Hydrogen Peroxide and Nitric Oxide Crosstalk Mediates Brassinosteroids Induced Cold Stress Tolerance in Medicago truncatula

Brassinosteroids (BRs) play pivotal roles in modulating plant growth, development, and stress responses. In this study, a Medicago truncatula plant pretreated with brassinolide (BL, the most active BR), enhanced cold stress tolerance by regulating the expression of several cold-related genes and antioxidant enzymes activities. Previous studies reported that hydrogen peroxide (H₂O₂) and nitric oxide (NO) are involved during environmental stress conditions. However, how these two signaling molecules interact with each other in BRs-induced abiotic stress tolerance remain largely unclear. BL-pretreatment induced, while brassinazole (BRZ, a specific inhibitor of BRs biosynthesis) reduced H₂O₂ and NO production. Further, application of dimethylthiourea (DMTU, a H₂O₂ and OH^- scavenger) blocked BRs-induced NO production, but BRs-induced H₂O₂ generation was not sensitive to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a scavenger of NO). Moreover, pretreatment with DMTU and PTIO decreased BL-induced mitochondrial alternative oxidase (AOX) and the photosystem capacity. However, pretreatment with PTIO was found to be more effective than DMTU in reducing BRs-induced increases in V, V, and MtAOX1 gene expression. Similarly, BRs-induced photosystem II efficiency was found in NO dependent manner than H₂O₂. Finally, we conclude that H₂O₂ was involved in NO generation, whereas NO was found to be crucial in BRs-induced AOX capacity, which further contributed to the protection of the photosystem under cold stress conditions in Medicago truncatula.