Pathway-unit Transcriptome Analysis For The Wounding-induced Adventitious Root Formation In Populus Ussuriensis

Adventitious root(AR)induction by wounding has been an important regeneration technique particularly for the vegetative propagation of trees such as P.ussuriensis in afforestation practices,but poor understanding of its molecular mechanism of woundinginduced AR development in tree species is greatly limiting the afforestation practices.Recently,the transcriptome data mining tools such as DEG,clustering,GSEA,and WGCNA,have widely been applied to genomic studies on AR development in poplar trees and revealed the certain amount of evidences on the molecular mechanism of AR development in poplars including P.ussuriensis.However,those knowledge are fragmentary and non-comprehensive,which appears to be primarily due to the limitation of the current transcriptome data mining approaches.This study found the limitation of current transcriptome data mining approaches and modified the WGCNA(weighted gene co-expression network analysis)to improve the data mining efficiency,eventually succeeding in obtaining comprehensive and integrated insights into the molecular mechanism of wounding-induced AR development in P.ussuriensis.1.This study found the limitation of current transcriptome data mining approaches.Several rounds of preliminary analyses using the transcriptome data mining tools have been performed for a long period of time before this study and as a result,it was found that the difficulties in comprehensive understanding the molecular mechanism of a biological process actually come from the limitation of researchers themselves in handling the large amounts(thousands)of the genes generated by the transcriptome data.This was also observed for the DEG or clustering analysis results from this study.The DEG analysis showed that there were large amounts of DEGs present between the nine time points;based on the criteria of log FC >1 and p < 0.05,there found 5778,10073,10956,10172,8932,8213,9241,and 10077 DEGs at0.5,1,2,6,12,24,48,and 120 h,respectively.The clustering analysis also clustered the 27487 genes into several clusters,which were regrouped based on the expression trends,and as a result,two main groups contain 14612 and 8765 genes which were further annotated to 2948 and 2899 GO terms.From these large amounts of genes and pathways,it was extremely hard to find key pathways or genes that can reflect the whole aspects of biological processes comprehensively.2.This study modified the functional unit of the conventional WGCNA analysis,from genes to genesets,to address the limitation of current transcriptome data mining approaches.Similar modification has been already applied in GSEA or GSVA analyses,but not yet for the WGCNA.In this study,the modified WGCNA was applied as follows.Total 694 GO and KEGG terms were proven as significantly enriched at specific time points during AR development of P.ussuriensis in the GSEA results,and further used as the pre-defined genesets.WGCNA analysis was then applied to these 694 genesets,instead of the 27487 genes,thus,this study named this process the ‘weighted geneset co-enrichment network analysis’.Consequently,the hub pathways were identified at specific time points.Next,only the genes in the identified hub pathways,which are much smaller than in conventional WGCNA,were further subject to the second-round WGCNA analysis,thereby enabling easier identification of key genes driving the hub pathways and construction of the regulatory networks.This study called these WGCNA processes the ‘stepwise WGCNA’.Compared to the DEG,clustering,and GSEA/GSVA,the ‘stepwise WGCNA’ analysis revealed integrated and comprehensive knowledge on the time-specific key pathways,genes,and regulatory networks in this study.3.According to the ‘stepwise WGCNA’ results,wounding stress induced rapid upregulation of JAZ proteins and ERF109 transcription factor which regulated the JA biosynthesis and signaling at 0.5 h(ERF109 was de novo expressed).ERF109 then activated the auxin biosynthetic genes to promote auxin accumulation at 1 h.After 2 h,JA biosynthesis stopped,while auxin biosynthesis also stopped after 6 h,due probably to the negative feedback regulatory action of E3 ubiquitin ligases upon perception of JA or auxin levels.After 6 h,WOX11,a gene encoding the master regulator for cell fate transition in AR development,was de novo expressed and regulated downstream pathways for auxin transport and signaling,cell division,and root meristem growth,and root morphogenesis throughout long period of time(6h to 48 h).At 120 h,the pathways associated with root meristem growth and root morphogenesis were highly activated and PLT1 transcription factor played a central role in the pathways.Overall,the application of novel data mining approach provided novel,comprehensive,and integrated insights into wounding-induced AR development in P.ussuriensis,thereby not only greatly advancing genomic studies for the developmental processes,but also promoting the AR-based proliferation practices in P.ussuriensis.