Identification And Functional Analysis Of Genes Involved In Secondary Growth In Neolamarckia Cadamba

Neolamarckia cadamba is one of the most important fast-growing timber trees with fine texture in south China.Its rapid secondary growth mainly depends on the cell activity of vascular cambium,including cambium cell proliferation and differentiation.However,the molecular mechanism of the secondary growth and development regulation in woody plants remains unclear because of the difficulty in isolating the cambial cells accurately.It is an efficient method to identify the key genes involved in the regulation of secondary growth and development by comparative transcriptomic analysis of different cell types in vascular tissue,which would be benefit for exploration of the regulatory mechanism of secondary growth and development.In this study,we developed vascular tissue cells capture system by laser microdissection using N.cadamba seedlings as materials through the optimization of paraffin section system and the adjustment of LMD parameters,and high-quality RNA was isolated from dissected xylem,phloem and cambium cells accordingly.The different expressed genes(DEGs)were identified by RNA-seq,followed by gene ontology(GO)terms and KEGG pathways enrichment analysis,and key genes associated with secondary growth were therefore investigated.Our study would provide clues for understanding the genetic improvement of fast-growing and high-quality traits in forest tree species.The results are as follows:1.The growth nodes from primary to secondary growth were determined.The first to fourth stems of N.cadamba seedlings were prepared into paraffin sections.Then,the development of vascular tissue in different internode was observed with saffron staining.The results show that the peripheral vascular bundle of the first stem node was incompletely developed with only 2-3 layers of the xylem and phloem cells,which was defined as the development stage of the primary growth.In the second stem node,the peripheral vascular bundle was basically formed,and xylem and phloem tissues were thickened to form secondary xylem and secondary phloem,which was a transitional stage from primary to secondary growth.The peripheral vascular bundles of the third and fourth stem nodes were fully developed,and the secondary xylem and phloem were structurally intact,which was defined as the secondary growth stage.2.The vascular tissue cells capture system by laser microdissection(LMD)was established in N.cadamba.In this study,the paraffin sectioning was optimized by comparison of different combinations of fixative,dehydration and infiltration,and the LMD parameters of vascular tissue cells in N.cadamba were set with magnification,energy,aperture and speed.Accordingly,the vascular tissue cells capture system by laser microdissection was established in N.cadamba.High-quality RNA was isolated from dissected cambium,phloem and xylem cells,respectively,which can be used for subsequent RNA-seq analysis.3.Through transcriptome sequencing analysis,803,109 and 1457 DEGs were identified in the secondary xylem,secondary phloem,and cambium in the transitional stage from primary to secondary growth.Interestingly,among the different expressed genes in cambium,WOX4.1 and WOX4.2 were highly expressed during the transition from primary to secondary growth.4.Identification and expression pattern analysis of WOX gene family in N.cadamba.19 members of the WOX family were identified in the genome of N.cadamba.Among them,expression of WOX4.1 and WOX4.2 were significantly higher in cambium than in xylem and phloem,and WOX13.1 and WOX13.2 were expressed in high levels in cambium and phloem,but low in the xylem expression.5.Heterologous expression of Nc WOX4.1,Nc WOX4.2,Nc WOX13.1 and Nc WOX13.2.The full-length CDS and promoter of WOX4.1,WOX4.2,WOX13.1 and WOX13.2 genes of N.cadamba and the promoter of WOX4 and WOX13 from Arabidopsis were cloned.The N.cadamba native promoter-driven overexpression vectors of the above four genes were constructed,and as well as the promoter from homozygous WOX in Arabidopsis driven the corresponding genes.These constructs were subsequently transformed into Arabidopsis.So far,T0 generation seeds of transgenic Arabidopsis have been obtained.