PagMIR166c targets PagECH2 to regulate cambial differentiation into secondary xylem, cell expansion, and lignin deposition via different auxin signaling pathways in poplar stems.

Secondary xylem, a key trait of trees, is the main source of tree biomass. MicroRNAs (miRNAs) play important regulatory roles in the secondary xylem formation; however, the underlying regulatory mechanisms are far from being completely elucidated. In this study, we provide the first evidence that PagMIR166c is involved in the secondary xylem formation in poplar stems. Overexpression of PagMIR166c inhibited cambial differentiation into secondary xylem and cell expansion, but promoted lignin deposition and cell wall thickening in poplar stems. Consistently, CRISPR/Cas9-mediated pagmir166c mutants showed an opposite phenotype. 5' RNA ligase-mediated rapid amplification of cDNA ends, degradome sequencing, and dual-luciferase reporter assay showed that PagMIR166c targets PagECH2, a key gene for IAA biosynthesis. PagECH2-overexpressing (PagECH2-OE) plants showed a phenotype opposite to that of PagMIR166c-overexpressing (PagMIR166c-OE) plants, but similar to that of pagmir166c mutants. It was also found that the expression levels of auxin response factor genes (PagARF2/PagARF3/PagARF4/PagARF5/PagARF7) were significantly down- or upregulated in PagMIR166c-OE and PagECH2-OE plants as well as pagmir166c mutants compared to wild-type. Taken together, we identified a novel module, PagMIR166c-PagECH2, and elucidated the mechanism by which this module regulates the secondary xylem formation via different auxin signaling pathways in poplar stems.