Spatial–Temporal Response of Reactive Oxygen Species and Salicylic Acid Suggest Their Interaction in Pumpkin Rootstock-Induced Chilling Tolerance in Watermelon Plants

Grafting with pumpkin rootstock could improve chilling tolerance in watermelon, and salicylic acid (SA) as a signal molecule is involved in regulating plant tolerance to chilling and other abiotic stresses. To clarify the mechanism in pumpkin rootstock-induced systemic acquired acclimation in grafted watermelon under chilling stress, we used self-grafted (Cl/Cl) and pumpkin rootstock-grafted (Cl/Cm) watermelon seedlings to study the changes in lipid peroxidation, photosystem II (PSII) activity and antioxidant metabolism, the spatio–temporal response of SA biosynthesis and H2O2 accumulation to chilling, and the role of H2O2 signal in SA-induced chilling tolerance in grafted watermelon. The results showed that pumpkin rootstock grafting promoted SA biosynthesis in the watermelon scions. Chilling induced hydrolysis of conjugated SA into free SA in the roots and accumulation of free SA in the leaves in Cl/Cm plants. Further, pumpkin rootstock grafting induced early response of antioxidant enzyme system in the roots and increased activities of ascorbate peroxidase and glutathione reductase in the leaves, thus maintaining cellular redox homeostasis. Exogenous SA improved while the inhibition of SA biosynthesis reduced chilling tolerance in Cl/Cl seedlings. The application of diphenyleneiodonium (DPI, inhibitor of NADPH oxidase) and dimethylthiourea (DMTU, H2O2 scavenger) decreased, while exogenous H2O2 improved the PSII activity in Cl/Cl plants under chilling stress. Additionally, the decrease of the net photosynthetic rate in DMTU- and DPI-pretreated Cl/Cl plants under chilling conditions could be alleviated by subsequent application of H2O2 but not SA. In conclusion, pumpkin rootstock grafting induces SA biosynthesis and redistribution in the leaves and roots and participates in the regulation of antioxidant metabolism probably through interaction with the H2O2 signal, thus improving chilling tolerance in watermelon.

Melatonin Promotes the Chilling Tolerance of Cucumber Seedlings by Regulating Antioxidant System and Relieving Photoinhibition

Chilling adversely affects the photosynthesis of thermophilic plants, which further leads to a decline in growth and yield. The role of melatonin (MT) in the stress response of plants has been investigated, while the mechanisms by which MT regulates the chilling tolerance of chilling-sensitive cucumber remain unclear. This study demonstrated that MT positively regulated the chilling tolerance of cucumber seedlings and that 1.0 μmol⋅L–1 was the optimum concentration, of which the chilling injury index, electrolyte leakage (EL), and malondialdehyde (MDA) were the lowest, while growth was the highest among all treatments. MT triggered the activity and expression of antioxidant enzymes, which in turn decreased hydrogen peroxide (H2O2) and superoxide anion (O2⋅–) accumulation caused by chilling stress. Meanwhile, MT attenuated the chilling-induced decrease, in the net photosynthetic rate (Pn) and promoted photoprotection for both photosystem II (PSII) and photosystem I (PSI), regarding the higher maximum quantum efficiency of PSII (Fv/Fm), actual photochemical efficiency (ΦPSII), the content of active P700 (ΔI/I0), and photosynthetic electron transport. The proteome analysis and western blot data revealed that MT upregulated the protein levels of PSI reaction center subunits (PsaD, PsaE, PsaF, PsaH, and PsaN), PSII-associated protein PsbA (D1), and ribulose-1,5-bisphosphate carboxylase or oxygenase large subunit (RBCL) and Rubisco activase (RCA). These results suggest that MT enhances the chilling tolerance of cucumber through the activation of antioxidant enzymes and the induction of key PSI-, PSII-related and carbon assimilation genes, which finally alleviates damage to the photosynthetic apparatus and decreases oxidative damage to cucumber seedlings under chilling stress.

H2O2 Functions as a Downstream Signal of IAA to Mediate H2S-Induced Chilling Tolerance in Cucumber

Hydrogen sulfide (H2S) plays a crucial role in regulating chilling tolerance. However, the role of hydrogen peroxide (H2O2) and auxin in H2S-induced signal transduction in the chilling stress response of plants was unclear. In this study, 1.0 mM exogenous H2O2 and 75 μM indole-3-acetic acid (IAA) significantly improved the chilling tolerance of cucumber seedlings, as demonstrated by the mild plant chilling injury symptoms, lower chilling injury index (CI), electrolyte leakage (EL), and malondialdehyde content (MDA) as well as higher levels of photosynthesis and cold-responsive genes under chilling stress. IAA-induced chilling tolerance was weakened by N, N′-dimethylthiourea (DMTU, a scavenger of H2O2), but the polar transport inhibitor of IAA (1-naphthylphthalamic acid, NPA) did not affect H2O2-induced mitigation of chilling stress. IAA significantly enhanced endogenous H2O2 synthesis, but H2O2 had minimal effects on endogenous IAA content in cucumber seedlings. In addition, the H2O2 scavenger DMTU, inhibitor of H2O2 synthesis (diphenyleneiodonium chloride, DPI), and IAA polar transport inhibitor NPA reduced H2S-induced chilling tolerance. Sodium hydrosulfide (NaHS) increased H2O2 and IAA levels, flavin monooxygenase (FMO) activity, and respiratory burst oxidase homolog (RBOH1) and FMO-like protein (YUCCA2) mRNA levels in cucumber seedlings. DMTU, DPI, and NPA diminished NaHS-induced H2O2 production, but DMTU and DPI did not affect IAA levels induced by NaHS during chilling stress. Taken together, the present data indicate that H2O2 as a downstream signal of IAA mediates H2S-induced chilling tolerance in cucumber seedlings.