Research On The Function And Mechanism Of Second Messenger CGMP Involvement In Auxin Regulated Growth And Development Of Arabidopsis Roots

Second messenger cyclic guanosine 3’,5’-monophosphate (cGMP) plays an important role in plant development and responses to stresses. Studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation and gravitropic bending. Nonetheless, the mechanism of the cGMP involvement in auxin signalling to affect plant growth and developmental processes is largely unknown. Therefore, in this study, we used the model plant Arabidopsis as materials and studied the mechanism of auxin and cGMP signalling on Arabidopsis root growth and development using related mutants and transgenic plants. Our main results are as follows:1. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development.In this study, we found that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell membrane-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth and the expression of DR5-GUS reporter gene and auxin responsive genes. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin, suggesting that cGMP plays an important role in auxin-regulated root development and gene expression. Further studies showed that auxin signalling mutants tirl-1, axr1-3 and axrl-12 displayed reduced sensitivity to LY83583 and 8-Br-cGMP in auxin-inhibited primary root elongation. Data also showed that 8-Br-cGMP enhances the auxin-induced degradation of Aux/IAAs protein modulated by the SCFTIR1 ubiquitin-proteasome pathway, suggesting that cGMP may regulate auxin signalling by affecting the activity of SCF complex. Given the important role of interactions between auxin receptors (TIR1) and transcriptional suppression protein (AUX/IAAs) in auxin signalling, we found that 8-Br-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAAs interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provided evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKGs). Our results suggested that cGMP acts as a mediator in participating in auxin signalling and may exert its role in this process by PKGs activity via its influence on auxin-regulated gene expression and AUX/IAAs degradation.2. Effects of cGMP on auxin biosynthesis and polar transport in Arabidopsis roots.DR5::GUS reporter gene showed that inhibition of endogenous cGMP synthesis reduced the auxin content in Arabidopsis roots, even disappeared. Removal of LY83583 after a certain time (0-12 h) of LY83583 treatment, the auxin content and root growth can be restored in Arabidopsis roots. Further qRT-PCR analysis indicated that cGMP influences the expression of Trp-dependent auxin biosynthesis genes, suggesting that cGMP may regulate auxin levels and distribution in Arabidopsis roots by influencing the genes expression of auxin biosynthesis. In addition, using the transgenic plants of PINs-GFP/GUS and A UX1-YFP as well as qRT-PCR analysis, we found that the change of cGMP levels influences auxin efflux carrier PINs and influx carrier AUX1 expression abundance at both protein and transcription levels in Arabidopsis roots. Moreover, DR5::GFP results showed that the change of cGMP levels influences the gravity-induced downside auxin accumulation in Arabidopsis root tips. These results suggested that the cGMP modulates the auxin polar transport in Arabidopsis roots.3. cGMP regulates primary root growth of Arabidopsis by affecting the columella stem cell division.In this study, we found that LY83583 treatment inhibits the primary root growth of Arabidopsis in a dose-dependent way.8-Br-cGMP treatment modestly restores the effect of LY83583, suggesting that cGMP is essential for the normal growth of Arabidopsis primary roots. Further studies demonstrated that 8-Br-cGMP promotes and LY83583 inhibits the length of meristem and the expression of cell cycle proteins in Arabidopsis primary roots, suggesting that cGMP may affect primary root growth of Arabidopsis by regulating the activity of root meristem. However, we found that auxin polar transport and ethylene signalling may not be the main causes in cGMP-influenced primary root growth of Arabidopsis. Because the quiescent center (QC) and the surrounding stem cell activity play an important role in the meristem and primary root normal growth, we examined the change of columella stem cell (CSCs) division and starch grain accumulation in Arabidopsis primary root tips. The results showed that LY83583 treatment suppresses the division of CSCs and the accumulation of starch grains in Arabidopsis primary root tips. Furthermore, CSCs-specific markers J2341, QC-specific markers QC25 and WOX5pro::GFP provide further evidence to support the conclusion. Moreover, using the 35S::WOX5-GR transgenic plants, we found that 8-Br-cGMP increases and LY83583 prevents the WOX5-induced division of CSCs, suggesting that cGMP can regulate the division of CSCs in Arabidopsis primary root tips. Further analysis suggested that cGMP also regulates the expression of transcription factors(PLTs, MP and SCR/SHR), which are involved in the stem cell niche (SCN) maintenance, at both protein and transcription levels.