The Transcription Factor GmbHLH120 Is Involved In Soybean Response To Drought Stress

In recent years,the annual demand of soybean in China exceeds 100 million tons,but the self-sufficient rate is less than 20%,and it is highly dependent on import.Drought stress affects all stages of soybean growth and eventually leads to its yield reduction.Therefore,it is very urgent to understand the mechanism of soybean responses to drought and cultivate drought-tolerant and high-yielding soybean varieties.Drought response in plants can be divided into three processes: the perception of drought signal,the transmission of drought signal and the expression of droughtresponsive genes.The expression of drought-responsive genes in plants can be divided into ABA-dependent signaling pathway and ABA-independent signaling pathway,transcription factors play important roles in both ABA-dependent and ABAindependent signaling pathways in response to drought stress.Previous studies have shown that the bHLH transcription factor family responds to drought stress by regulating stomatal development,root hair and epidermal trichome formation,and participating in ABA-dependent signaling pathways.However,the mechanism of bHLH family in the regulation of soybean responses to drought stress is rarely reported.Earlier studies in our laboratory shown that GmbHLH120 was artificially selected during genetic domestication.And its homologue in Arabidopsis thaliana,AtbHLH018 could negatively regulated ABA-dependent drought stress response,implying that GmbHLH120 may also be involved in drought stress response in soybean.Taking soybean transcription factor GmbHLH120 as the research object in this study,we found that GmbHLH120 was involved in the regulation of drought stress response in soybean by applying PEG simulation of drought stress experiment,analyzing GmbHLH120 gene expression under drought induction,measuring stomatal opening and soil drought treatment.Our main results are as follows:(1)Analysis of GmbHLH120 promoter revealed the presence of several homeopathic elements,including two ABRE cis-regulatory element related to ABA specific,these results suggest that GmbHLH120 is involved in abiotic stress response.(2)Expression patterns of GmbHLH120 in different soybean tissues,circadian rhythm and dehydration have been analyzed.It was found that GmbHLH120 was highly expressed in apical growth point,adventitious leaf and root,and its expression wasregulated by circadian rhythm.Under natural dehydration,GmbHLH120 is transiently activated and then continuously suppressed.Moreover,GmbHLH120 protein is expressed in the nucleus of Arabidopsis protoplasts.The results of dual luciferase reporter assay showed that GmbHLH120 has transcriptional activator activity,which suggests that GmbHLH120 may regulate the expression of downstream genes at the transcriptional level.(3)GmbHLH120 is involved in the regulation of drought tolerance in soybean plants.The sensitivity of transgenic hairy roots overexpressing GmbHLH120 to PEG was enhanced on 1/2 MS medium supplemented 25% PEG.The changes of stomatal opening of gmbhlh120 loss-of-function mutant and GmbHLH120-OE transgenic plants were not significantly increased or decreased compared to that of wild type under dehydration treatment,however,except gmbhlh120-1 and GmbHLH120-OE-40,the stomatal opening of the other materials was not significantly different from that of the wild type the water loss rate of GmbHLH120 mutant and over-expression transgenic materials in vitro was measured.The results showed that there was no significant difference in water loss rate of detached leaves between the wild type and the mutant,but the water loss rate of overexpression transgenic materials was faster than that of wild type.Both the loss-of-function mutant gmbhlh120 and the GmbHLH120-overexpressing transgenic lines were less drought-tolerant than the wild type.These results suggest that GmbHLH120 is involved in the regulation of drought tolerance in soybean plants.(4)GmbHLH120 may negatively regulate ABA-mediated responses to drought stress in soybean.The transgenic hairy roots overexpressing GmbHLH120 were insensitive to ABA on 1/2MS medium treated with ABA,the change of relative stomatal opening of gmbhlh120 mutant was more sensitive to ABA than that of wild type,but the change of relative stomatal opening opening of GmbHLH120 transgenic material was less sensitive to ABA than that of wild type,these results suggest that GmbHLH120 may negatively regulate ABA-mediated drought stress response.In conclusion,our study preliminarily found that the transcription factor GmbHLH120 plays a negative regulatory role in the response to drought stress and may be mediated by ABA.It not only provides a basis for further elucidating the specific molecular mechanism of GmbHLH120 transcription factor in soybean drought stress but also diversifies the drought-resistant molecular regulatory network of soybean.