Functional Verification Of Salt Stress-responsive Genes GhMYB73 And GhSCL4

Upland cotton is widely cultivated as an important fiber and oil crop.It is important for improving cotton yield through uncovering the molecular mechanism of cotton in response to stress.Transcriptional regulation network of plant salt stress response is complex,in which transcription factors play an important role.MYB and GRAS transcription factor family genes have been reported to participate in the response to salt stress.To identify the salt stress response genes of these two families in cotton and reveal their molecular mechanisms,salt stress related transcriptome data was analyzed and two salt induced genes?MYB family gene Gh MYB73 and GRAS family gene GhSCL4?were selected for further study.Genetic analysis and biochemical experiments were performed to reveal their function and regulatory mechanism in response to salt stress.The main findings are as follows:1. Overexpression of Gh MYB73 increased salt tolerance in transgenic plantsThe result of qRT-PCR showed that the transcript level of Gh MYB73 was up-regulated after treatment with ABA and salt.Subcellular localization and transactivation activity assay showed that Gh MYB73 acts as a transcriptional activator.Two single copy insert-overexpression?OE?lines of Gh MYB73 transgenic cotton of T₁generation were obtained,and their root phenotype was observed at the harvest season.The results showed that the lateral root number of the OE lines was significantly higher than that of the control ZM24.Furthermore,the plants of OE lines were subjected to salt treatment and the result showed that salt tolerance of OE lines was significantly higher than that of ZM24.The VIGS assay showed that a more salt-sensitive phenotype was observed in Gh MYB73-silenced plants than the control plants.The salt tolerance of homozygous Gh MYB73 transgenic Arabidopsis in T₃ generation was also investigated.The results show that when the plants were grown in MS plates containing NaCl,both the opening cotyledon rate and germination rate of the Gh MYB73 over-expression lines?OE?were significantly higher than those of wild type.Moreover,we observed the tolerance to salt stress in adult seedlings,and the OE lines exhibited lower sensitivity to salt than WT.Thus,Gh MYB73 is a positive regulator in response to salt stress.The seeds of Gh MYB73 OE lines and WT were also grown in MS plants containing mannitol for 10 days and the opening cotyledon rates of the Gh MYB73 over-expression lines?OE?were also significantly higher that those of wild type as the NaCl treatment.Additionally,the proline content and expression of osmotic stress-induced genes?At SOS3,At P5CS1 and At NHX1?were significantly higher than those of WT after salt treatment.Thus Gh MYB73 may regulate the expression of the osmotic stress-induced genes to increase the osmotic tolerance of plants,which finally improve the slat tolerance of plants.For ABA sensitivity asssy,the results showed that the OE lines showed higher tolerance to ABA than WT.Additionally,the transcription levels of ABA-dependent genes,including At PYL8,At ABI5,At ABF3,and At RD29B,were substantially higher in the OE lines than in WT.The interaction between Gh PYL8 and Gh MYB73 was confirmed by yeast two-hybrid and Bi FC assays.Therefore,the positive regulation of Gh MYB73 in response to salt may be associated with ABA signalling pathway.The interaction between PYL8 and Gh MYB73 may act as an important role for Gh MYB73 in response to ABA.2. Over-expression of GhSCL4 increases tolerance to salt stress in plantsqRT-PCR assay showed that transcript levels of GhSCL4 were up-regulalted after slat and drought treatment.Three overexpression lines?OE1,OE2,and OE3?of GhSCL4transgenic Arabidopsis were obtained through genetic transformation.We used NaCl to simulate salt stress and mannitol to simulate osmotic stress,and then investigate the salt and osmotic tolerance of GhSCL4 transgenic plants during germination period.The cotyledon greening rates of GhSCL4 overexpression lines?OE?were significantly higher than those of wild type either in MS plates containing NaCl or mannitol.The VIGS assay was also performed to interfere the expression of GhSCL4 and higher water loss rate and more salt/drought-sensitive phenotype were observed in GhSCL4-silenced plants than the control plants.To investigate the molecular mechanism of GhSCL4 in response to salinity,the co-expression genes were obtained from co-expression network internet.The GO enrichment analysis showed that most co-expression genes were enriched in the calcium ion binding pathway.What?s more,Bi FC and Luciferase-assay confirmed the interaction between GhSCL4 and Gh Ca M7.And the results of VIGS assay showed the Gh Ca M7–silenced plants showed increased sensitivity to salt stress than those of control,which indicated Gh Ca M7 is a positive regulator of plants in response to salinity.In conclusion,GhSCL4 may respond to salt stress via calcium signalling by interacting with Gh Ca M7,which can increase the salt tolerance of plants.Furthermore,Bi FC assay indicated that Gh Ca M7 can interact with a key enzyme?Gh GSNOR?in the NO removal system,and we speculate that the interaction between Gh Ca M7 and GhSCL4 can enhance the activity of Gh Ca M7 for inhibition of Gh GSNOR activity,and increase the NO content in plants under salt stress.