| ROPs GTPases (Rho of plant, ROPs) are a group of plant-specific Rho-related small G proteins, which are regulated by their shuttling between the inactive GDP-bound form and the active GTP-bound form. They play critical roles in plant growth and development, and also participate in diverse cellular pathways while responding to a broad range of extracellular stimuli. Act as ROPs regulators RopGEFs stimulate the exchange of GDP to GTP for ROPs, in turn, to activate ROP proteins. Numerous studies demonstrate the importance of RopGEFs during plant development. However, the roles of RopGEFs which played in ABA signaling are poorly understood. Specifically, their regulatory mechanisms in the ABA-suppressed seed germination and ABA-induced guard cell movement are remained elusive. In this work, we characterized two Arabidopsis RopGEFs (AtRopGEF2 and AtSPIKE1), and determined their functions through analyzing the loss-of-function mutant ropgef2-ko and the defective microRNA lines such as amiR-SPK1. Results showed that RopGEF2 and SPIKE1 are involved in ABA-regulated seed germination and guard cell movement respectively, which revealed the important functions of RopGEF2 and SPIKE1 in ABA signaling. In the seed germination assay, our results showed that RopGEF2 may act as an important negative component in ABA-suppressed seed germination. And, ABA could regulate RopGEF2 via ubiquitin-26S proteasome mediated ubiquitination and degradation. When RopGEF2 is bound with ROPs, the ABA-mediated degradation process could be prevented. Notably, the localization of RopGEF2 is another interesting point, and RopGEF2 itself is mainly localized at mitochondria, when it is bound with its target ROPs, it is observed at the cell periphery, along with ROPs. In order to determine the mechanism of ABA-inhibited ROP activity during guard cells movement, we also characterized the role of SPK1, a unique Dock180 family RopGEF in Arabidopsis. We found that SPK1 was involved in ABA-induced dynamic reorganization of F-actin in guard cell. Our results also indicate that the calcium-dependent protein kinase CPK3 could phosphorylate SPK1 which is essential for the function of SPK1. Further analyses determined that, clearly, ABA could initiate downstream signaling through its receptor PYR1l-mediated releasing inhibitory effect of ABIl to a protein kinase, such as CPK3; in such, CPK3 could phosphorylate SPK1, in turn, to alter the ROP-signaling. The suppression of ROPs signaling ultimately affects the reorganization of F-actin and changes the ABA response of guard cell movement. The main results and significance in our work are described as followed:1. The loss-of-function mutant ropgef2-ko exhibits enhanced sensitivity to ABA during seed germination. Transgenic lines (Com-8 and Com-10) harboring RopGEF2 in ropge/2-ko background showed similar germination phenotype to that in WT. These results demonstrate that disruption of RopGEF2 function could induce ABA-hypersensitive response during seed germination. It suggests that RopGEF2 might serve as a negative regulator in the ABA-suppressed seed germination.2. In the quantitative RT-PCR analytic experiment, it showed that expression of RopGEF2 could be measured in most of tissues. Further analyzing RopGEF2pro-GUS expression pattern, results showed that RopGEF2 mainly expressed in developing embryos and germinating seeds, reflecting the functional preference of RopGEF2 for the seed germination.3. YFP-RopGEF2 fusion protein was detected near plasma membrane, in cytosol and at mitochondria. The PRONE2 domain is critical and necessary for the mitochondrial localization of RopGEF2. These results indicate the unique regulation mechanism of RopGEF2.4. Through Y2H, BiFC and pull-down assays, we proved that RopGEF2 interact with multiple ROPs (ROP2, ROP6 or ROP 10) whose involvement in the ABA signaling has been demonstrated. Further analyses suggest that the interaction between RopGEF2 and ROPs could not only affect localization of RopGEF2, but also altered its protein stability. Thus, the role of RopGEF2 is tightly linked to its targetting ROPs.5. ABA treatment does stimulate RopGEF2 protein degradation, via the ubiquitin-26S proteasome pathway. Interstingly, the ABA-induced degradation of RopGEF2 occurs mainly in the cytosol, suggesting that the localization of RopGEF2 is related with its stability.6. In order to determine the role of a RopGEF played in the ABA-induced guard cell movement, we generated artificial microRNA lines, amiR-SPK1 transgenic lines which are of reduced expression level of SPIKE1 transcript. We found that guard cells in amiR-SPK1 lines are of enhanced sensitivity to ABA. Results from water loss assay indicated that SPK1 could response to the ABA treatment in guard cells. CA-rop6 could rescue the hypersensitive stomatal closure in amiR-SPK1 which was caused by ABA treatment. These data suggest that SPIKE1 may mediate ABA signaling in guard cells via modulating ROP6 activity.7.In addition, the dynamics of actin microfilaments (MFs) in guard cells of amiR-SPK1 lines showed enhanced sensitivity to ABA as well. Less radial arrayed actin MFs was observed in most of guard cells of amiR-SPK1 plants upon treated by ABA.8. Through semi-in vivo pull-down assays and in vitro kinase assay, we demonstrated that CPK3 could interact with, and, phosphorylate SPK1. We also determined that the amino acid S408 in SPK1 protein is the main phosphorylation targeting site for CPK3.9. The results from pull-down assay showed that the affinity of SPK1 to ROP6 was reduced by ABA treatment. However, application of W-7 which is a calcium dependent protein kinase antagonist could restrain the ABA-induced reduction in binding affinity between SPK1 and ROP6. These results suggested that ABA could suppress the interaction between SPK1 and ROP6, and the inhibition effect of ABA was dependent on the kinase activity of CPKs.10. At final, we could reconstitute the ABA-PYR1-ABI1-CPK3 signaling pathway, in which, we confirmed that, the CPK3 kinase activated SPK1 is required for governing the ABA signal transduction in Arabidopsis. |
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