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 plants development and responses to biotic/abiotic stresses. In this study, we functionally characterized the WRKY transcription factor PagWRKY39a as a repressor of stem secondary growth in Populus. PagWRKY39a expression level is significantly higher in secondary phloem and cambium compared to early expanding secondary xylem. Compared to wild type (WT), overexpression of PagWRKY39a (PagWRKY39a-OE) significantly decreased stem diameter and plant height while knockout of PagWRKY39a (PagWRKY39a-KO) are not significantly different from WT. Further analysis showed that the vascular cambium cell layers, secondary xylem width, and secondary cell wall (SCW) thickness were significantly reduced in PagWRKY39a-OE but significantly increased in PagWRKY39a-KO. Transcriptome analysis revealed that genes related to mitotic cell cycle and SCW biosynthesis are significantly down-regulated in PagWRKY39a-OE plants while no significant changes in PagWRKY39a-KO plants. We also performed DNA affinity purification followed by sequencing (DAP-seq) assays, in 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.