Objective  The properties of wood are determined by the composition and characteristics of xylem cell wall. It is important for wood improvement to study the molecular regulation mechanism of xylem cell wall formation. In this study, the molecular regulatory mechanisms in the process of regeneration of primary wall in the protoplast of birch (Betula platyphylla) xylem were analyzed and the important regulatory genes were identified, aiming to provide data and materials for the study of wood properties.

  Method  The protoplasts of birch xylem cultured for 0 h and 2 h were used as materials, respectively, and the regeneration process of primary cell wall was observed by staining with calcofluor white. The differentially expressed genes (DEGs) and differential metabolic pathways participating before and after the regeneration of the primary wall were compared by transcriptomic analysis. The detected DEGs were aligned with GO, KEGG and PlantTFDB.

  Result  Observation by fluorescence microscopy showed that protoplasts had no cell walls after enzymatic digestion and the primary cell walls had been regenerated after 2 h culture. The DEGs were screened using |log₂(FC)| ≥ 1(fold change, FC) and q < 0.05 as the parameters. The results showed that compared with the protoplasts without cell wall, 4396 up-regulated genes and 4056 down-regulated genes were detected in the protoplasts cultured for 2 h, with a total number of 8452 DEGs. In the GO database, there were 10 significantly up-regulated terms. KEGG analysis noted 10 significantly different metabolic pathways. Total of 360 differentially expressed transcription factors from 16 families was annotated in PlantTFDB database. The GO database annotation results showed that DNA replication and cell cycle related genes were up-regulated in the cell wall regeneration lines compared with that in control. The results of KEGG database annotation showed that genes involved in glutathione, α-linolenic acid and other stress-related metabolism genes were down-regulated, and pectinester related genes were up-regulated. PlantTFDB annotation results showed that bHLH, NAC, MYB, bZIP and other transcription factors closely related to cell wall biosynthesis were differentially expressed.

  Conclusion  Above results show that xylem protoplasts are in a state of cell wall regeneration and division preparation at 2 h culture. DNA replication, cell cycle, polysaccharide biosynthesis and other related genes play a regulatory role in xylem protoplast culture and primary cell wall formation of B. platyphylla.