Brassinosteroids enhance cold tolerance in Elymus nutans via mediating redox homeostasis and proline biosynthesis

ATSTRACT Brassinosteroids (BRs) are a class of polyhydroxylated steroidal phytohormones that induce plant tolerance to a wide variety of stresses. Cold stress is a major environmental factor that limits plant growth. However, the mechanisms by which BRs regulate cold tolerance in alpine plants remains unclear. We analyzed the effects of 24-epibrassinolide (EBR) on growth, antioxidant defense, proline metabolism, and cold stress-related genes in Elymus nutans seedlings exposed to cold stress. Cold stress increased the transcript levels of BR-biosynthetic genes and the endogenous levels of BRs in E. nutans, which indicates the role of BRs in regulating cold tolerance. In addition, cold stress inhibited plant growth and increased malondialdehyde and reactive oxygen species (ROS) accumulation. However, application of EBR (1 μM) triggered catalase (CAT) activity and ascorbate (AsA)-reduced glutathione (GSH) cycle, thereby leading to lower ROS levels and plant growth inhibition. BR-induced cold tolerance was associated with an increase in proline levels. Pretreatment with EBR prior to cold stress improved the activities of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (P5CR), while slightly reduced the activities of proline dehydrogenase (PDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). Increased transcript levels of P5CS and P5CR genes and decreased transcript level of PDH1 were detected in EBR-pretreated seedlings. Exogenous proline could alleviate the plant damage caused by cold stress in E. nutans and Arabidopsis bak1 mutant. Furthermore, bak1 mutant accumulated less proline compared with wild type plants, which indicates the close association between BR-mediated cold tolerance and proline metabolism. EBR Pretreatment also induced the expression of cold stress-related genes. These findings suggest that BRs enhance cold tolerance by inducing cold stress-related genes, which further regulates antioxidant defense and proline accumulation.