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Abstract Diketopiperazines (DKPs) are chemically and functionally diverse cyclic dipeptides associated primarily with microbes. Few DKPs have been reported from plants and animals; the best characterized is cyclo(His-Pro), found in the mammalian central nervous system, where it arises from the proteolytic cleavage of a thyrotropin-releasing tripeptide hormone. Herein, we report the identification of cyclo(His-Pro) in Arabidopsis (Arabidopsis thaliana), where its levels increase upon abiotic stress conditions, including high salt, heat, and cold. To screen for potential protein targets, we used isothermal shift assays, which examine changes in protein-melting stability upon ligand binding. Among the identified proteins, we focused on the glycolytic enzyme, cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC1). Binding between the GAPC1 protein and cyclo(His-Pro) was validated using nano-differential scanning fluorimetry and microscale thermophoresis, and we could further demonstrate that cyclo(His-Pro) inhibits GAPC1 activity with an IC50 of ∼200 μm. This inhibition was conserved in human GAPDH. Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity has previously been reported to reroute carbon from glycolysis toward the pentose phosphate pathway. Accordingly, cyclo(His-Pro) supplementation augmented NADPH levels, increasing the NADPH/NADP+ ratio. Phenotypic screening revealed that plants supplemented with cyclo(His-Pro) were more tolerant to high-salt stress, as manifested by higher biomass, which we show is dependent on GAPC1/2. Our work reports the identification and functional characterization of cyclo(His-Pro) as a modulator of glyceraldehyde-3-phosphate dehydrogenase in plants. 

Abstract Posttranscriptional regulation of mRNA mediated by methylation at the N6 position of adenine (N6-methyladenosine [m6A]) has profound effects on transcriptome regulation in plants. Focused studies across eukaryotes offer glimpses into the processes governed by m6A throughout developmental and disease states. However, we lack an understanding of the dynamics and the regulatory potential of m6A during biotic stress in plants. Here, we provide a comprehensive look into the effects of m6A on both the short-term and long-term responses to pathogen signaling in Arabidopsis (Arabidopsis thaliana). We demonstrate that m6A-deficient plants are more resistant to bacterial and fungal pathogen infections and have altered immune responses. Furthermore, m6A deposition is specifically coordinated on transcripts involved in defense and immunity prior to and proceeding the pathogen signal flagellin. Consequently, the dynamic modulation of m6A on specific stress-responsive transcripts is correlated with changes in abundance and cleavage of these transcripts. Overall, we show that the m6A methylome is regulated prior to and during simulated and active pathogen stress and functions in the coordination and balancing of normal growth and pathogen responses.