷物发育而变化。这将为发现和表征参与A型和b型颗粒起始的新成分提供重要机会。我们还将探索颗粒起始和淀粉体结构之间的联系，使用活细胞成像来可视化我们起始蛋白突变体中的淀粉体数量和结构。最后，我们将研究转录调控在颗粒起始时间控制中的重要性。初步数据表明，起始蛋白遵循不同的表达模式。我们将探讨起始蛋白的转录水平如何在谷物发育过程中发生变化，以及这如何反映在蛋白质丰度上。一个诱导启动子系统将被用来改变起始蛋白表达的时间，随后对颗粒起始时间的影响将被检查。我们还将尝试确定控制起始蛋白不同表达模式的候选转录因子。总的来说，我们的工作将揭示控制小麦颗粒形成独特时空模式的机制，大大提高我们对这一重要作物淀粉合成的认识。这一发现将有可能通过改变淀粉粒数和形态来改善小麦品质。

This project aims to develop a full mechanistic understanding of how starch granules are initiated in developing wheat grains. Starch is the main calorific component of our staple cereal crops. It is synthesised in plastids (amyloplasts) of the endosperm as semi-crystalline insoluble granules composed of the glucose polymers, amylopectin and amylose. While the synthesis of these polymers is relatively well understood, we are only beginning to understand how starch granule formation is initiated within plastids, and the factors determining granule shape and size. These traits are important determinants of starch functionality, and thus influence crop quality. Diverse starch granule shapes and sizes are observed across different cereal crops, and this is at least partially determined by differences in granule initiation patterns during grain development. In particular, grains of the Triticeae (wheat, barley, rye) produce two distinct populations of starch granules in the endosperm: large A-type granules, and small B-type granules. These arise from two spatially and temporally separated waves of granule initiation: A-type granules initiate early in grain development in amyloplasts, and B-type granules initiate in late grain development in amyloplast stromules. Our recent research in Arabidopsis leaves identified conserved "initiation proteins" required to initiate the correct number of granules per plastid. Interestingly, we also discovered that these proteins are involved in different aspects of granule initiation in wheat, with some required for correct A-type granule formation, and others for the correct number and timing of B-type granule initiation. How these individual proteins act together in an overall mechanism to orchestrate the initiation of granules during grain development is not understood.We aim to develop a full mechanistic model of the spatial and temporal control of granule initiation in developing wheat grains. In a genetic approach, we will perform crosses using our mutants of tetraploid wheat defective in individual initiation proteins. Starch granule phenotypes will be analysed in the mutants lacking multiple initiation proteins to reveal functional dependencies. In a complementary biochemical approach, we will profile how the abundance and interactions of initiation proteins change through grain development. This will provide an important opportunity to discover and characterise novel components involved in A- and B-type granule initiation. We will also explore the links between granule initiation and amyloplast structure, using live-cell imaging to visualise amyloplast number and structure in our initiation protein mutants. Finally, we will investigate the importance of transcriptional regulation in the temporal control of granule initiation. Preliminary data suggest that initiation proteins follow distinct expression patterns. We will explore how transcript levels of initiation proteins change through grain development, and how this is reflected in protein abundance. An inducible promoter system will be used to alter the timing of initiation protein expression, and subsequent effects on the timing of granule initiations will be examined. We will also attempt to identify candidate transcriptional factors that control the distinct expression patterns of initiation proteins. Overall, our work will reveal mechanisms governing the unique spatiotemporal pattern of granule initiation in wheat, greatly advancing our knowledge of starch synthesis in this important crop. The findings will potentially lead to novel approaches to improve wheat quality by modifying starch granule number and morphology.