Fulvic acid and hydrogen sulfide synergistically mitigate chromium stress in tobacco seedlings by regulating oxidative stress indicators and antioxidant defense mechanisms

Chromium (Cr) toxicity poses a significant threat to crop growth and agricultural productivity. Fulvic acid (FA) and hydrogen sulfide (HS) have emerged as promising agents for mitigating metal-induced toxicity; however, their combined effects on physiological and biochemical responses under Cr stress remain largely unexplored. This study investigated the synergistic effects of FA and HS on Cr accumulation, seedling growth, physio-biochemical attributes, leaf ultrastructure, and the expression of photosynthesis-related genes in tobacco seedlings subjected to Cr stress. Cr stress significantly inhibited plant growth, physiological and biochemical performance, gas exchange parameters, and chlorophyll synthesis, while increasing Cr accumulation in soil, roots, and shoots. In contrast, FA and HS applications, particularly in combination, significantly alleviated Cr toxicity. The combined treatment improved seedling growth, enhanced chlorophyll fluorescence efficiency, and promoted gas exchange under Cr stress. It also strengthened antioxidant defense, as indicated by increased activities of superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%), and ascorbate peroxidase (APX; 49%), which are crucial for reactive oxygen species (ROS) scavenging. Transmission electron microscopy analysis confirmed that FA and HS mitigated Cr-induced ultrastructure damage in leaf cells, whereas scanning electron microscopy revealed improved stomatal characteristics under combined treatment. Moreover, Cr accumulation in roots and shoots was significantly reduced. qRT‒PCR analysis showed that FA and HS regulated chlorophyll biosynthesis- and chloroplast-related genes, including CHLG (32.35%), CCD4 (71.24%), PsbO (93.30%), and PsbQ (105.90%), compared with the Cr-stressed control. Overall, the combined application of FA and HS effectively alleviates Cr-induced toxicity by reducing Cr accumulation, strengthening antioxidant defense systems, preserving leaf ultrastructure, and enhancing photosynthetic capacity and overall growth performance.