Functional Characterization and Promoter Analysis of a MeJA-Responsive Squalene Synthase Gene CpSQS from Cyclocarya paliurus (Batal.) Iljinsk.

Cyclocarya paliurus leaves are rich in bioactive triterpenoids, yet their regulatory mechanisms, particularly in response to methyl jasmonate (MeJA), remain unclear. This study aimed to characterize the spatial and temporal effects of MeJA on triterpenoid accumulation, identify MeJA-responsive genes involved in triterpenoid biosynthesis, and functionally evaluate CpSQS and its promoter regulatory regions. Upper and lower leaves were sampled at multiple time points after MeJA treatment. Six triterpenoid monomers were quantified by HPLC, and transcriptomes were analyzed at 5 and 10 days. MeJA altered triterpenoid accumulation in a time- and leaf-position-dependent manner, with stronger responses in upper leaves. Differentially expressed genes were enriched in terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, plant hormone signaling, and MAPK pathways, while HMGR, FPPS, SQS, and SE were generally upregulated. CpSQS was induced by MeJA and showed a spatial expression pattern associated with triterpenoid accumulation. Heterologous overexpression of CpSQS in tobacco calli significantly increased CpSQS transcript abundance and total triterpenoid content, supporting its positive role in triterpenoid biosynthesis. Promoter deletion analysis further identified a 188 bp region from -961 to -773 bp that contributed substantially to promoter activity, with its deletion reducing GUS activity by 56%. These findings reveal the spatiotemporal regulation of triterpenoid biosynthesis by MeJA and identify CpSQS and its transcriptionally active promoter region as potential targets for further functional studies and metabolic improvement in C. paliurus.