Rop9GTPase Mediates Auxin-regulated Lateral Root Development In Arabidopsis

Lateral root (LR) formation is very important in a plant’s development and adaptation to theenvironment. The auxin indole-3-acetic acid is the major plant hormone regulating LR development. Inthis study, we show that ROP9, a member of the Arabidopsis Rop subfamily of Rho GTPase, expressinghighly in the LR promordia. A null rop9mutant exhibits a decreased number of LRs. In addition, theexpression of Rop9in LR and root tip is upregulated by Auxin. We also show that rop9rop10doublemutants produce more significant phenotype than single mutant due to rop proteins functionlyredundancy. Futhermore, transgenic expression of a constitutively active (CA) form of ROP9activatesauxin-responsive gene expression, whereas dominant-negative (DN) ROP9supresses auxin-inducedgene expression. Together, our results suggest that ROP9regulates LR development by mediating auxinin Arabidopsis. Research results were as follows:1. Through the analysis of ROP9pro::GUS transgenic plants, we found GUS activity mainly restricted tothe meristematic and LR cells of one-week seedings. To further determine which developmental stagesin which ROP9expression was predominantly detected in the LR development. We focused on thedetail of LRs formation and strong GUS activity was detected at the stage of LRs initiation, two celllayers, more cell layers of LR primordium (LRP), immediately after emerged LRP. A weak GUS signalwas observed in the emerged LRs, like the root tip staining.2. We used transgenic plants expressing ROP9:GUS construct and determined the effect of auxin on theROP9expression level by detecting histologically the activity of GUS reporter gene with the applicationof oxgenous auxin indole-3-acetic acid (IAA), a native auxin.we found treatment with10μM IAA for0.5,1,2,4,8hours dramatically increased the level of ROP9compared with that for0hour (control) inLRs and PRs.we also detected quantitatively the ROP9GUS activity by cutting and assaying the roots,which revealed a significant increase in comparison with the control. Thus, ROP9expression wasinduced by the auxin, the LR-promoting plant hormone.3. We isolated a T-DNA insertional mutant of ROP9, designated rop9-ko4. Because rop9-ko4does notshow any visible whole plant phenotype, including LRs, We firstly determined the optimal exogenousauxin1-naphthalene acetic acid (NAA), a synthetic and extremely stable auxin, concentration bycompared the lateral root induction rate from10nM to10μM NAA–containing Murashige and Skoog(MS) medium. We found application of1μM NAA caused～37%reduction in LR density in rop9-ko4compared with that in the wild-type ecotype Landsberg erecta (Ler) and the lower and higher NAAlevels exhibited similar effect on LR formation (data not shown). rop9-ko4plants were transformedwith a35S:ROP9cDNA construct. Seedings from three independent transgenic lines expressing theROP9transgene showed similar NAA promotion of LR density with Ler, which indicated that ROP9isinvolved in the regulated-auxin LR formation.4. We isolated and identified a homozygous Rop9Rop10mutant, rop9rop10-1, by crossing rop9-ko4androp10-1, a mutant enhancing ABA responses (in ws-2background). To subtract the background, we gotthe rop9rop10-2through backcross with rop10-1. The phenotype of reduced auxin effect on LR formation was particully apparent in the rop9rop10double mutants, causing respectively～60%and～70%reduction in LR density compared with～37%of rop9-ko4single mutant to Ler in the1μM NAA. More interestingly, few emerged LRs were observed in the roots of the double mutants afterapplication of5μM NAA in comparison with wild-type (Ler and Ws-2) and single mutants (rop9-ko4and rop10-1) showing the normal auxin-promoted LR formation. Additionally, we also tested the ABArepsonses of rop9rop10mutants and as expected, the double mutants show much stronger insenstitivityto0.8μM ABA in contrast to that of single mutants to wild-type, respectively (data notshown).Therefore, these findings suggested the possibility that ROP10and ROP9act as redundancy tomediate auxin-promoting LR formation.5. We next investigate the subcellular localization of ROP9protein in Arabidopsis cells by fusing it withGFP. The GFP-ROP9fusion protein was expressed under the35S promoter of Cauliflower mosaic virusin transgenic Arabidopsis plants. Subcellular localization of the fusion protein was visualized byconfocal imaging of green fluorescence in the root cells of transgenic seedings. GFP-ROP9waslocalized to the cell periphery and absent from the nucleus, suggesting that ROP9is localized to the PM,which is critical for its fuction.