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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 alt  , V t  , 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 .

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