The gasotransmitters hydrogen sulfide and nitric oxide independently regulate growth, physiology, and secondary metabolism in fenugreek (Trigonella foenum-graecum L.).

Fenugreek (Trigonella foenum-graecum L.) is a highly valued aromatic herb known for its therapeutic, cosmeceutical, and pharmacological uses. The key secondary metabolites of fenugreek, including trigonelline and diosgenin, exhibit noteworthy antidiabetic and anticarcinogenic properties. The present study examines the putative roles of the signaling molecules, such as hydrogen sulfide (H2S) and nitric oxide (NO), in regulating plant growth and development. The foliar application at concentrations of 0, 25, 50, 100, and 200 μM significantly improved the physiological and agronomic performance of fenugreek. Furthermore, the greatest improvements in growth, photosynthetic efficiency, physiological traits, and secondary metabolite production occurred at optimal concentrations of H2S (100 μM) and NO (200 μM) compared with the control. The applied concentrations significantly enhanced plant biomass, seed yield, and key traits, including total chlorophyll, carotenoid, and chlorophyll fluorescence. Both elicitors independently accelerate carboxylation efficiency and nitrogen metabolism by stimulating carbonic anhydrase and nitrate reductase activities in fenugreek plants. They also induce the production of antioxidant enzymes (SOD, CAT, POX, APX, and PPO), osmolytes (proline and glycine betaine), phenolic, flavonoid, and alkaloid content in fenugreek. Additionally, H2S and NO significantly increased trigonelline production compared with the control group, likely through the modulation of redox homeostasis and elicitor-mediated stimulation of antioxidants and secondary metabolite biosynthetic pathways. Overall, the findings provide new evidence that both signaling molecules autonomously regulate primary and secondary metabolism in fenugreek's growth and biosynthetic yield, highlighting their independent roles beyond their well-known interactive effects. Therefore, H2S and NO can be employed as effective elicitors in a wide range of crops.