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

植物在一生中形成器官，生长和发育，有些树的寿命可能超过一千年。为此，它们在分生组织中维持多能体细胞干细胞，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不太活跃的组织中心，称为静止中心（QC），它们一起形成干细胞生态位。静止中心是由两个基因组的组合作用决定的。这些过多的基因转录是为了响应植物激素和生长素的积累，并且是在根中定位QC所必需的，而以突变体命名的基因，SCARECROW （SCR）和SHORT-ROOT （SHR）的独立发育途径，为QC建立了径向位置。虽然我们开始了解分生组织的模式，但我们不知道在QC区域周围保持细胞未分化的机制，以及当细胞离开分生组织区时是什么建立了分化场。生长素长期以来一直被认为是培养细胞分裂所必需的，并参与广泛的发育过程，包括依赖于细胞分裂和分化之间平衡调节的器官的维持和启动。通道被精心定位和调节，以允许生长素进出细胞，并确定生长素在细胞间流动的方向性，从而形成生长素梯度的高动态场。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细胞分裂，但确切的机制尚不清楚。然而，我们知道，不同的生长素浓度是由一种叫做TIR1的蛋白质感知的，它能够在大量的基因上操作复杂的开关，这些基因依赖于生长素调节的蛋白质降解来提升抑制分子。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节因子，一个是E2Fb，它促进细胞增殖并与细胞生长协调；而另一种叫做E2Fc，阻止细胞分裂，并促进细胞获得特定的功能，称为细胞分化。我们惊讶地发现，这些蛋白的丰度受到生长素的相反调节，E2Fb稳定，而E2Fc不稳定。我们提出了一个工作假设，即生长素浓度被转化为这些正、负细胞周期调节因子的相反浓度梯度，并构成分裂或延长和分化决定之间的切换。E2Fs是由一种叫做视网膜母细胞瘤（RB）的口袋蛋白控制的，因为在动物身上发现它会导致眼睛里不受控制的肿瘤生长。最近发现，在拟南芥中，RB相关蛋白对维持根内干细胞生态位对发育调节因子稻草人的响应至关重要。因此，e2f为发育调节因子和生长素提供了一个交汇点，以确定干细胞的功能。我们建议通过观察E2F基因与RB相关基因结合的突变体和生长素产生或运输调节体的突变体的表型，从遗传学上验证这一模型。我们还计划可视化与生长素梯度相关的E2F蛋白分布。由于E2Fs通过控制大量基因而起作用，因此确定E2Fs结合的基因并确定这些基因如何被调节是至关重要的。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.