Functional Characterization Of Arabidopsis Transcription Factor AtLBD15 In Plant Response To Drought Stress

Due to the global climate change,drought has become a major environmental factor that limits plant growth and development.As sessile organisms,plants have to adjust growth and development to cope with drought stress for survival.Plant hormone Abscisic acid(ABA)is one of the important hormones that regulates various physiological processes.For example,under drought stress,ABA is rapidly accumulated in plants and moves from roots to shoots to induce stomatal closure to protect water loss and expression of numerous stress-responsvie genes.Meanwhile,ABA promotes metabolites accumulation to adjust osmotic potential.In this study,we analyzed the seed germination and primary root elongation growth of LBD15 loss of function mutants and overexpressing plants on the MS medium with or without ABA.Using biochemistry and molecular biology methods combined with bioinformatics analysis confirmed the LBD15 targeted gene ABI4,and regulatory relationships were verified by genetic experiments.Finally,Arabidopsis thaliana AtLBD15 confers plant drought resistance through ABA signaling pathway.The results are as follows:1.The seeds of lbd15 and overexpressing LBD15 transgenic plants were used to analyze the germinate rate in the MS medium with or without ABA.In the absence of ABA,the germinate rate of all lines were similar.In the presence of ABA,the germinate rate of overexpressing lines were significantly inhibited,but the lbd15 seeds were more resistant to ABA inhibition.2.3-day-old seedlings lbd15 and overexpressing LBD15 transgenic plants grown on MS medium were transferred to MS medium with different concentrations of ABA for 2 days.Root growth of lbd15 was shown ABA-hyposensitive compared with WT seedlings,while transgenic lines displayed an ABA-sensitive phenotype compare with WT seedlings3.WT,lbd15 and overexpression lines were simultaneously grown for 6-week in pots under normal growth conditions,and then withheld water for 2 weeks.Survival rate were examined 3 d after re-watering.Overexpression lines were obviously more tolerant to drought stress and their survival rate were approximately 60%,whereas the survival rate of lbd15 mutants was just ~1.5%.We also examined the rate of water loss and stomatal aperture in WT,lbd15,OX-L15-1 and OX-L15-2.The detached leaves of lbd15 showed more water loss than those of WT,whereas OX-L15-1 and OX-L15-2 were less water loss than WT.After drought treatment,stomatal aperture of lbd15 mutant decreased to a much less degree,with respect to the WT,whereas it decreased to a greater extent in the OX-L15-1and OX-L15-2.4.We analyze the promoter of LBD15 found that the region from-300 to-400 exists ABA response elements.To evaluate the function of the region,ProLBD15:GUS and ProLBD15(300-400):GUS transgenic plants were treated with ABA.In the absence of ABA,the expression levels of GUS were similar.In the presence of ABA,the expression levels of GUS in ProLBD15:GUS transgenic plants were higher than those in ProLBD15(300-400):GUS transgenic plants.Overexpression of LBD15 results into the leaf smaller and curls,but the Lz region of LBD15 substituted by the Lz of AS2 or LBD40 has no obvious phenotype compared with wild type.Thses results indicates thatthe Lz region of LBD15 plays an important role in plant development.5.ChIP-on-chip was performed to screen the target genes of LBD15.And then LBD15-binding motif ?CATTTAT‘ was used to search the ABA response gene in the ChIP-on-chip.The ?CATTTAT‘ cis element was found in the promoter region of 1794 bp upstream of ATG of ABI4.We then examine whether LBD15 directly activates the expression of ABI4 by binding the promoter of ABI4.qRT-PCR was performed to analyze the expression levels of ABI4 in different lines.qRT-PCR was performed to analyze the expression levels of ABI4 in different lines.The results showed ABI4 expression levels were obviously upregulated in LBD15-overexpression lines and repressed in lbd15 mutants.This conclusion was further verified by the EMSA,ChIP-qPCR and transient expression assay experiments.Taken together,LBD15 can directly bind to ABI4 promoter to activate its expression6.To study the genetic relationship between LBD15 and ABI4 in ABA signaling pathway.lbd15abi4 and lbd15OX-ABI4 were generated to study the genetic interaction.ABA sensitivity was reduced in the lbd15,abi4 and lbd15abi4.Whereas the phenotype of lbd15OX-ABI4 was similar to OX-ABI4.These results suggested that LBD15 acted upstream of ABI4,and directly modulate ABI4 transcription.