Gravitropic setpoint angle control in higher plants

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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.