RGIs-mediated root apical meristem development is essential for root hydrotropic response in Arabidopsis thaliana

Hydrotropism refers to the tendency of roots which prefer to grow towards a region with more water availability. We previously detected higher responses of cytokinins on the drier sides of Arabidopsis root tips, which lead to more cell division activity there and the growth of roots towards higher water potential areas. To further verify the significance of cell division activity in controlling root hydrotropism, we analyzed the hydrotropic responses of a number of well-characterized short root mutants with reduced root apical meristem activities. The mutants used were from a RGF1-RGIs signaling pathway, including a quadruple and a quintuple mutant for the receptors of an RGF1 peptide hormone, rgi1234 and rgi12345, a tyrosyl protein sulfotransferase mutant tpst, and a double mutant for two transcription factors, plt1 plt2. All these mutants showed a greatly reduced or even undetectable hydrotropic response. The expression levels of TCSn::GFP, a reporter system for monitoring the response of cytokinins, were drastically down-regulated in the root tips of rgi1234, rgi12345, tpst, and plt1 plt2. The introduction of pRGI2::MKK4^DD, a construct encoding a constitutively active MKK4, can significantly rescue not only the short root phenotype but also the defective responses of cytokinins and hydrotropism in rgi12345. Interestingly, the hydrotropic response of tie1 tie2, a short root double mutant with normal cell division activity in the root apical meristem but defects in the elongation region, is relatively normal. Our results substantiate the key roles of the RGF1-RGIs signaling pathway and cell division activity in determining root hydrotropism. These analyses also reveal the crosstalk between the RGF1-RGIs signaling pathway and the responses of cytokinins, which are worth being further investigated in the future.