Functional Characterization Of ASR1 And ASR2,two ABA Responsive Transcription Repressors In Arabidopsis Thaliana

The plant hormone abscisic acid(ABA)regulates several aspects of plant growth and development.Most importantly,ABA is able to regulate the expression of ABA-responsive genes through signal transduction,thus play a significant role in regulating plant responses to abiotic stresses.Therefore,characterization of unknown function of ABA responsive genes may lead to the identification of novel regulators of ABA and abiotic stress responses.In this study,we found that the expression levels of ABA-induced Serine-rich Repressor1(ASR1)and ASR2,two closely related unknown function genes,were increased in response to ABA treatment.The amino acid sequences analysis shows that only ASR1 contains L×L×L EAR motif but both ASR1 and ASR2 are significantly enriched in serine amino acid.Arabidopsis leaf protoplast transfection assays show that both ASR1 and ASR2 localized predominantly in the nucleus and mainly expressed in the roots,and were able to repress the expression level of the reporter gene,indicating that ASR1 and ASR2 function as transcription repressors.By using homologs identification analysis,we found that there are a total of 164 homologs of ASRs widely distributed in 41 different angiosperms plant species,suggesting that ASRs may be a novel transcription repressor family in plant.To investigate the possible roles of ASRs in regulating ABA responses,Arabidopsis transgenic overexpression plants as well as CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2 genes were generated.We found that the 35S:ASR1 and35S:ASR2 transgenic plants produced relatively longer roots compare to the Col wild-type,suggesting that ASR1 and ASR2 are involved in the regulation of primary root length in Arabidopsis.On the other hand,in both the seed germination and cotyledon greening assays,ABA sensitivity in the 35S:ASR1 and 35S:ASR2 transgenic plants and the asr1 and asr2 single mutants remained largely unchanged,whereas ABA sensitivity was greatly increased in the asr1 asr2 double mutants.In root elongation assays,however,reduced ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants,but increased ABA sensitivity was observed in the asr1 and asr2 single mutants and further increased ABA sensitivity was observed in the asr1 asr2 double mutants.These results suggest that ASR1 and ASR2 function redundantly to regulate ABA response in Arabidopsis.By using the transgenic plants and the mutants generated,we also examined if ASR1 and ASR2 may play a role in regulating salt stress response in Arabidopsis.However,we found that in both seed germination and cotyledon greening assays,effects on mature plants the 35S:ASR1 and35S:ASR2 transgenic plants,the asr1 and asr2 single mutants,as well as the asr1 asr2 double mutants showed a wild type response to NaCl treatment.Considering that ASRs function as transcription repressors,to investigate how ASR1 and ASR2 may regulate ABA response in Arabidopsis,differentially expressed genes(DEGs)that were down-regulated in the 35S:ASR1 transgenic plant seedlings,but up-regulated in the asr1asr2 double mutant seedlings were identified by using transcriptome analysis.We found that the DEGs were highly enriched in several biological processes including development,responses to plant hormones and stress stimuli.Taken together,our findings indicate that ASR1 and ASR2 are closely related ABA response genes that ASR1 and ASR2 are serine-rich novel transcription repressors,and their homologs are widely distributed in different plant species,therefore may represent a novel family of transcription repressors.Our findings also indicate that ASR1 and ASR2 negatively regulate ABA responses in Arabidopsis in a redundant manner,and they may regulate the expression of some hormone signaling and abiotic stress response-related genes.