Drought tolerance mechanisms across C3 and C3–C4 intermediate photosynthetic types revealed by physiological and gene expression profiling

Abiotic stress, particularly drought, significantly reduces crop yields and threatens global agricultural sustainability. This study investigated drought and recovery responses in four plant species with contrasting photosynthetic types: Triticum aestivum (C3), Helianthus annuus (C3), Chenopodium album (intermediate-C4), and Alternanthera brasiliana (C4-like). Drought markedly reduced plant fresh biomass (up to 80% in H. annuus) and relative water content, particularly in C. album. Oxidative damage intensified, with H. annuus showing the greatest increase in hydrogen peroxide (258%) and C. album exhibiting the highest malondialdehyde accumulation (284%). Antioxidant enzymes were strongly activated; catalase activity increased dramatically in C. album (837%) and H. annuus (630%). Proline levels increased sharply, particularly in T. aestivum and C. album, indicating enhanced osmotic adjustment. Carotenoid content also rose significantly in H. annuus (141%), suggesting photoprotective adaptation. Gene expression analysis revealed upregulation of TaP5CS in T. aestivum, correlating with proline accumulation, and CaHSP26 in C. album, potentially stabilizing photosystem II. Principal component analysis identified catalase activity, root-to-shoot ratio, hydrogen peroxide, and proline as major contributors to drought response variance. These findings highlight species-specific strategies for drought tolerance and recovery, with C3 species showing strong enzymatic and osmotic adjustments, and intermediate-C4 and C4-like species exhibiting greater tissue integrity and ROS balance. This comparative framework provides valuable insights for developing drought-resilient crops.