Abstract:

Objective: The secondary vascular system(SVS) regeneration can regenerate vascular tissue through tissue repairing such as past differentiation, re-differentiation or transdifferentiation after the loss of vascular system. In order to identify and study the important genes and regulatory elements related to the development of SVS, we analyzed the gene expression patterns at different regeneration stages of SVS regeneration of Populus tomentosa. It laid a foundation for further revealing the gene regulation mechanism of wood formation. Method: Four-year-old P. tomentosa clones were girdled and sampled during the active period of cambium. High throughput transcriptome analysis was performed on the inner bark and trunk surface samples after girdling, as well as the regenerated tissue obtained on the 7^th, 10^th, 14^th, 18^th and 21^st days after the girdling. Weighted gene co-expression network analysis(WGCNA) was used to obtain the modules closely related to different regeneration stages, and the expression network and biological function of these specifically expressed genes were analyzed. Result: The 14 202 differentially expressed genes, obtained by comparing differences between the samples of inner bark and trunk surface, 7^th day and trunk surface, and adjacent regeneration periods, were analyzed by WGCNA. The gene co-expression network was constructed and 10 modules related to the regeneration of SVS were obtained. Among them, the genes in Grey60 module, which were specifically expressed in the samples on the 7^th day of regeneration and inner bark, mainly involved in DNA replication, mitosis, cell division and microtubule movement. The module also contained a large number of genes related to epigenetics and cell cycle. Their high expression on the 7^th day of regeneration may activate the proliferation of dedifferentiated xylem cells and the change of tissue type, which enabled them to re-differentiate. In addition, the expression values of genes in the Pink module were very low in the mature vascular tissue samples on day 0, but they showed an upward trend in the process of regeneration, and reached the highest values on 21^th day. These genes were mainly involved in cell division, cell wall modification, phloem development, carbohydrate metabolism, DNA transcription regulation and other related biological pathways. In the Pink module, the expression of some phloem related marker genes, such as APL, NAC45/86, DOF, BSPA, PP2 and SUS, were reactivated. Same as CLE41/44 and CLV1 that can regulate the proliferation and differentiation of cambium cells, their expression level increased gradually from the 10^th day. At the late stage of SVS regeneration on the 18^th and 21^st day, the re-differentiated cambium had completed structural reconstruction and could undergo cell division and differentiation. The xylem marker genes and secondary wall regulatory factors were highly expressed at this stage. Conclusion: At the early stage of SVS regeneration of P. tomentosa, the dedifferentiated xylem cells regained the ability of cell division through epigenetic and cell cycle regulation; then the cell differentiation of phloem was started, and the regeneration cambium began cell proliferation and differentiation; at the late stage of regeneration, the cambium re-differentiated into xylem and phloem through the regulation of related genes. Through the analysis of gene expression and identification of regulatory modules in the process of SVS regeneration, we further verified the genetic regulatory basis of SVS regeneration, and also laid a foundation for revealing the regulatory mechanism of plant vascular tissue regeneration.

Key words: poplar, secondary vascular system, regeneration, gene expression, WGCNA