Molecular Mechanism Of Long Noncoding RNA LncRNAW20 Regulating Drought Tolerance In Betula Platyphylla

In response to drought conditions,plants have evolved complex regulatory mechanisms.Therefore,it is important to understand how plants respond to drought stress and the molecular mechanisms in depth.Long noncoding RNAs（lnc RNAs）are ubiquitously distributed in plant genomes,but there are few studies to stress tolerance mechanism of lnc RNAs in woody plants.This study systematically studied a previously identified lnc RNA（lncW20）that might be involved in the adaptation of birch to abiotic stress.Analysis of the regulatory mechanism of lncW20 in drought tolerance of birch.The results are shown as followings:1.Cloning of the full-length of lncW20 and analysis of its expression patternLnc W20 is located on chromosome 8 of birch,which is an intronic lnc RNA with a full length of 1953 bp without an ORF encoding a long peptide.The expression of lncW20 was induced by Na Cl,ABA,JA and drought stress,and the expression level of lncW20 was the highly induced by drought stress.The GUS gene under the control of the promoter of lncW20was transformed into birch,and the transgenic birch was subjected to 4%PEG6000s.GUS staining analysis showed that lncW20 was mainly induced and expressed in roots,young leaves and terminal buds after drought stress.The nucleocytoplasmic separation and RT-qPCR confirmed that lncW20 was mainly enriched in the nucleus.2.lncW20 improves the drought tolerance of birchTwelve lncW20-overexpressing（lncW20-OE）birch transgenic lines,and ten mutant birch transgenic lines（lncw20^#）mediated by CRISPR/Cas9 were generated.Three overexpression lines（lncW20-OE5,lncW20-OE7 and lncW20-OE8）with the highest expression levels,the three mutant plants（lncw20^#-1,lncw20^#-2,lncw20^#-5）with altered secondary structure and WT plants were used for phenotypic observation and biomass determination.It was found that lncW20-OE plants had developed root system and more lateral roots,and had the same performance under drought conditions.However,lncw20^#mutant plants had weak root growth and sparse lateral roots.Physiological determination showed that lncW20-OE plants were more tolerance to drought stress than WT plants did,and could reduce the degree of lipid peroxidation and the accumulation of H₂O₂ by increasing the activities of CAT and POD enzymes in roots.In contrast,mutants showed sensitivity to drought stress.3.BpMYB73 positively regulates the expression of lncW20Yeast one-hybrid technique was used to screen out the upstream regulator BpMYB73 that might regulate lncW20.Through chromatin Ch IP and EMSA experiments,it was verified that BpMYB73 could directly bind to MBS and ABRE elements of lncW20 promoter in vivo and in vitro.BpMYB73 could promote the transcription of lncW20 by dual luciferase experiment.RT-qPCR results showed that the expression of lncW20 was significantly increased in Overexpression of BpMYB73 plants.These results suggest that BpMYB73 could directly bind to the lncW20 promoter to positively regulate lncW20 expression.4.BpMYB73 positively regulates the tolerance of birch to drought stressThe CDS sequence of BpMYB73 is 933 bp,encoding 310 amino acids.BpMYB73 is localized in the nucleus.The N-terminal of BpMYB73 has a typical R2R3 domain.Analysis of transcriptional activation experiments showed that the transcriptional activation domain of BpMYB73 is located at the C-terminus（193-310aa）.The expression of BpMYB73 was significantly induced in roots and leaves under drought stress conditions.Further studies found that BpMYB73-OE could promote root growth and increase root biomass.Furthermore,this phenotype was more pronounced under stress conditions.However,the mutant plants（bpmyb73^#）showed decreased tolerance to drought stress,which mainly showed that the activities of CAT and POD enzymes in roots were reduced,and the degree of lipid membrane peroxidation and H₂O₂ accumulation were significantly increased compared with wild type.The drought tolerance of BpMYB73-OE was improved compared with that of wild type.Therefore,BpMYB73 can improve the drought resistance of birch.5.BpMYB73 interacts with BpWRKY7Three proteins were identified that could interact with BpMYB73 using yeast two-hybrid technique,including abscisic acid receptor protein Bp PLY4,phenylalanine ammonia lyase Bp PAL1 and BpWRKY7 transcription factors.Bi FC,GST pull-down and tobacco transient transcriptional activation experiments confirmed that BpMYB73 can interact with BpWRKY7to form a heterodimer to promote lncW20 transcription.6.Screening and identification of target genes of lncW20The DNA sequences with affinity for lncW20 were captured by Ch IRP-seq.The results showed that the main binding site of lncW20 was in the promoter region.Among the genes enriched in the promoter region,11 genes might be involved in abiotic stress,and they might be regulated by lncW20.These 11 genes were investigated by RT-qPCR,and the results showed that 8 genes were significantly up-regulated,including the peroxidase（Bp POD59）and cytochrome P450 monooxygenase（Bp CYP76G1）genes.Dual-luciferase experiments demonstrated that lncW20 could activate the transcription of Bp POD59 and Bp CYP76G1 genes,indicating that lncW20 bind to the promoters of Bp POD59 and Bp CYP76G1 genes to induce their expression,thereby reducing the accumulation of H₂O₂ and improving the drought resistance of birch.In conclusion,the heterodimer formed by the interaction of BpMYB73 and BpWRKY7can bind to the promoter of lncW20 and promote the expression of lncW20.In addition,lncW20 positively regulates the expression of Bp POD59 and Bp CYP76G1 by binding to its promoter,and enhances the antioxidant capability of the root system,thereby improving the drought resistance of birch.