Poplar NF-YA11 alters lignin composition and increases methane yield by upregulating the FERULIC ACID 5-HYDROXYLASE 2 gene

Lignin impacts the cost and efficiency of poplar’s development and utilization. Transcription factors (TFs) play a crucial role in regulating lignin biosynthesis. However, investigations into the molecular mechanism involving NUCLEAR FACTOR Y subunit A (NF-YA) TFs in poplar lignin biosynthesis have yet been widely conducted. Here, PtNF-YA11 was identified in Populus trichocarpa. It is predominantly expressed in the xylem and is responsible for regulating S-lignin biosynthesis in poplar. Further investigation revealed that overexpression of PtNF-YA11 leads to an increase in S-lignin content, while the CRISPR-Cas9-edited nf-ya11 poplars exhibit a decrease in S-lignin composition. Reverse transcription quantitative PCR data revealed that several genes associated with lignin biosynthesis, including phenylalanine ammonia-lyase (PtPAL1), 4-hydroxycinnamoyl CoA ligase (Pt4CL1), caffeoyl CoA O-methyltransferase (PtCCoAOMT), and ferulate 5-hydroxylase (PtF5H1 and PtF5H2), were upregulated in PtNF-YA11-overexpressing poplars but downregulated in nf-ya11 poplars. Biochemical experiments have validated that PtF5H2 is a direct target gene of PtNF-YA11. The 2D nuclear magnetic resonance assay revealed that the S/G lignin ratio was indeed increased in PtNF-YA11-overexpressing poplars and decreased in nf-ya11 poplars, confirming that PtNF-YA11 regulates S-lignin biosynthesis by activating the expression of PtF5H2. Furthermore, compared with wild-type poplar, PtNF-YA11-overexpressing poplar showed a 17.8 % increase in methane yield during anaerobic digestion. Overall, this study provides a comprehensive understanding of the functions of poplar NF-YA transcription factors in regulating lignin biosynthesis and enhancing biofuel yields.