Exogenous Melatonin and CaCl2 Alleviate Cold-Induced Oxidative Stress and Photosynthetic Inhibition in Cucumber Seedlings

Melatonin (Mel) and CaCl2 have been demonstrated to play significant roles in plant cold tolerance. However, studies on their mechanisms of action are relatively limited. In this study, the effects of Mel and CaCl2 on the growth of cucumber seedlings under low-temperature stress (10 degrees C/6 degrees C, day/night) and the possible interplay between Mel and CaCl2 in this process were investigated. The results indicated that Mel and CaCl2 at different concentrations inhibited low-temperature-induced reactive-oxygen species burst and decreased lipid peroxidation levels when compared with low-temperature stress alone, with the most significant effects being observed in the treatment with 100 mu M Mel and 10 mM CaCl2 (P < 0.05). The foliar application of 100 mu M Mel and 10 mM CaCl2 also increased the accumulation of osmoprotective compounds in different organs and the root vigor of cucumber seedlings under low-temperature conditions. However, the above favorable effects of Mel and CaCl2 were significantly negated by the combined treatment of p-chlorophenylalanine (p-CPA, inhibitor of Mel biosynthesis) with Mel and that of lanthanum chloride (LaCl3, Ca2+ channel blocker), ethylene glycol tetraacetic acid (EGTA, Ca2+ chelator), trifluoperazine dihydrochloride (TFP, calmodulin antagonist and CDPK inhibitor), or N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, calmodulin antagonist and CDPK inhibitor) with CaCl2. Furthermore, increased photosynthetic efficiency (confirmed by chlorophyll fluorescence parameters) and expression levels of photosynthesis-related genes (Rbc L, Rbc S, RCA, FBPase, and SBPase) were observed in cucumber seedlings pretreated with Mel and CaCl2 under low-temperature stress. Intriguingly, co-treatment of LaCl3/EGTA or TFP/W-7 with Mel significantly attenuated the effects of Mel on the growth and photosynthesis of cucumber seedlings under low-temperature stress. In contrast, p-CPA had slight or no effect on the growth and photosynthesis by CaCl2. These results suggested that Mel and CaCl2 could improve the cold tolerance of cucumber seedlings by maintaining redox homeostasis and osmotic balance and improving photosynthetic efficiency. In addition, Mel confers cold tolerance to cucumber seedlings in a Ca2+ signaling (Ca2+/CDPKs and Ca2+/CaM)-dependent manner. In conclusion, this study revealed the positive regulatory effects of exogenous Mel and CaCl2 on the cold tolerance of cucumber seedlings and their interactions during this process. This work could also promote the application of these two substances in agriculture and provide a reference for the study of their mechanisms of action.