Identification And Functional Study Of LncRNA Related To Adventitious Root Development Of Poplar

Adventitious root formation is not only the key to forest clonal breeding and populationization,but also the most basic scientific theory in the field of plant developmental biology.The molecular regulation mechanism of adventitious root formation remains to be further studied and explained.Unlike mRNA and miRNA,which have been studied in depth,long noncoding RNA(lncRNAs)have been shown to be widely involved in plant growth and development and stress response,but they still lack in-depth and systematic research.The lncRNAs in forest trees have also been predicted in large numbers,but their authenticity and biological functions have rarely been reported.In this study,the adventitious roots of model tree poplars were used as research objects.A large number of lncRNAs related to adventitious root development were identified by high-throughput transcriptome sequencing,and lnc12 and lncWOX11 were further systematically cloned and verified.The main conclusions are as follows:(1)The 5952 lncRNAs and 45,778 protein-coding genes were obtained by constructing standspecific cDNA libraries and performing high-throughput sequencing on the tissues related to adventitious root formation of poplar.Compared with protein-coding genes,most lncRNAs are shorter in length,fewer in exons,significantly lower in expression levels,and less conserved in different plant species.There were significant differential expression of 933 lncRNAs(DELs)and 1,8451 protein-coding genes(DEGs)during the adventitious root formation of poplar.Based on indepth analysis of bioinformatics,many classical signaling pathways involved in adventitious roots(such as the auxin signal transduction pathway)and important transcription factors(such as WOX11)may be regulated by lncRNAs.(2)Lnc12 was cloned using RACE technology,which is a lncRNA that may involve in the auxin signal transduction pathway.The full-length cDNA of lnc12 is 1372 bp with a typical 5' cap structure and a 3' poly A,which is predicted to have a short open reading frame(sORF)of 159 bp in length.By constructing 35S::lnc12 sORF-GFP and 35S::lnc12 5'UTR+sORF-GFP vectors,and transient expression verification in poplar protoplasts,it was found that lnc12 had no coding ability.After lnc12 overexpression vector construction,stable genetic transformation of poplar and positive plant selection,lnc12-overexpressing transgenic poplars were obtained.The rooting time of lnc12-overexpressing transgenic poplars was significantly delayed,the rooting rate was significantly reduced,the number of adventitious roots and root hairs was also lower than that of the control plants.The addition of NAA to the medium can restore these rooting defects to a large extent.It is speculated that lnc12 may inhibit the formation and development of adventitious roots and root hairs of poplar by regulating the expression of related genes in the auxin signal transduction pathway.(3)Previous studies in our laboratory have shown that PeWOX11 plays a crucial role in the formation of adventitious roots of poplar.The adventitious roots of PeWOX11-overexpressing poplar increased significantly,and a large number of ectopic roots were also produced on stems and leaves.In this study,lncWOX11 was cloned,which is a lncRNA that may regulate the PeWOX11 gene.The full-length cDNA of lncWOX11 is 215 bp and contains a short ORF of 51 bp in length.Both bioinformatics analysis and transient expression detection revealed that lncWOX11 has no coding ability.LncWOX11 has a dynamic expression pattern in different tissues of poplar,and has a high expression abundance in 1-week old adventitious roots.The adventitious roots of lncWOX11-overexpressing poplar was significantly reduced,indicating that lncWOX11 inhibited the formation and development of adventitious roots of poplar.(4)It is predicted that lnc12 may regulate 20 protein-coding genes by cis-acting,and may also regulate 12 protein-coding genes by trans-acting.LncWOX11 may encode eight protein-coding genes by cis-acting.To explore the regulatory relationship between these two lncRNAs and their target genes,not only the expression levels of these target genes in lnc12 and lncWOX11-overexpressing poplar were detected,but also the multi-target CRISPR/Cas9 knockout vectors of lnc12 and lncWOX11 were constructed,and transiently expressed in poplar protoplasts.Based on the above-mentioned complementarity verification of gain-of-function poplars and loss-of-function poplars,it was revealed that lnc12 positively regulates PeSCE1 and negatively regulates the expression of PeSAUR72,while lncWOX11 negatively regulates the expression of PeWOX11.(5)As mentioned above,the phenotype of the lncWOX11-overexpressing transgenic poplar and the PeWOX11-overexpressing transgenic poplar is complementary,which confirms the negative regulation of lncWOX11 on the functional gene PeWOX11.However,the biological functions of PeSCE1 and PeSAUR72 in poplar are unknown.It is impossible to judge whether the phenotypic traits produced by lnc12 and these two target genes during poplar development are consistent or complementary.Therefore,we cloned and studied the functions of these two genes.The results showed that the number of adventitious roots root hairs of PeSCE1-overexpressing poplar were significantly decreased,while the number of adventitious roots and root hairs of PeSAUR72-overexpressing poplar were significantly increased.These results further reveal the regulation of lnc12 on PeSCE1 and PeSAUR72.In summary,lnc12 and lncWOX11 mediate the formation and development of adventitious roots of poplar by regulating the expression of functional genes.