Expression and functional analysis of NAC transcription factors under five diverse growth stages reveal their regulatory roles during wood formation in Chinese cedar (Cryptomeria fortunei Hooibrenk)

Abstract

NAC transcription factor (TF), the most prominent plant-specific gene family, is involved in various metabolic pathways during the growth and development of the plant. However, few reports have been published concerning the NAC gene family in gymnosperms such as Cryptomeria fortunei Hooibrenk. As an essential native coniferous timber tree, C. fortunei is widely cultivated in southern China due to its significant economic and ecological value. Here, we aimed to identify and characterize the CfNACs in C. fortunei vascular development. A total of 33 CfNACs were identified from C. fortunei vascular cambium. They all shared the conserved NAC motifs. Phylogenetic evolutionary indicated that CfNACs were classified into various evolutionary branches, including secondary xylem development, lignin and secondary cell wall (SCW) biosynthesis, and various stress response. In addition, the expression profiles of CfNACs under diverse developmental stages suggested that several CfNACs were involved in multiple metabolic pathways during C. fortunei cambium development. Moreover, the co-expression network revealed that numerous CfNACs were involved in vascular and xylem development, and lignin biosynthesis as key upstream regulators. There were significant correlations between CfNACs and lignin biosynthetic genes, phytohormone synthase genes. Transient expression analysis implied that CfNAC5 and CfNAC16, targeted to the nucleus, acted as activators. Genetic transformation analysis demonstrated that overexpression of CfNAC5 dramatically affected the secondary xylem development and elevated the lignin biosynthesis and SCW thickness in transgenic plants. Furthermore, the expression levels of lignin biosynthetic genes were markedly increased in transgenic plants. In conclusion, these findings would help provide insight into the functional differentiation and evolutionary relationships of CfNACs and elucidate the regulatory roles of CfNACs during C. fortunei vascular development and wood formation.