Expression Differtiation Of Poplar Non-coding RNA In Response To Abiotic Stress

Non-coding RNA(nc RNA)play an important regulatory role in biological growth,development,mainly including circular RNA(circ RNA),long non-coding RNA(lnc RNA)and small RNA(mi RNA),etc.The regulation of target gene expression by these nc RNAs occurs at the transcriptional,post-transcriptional and epigenetic levels.The study on nc RNAs in plants remainly still largely unknown,mainly due to limited identification tools,limited data of genome and transcriptome,difficulties in interspecies comparison and experimental validation.Poplar species distribute widely,and have strong adaptability to the environment,short differentiation time,which all define poplars as ideal plant material for studying interspecific differentiation of plant nc RNAs.In this study,we analyzed the regulatory function of nc RNAs during salt stress in poplar by comparing the 108 strand-specific transcriptomes under salt stress in three closely related salt-tolerant(Populus euphratica and P.pruinosa)and saltsensitive(P.alba var.pyramidalis)poplars,and the RNase R+ transcriptomic data under drought stress in P.alba var.pyramidalis,screened a series of nc RNAs involved in salt tolerance and drought resistance in poplar.We also screened a lnc ERF024 with conserved sequence but diversification in expression pattern in three poplar species,and explored its possible molecular regulatory mechanism in the adaptation process of salt-tolerant poplar.The main findings were as follows.1.Induced expression differentiation analysis of poplar circ RNA under salt stressBased on Find＿circ software,we developed a new filtering pipeline for plant circ RNAs and validated it in model plants,such as rice and Arabidopsis.The filter pipeline was found to significantly reduce the identification of false positive circ RNA in plants.Through new filtering pipeline,poplar circ RNAs were identified from 108strand-specific transcriptomes of three poplar species under salt stress,and the differentiation of circ RNA among closely related poplar species was compared.The results showed that most poplar circ RNAs were transcribed from exonic regions,induced by longer flanking intron sequences for cyclization,and expressed at low levels in specific tissues/species.Furthermore,based on the comparison of homology of circ RNA parental gene,we found that most circ RNAs were species-specific or moderately conserved in origin,and only a small number of circ RNAs were conserved in three poplar species.However,the expression patterns,homologous gene transcription positions,and numbers of these conserved circ RNAs diverged significantly;in addition,comparing the parental genes of species-specific circ RNAs,we found that the parental genes with stress-responsive functions differed significantly in structural domain types and numbers,which may be important factors contributing to the diversification of salt tolerance in different poplar species.Finally,15 circ RNAs with mi RNA binding sites were revealed in poplar,which respond to salt stress by promoting mi RNA target gene expression.These results suggest that circ RNA may regulate the gene expression of woody poplars very finely and the potential regulatory functions of circ RNA in salt tolerance of different poplars.2.Analysis of drought stress-induced expression of circ RNAs in P.alba var.pyramidalisTo verify the molecular response mechanism of poplar circ RNA to drought stress,we applied sequencing approach specifically for circ RNAs to identify 799 high confidence circ RNAs in leaf tissues of P.alba var.pyramidalis under drought stress,of which 65% were differentially expressed induced by drought stress.In addition,the expression patterns of some circ RNAs and their parental genes showed positive correlation;and the prediction of ce RNA regulatory network indicated that differentially expressed circ RNAs regulated the expression of homologous m RNAs by interacting with mi RNAs,speculating that circ RNA may be involved in poplar response to drought stress in multiple ways.In addition,the analysis revealed an increase in the number of circ RNA transcripts under drought stress,which may be related to the increased frequency of alternative splicing of homologous m RNAs under stress.3.Induced expression differentiation analysis of poplar lnc RNA under salt stressWe utilized comparative strand-specific transcriptome analysis between three closely related salt-tolerant and salt-sensitive poplars to identify the differentiation of lnc RNAs during salt stress.Based on sequence similarity,the 34,363 sequences of lnc RNAs identified in the three poplars were clustered into 26,288 homology groups(HGs),and the sequences of lnc RNAs within 248 HGs were highly similar in the three poplars and named as conserved lnc RNAs.Further studies revealed that the gene copy and mode of origin of some conserved lnc RNAs were rapidly evolving in the three poplars.Notably,the expression patterns of conserved lnc RNAs with consistent mode of origin diverged under different tissues and salt concentration treatments in poplar,especially in salt-tolerant poplars(P.euphratica and P.pruinosa)showing a more consistent up-or down-regulation trend.This suggests that the divergence of the expression patterns of conserved lnc RNAs between salt-tolerant and salt-sensitive poplars may be one of the main mechanisms to improve salt resistance in salt-tolerant poplars.4.Peulnc ERF024 overexpression improves salt resistance in poplarTo verify the important role of lnc RNA expression diversity for ecological adaptation in poplar,the study selected the conserved lnc ERF024 in poplar,which is transcribed by the antisense strand of ERF024(encoding ethylene-responsive transcription factor)and expressed at a higher level in salt-tolerant poplar than saltsensitive poplar,thus presumably its high expression is related to the salt resistance of poplar.Transgenic experiments verified that overexpression of Peulnc ERF024 significantly improved the salt tolerance of poplar in P.alba var.pyramidalis;the results of RNA pull＿down and RNA-EMSA experiments revealed that Peulnc ERF024 could directly bind photosynthesis-related target proteins(including photosynthetic membrane protein D1,ATPase subunit,etc.).In addition,RNA-seq analysis revealed that the expression of genes related to stress response and photosynthesis were significantly up-regulated.Thus,we hypothesized that lnc ERF024 may improve salt tolerance in poplar by regulating the expression of genes related to stress response and photosynthesis or by altering the activity,localization or assembly of proteins in the cell.In summary,this study identified a large number of nc RNAs induced by abiotic stress in poplar,explored the differentiation of nc RNAs induced by salt stress among poplar species,and investigated the function and molecular mechanism of Populus Peulnc ERF024 involved in salt stress response.The results provide an important reference on the molecular regulatory mechanisms of stress resistance in plants,especially in forest trees,and provide candidate gene resources for poplar stress resistance molecular breeding.