The role of the E2F transcription factors in regulating stem cell functions during Arabidopsis root development

植物在其一生中形成器官、生长和发育，就某些树木而言，其寿命可以超过一千年。为此，它们在分生组织中维持多能体细胞，分生组织是有丝分裂活跃细胞的局部池。在根中，这些干细胞围绕着有丝分裂活性较低的组织中心，称为静止中心(QC)，它们共同形成一个干细胞利基。静止中心是由两组基因的组合作用确定的。过量基因的转录响应于植物激素、生长素的积累，并需要在根中定位QC的基础位置，而以突变体命名的稻草人(SCR)和短根(SHR)基因的一个独立的发育途径为QC建立了径向位置。虽然我们开始了解分生组织中的模式，但我们不知道QC区域周围保持细胞未分化的机制，也不知道是什么建立了细胞离开分生组织区域时的分化领域。长期以来，生长素一直被认为是细胞分裂所必需的，并参与广泛的发育过程，包括器官的维持和启动，这依赖于细胞分裂和分化之间的平衡调节。通道被仔细定位和调节，以允许生长素进出细胞，并确定生长素在细胞之间流动的方向性，从而形成高度动态的生长素梯度场。这个想法是，细胞能够区分这些不同的生长素浓度，并对它们做出不同的反应，例如通过伸长生长或通过细胞分裂，但确切的机制尚不清楚。然而，我们知道，一种名为TIR1的蛋白质感受到不同的生长素浓度，它能够在大量基因上操作复杂的开关，这些基因依赖于通过生长素调节的蛋白质降解来提升阻遏分子。最近我们发现了两个在细胞分裂中具有拮抗作用的转录调控因子，一个称为E2Fb，促进细胞增殖并协调其与细胞生长；另一个称为E2Fc，它阻止细胞分裂，促进细胞达到特定的功能，称为细胞分化。我们惊讶地发现，这些蛋白质的丰度相反地受到生长素的调节，E2Fb稳定，而E2Fc不稳定。我们提出了一个工作假说，即生长素浓度被转化为这些正向和负向细胞周期调节因子的相反浓度梯度，并构成了分裂或伸长和分化决定之间的切换。E2F由一种名为视网膜母细胞瘤(RB)的口袋蛋白控制，因为它是在动物身上发现的，会导致眼睛中肿瘤的不受控制的生长。最近发现，在拟南芥中，一种与Rb相关的蛋白对于维持根中的干细胞生态位是必不可少的，以响应稻草人的发育调节。因此，E2F为发育调节因子和生长素提供了一个确定干细胞功能的交汇点。我们建议通过观察E2F基因突变与Rb相关基因的表型以及生长素生产或运输调节突变的表型来从遗传学上检验这一模型。我们还计划可视化与生长素梯度相关的E2F蛋白分布。因为E2F通过控制大量的基因来运作，所以我们识别E2F结合的基因并确定这些基因是如何调控的是至关重要的。我们的工作应该揭示生长素如何调节细胞分裂，从而调节植物生长。

Plants form organs, grow and develop throughout their lifetime, which can be over a thousand years in the case of some trees. For that purpose they maintain pluripotent somatic stem cells within the meristems, local pools of mitotically active cells. In roots these stem cells surround the mitotically less active organising centre, called the quiescent centre (QC) and together they form a stem cell niche. The quiescent centre is specified by combinatorial action of two gene sets. The PLETHORA genes are transcribed in response to the phytohormone, auxin accumulation and are required to position QC basal in roots while an independent developmental pathway of the genes named after the mutants, SCARECROW (SCR) and SHORT-ROOT (SHR), sets up the radial position for QC. Although we are beginning to understand the patterning in meristems, we do not know the mechanisms that keep cells undifferentiated around the region of QC and what sets up the field of differentiation as cells leave the meristematic zone. Auxin has long been known to be essential for cell division in culture and to be involved in a wide range of developmental processes including the maintenance and initiation of organs that depend on the regulation of the balance between cell division and differentiation. Channels are carefully positioned and regulated to allow auxin to get in and out from cells and to determine the directionality of auxin flow from cell to cell that leads to the formation of highly dynamic fields of auxin gradients. The idea is, that cells are able to distinguish these different auxin concentrations and respond to them differently, for instance by growth through elongation or by cell division, but the exact mechanism is not known. We know however, that different auxin concentrations are sensed by a protein known as TIR1, which is able to operate sophisticated switches on large sets of genes that rely on lifting repressor molecules by auxin-regulated protein degradation. Recently we have discovered two transcriptional regulators in cell division with antagonistic roles, one called E2Fb promotes cell proliferation and co-ordinates it with cell growth; while the other, called E2Fc, blocks cell division, and promote cells to attain specific functions, termed cell differentiation. We were surprised to find that the abundance of these proteins are oppositely regulated by auxin, E2Fb being stabilised while E2Fc is destabilised. We formulated a working hypothesis that auxin concentrations are converted into opposing concentration gradients of these positive and negative cell cycle regulators and constitute the switch between decisions to divide or elongate and differentiate. E2Fs are kept under control by the pocket protein called retinoblastoma (RB), because it was discovered in animals to cause uncontrolled tumour growth in the eye. It was recently discovered that in Arabidopsis an RB related protein is essential to maintain the stem cell niche in roots in response to the developmental regulator SCARECROW. Thus, E2Fs provide a converging point for developmental regulators and auxin to determine stem cell functions. We propose to test this model genetically by observing the phenotypes of mutants in E2F genes in combination with the RB related gene and mutants in regulators of auxin production or transport. We also plan to visualise the E2F protein distribution in relation to auxin gradients. Because E2Fs operate by controlling large number of genes, it is vital that we identify the genes E2Fs bind to and determine how these genes are regulated. Our work should uncover how auxin regulates cell division, and thus plant growth.