R2R3 MYB Transcription Factors MYB71,MYB79 And MYB121 Regulate ABA And Drought Stress Responses In Arabidopsis Thaliana

Plants have evolved so many mechanisms to adapt to the hostile enviornment,because of they can’t seek advantages and avoid disadvantages,and can’t escape from the undesirable growth environment,due to its sessile growth characteristics.As one of the five classical plant hormone,abscisic acid(ABA)plays an important role in regulating plant growth and development including seed germination,the transition from vegetable growth to reproductive growth,flowering time and senescence.Most importantly,ABA regulates plant response to abiotic stresses by activating or inhabiting the expression of ABA-responsive genes through signal transduction.However,the functions of many ABA-responsive genes are still unclear.As one of the largest transcription factor families,MYB transcription factors also play an important role in regulating plant growth and development as well as plant adaptation to the environment.Yet the functions of a large portion of MYB transcription factors remained uncharacterized.In this study,we characterized the functions of three closely related ABA responsive MYB transcription factors,i.e.,MYB71,MYB79 and MYB121 in regulating plant response to ABA and drought stress.RT-PCR and GUS staining of promoter-GUS transgenic plants showed that the expression of MYB71,MYB79 and MYB121 was induced dramatically by ABA.The phylogenetic tree and amino acid sequence alignment analysis showed MYB71 and MYB79 were most closely related among the three MYBs.However,results in Arabidopsis protoplast transient transfection showed that MYB71,MYB79 and MYB121 were all localized in the nucleus,and had transcriptional activation activity.In order to investigate the function of MYB71,MYB79 and MYB121 in regulating plant response to ABA and abiotic stress,we generated overexpression transgenic plants and single,double and triple mutants for MYB71,MYB79 and MYB121.In both seeds germination and green seedlings assays,the 35S:MYB71 and35S:MYB79 transgenic plants showed enhanced sensitivity,whereas myb71 and myb79 single mutants showed decreased sensitivity to ABA,indicating that MYB71 and MYB79 positively regulated the ABA response in Arabidopsis.On the other hand,the35S:MYB121 transgenic plants showed decreased sensitivity,whereas the myb121 single mutants showed enhanced sensitivity to ABA,indicating that MYB121 negative regulates ABA response in Arabidopsis.Seeds germination and green seedlings assays also showed that ABA sensitivity in the myb71 myb121 double and myb71 myb79 myb121 triple mutants were increased,but to a degree less than that in the myb121 single mutants,indicating that MYB121 maybe function downstream of MYB71 and/or MYB79,or function via different pathways to regulate ABA response in Arabidopsis.The results of drought treatment showed that drought tolerance was reduced in the35S:MYB79 overexpression transgenic plants,but remained largely unchanged in the myb79 single mutants,indicating that MYB79 negatively regulate drought tolerance in Arabidopsis.On the other hand,drought tolerance was reduced in the myb121 single mutants but remained largely unchanged in the 35S:MYB121 overexpression transgenic plants,indicating that MYB121 positively regulates drought tolerance in Arabidopsis.Drought treatment with also showed that drought tolerance was reduced in both the myb71 myb121 double mutants and myb71 myb79 myb121 triple mutants.However,the triple mutants were more tolerant to drought than the myb121 single and the myb71myb121 double mutants.These results indicate that MYB121 may be function downstream of MYB79,or function via different pathways to regulate drought tolerance in Arabidopsis.To explore the function mechanism of MYBs,35S:MYBs-GR transgenic Arabidopsis was constructed and analyzed by transcriptome sequencing.After comparing the transcriptome sequencing results in 35S:MYBs-GR transgenic plants treated by DEX,ABA,ABA+DEX and solvent methanol as the control,it was found that the expression levels of 26 genes were up-regulated at least 2-fold after DEX treatment compare with the solvent methanol group.The GO annotation and GO enrichment analysis of these genes showed that 18 genes were involved in stress responses.After ABA+DEX treatment,the expression levels of 112 genes were upregulated more than 2-fold,compared with ABA treatment group,among which 46 genes were involved in stress responses.These statistical results showed that GO annotation was significantly enriched in genes related to stress responses.Then we verified the expression of some candidate target genes and found that a few genes,PER4,At1g14550,CYP82C2 and LURP1,whose function related to seed germination regulation and plant responses to stress stimuli,were up-regulated in 35S:MYB71overexpression transgenic plants.Therefore they may be the target genes of MYB71.In addition,we found that transcription factor genes WRKY6 and ABI3 two positive regulator genes of ABA signaling were also increased in ABA+DEX group compared with ABA treatment in the results of RNA-seq.We detected the expression of WRKY6 and ABI3 in MYB71,MYB79 and MYB121 transgenic plants and mutants.We found that the expression of WRKY6 was decreased in 35S:MYB71 and 35S:MYB79 transgenic plants,but remained larged unaffected in the 35S:MYB121 transgenic plants and single mutants.We also found that the ABA induced expression level of ABI3 in the35S:MYB71 transgenic plants and myb71 myb79 myb121 triple mutants was decreased.These results may partially explain the difference between MYB121 from MYB71 and MYB79 in regulation of ABA response in Arabidopsis,and suggest that MYB71,MYB79 and MYB121 may be involved in the regulation of ABA response in plants by regulating ABI3 and WRKY6.Considering that transcription factors may act by interacting with other proteins to perform their functions,we also investigated the proteins may interact with MYB71,MYB79 and MYB121.Based on the prediction of protein interaction,we found several proteins that may interact with MYB71,MYB79 and MYB121,and proved by Bi FC that MPK6,which is involved to regulation plants defense against pathogens,osmotic stress,ABA response,cold and oxidative responses,could interact with MYB79.These results suggest that MYB79 may play a role in regulating plant response to stress through the interaction with MPK6.In conclusion,the results of this study indicated that MYB71,MYB79 and MYB121 are ABA responsive genes,and MYB71,MYB79 and MYB121 are all transcriptional activators and are involved in regulation of ABA and drought response in Arabidopsis.However,MYB121 may has opposite functions to MYB71 and MYB79,and it may act downstream of MYB71 and MYB79,or functions in different pathways to regulate ABA and drought responses in Arabidopsis.