Abstract: Precise phenological regulation is critical for temperate trees to survive the winter. However, the underlying mechanism is still unclear. Here, we found that Pag4CL3 coordinately modulates lignin biosynthesis and melatonin accumulation in 84 K poplar (Populus alba × P. glandulosa). Overexpression of Pag4CL3 or Pag4CL5 increased the lignin content in stem but reduced plant growth. In contrast, knockout of either gene reduced stem lignin monomers, promoted growth, and improved cold tolerance, with Pag4CL3 mutants (4cl3) exhibiting more pronounced resistance. PagSNAT2, which encodes a key enzyme in melatonin (MT) biosynthesis, is markedly upregulated in the 4cl3 mutant. Consistent with this, overexpression of PagSNAT2 promoted MT accumulation in 84 K poplar, and the 4cl3 mutant exhibited significantly higher MT levels in both autumn dormant and spring sprouting buds compared to the wild-type. Yeast two-hybrid (Y2H) and luciferase complementation assays further confirmed that Pag4CL3 directly interacts with PagSNAT2. Additionally, low temperature inhibited the binding of transcription factor PagDRS1 to the Pag4CL3 promoter and attenuating its suppression of melatonin synthesis. This study thus unveils a cold-responsive PagDRS1–Pag4CL3–PagSNAT2 regulatory module that balances structural formation and stress adaptation in trees, providing a theoretical basis and breeding strategy for developing poplar varieties with enhanced biomass and winter resilience.

Key words: Pag4CL3, Pag4CL5, 84 K poplar, Melatonin, Cold