Exogenous or endogenous caffeine application confers resistance to diverse biotic stresses in plants. In this study, we demonstrate that endogenous caffeine in caffeine-producing rice (CPR) increases tolerance even to abiotic stresses such as water deficit. Caffeine produced by CPR plants influences the cytosolic Ca2+ ion concentration gradient. We focused on examining the expression of Ca2+-dependent protein kinase genes, a subset of the numerous proteins engaged in abiotic stress signaling. Under normal conditions, CPR plants exhibited increased expressions of seven OsCPKs (OsCPK10, OsCPK12, OsCPK21, OsCPK25, OsCPK26, OsCPK30, and OsCPK31) and biochemical modifications, including antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase) activity and non-enzymatic antioxidant (ascorbic acid) content. CPR plants exhibited more pronounced gene expression changes and biochemical alterations in response to water-deficit stress. CPR plants revealed increased expressions of 16 OsCPKs (OsCPK1, OsCPK2, OsCPK3, OsCPK4, OsCPK5, OsCPK6, OsCPK9, OsCPK10, OsCPK11, OsCPK12, OsCPK14, OsCPK16, OsCPK18, OsCPK22, OsCPK24, and OsCPK25) and 8 genes (OsbZIP72, OsLEA25, OsNHX1, OsRab16d, OsDREB2B, OsNAC45, OsP5CS, and OsRSUS1) encoding factors related to abiotic stress tolerance. The activity of antioxidant enzymes increased, and non-enzymatic antioxidants accumulated. In addition, a decrease in reactive oxygen species, an accumulation of malondialdehyde, and physiological alterations such as the inhibition of chlorophyll degradation and the protection of photosynthetic machinery were observed. Our results suggest that caffeine is a natural chemical that increases the potential ability of rice to cope with water-deficit stress and provides robust resistance by activating a rapid and comprehensive resistance mechanism in the case of water-deficit stress. The discovery, furthermore, presents a new approach for enhancing crop tolerance to abiotic stress, including water deficit, via the utilization of a specific natural agent.
Keywords: antioxidants; caffeine; calcium-dependent protein kinase; transcription factors; water-deficit stress tolerance.