Integrated phenotypic, physiological, transcriptomic, and metabolomic analyses reveal water–nitrogen coupling regulation of lignin biosynthesis in Larix principis–rupprechtii

Water and nitrogen are key environmental factors that influence wood formation in trees, a process that depends largely on lignin deposition in the cell wall. However, regulation of lignin biosynthesis at the molecular level by water–nitrogen coupling in conifers remains largely unknown. In this study, three-year-old Larix principis–rupprechtii seedlings were subjected to three water regimes and three nitrogen levels. Growth measurements, anatomical observations, cell wall component assays, and enzyme activity analyses were integrated with transcriptomic and metabolomic profiling. Drought emerged as the primary factor limiting seedling growth and wood formation, and its inhibitory effect intensified with increasing water deficit. Nitrogen only modulated growth within the limits set by water availability. Lignin content declined progressively with increasing drought intensity, accompanied by reduced tracheid wall thickness, whereas cellulose and hemicellulose contents remained unchanged. Integrated omics analyses identified 6827 differentially expressed genes and 2122 differentially accumulated metabolites, with the phenylpropanoid pathway significantly associated with both lignin content and tracheid wall thickness. Within this pathway, POD93 emerged as a key responsive node at the terminal polymerization step of lignin biosynthesis, and its transcript levels dropped sharply under drought, with this decline further exacerbated by reduced nitrogen supply. Co–expression analysis and promoter cis-element prediction pinpointed MYB53 as a candidate regulator of POD93 ; subsequent yeast one-hybrid and dual-luciferase assays confirmed that the upstream transcription factor (TF) MYB53 activates POD93 transcription. Collectively, these findings establish a regulatory pathway linking water–nitrogen signals, a MYB TF, phenylpropanoid pathway genes, and the association between lignin content and tracheid wall thickness in a conifer species. Our work provides mechanistic insights into environment-dependent secondary metabolism and offers candidate gene resources for wood quality improvement in L. proncipis-rupprechtii .