Molecular Mechanism Of Tension Wood Formation In Catalpa Bungei And Functional Analysis Of CbFLA1

Wood is an important and indispensable natural resource for human life.It is widely used in construction,furniture production,pulp and paper fields.With the social development,the demand for high-quality wood products is increasing.How to obtain high-quality and special wood has become the focus of the tree breeders.Wood formation is controlled by complex molecular network,which included the secondary wall cellulose synthesis,lignin deposition and the proliferation and elongation of fiber cells.It is important to understand the biological mechanism of wood formation for the genetic improvement of wood properties.In this study,Catalpa bungei was used as the research object to study the induced tension wood(TW)formation by artificial bending.Transcriptome(lnc RNA,m RNA and mi RNA),proteome and confocal Raman spectral imaging techniques were performed to analyze the key molecular regulation networks of the TW formation from multiple levels of gene transcription,protein translation and metabolism.The main results and conclusions of this study are summarized as follows:(1)There was no obvious gelatinous fiber in the TW fiber cavity of C.bungei.The thickness of the secondary wall of fiber was significantly thinner than that of the opposite wood(OW)and normal wood(NW).Compared with OW and NW,the length to width ratio of TW vessels increased significantly,showing an oval shape,and the number of vessels in TW was significantly less than that of OW and NW.In addition,tissue staining showed that the lignification degree of TW was lower than that of OW and NW.(2)The results of endogenous hormone content determination indicated that various plant hormones were involved in the TW formation.A regulatory network of TW formation mediated by multi-phytohormone was constructed,based on the data of LC-MS and transcriptome.The results of this study showed the increase of IAA and c Z of TW further induced the expression of genes involved in IAA,c Z and BR signal transduction,which could be regulated by some ATHB transcription factors and lnc RNAs.Finally,the functional genes located downstream were activated to regulate the development of vessels and fibers in TW.What is more,some lnc RNAs and transcription factors also participate in this process.(3)The results of tissue section staining and Raman spectrum showed that the lignification degree of TW was significantly lower than that of OW and NW.LC-MS results indicated that TW had higher contents of p-coumaraldehyde,coniferyl aldehyde and sinapinaldehyde,and lower contents of coniferly alcohol compared with OW and NW.In the lignin monomer biosynthesis pathway,the expression trend of DEGs and the corresponding protein between TW/OW and TW/NW was consistent.The transcriptional level of three cinnamyl alcohol dehydrogenase genes(CADs)in TW was higher than that in OW and NW,but their protein level was significantly lower.Meanwhile,mi RNAs sequence and target gene prediction of mi RNA revealed that mi R860,novel-m3749-5p,novel-m0132-5p and novel-m4121-3p could regulate CADs at the post-transcriptional level by translational inhibition.In conclusion,it is speculated that the expression of CAD gene regulated by the above mi RNAs in translation inhibition may be the potential molecular mechanism of the limitation of lignin monomer synthesis during the TW formation.(4)The results of transcriptome showed that the functions of most of the differentially expressed genes(DEG)in TW/OW and TW/NW were involved in metabolism related to carbohydrate and biosynthesis of secondary metabolites.Two endoglucanase genes(CELs)involved in cellulose metabolism were significantly higher expressed in TW than OW or NW.Five Glucosidase transcripts(BGLU)were significantly highly expressed in TW/OW,and two BGLU proteins level were also significantly increased in TW.Five xyloglucan glucosyltransferase(XTH)genes and one glucomannan-β-mannose transferase(CLSA9)gene related to hemicellulosic synthesis and metabolism were highly expressed in TW.In pectin metabolism,five polygalacturonase(PG)transcripts and three PG proteins were up-regulated in TW.In addition,the transcription and protein levels of galacturonase(GAUT),glucuronyltransferase(IRX7),and glucuronyl-4-O-methyltransferase were significantly upregulated in TW.Meanwhile,the results of Raman spectra showed that the contents of cellulose and non-cellulose polysaccharides in TW were lower than those in OW and NW.(5)This study preliminarily found that Cb FLAs might be the key gene affecting TW formation of C.bungei according to the results of multi-omics.To verify our hypothesis,we analyzed the gene structure,protein structure and phylogenetic relationship of the Cb FLAs gene family by bioinformatics method.The results showed that all members of the Cb FLAs family had conserved glycosylated sites,and the functional localization of most members at the subcellular level was shown in the membrane system.The tissue expression profile of Cb FLAs family showed diversity.Cb FLA1,7,8 and 11 were highly expressed only in the xylem.At the same time,Cb FLA1 and Cb FLA11 had higher expressed and protein levels in TW rather than in OW and NW.Based on the above results,we screened and cloned Cb FLA1 which had a potential influence on xylem development of C.bungei.Further,the Cb FLA1 overexpression vector was constructed and a heterologous transformation experiments were carried out in Arabidopsis thaliana.The results of transgenic experiments showed that Cb FLA1 expression promoted xylem differentiation and fiber development of A.thaliana.This preliminarily confirmed the hypothesis that Cb FLA1 may affect the wood formation of C.bungei.In summary,my Ph D project explored the potential transcriptional regulation mechanism of wood formation of C.bungei using the TW model in C.bungei.It was found that(1)the significant change of gene expression in the biosynthesis pathway of cellulose and pectin might contribute to the thin fiber cell wall in TW;(2)The negative regulation of CAD protein translation by mi RNA860 might lead to the limitation of lignin monomer biosynthesis in TW.(3)The synergistic regulation of various endogenous hormones,especially IAA and c Z,in the wood has a crucial role in the TW formation in C.bungei.(4)Cb FLA1 is highly expressed in TW.Overexpression of Cb FLA1 can promote xylem and fiber development in A.thaliana.These results enriched our knowledge of the TW formation and would help to improve the theoretical foundation for the molecular mechanism of wood formation and genetic improvement of wood quality in C.bungei.