A dual role for a member of the WRYK family in regulating both cambial cell division and secondary cell wall formation in Populus

WRKY transcription factors are unique to plants and play diverse roles in plant development and responses to biotic and abiotic stresses. In this study, we functionally characterized the WRKY transcription factor PagWRKY39a as a repressor of stem secondary growth in Populus (Populus alba×Populus glandulosa). PagWRKY39a expression level was significantly higher in secondary phloem and cambium compared with early expanding secondary xylem. Compared with wild type (WT), overexpression of PagWRKY39a (PagWRKY39a-OE) significantly decreased stem diameter and plant height, while plants with knockout of PagWRKY39a (PagWRKY39a-KO) were not significantly different from WT. Further analysis showed that the vascular cambium cell layers, secondary xylem width, and secondary cell wall thickness were significantly reduced in PagWRKY39a-OE but significantly increased in PagWRKY39a-KO plants. Transcriptome analysis revealed that genes related to mitotic cell cycle and secondary cell wall biosynthesis were significantly down-regulated in PagWRKY39a-OE plants while showing no significant changes in PagWRKY39a-KO plants. We also performed DNA affinity purification followed by sequencing assays, coupled with yeast one-hybrid and expression analysis, to identify PagWRKY39a direct target genes during secondary growth. Our results showed that PagHB4, a key regulatory gene of secondary growth, was directly bound and repressed by PagWRKY39a. Together, our results demonstrated that PagWRKY39a plays an important role in regulating cambium activity and secondary xylem differentiation in Populus, which may have a significant effect on plant biomass production.