Integrating Metabolic and Gene Expression Profiling of Glucosinolate Biosynthesis Under Drought Stress in Brassica oleracea

Drought stress induces pronounced metabolic and transcriptional reprogramming of glucosinolate (GLS) biosynthesis in <i>Brassica oleracea</i>. An integrative approach combining HPLC-based quantification of individual GLSs, quantitative real-time PCR of core biosynthetic and regulatory genes, correlation-based network analysis, and in silico promoter characterization was applied to evaluate drought responses across genetically diverse accessions. Drought triggered strong, accession-specific shifts in GLS composition, with sinigrin content increasing from 35.9% to 55.1% in BR1 and glucoerucin reaching up to 80.2% in CCP1, while indolic GLSs such as glucobrassicin and neoglucobrassicin accounted for >75% of total GLSs in CV2 and CCP3. Hierarchical clustering separated accessions into four distinct drought response clusters independent of morphotype. Correlation analysis revealed drought-induced rewiring of GLS interdependencies, characterized by strengthened positive associations among aliphatic GLSs (r > 0.75). Gene expression profiling identified conserved MYB-centered regulatory modules (<i>MYB28</i>, <i>MYB29</i>, <i>MYB34</i>, <i>MYB122</i>) alongside strong accession-specific induction of <i>CYP79F1</i> (up to 6.3-fold), <i>FMOGS-OX5</i> (up to 4.8-fold), and <i>ST5a</i> (up to 5.1-fold). Promoter analysis revealed enrichment of ABA- and stress-responsive cis-regulatory elements. These findings delineate a genotype-dependent regulatory framework underlying GLS plasticity and identify quantitative metabolic and transcriptional markers relevant for breeding drought-resilient <i>Brassica</i> cultivars.