Expressional Regulation And Dissection Of Genetic Effects Of Long Non-coding RNAs In Response To Lead Stress In Populus Tomentosa

Lead(Pb)is one of the most abundant toxic heavy metals in soil.It has toxic effects on every aspect of plant growth and development,and can eventually accumulate in the human body through the food chain,seriously endangering agricultural and forestry production and human health.Perennial woody plants such as poplars have large biomass,well-developed root systems and less connection with the food chain.All these characteristics make forest trees more suitable for bioremediation and soil environment improvement.At present,many genes related to heavy metal uptake and transport have been identified in the analysis of heavy metal tolerance mechanism in plants,However,heavy metal tolerance is a complex quantitative trait,and a single gene is not enough to explain the underlying mechanism.In recent years,researchers have discovered that lncRNAs,as an important regulator,plays an important role in plant growth and stress tolerance.However,little is known about the genetic regulation of lncRNAs on heavy metal tolerance in woody plants.Therefore,lncRNAs may be involved in the genetic regulatory network related to lead stress tolerance.In this study,one year old Populus tomentosa cv.1316 was treated with Pb,transcriptome sequencing analysis was used to reveal the basic characteristics and expression patterns of lncRNAs in response to lead stress in Populus tomentosa.Target gene prediction and SNP-based association analysis were used to explore the biological functions and transcriptional regulation of stressresponsive lncRNAs,and detecte the additive,dominant and epistatic genetic effects of stress-responsive lncRNAs on photosynthesis and growth traits.Molecular biology methods and genetic transformation were used to analyze the functions of candidate lncRNAs and their target genes and the underlying transcriptional regulation mechanisms.This paper provides a theoretical basis for the study of forest lncRNAs in the genetic regulation of heavy metal tolerance in woody plants,and provides theoretical guidance for the cultivation of comprehensive and excellent new varieties with bioremediation functions.The main results and conclusions are as follows:(1)The photosynthetic system of P.tomentosa was extremely sensitive to high-concentration lead stress,stress treatment for 6h is the key transition time node of physiological response.Leaves samples from 6-h Pb treated and control plants were used for transcriptome sequencing.In total,226 lncRNAs were found in response to Pb stress,Target gene prediction and gene function annotation results showed that these stress-responsive DE-lncRNAs mainly involved in biosynthesis of secondary metabolites,carbon metabolism,energy metabolism,and signal transduction pathways by target their DE-PTGs.(2)Association analysis revealed that 5 DE-lncRNA and 6 DE-PTGs significantly associated with2 to 4 phenotypic traits at the genomic level,which were pleiotropic genes.Remarkably,4 DE-lncRNAtarget pairs were significantly associated with the same phenotypic traits,suggesting that these DElncRNAs affect the physiology and leaf phenotypes of poplar under Pb treatment by regulating their target genes.The examination of epistatic genetic effects of the significant associated SNPs found that two DElncRNA-target pairs,PMAT-PtoMYB46 and PCIT-PtoMYB43,simultaneously had epistatic effects on different phenotypic traits.So we speculated that these DE-lncRNA-target pairs may play crucial biological functions in poplar growth under Pb treatment.(3)In Populus tomentosa,both PtoMYB46 and PtoMYB43 can respond to diverse abiotic stresses.In Arabidopsis,overexpressing PtoMYB46 increase the chlorophyll content(Chl),leaf area(LA),leaf dry weight(LDW),net photosynthetic rate(Pn)and transketolase activity(TKT),and can Improve the absorption of Pb and resistance to salt,drought and lead.In poplar,can promote growth,increase Pb tolerance and Pb absorption;In Arabidopsis,the overexpression PtoMYB43 can increase LA,LDW,Pn and FDX in addition to the Chl content,and can promotes the absorption of Pb,and enhances the tolerance to salt and lead stress,but it has no obvious effect on drought stress tolerance.In poplar,the overexpression of PtoMYB43 promoted the plant growth,enhanced the tolerance to Pb stress and increased the absorption of Pb.(4)The DAP-seq sequencing analysis identified that the most significant motif sequence of PtoMYB46 protein was "ACCWAMY";and the most significant motif sequence of PtoMYB46 protein was "TYACCWAMYWH".Combined with RNA-seq and DAP-seq data,a total of 123 DE-PTGs and 88DE-PTGs of PtoMYB46 and PtoMYB43 were identified in response to Pb stress respectively.It is speculated that PtoMYB46 and PtoMYB43 may respond to lead stress by regulating these target genes.(5)Based on the results of bioinformatics and molecular biology experiments,a model of Pb uptake and growth regulation in poplar was established.According to this model,three molecular pathways regulate Pb uptake and plant growth in poplar:(1)PtoMYB46 is downregulated by Pb,and PtoMYB46 represses Pto MATE expression by reducing its promoter activity and acting on its 3′ UTR.PtoMYB46 inhibits the expression of Pto MATE;this repressive effect is weakened under Pb treatment,leading to reduced Pb uptake.(2)PMAT,a target gene of PtoMYB46,modulates Pb uptake.In this pathway,due to a decrease in PtoMYB46 expression,the positive regulatory effect of PtoMYB46 on PMAT expression is weakened,and the inhibitory effect of PMAT on Pto MATE is also weakened.This leads to the upregulation of Pto MATE,which subsequently inhibits Pb uptake.(3)PtoMYB46 targets Pto ARF2 and inhibits its expression,thereby positively regulating plant growth in the presence of Pb.