The PtoKNAT1-PtomiR6438a-PtoPOD38 axis controls lignin accumulation in Populus tomentosa

Through their ability to posttranscriptionally regulate gene expression in a targeted manner, microRNAs (miRNAs) can shape the growth and development of plants while also influencing their ability to respond to specific stressors. The species-specific evolution of miRNAs is thought to confer unique traits to individual plants, but further research is necessary to highlight the roles of these miRNAs in perennial woody plants. To address this knowledge gap, the genetic roles of Populus-specific miRNAs as regulators of wood development were herein explored through analyses of data derived from a natural population of 435 unrelated Populus tomentosa Carr. specimens. Initial analyses identified PtomiR6438a as a Populus-specific miRNA as a key candidate associated with the regulation of lignin accumulation. Further degradome sequencing revealed that PtomiR6438a can function as a negative regulator of PtoPOD38, which encodes a peroxidase gene involved in the polymerization of lignin monomers. Dual-luciferase assays confirmed the ability of PtoKNAT1 to bind the PtomiR6438a promoter region, suppressing the expression of this miRNA. In total, single nucleotide polymorphism (SNP)-based association analyses of the PtoKNAT1-PtomiR6438a-PtoPOD38 axis led to the identification of 34 significant SNPs (P < 0.01, Q < 0.05) related to five wood-associated traits, supporting the importance of this module as a regulator of wood formation. Subsequent epistasis analyses revealed 50 significant pairwise epistatic associations related to eight different wood formation-associated traits. Heterologously expressing PtoMIR6438a in Arabidopsis thaliana confirmed the ability of this miRNA to serve as a negative regulator of PtoPOD38 and thereby decrease peroxidase expression and lignin content in these cells. These results support the hypothesis that the PtoKNAT1-PtomiR6438a-PtoPOD38 axis regulates peroxidase activity, thus influencing lignin monomer polymerization and contributing to a reduction in lignin content. Overall, these findings will serve as a valuable foundation for future research aimed at clarifying the importance of transcription factor-miRNA-target gene networks as regulators of tree development.