OPLS-DA-guided physiological trait dissection and WGCNA-driven network analysis uncover key metabolic pathway and candidate genes under salt stress in Taxodium mucronatum

Soil salinization increasingly constrains global forestry and agricultural productivity; breeding high-yielding, salt-tolerant cultivars is therefore the most effective strategy to mitigate yield losses. Taxodium mucronatum , possesses significant ecological and economic value; however, the molecular mechanisms underlying its salt stress response remain unknown. In this study, we exposed T. mucronatum seedlings to soil augmented with 3 ‰ NaCl and analyzed their responses at 0,15, 30, and 60 days post-treatment (DPT) through growth phenotyping, physiological assays, and RNA sequencing. The results revealed that salt stress reduced the relative increase in seedling height and aboveground biomass, whereas no significant difference was observed in root growth. Membrane damage, quantified by malondialdehyde (MDA) and electrolyte leakage (REL), peaked at 30 DPT. Proline (PRO) content increased monotonically, whereas soluble sugars (SS) displayed a fluctuating pattern (decrease-rise-decline); soluble proteins (SP) declined sharply at 15 DPT and remained low thereafter. Peroxidase (POD) and superoxide dismutase (SOD) activities exhibited a unimodal response, peaking at 15 and 30 DPT, respectively, whereas catalase (CAT) activity increased steadily until 60 DPT. OPLS-DA identified POD, PRO, and SOD as the most critical physiological indicators. RNA sequencing across the 12 libraries generated 47,536 de novo assembled unigenes, among which 4691 were consistently differentially expressed across all time points. Weighted gene co-expression network analysis (WGCNA) identified key modules (greenyellow, indianred4, ivory) strongly correlated with the crucial physiological traits. A total of 466 hub genes were screened by integrating consistently differentially expressed genes. Comprehensive KEGG enrichment analysis indicated that phenylpropanoid biosynthesis is a major pathway involved in salt adaptation. Within this pathway, key genes including PER , CAD , OMT , HCT , CCR , PAL and C4H were identified, linking lignin deposition with reactive oxygen species scavenging. These findings provide systematic insights for molecular breeding of salt-tolerant T. mucronatum and offer new directions for enhancing stress resistance in woody crops. • First time-series of salt stress integrating physiology & transcriptome in T. mucronatum. • POD, PRO and SOD identified as top salt indicators by OPLS-DA. • WGCNA pinpoints 466 hub genes tightly linked to protective traits. • Phenylpropanoid pathway drives ROS scavenging and lignification.