Coordination of meiotic recombination and prophase I progression in plants: the role of retinoblastoma (RBR)

据预测，由于人口增长、工业化和气候变化，全球粮食需求到2050年将翻一番（《收获利益：皇家学会评论》，2009年）。为了迎接这一挑战，有必要开发新的作物品种，以各种方式加以改进，例如增加营养价值和产量以及对生物和非生物胁迫的耐受性。虽然转基因也有其作用，但新品种的开发仍将高度依赖于传统育种方法的方法，传统育种方法依赖于减数分裂重组，通过形成基因交叉产生变异，从而产生新的基因组合。了解控制减数分裂重组的因素对于改进作物育种具有重要意义，因为现在很清楚，许多物种特别是谷物，在其染色体上具有很少重组的大区域。这对新的遗传性状的渐渗提出了显著的障碍。因此，为了克服这个问题，我们需要知道如何控制CO的频率和分布。此外，估计有50%的植物是多倍体，这创造了一个额外的减数分裂调节水平，我们需要了解。研究表明，控制CO的形成是依赖于蛋白质之间的相互作用，催化重组和那些在减数分裂前期I调节染色体的广泛重塑。人们对这些进程如何协调仍知之甚少。最近，我们在任何生物体中获得了第一个证据，即视网膜母细胞瘤蛋白Rb（植物中的RBR）在减数分裂重组的控制中起着至关重要的作用。Rb在有丝分裂细胞周期调控中的作用以及作为肿瘤抑制因子的作用已被广泛研究。然而，通常很难研究其在成体生物体发育中的作用，因为Rb的缺失导致胚胎发生期间的致死性。使用一种特定的rbr突变体，我们已经能够克服这个问题。我们的研究揭示了一个重要的协调作用，RBR在减数分裂中通过直接相互作用的减数分裂染色体的重组位点。我们现在建议调查RBR如何发挥这种协调作用。特别是，我们的目标是建立RBR如何与细胞周期机制的组成部分，以确保染色体重塑发生在一个及时的方式与减数分裂重组和调查，如果它的功能作为“传感器”连接减数分裂与温度的变化。虽然研究将主要在模式植物拟南芥中进行，因为这是最容易实验的系统，但这些研究将通过在作物物种中的额外工作来补充。我们的实验策略将基于分子细胞遗传学，使用识别关键减数分裂蛋白的抗体结合高分辨率光学显微镜来研究野生型植物和一系列减数分裂突变体（包括缺乏RBR的品系）中的减数分裂前期I。RBR，减数分裂蛋白质和细胞周期组分之间的相互作用将使用质谱法进行研究，以分析使用抗RBR抗体从性母细胞中沉淀的蛋白质复合物。酵母双杂交分析将用作替代策略，并确认推定的相互作用。分析的另一个方面是研究RBR在多倍体生物中协调染色体配对和重组的作用。具体而言，我们将调查RBR和小麦中的Ph1位点的功能，这是重要的染色体配对在这个六倍体物种的控制之间的关系。我们预计这些研究将提供重要的新的见解减数分裂过程中重组的控制，将有利于植物育种提供的方法，使CO的频率和分布的变化。

It is predicted that as a result of population increase, industrialization and climate change global demand for food will double by 2050 (Reaping the Benefit: Royal Society Review 2009). To meet this challenge it will be necessary to develop new crop varieties that are improved in various ways, for example, increased nutritional value and yield and tolerance to biotic and abiotic stresses. Although GM has its part to play, the development of new varieties will remain highly dependent on methodologies derived from traditional breeding methods which are reliant on meiotic recombination to generate variation through the formation of genetic crossovers (COs) which results in new combinations of genes. Understanding the factors that control meiotic recombination is of great significance for the improvement of crop-breeding since it is now clear that many species notably cereals, possess large regions on their chromosomes that rarely recombine. This presents a significant barrier for the introgression of new genetic traits. Hence, to overcome this problem we need to know how the frequency and distribution of COs are controlled. In addition an estimated 50% of plants are polyploid, which creates an additional level of meiotic regulation that we need to understand. Studies indicate that the controlled formation of COs is dependent on the interplay between the proteins that catalyse recombination and those that regulate the extensive remodelling of chromosomes during prophase I of meiosis. How these processes are coordinated remains poorly understood. Recently, we obtained the first evidence in any organism, that the retinoblastoma protein Rb (RBR in plants) plays an essential role in the control of meiotic recombination. The function of Rb in mitotic cell-cycle control and as a tumour-suppressor has been extensively studied. However, it is generally difficult to study its role in development in adult organisms as loss of Rb results in lethality during embryogenesis. Using a specific rbr mutant we have been able to overcome this problem. Our studies reveal an important coordinating role for RBR in meiosis through a direct interaction with the meiotic chromosomes at the sites of recombination. We now propose to investigate how RBR exerts this coordinating role. In particular we aim to establish how RBR links with components of the cell-cycle machinery to ensure that chromosome remodelling occurs in a timely fashion in relation to meiotic recombination and investigate if it functions as "sensor" to link meiosis with changes in temperature. Although studies will primarily be conducted in the model plant Arabidopsis, as this is the most experimentally tractable system, these will be complimented by additional work in crop species. Our experimental strategy will be based around molecular cytogenetics using antibodies that recognize key meiotic proteins combined with high resolution light microscopy to study meiotic prophase I in wild-type plants and a range of meiotic mutants, including a line lacking RBR. Interactions between RBR, meiotic proteins and cell-cycle components will be studied using mass-spectrometry to analysis protein complexes that have been precipitated from meiocytes using an anti-RBR antibody. Yeast two-hybrid analysis will be used as an alternative strategy and to confirm putative interactions. A further aspect of the analysis will be to investigate the role of RBR in coordinating chromosome pairing and recombination in polyploid organisms. Specifically, we will investigate the relationship between RBR and the function of the Ph1 locus in wheat which is important for the control of chromosome pairing in this hexaploid species. We anticipate these studies will provide important new insights into the control of recombination during meiosis that will be of benefit for plant breeding by providing approaches to enable changes in CO frequency and distribution.