Huangai Bi

Key message MT mitigates chilling damage by enhancing antioxidant system and photosystem activities, and cold-responsive genes expression in cucumbers. H₂O₂ may act as a downstream signaling molecule in the MT-induced chilling tolerance. Abstract Melatonin (MT) and hydrogen peroxide (H₂O₂) are important endogenous signaling molecules that play multifaceted roles in plant responses to abiotic stress. However, the interactive mechanism by which MT and H₂O₂ regulate chilling tolerance remains unclear. Here we found that MT exhibited a positive regulatory effect on the chilling tolerance of cucumbers, with an optimum concentration of 100 µM. MT markedly enhanced RBOH1 mRNA expression, activity and endogenous H₂O₂ accumulation in cucumber seedlings. However, 1.0 mM H₂O₂ had no significant effect on mRNA levels of TDC and ASMT, the key genes for MT synthesis, and endogenous MT content. Both MT and H₂O₂ significantly decreased malondialdehyde (MDA), electrolyte leakage (EL) and chilling injury index (CI) by activating the antioxidant system, thereby alleviating chilling damage in cucumber seedlings. MT and H₂O₂ improved photosynthetic carbon assimilation, which was primarily attributed to an increase in activity, mRNA expression, and protein levels of RuBPCase and RCA. Meanwhile, MT and H₂O₂ induced the photoprotection for both PSII and PSI by enhancing the QA’s electron transport capacity and elevating protein levels of the photosystems. Moreover, MT and H₂O₂ significantly upregulated the expression of cold response genes. MT-induced chilling tolerance was attenuated by N′, N′-dimethylthiourea (DMTU), a H₂O₂ specific scavenger. Whereas, the MT synthesis inhibitor (p-chlorophenylalanine, p-CPA) did not influence H₂O₂-induced chilling tolerance. The positive regulation of MT on the antioxidant system, photosynthesis and cold response gene levels were significantly attenuated in RBOH1-RNAi plants compared with WT plants. These findings suggest that H₂O₂ may functions as a downstream signaling molecule in MT-induced chilling tolerance in cucumber plants.

Melatonin (MT) is a key regulator in plants’ response to leaf senescence induced by aging or various abiotic stresses. Here, we demonstrated that darkness and leaf aging enhance endogenous MT levels by upregulating key genes involved in MT synthesis. Additionally, exogenous MT application significantly mitigated leaf senescence induced by darkness and leaf aging in tomato plants, leading to higher chlorophyll content and maximum photochemical efficiency (Fv/Fm) compared to the control. Using Solanum lycopersicum L. tryptophan decarboxylase (SlTDC)-overexpressed and -knockout transgenic tomato seedlings, we found that SlTDC overexpression increased endogenous MT content and suppressed mRNA levels of chlorophyll degradation-related genes: pheophorbide a oxygenase (PAO), pheophytinase (PPH), and non-yellow coloring1 like (NOL). Furthermore, SlTDC overexpression alleviated photosystem II complex (PSII) photoinhibition, increased ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco activase (RCA) activities and mRNA levels, and maintained higher photosynthetic efficiency in leaves under darkness and in leaves aged over 35 d compared to wild-type plants. Conversely, SlTDC knockout accelerated darkness- or leaf aging-induced leaf senescence in tomatoes. Our findings suggest that MT application or SlTDC overexpression can effectively alleviate leaf senescence by regulating photosynthesis in tomato plants.

Melatonin (MT) is an important regulator that participates in plant growth and stress tolerance. The aim of this study was to determine how MT alleviates the decline of photosynthesis in cucumber seedlings under high-temperature stress. The results showed that foliar spraying of MT significantly alleviates the heat symptoms of cucumber by reducing the electrolyte leakage (EL) and increasing the net photosynthetic rate (P_n), light saturation CO₂ assimilation rate (A_sat), actual photochemical efficiency under light (Φ_PSII), and maximum photochemical efficiency of photosystem II under dark (F_v/F_m). The mitigative effect of MT depended on the concentration and 100 μmol·L⁻¹ MT was the optimal concentration. Moreover, we found that overexpression of N-acetyl-5-serotonin-methyltransferase (ASMT) significantly increased endogenous MT content and heat tolerance of cucumber. Furthermore, compared to WT, OE-CsASMT plants displayed higher P_n, A_sat and the activities and relative expression of photosynthetic enzymes under high-temperature stress. Meanwhile, overexpression of CsASMT significantly alleviated the damage of the PSII donor side and acceptor side and the degradation of D1 and PsaD protein under heat stress, thereby reducing the photoinhibition of PSI and PSII and enhancing the photosynthetic capacity of cucumber. While, suppression of CsASMT aggravated the damage of photosynthetic apparatus. Furthermore, the suppression of proton gradient regulation 5 (PGR5) aggravated the decline of photosynthesis caused by heat stress, which could not be reversed with the application of MT. Specifically, MT alleviated photoinhibition caused by heat stress via regulating PGR5 depended-CEF. Taken together, our results help to reveal the regulation mechanism of MT on photosynthesis in cucumber under heat stress.

Melatonin (MT) and abscisic acid (ABA) are crucial in regulating abiotic stress tolerance in cucumber. However, their roles as signaling molecules in regulating chilling tolerance remain ambiguous. The results showed that neither applying ABA nor removing endogenous ABA with sodium tungstate (Na₂WO₄, an inhibitor of ABA synthesis) has no effect on endogenous MT levels, however, MT-induced endogenous ABA content via activating the activities and mRNA levels of 9-cis-epoxycarotenoid dioxygenase (NCED) under normal conditions and inhibiting endogenous MT with p-chlorophenylalanine (p-CPA) blocked this effect. Further studies indicated that MT and ABA had a noteworthy effect on enhancing the chilling tolerance of cucumber seedlings in terms of lower chilling injury, EL, and MDA contents. Moreover, cucumber seedlings pretreated with MT and ABA showed lower ROS contents and higher antioxidant capacity than those in H₂O treatment under chilling stress. Meanwhile, MT and ABA alleviated the decline of chlorophyll content, photosynthesis, gene expression, and protein level of Rubisco and RCA caused by chilling stress as well as maintained photosynthetic electron transport via increasing heat dissipation, the levels of proteins PsbS and VDE as well as decreasing the chilling damage to donor and recipient side of electron transport chain under chilling stress. However, it was discovered that Na₂WO₄ and fluridone (Flu) blocked the chilling tolerance induced by MT, whereas p-CPA had little impact on ABA promotion on the chilling tolerance of cucumber. These findings imply that ABA may act as a downstream signal of MT in the regulation of chilling tolerance in cucumber.

Grafting with pumpkin as rootstock could improve chilling tolerance of cucumber; however, the underlying mechanism of grafting-induced chilling tolerance remains unclear. Here, we analyzed the difference of physiological and transcriptional level between own-rooted (Cs/Cs) and hetero-grafted (Cs/Cm) cucumber seedlings under chilling stress. The results showed that grafting with pumpkin significantly alleviated the chilling injury as evidenced by slightly symptoms, lower contents of electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂^-) and higher relative water content in Cs/Cm seedlings compared with Cs/Cs seedlings under chilling stress. RNA-seq data showed that grafting induced more DGEs at 8 °C/5 °C compared with 25 °C/18 °C. In accordance with the increase of the activities of antioxidant enzymes (SOD, POD, CAT, APX), grafting upregulated the expression of the regulated redox-related genes such as GST, SOD, and APX. Moreover, grafting increased the expression of genes participated in central carbon metabolism to promote the conversion and decomposition of sugar, which provided more energy for the growth of Cs/Cm seedlings under chilling stress. In addition, grafting regulated the genes involved in the intracellular signal transduction pathways such as calcium signal (CAML, CML, and CDPK) and inositol phospholipid signal (PLC), as well as changed the gene expression of plant hormone signal transduction pathways (ARF, GAI, ABF, and PYR/PYL). These results provide a physiological and transcriptional basis for the molecular mechanism of grafting-induced chilling tolerance of cucumber seedlings.

Grafting is widely applied to enhance the tolerance of some vegetables to biotic and abiotic stress. Salicylic acid (SA) is known to be involved in grafting-induced chilling tolerance in cucumber. Here, we revealed that grafting with pumpkin (Cucurbita moschata, Cm) as a rootstock improved chilling tolerance and increased the accumulation of SA, abscisic acid (ABA) and hydrogen peroxide (H₂O₂) in grafted cucumber (Cucumis sativus/Cucurbita moschata, Cs/Cm) leaves. Exogenous SA improved the chilling tolerance and increased the accumulation of ABA and H₂O₂ and the mRNA abundances of CBF1, COR47, NCED, and RBOH1. However, 2-aminoindan-2-phosphonic acid (AIP) and L-a-aminooxy-b-phenylpropionic acid (AOPP) (biosynthesis inhibitors of SA) reduced grafting-induced chilling tolerance, as well as the synthesis of ABA and H₂O₂, in cucumber leaves. ABA significantly increased endogenous H₂O₂ production and the resistance to chilling stress, as proven by the lower electrolyte leakage (EL) and chilling injury index (CI). However, application of the ABA biosynthesis inhibitors sodium tungstate (Na₂WO₄) and fluridone (Flu) abolished grafting or SA-induced H₂O₂ accumulation and chilling tolerance. SA-induced plant response to chilling stress was also eliminated by N,N′-dimethylthiourea (DMTU, an H₂O₂ scavenger). In addition, ABA-induced chilling tolerance was attenuated by DMTU and diphenyleneiodonium (DPI, an H₂O₂ inhibitor) chloride, but AIP and AOPP had little effect on the ABA-induced mitigation of chilling stress. Na₂WO₄ and Flu diminished grafting- or SA-induced H₂O₂ biosynthesis, but DMTU and DPI did not affect ABA production induced by SA under chilling stress. These results suggest that SA participated in grafting-induced chilling tolerance by stimulating the biosynthesis of ABA and H₂O₂. H₂O₂, as a downstream signaler of ABA, mediates SA-induced chilling tolerance in grafted cucumber plants.

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 (ΦPS_II), 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.

As an important gas signaling molecule, hydrogen sulfide (H₂S) plays a crucial role in regulating cold tolerance. H₂S cooperates with phytohormones such as abscisic acid, ethylene, and salicylic acid to regulate the plant stress response. However, the synergistic regulation of H₂S and auxin in the plant response to cold stress has not been reported. This study showed that sodium hydrosulfide (NaHS, an H₂S donor) treatment enhanced the cold stress tolerance of cucumber seedlings and increased the level of auxin. CsARF5, a cucumber auxin response factor (ARF) gene, was isolated, and its role in regulating H₂S-mediated cold stress tolerance was described. Transgenic cucumber leaves overexpressing CsARF5 were obtained. Physiological analysis indicated that overexpression of CsARF5 enhanced the cold stress tolerance of cucumber and the regulation of the cold stress response by CsARF5 depends on H₂S. In addition, molecular assays showed that CsARF5 modulated cold stress response by directly activating the expression of the dehydration-responsive element-binding (DREB)/C-repeat binding factor (CBF) gene CsDREB3, which was identified as a positive regulator of cold stress. Taken together, the above results suggest that CsARF5 plays an important role in H₂S-mediated cold stress in cucumber. These results shed light on the molecular mechanism by which H₂S regulates cold stress response by mediating auxin signaling; this will provide insights for further studies on the molecular mechanism by which H₂S regulates cold stress. The aim of this study was to explore the molecular mechanism of H₂S regulating cold tolerance of cucumber seedlings and provide a theoretical basis for the further study of cucumber cultivation and environmental adaptability technology in winter.

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.

Melatonin (MT) and nitric oxide (NO) are two multifunctional signaling molecules that are involved in the response of plants to abiotic stresses. However, how MT and NO synergize in response to cold stress affecting plants is still not clear. In this study, we found that endogenous MT accumulation under cold stress was positively correlated with cold tolerance in different varieties of cucumber seedlings. The data presented here also provide evidence that endogenous NO is involved in the response to cold stress. About 100 μM MT significantly increased the nitrate reductase (NR) activity,NR-relative messenger RNA (mRNA) expression, and endogenous NO accumulation in cucumber seedlings. However, 75 μM sodium nitroprusside (SNP, a NO donor) showed no significant effect on the relative mRNA expression of tryptophan decarboxylase (TDC), tryptamine-5-hydroxylase (T5H), serotonin-N-acetyltransferase (SNAT), or acetylserotonin O-methyltransferase (ASMT), the key genes for MT synthesis and endogenous MT levels. Compared with H₂O treatment, both MT and SNP decreased electrolyte leakage (EL), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation by activating the antioxidant system and consequently mitigated cold damage in cucumber seedlings. MT and SNP also enhanced photosynthetic carbon assimilation, which was mainly attributed to an increase in the activity and mRNA expression of the key enzymes in the Calvin–Benson cycle. Simultaneously, MT- and SNP-induced photoprotection for both photosystem II (PSII) and photosystem I (PSI) in cucumber seedlings, by stimulating the PsbA (D1) protein repair pathway and ferredoxin-mediated NADP⁺photoreduction, respectively. Moreover, exogenous MT and SNP markedly upregulated the expression of chilling response genes, such as inducer ofCBFexpression (ICE1), C-repeat-binding factor (CBF1), and cold-responsive (COR47). MT-induced cold tolerance was suppressed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a specific scavenger of NO). However, p-chlorophenylalanine (p-CPA, a MT synthesis inhibitor) did not affect NO-induced cold tolerance. Thus, novel results suggest that NO acts as a downstream signal in the MT-induced plant tolerance to cold stress.