Drought stress differentially suppresses growth and triggers antioxidant and anthocyanin pathways in Brassica oleracea (C3) and Echinochloa crusgallii (C4).

Although drought responses of C3 and C4 plants have been widely investigated, direct comparative analyses of their physiological and detailed metabolic biosynthetic adaptations remain limited. This study provided a detailed pathway-level analysis of drought stress responses in a Brassica oleracea (kale, C3) versus Echinochloa crus-galli (barnyard grass, C4) moving beyond previous comparisons that typically focus on broad physiological differences. Our study integrates multi-level physiological, molecular, biochemical, and metabolic data, emphasizing specific drought-adaptive biosynthetic pathways such as anthocyanin and glucosinolate synthesis. Drought stress significantly reduced biomass and photosynthetic efficiency (p < 0.05) in both species, with kale exhibiting greater declines. Kale also accumulated higher oxidative stress indicators, with H₂O₂ and lipid peroxidation (MDA) increasing by 21.81% and 113%, respectively, relative to barnyard grass, indicating stronger oxidative damage. Both species significantly upregulated antioxidants metabolites and enzymes, reflecting activation of protective mechanisms. Anthocyanin content rose by 83% in barnyard grass and 113% in kale under drought. Detailed pathway analysis revealed elevated precursor levels (phenylalanine and p-coumaroyl-CoA) and increased activities and gene expression of key enzymes in the phenylpropanoid pathway, including cinnamate-4-hydroxylase (C4H), chalcone synthase (CHS), and 4-coumarate-CoA ligase (4CL), highlighting the specific C3-C4 photosynthetic-type biochemical strategies for drought adaptation. The strong activation of defense pathways such as anthocyanin and glucosinolate biosynthesis suggests that kale compensates for its lower drought tolerance by increasing metabolic flexibility. Molecular evidence further supports induced expression of anthocyanin related biosynthetic genes. The detailed, multi-level metabolic analysis uncover the -specific C3-C4 photosynthetic-type biochemical strategies for drought adaptation. This work advances plant physiology knowledge by revealing how C3 and C4 plants differ in metabolic drought adaptations, offering practical relevance for both crop improvement and weed management. This reveals key physiological and biochemical differences, with kale (C3) needing more metabolic adjustment to handle drought.