Gravitropic setpoint angle control in higher plants

植物的整体形状，即它们在地上和地下所占的空间，主要由它们的枝条的数量、长度和角度决定。有趣的是，许多侧枝生长的角度是相对于重力而不是主根轴来设定和保持的。这些角度被称为重力设定点角度或GSA。尽管GSA是整个自然界中观察到的植物结构奇妙变化的一个基本组成部分，但直到最近，GSA调控的机制还不清楚。在主要的初级根冠轴线上维持垂直的GSA，也称为向重力性，是很好地理解的：主茎或根的移位是在根和枝条中专门的重力感应细胞内感知的。这导致一种名为生长素的植物激素从这些细胞活跃地运输到移位的器官的下侧。在根中，生长素抑制细胞的伸长，导致向下弯曲到垂直方向，而在芽中，生长素则相反，促进向上的曲率。在我们以前关于GSA控制的工作中，我们表明在非垂直的根和地上部枝中发生了相同的潜在的重力反应，但被另一个生长成分，反重力偏移所抵消。我们发现，垂直程度较低的分支有更强的反重力偏移量，这种偏移量的大小也直接在重力感应细胞内调节。我们还表明，与向重力性一样，反向重力性依赖于生长素的运输，但在这种情况下，来自重力感应细胞的上部，而不是下部。反重力活动背后的分子机制以及这种活动如何被限制在非垂直分支上尚不清楚，这是本研究的主题。该项目包括三个部分。首先，我们专注于生长素转运蛋白的调控，称为PIN蛋白，它负责将生长素移出细胞。在重力敏感细胞中，两个特殊的PIN3和PIN7似乎同时调节向重力和向反重力的生长素流量。PIN的亚细胞定位和活性已被证明受其磷酸化的调节。我们的初步工作表明，突变的PIN3的侧根较少，而不能磷酸化的突变的PIN3有更多的垂直侧根。对这些数据最简单的解释是，重力敏感细胞中未磷酸化的PIN有助于向下的重力生长素通量，而磷酸化的PIN介导向上的反重力通量。重要的是，虽然如果植物被重新定位，重力感应细胞外的重力通量的方向可以非常迅速地重新设置，但我们的数据表明，驱动反重力活动的生长素通量方向的变化需要更长的时间才能重新建立。因此，通过跟踪经过简单重新定位的侧根重力敏感细胞内的分子和细胞生物学事件，我们可以观察到哪些PIN和PIN磷调节因子与曾经运输生长素以维持向重力或向反重力生长的细胞表面相关联，然后观察到向重力和向反重力生长素通量的新极性重新建立。向重力性的一个中心特征是，重力反应的幅度越大，器官离垂直越远。这一现象对GSA的控制至关重要，因此在项目的第二部分，我们使用先进的显微镜来了解这种反应的生物物理和分子基础。在项目的最后部分，我们将筛选一组突变的拟南芥植物，以寻找侧枝很少或没有非垂直生长的植物。这将使我们能够识别将反重力活动限制在侧枝上的基因，并允许我们对植物中GSA的控制形成一致的理解。

The overall shape of plants, the space they occupy above and below ground, is determined principally by the number, length, and angle of their branches. Interestingly, the angles at which many lateral branches grow out are set and maintained relative to gravity rather than the main root-shoot axis. These angles are known as a gravitropic setpoint angles or GSAs. Despite being a fundamental component of the wonderful variation in plant architecture observed throughout nature until recently the mechanisms underlying GSA regulation were not known. The maintenance of vertical GSAs in the main primary root-shoot axis, otherwise known as gravitropism, is well understood: displacement of the main stem or root is perceived within specialised gravity-sensing cells in roots and shoots. This leads to the active transport of a plant hormone called auxin from these cells and to the lower side of the displaced organ. In roots, auxin inhibits cell elongation, causing bending downwards to the vertical while in the shoot, auxin does the opposite, promoting upward curvature. In our previous work on GSA control we showed that the same underlying gravitropic response occurs in non-vertical root and shoot branches but is counteracted by another growth component, the antigravitropic offset. We showed that branches that are less vertical have a stronger antigravitropic offset and that the magnitude of this offset is also regulated directly within the gravity-sensing cell. We have also shown that like gravitropism, antigravitropism depends on auxin transport but in this case from the upper, rather than the lower side of the gravity-sensing cell. The molecular mechanisms underlying antigravitropic activity and how that activity is restricted to non-vertical branches is not known and is the subject of this study.The project has three parts. In the first, we focus on the regulation auxin transporters called PIN proteins that are responsible for moving auxin out of cells. In gravity-sensing cells, two particular PINs, PIN3 and PIN7, appear to mediate both gravitropic and antigravitropic auxin fluxes. The subcellular localisation and activity of PINs has been shown to be regulated by their phosphorylation. Our preliminary work has shown that a mutated phosphomimic version of PIN3 has less vertical lateral roots while a mutated PIN3 that cannot be phosphorylated has more vertical lateral roots. The simplest interpretation of these data is that unphosphorylated PINs in gravity-sensing cells contribute to downward, gravitropic auxin flux while phosphorylated PIN mediates an upward, antigravitropic flux. Importantly, while the direction of the gravitropic flux out of the gravity-sensing cells can be reset very rapidly if the plant is reorientated, our data indicate that changes in the direction of auxin fluxes that drive antigravitropic activity take much longer to be re-established. Therefore, by tracking the molecular and cell biological events within gravity-sensing cells of lateral roots undergoing simple reorientations we can observe which PINs and PIN phosphoregulators are associated with the faces of the cell that were once transporting auxin to sustain gravitropic or antigravitropic growth and then watch as the new polarities for gravi- and antigravitropic auxin fluxes are re-established.A central feature of gravitropism is that the magnitude of graviresponse increases the further an organ is moved away from the vertical. This phenomenon is crucial for GSA control and so in the second part of the project we use advanced microscopy to understand the biophysical and molecular basis of this response. In the final part of the project we will screen a collection of mutated Arabidopsis plants to look for plants with little or no non-vertical growth in lateral branches. This will allow us to identify the genes that restrict antigravitropic activity to lateral branches and allow us to form a coherent understanding of GSA control in plants.