Mechanism Of Drought Stress Response Regulated By LincRNA DALIR In Arabidopsis Thaliana

Drought stress can affect the entire life cycle of plants,resulting in abnormal growth,development and reduction of crop yields.Plants have developed a variety of mechanisms to respond to drought stress,and long noncoding RNAs(lnc RNAs)have emerged as important players in drought response.However,the molecular mechanism of lnc RNA regulation of drought response in plant is still unknown.We previous identified a linc RNA,DALIR(drought associated linc RNA),which is involved in the plant drought response.The dalir mutants exhibited ABA insensitivity and reduced drought tolerance compared to wild-type.We have analyzed both biological function and underlying mechanism of DALIR through a variety of genetic and molecular biological methods.Our results are as follows:1.We found that there was a reduced drought tolerance in dalir mutants by soil drought experiment.Stomatal apertures of dalir mutants were larger than of Col-0,meanwhile the water loss rates of detached leaves from dalir mutants were higher than in Col-0.These results indicated that DALIR positively regulates the drought response by controlling stomatal opening in Arabidopsis thaliana.2.Three proteins including PABN1,PABN2 and PABN3 that interact with DALIR were identified by yeast three-hybrid assay.They belong to PABNs family,and may be involved in the regulation of m RNA alternative polyadenylation(APA).Interactions between PABN1,PABN2 and DALIR were verified by RNA pull-down experiment,respectively.Results of RNA immunoprecipitation also supported that PABN3 bound DALIR in vivo.Moreover,three PABNs were located in the nucleus,and they could interact with each other depend on yeast two-hybrid assay,suggesting that PABNs function as a protein complex.3.We successfully created loss of function mutants for three PABNs genes through applying CRISPR/Cas9 technology,and two independent mutant lines were produced for each gene.Phenotypic analysis showed that mutated pabn1,pabn2,and pabn3 could increase stomatal aperture,and pabn1 mutants were more insensitive to ABA treatment and sensitive to mannitol treatment compared with wild-type,indicating that those three PABNs proteins participate in the process of drought stress response by regulating the stomatal aperture of Arabidopsis.4.Based on transcriptome analyses between Col-0 and dalir mutant,we found that the expression level of NAC055,a transcription factor positively regulated drought response in Arabidopsis,was remarkably decreased in the dalir mutant.In addition,NAC055 expressions were down-regulated in three pabn mutants by q RT-PCR.Further analysis showed that NAC055 m RNA had two isoforms with different lengths through APA.We found that the proportion of short NAC055 transcript isoform was increased in dalir mutant based on q RT-PCR analysis.In summary,our study demonstrated that DALIR could regulate both expression level and alternative polyadenylation of NAC055,thereby affecting the drought stress response in Arabidopsis thaliana.This study can provide more theoretical basis of long non-coding RNA regulating plant drought resistance,and add new content to the signal network of plant stress response.