PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20 modules positively regulate cambial activity and its differentiation into secondary xylem in poplar.

Stem secondary xylem produced by cambial division and differentiation is the main source of tree biomass. The secondary xylem formation involves a complex transcriptional regulatory network, however, the underlying mechanism is still being explored. Here, we report that PagHAM4a and PagHAM4b are positive regulators of cambial differentiation into secondary xylem in hybrid poplar (Populus alba × Populus glandulosa clone 84K). Overexpression of PagHAM4a and PagHAM4b enhanced cambial activity and increased the number of secondary xylem cells in the stems of poplar. By contrast, single or double mutations of PagHAM4a and PagHAM4b by CRISPR-Cas9 decreased cambial activity, leading to a significant reduction of secondary xylem. Neither overexpression nor mutation of the two genes affected the size of vessels and fibers in xylem. Both PagHAM4a- and PagHAM4b-regulated gene networks were mainly centered in the stage when cambium had just initiated secondary growth, but the molecular networks regulated by the two genes were distinct. Further analysis revealed that PagSCL21 and PagTCP20 are direct targets of PagHAM4a and PagHAM4b, respectively, and their overexpression also promoted cambial differentiation into secondary xylem. Taken together, we identified two novel key regulatory modules in poplar, PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20, which provide new insights into the mechanism of secondary xylem formation in trees.