Abstract:

Objective: The aim of this study was to clone and identify NAC (NAM, ATAF1/2 and CUC) transcription factors related to secondary cell wall formation in Populus trichocarpa. Based on the analysis of tissue-specific expression pattern, the phenotype and downstream regulatory network of transgenic plants, our results provide some new insights into the mechanism of the gene regulatory network of secondary cell wall biosynthesis in poplar, laying a foundation for the use of this gene in forest germplasm innovation. Method: Based on the previous transcriptome data, PtrNAC128, which was preferentially expressed in the stems of P. trichocarpa, was cloned. Relative expression of PtrNAC128 in roots, stems and leaves were analyzed by qRT-PCR analysis. The subcellular localization of PtrNAC128 was analyzed by gene gun bombardment method; 35S∷PtrNAC128-GFP plant binary expression vector was constructed and transformed to Populus tomentosa. Anatomical cross-sections of transgenic and wild-type plants were observed by paraffin analysis method, and the effects of PtrNAC128 overexpressing on the components of poplar secondary cell wall were examined. The expression fluctuation of key enzyme genes and NAC, MYB transcription factors which regulating the biosynthesis of lignin, cellulose and hemicellulose synthesis were detected by qRT-PCR analysis. Result: PtrNAC128 clustered in one branch with Arabidopsis ANAC075, with typical domains and intact C terminal activation regions. PtrNAC128 was expressed in all tissues tested, with the highest expression level in old stems and the lowest expression level in leaves. The expression of PtrNAC128 in old and young stems was 23.49 folds and 11.44 folds higher than that in young leaves, respectively. Subcellular localization analysis indicated that PtrNAC128 was localized into the nucleus. The secondary xylem width of PtrNAC128-overexpression (PtrNAC128-OE) transgenic plants had significantly increased to 1.42-1.51 folds, while the number of secondary xylem cells increased to 1.22-1.31 folds compared to the wild-type plants. The Klason lignin contents in the stems of PtrNAC128-OE lines were significantly increased by 12%-22% compared to the wild-type plants. Moreover, no significant effect was detected on the hemicellulose biosynthesis, while cellulose content increased by 7.4%-13.1% in the PtrNAC128-OE lines. The qRT-PCR analysis revealed that the relative expression levels of key synthetic enzyme genes of lignin and cellulose as well as some secondary wall-associated NAC and MYB transcription factors were elevated in the PtrNAC128-OE lines. Conclusion: These results suggested that PtrNAC128 may positively regulate the secondary cell wall biosynthesis by activating the enzyme genes and transcription factors of lignin and cellulose biosynthesis in poplar.

Key words: Populus trichocarpa, PtrNAC128, transgenic, secondary cell wall component