Elevated proline and melatonin alter the methylation pattern in the promoter of their biosynthesis genes in rice

Salt stress upregulates osmoprotectants proline and melatonin in plants, and their exogenous application under salt stress can protect them from stress. While melatonin-induced plant epigenetic reprogramming is known in plants, such a mechanism is yet to be identified in the case of proline. The studies conducted so far used proline and melatonin along with one or more stress factors to look into their stress-alleviating effects. We investigated the impact of externally applied proline and melatonin on the methylation of promoter sequences of proline and melatonin biosynthesis genes in 14-day-old rice plants using a combination of biochemical, bioinformatics, and molecular techniques. Our findings demonstrate that exogenously applied proline and melatonin elevate endogenous levels of these compounds in rice, mimicking stress conditions in plants, as the biochemical assays corroborated the activation of antioxidant systems, particularly ascorbate peroxidase (APX) and catalase (CAT). Bioinformatics analysis unveiled multiple stress-responsive transcription factor binding sites on gene promoters associated with proline and melatonin biosynthesis pathways. Restriction analysis using the isoschizomer pair MspI/HpaII revealed distinct cytosine methylations at the restriction sites on the promoters analyzed in plants treated with proline and melatonin compared to the control. Differential methylation identified in the promoter sequences were matching with the biosynthesis of proline and melatonin and also the antioxidant enzyme levels. These observations are consistent with previous transcriptome data of proline and melatonin biosynthesis genes, providing insights into underlying regulatory mechanisms of proline and melatonin biosynthesis, their role in epigenome control during abiotic stress, and the evolution of various stress-tolerant varieties.