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Polyamine oxidases (PAOs) are key enzymes in polyamine metabolism and are related to the tolerance of plants to abiotic stresses. In this study, overexpression of cucumber (Cucumis sativus L.) PAO2 (CsPAO2) in Arabidopsis resulted in increased activity of the antioxidant enzyme and accelerated conversion from Put to Spd and Spm, while malondialdehyde content (MDA) and electrolyte leakage (EL) was decreased when compared with wild type, leading to enhanced plant growth under salt stress. Photosystem Ⅰ assembly 3 in cucumber (CsPSA3) was revealed as an interacting protein of CsPAO2 by screening yeast two-hybrid library combined with in vitro and in vivo methods. Then, CsPAO2 and CsPSA3 were silenced in cucumber via virus-mediated gene silencing (VIGS) with pV190 as the empty vector. Under salt stress, net photosynthetic rate (Pn) and transpiration rate (Tr) of CsPAO2-silencing plants were lower than pV190-silencing plants, and EL in root was higher than pV190-silencing plants, indicating that CsPAO2-silencing plants suffered more serious salt stress damage. However, photosynthetic parameters of CsPSA3-silencing plants were all higher than those of CsPAO2 and pV190-silencing plants, thereby enhancing the photosynthesis process. Moreover, CsPSA3 silencing reduced the EL in both leaves and roots when compared with CsPAO2-silencing plants, but the EL only in leaves was significantly lower than the other two gene-silencing plants, and conversion from Put to Spd and Spm in leaf was also promoted, suggesting that CsPSA3 interacts with CsPAO2 in leaves to participate in the regulation of salt tolerance through photosynthesis and polyamine conversion. [ABSTRACT FROM AUTHOR]

Soil salinization is the leading environmental factor that restricts crop growth. This study studied the effects of Bacillus cereus (B. cereus) on growth, photosynthesis, and antioxidant metabolism in salt stressed-cucumber seedlings. The results showed that B. cereus could maintain high activity in the high salt environment (4% NaCl). B. cereus significantly increased plant height, stem diameter, fresh weight, and dry weight of cucumber seedlings under salt stress, and increased root vitality, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of cucumber seedlings under salt stress. B. cereus significantly increased the maximum photochemical quantum yield of photosystem II (Fv/Fm), the actual photochemical quantum yield (ΦPSII), and the quantum yield of regulatory energy dissipation Y (NPQ) under salt stress, which were 9.31%, 20.44%, and 5.22% higher than those under salt stress, respectively. The quantum yield of non-regulatory energy dissipation Y (NO) was reduced by 19.81%. Superoxidase (SOD), peroxidase (POD), and catalase (CAT) activities in leaves and roots of cucumber seedlings were significantly increased by B. cereus under salt stress. Compared with salt stress, SOD activities in leaves were significantly increased by 1.70% and 6.32% on the first and third days after treatment. At 1 d, 3 d, and 5 d after treatment, SOD activity in roots increased by 3.06%, 11.24%, and 3.00%, POD activity in leaves increased by 113.38%, 38.81%, and 52.89%, respectively. The POD activity in roots increased by 56.79% and 10.92% on the third and fifth days after treatment, the CAT activity in leaves increased by 8.50% and 25.55%, and the CAT activity in roots increased by 30.59% and 84.45%. Under salt stress, the H2O2 and MDA contents of seedlings treated with B. cereus decreased significantly. Compared with salt stress, the proline content in leaves decreased by 12.69%, 3.90%, and 13.12% at 1 d, 3 d, and 5 d, respectively, while the proline content in roots decreased by 44.94% and 60.08% at 3 d and 5 d, respectively. These results indicated that B. cereus could alleviate salt-induced inhibition of growth and photosynthesis by regulating antioxidant metabolism of cucumber seedlings and thus enhancing salt tolerance of cucumber seedlings. [ABSTRACT FROM AUTHOR]

S-adenosylmethionine synthetase (SAMS) plays a crucial role in regulating stress responses. In a recent study, we found that overexpression of the cucumber gene CsSAMS1 in tobacco can affect the production of polyamines and ethylene, as well as enhancing the salt stress tolerance of tobacco, but the exact underlying mechanisms are elusive. The calcium-dependent protein kinase (CDPK) family is ubiquitous in plants and performs different biological functions in plant development and response to abiotic stress. We used a yeast two-hybrid system to detect whether the protein CDPK6 could interact with SAMS1 and verified their interaction by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. To further explore the function of cucumber CDPK6, we isolated and characterized CsCDPK6 in cucumber. CsCDPK6 is a membrane protein that is highly expressed under various abiotic stresses, including salt stress. It was also observed that ectopic overexpression of CsCDPK6 in tobacco enhanced salt tolerance. Under salt stress, CsCDPK6-overexpressing lines enhanced the survival rate and reduced stomatal apertures in comparison to wild-type (WT) lines, as well as lowering malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents and causing less relative electrolyte leakage. Moreover, repression of CsCDPK6 expression by virus-induced gene silencing (VIGS) in cucumber seedling cotyledons under salt stress increased ethylene production and promoted the transformation from putrescine (Put) to spermidine (Spd) and spermine (Spm). These findings shed light on the interaction of CsSAMS1 and CsCDPK6, which functions positively to regulate salt stress in plants. [ABSTRACT FROM AUTHOR]

7-hydroxymethyl chlorophyll (Chl) a reductase (HCAR) plays critical roles in the Chl cycle and degradation during leaf senescence, however, its function in horticultural crops remains unknown. Here, we identified an HCAR gene (CsHCAR) from cucumber (Cucumis sativus L.) and investigated its roles in response to dark-induced Chl degradation. CsHCAR encoded 459 amino acids, which were orthologous to Arabidopsis HCAR, had the conserved domains, and localized in the chloroplast. Gene expression analysis showed that CsHCAR expression was the highest in senescent leaves and was responsive to different stresses and phytohormone treatments. Overexpression of CsHCAR in tobacco accelerated dark-induced Chl degradation through enhancing the expression of Chl catabolic genes. After 10 d of darkness treatment, the biomass of CsHCAR overexpression plants was reduced. Furthermore, the value of net photosynthetic rate, maximum quantum yield of photosystem II, and effective quantum yield of photosystem II in CsHCAR overexpression plants was significantly reduced in comparison to that in wild-type (WT) plants. The photosynthetic protein content, including Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in CsHCAR overexpression plants exhibited a lower level as compared to that observed in WT plants. In addition, the expression of genes encoding these proteins in CsHCAR overexpression plants was significantly lower than that in WT plants. Moreover, CsHCAR overexpression plants inhibited the dark-induced accumulation of reactive oxygen species (ROS). These results indicate that CsHCAR affects the stability of photosynthetic proteins in chloroplasts, positively regulates Chl degradation, and plays an important role in maintaining ROS homeostasis in leaves. [ABSTRACT FROM AUTHOR]

High temperature is considered a critical abiotic stressor that is increasing continuously, which is severely affecting plant growth and development. The use of heat-resistant rootstock grafting is a viable technique that is practiced globally to improve plant resistance towards abiotic stresses. In this experiment, we explored the efficacy of bitter melon rootstock and how it regulates photosynthesis and the antioxidant defense system to alleviate heat stress (42 °C/32 °C) in cucumber. Our results revealed that bitter-melon-grafted seedlings significantly relieved heat-induced growth inhibition and photoinhibition, maintained better photosynthesis activity, and accumulated a greater biomass than self-grafted seedlings. We measured the endogenous polyamine and hydrogen peroxide (H2O2) contents to determine the inherent mechanism responsible for these effects, and the results showed that heat stress induced a transient increase in polyamines and H2O2 in the inner courtyard of grafted seedlings. This increment was greater and more robust in bitter-melon-grafted seedlings. In addition, the use of polyamine synthesis inhibitors MGBG (methylglyoxal bis-guanylhydrazone) and D-Arg (D-arginine), further confirmed that the production of H2O2 under heat stress is mediated by the accumulation of endogenous polyamines. Moreover, compared with other treatments, the bitter-melon-grafted seedlings maintained high levels of antioxidant enzyme activity under high temperature conditions. However, these activities were significantly inhibited by polyamine synthesis inhibitors and H2O2 scavengers (dimethylthiourea, DMTU), indicating that bitter melon rootstock not only maintained better photosynthetic activity under conditions of high temperature stress but also mediated the production of H2O2 through the regulation of the high level of endogenous polyamines, thereby boosting the antioxidant defense system and comprehensively improving the heat tolerance of cucumber seedlings. Taken together, these results indicate that grafting with a resistant cultivar is a promising alternative tool for reducing stress-induced damage. [ABSTRACT FROM AUTHOR]

The stomatal aperture is imperative for photosynthesis in higher plants. The function of polyamines (PAs) in stomatal regulation under a stressful environment has not been fully determined. In this study, we demonstrated the mechanism by which putrescine (Put) regulates stomatal changes in cucumber leaves under salt stress. The results showed that foliar application of Put alleviated the decrease of stomatal aperture and photosynthesis caused by salt stress and promoted plant growth. Exogenous Put caused a significant increase in endogenous PAs and hydrogen peroxide (H 2 O 2) levels by 105.43% and 27.97%, respectively, while decreased abscisic acid (ABA) content by 67.68% under salt stress. However, application of inhibitors of aminoguanidine hydrochloride (AG), 1, 8-diaminooctane (1, 8-DO), diphenyleneiodonium chloride (DPI) and salicylhydroxamic acid (SHAM) upregulated the 9-cis-cyclocarotenoid dioxygenase (NCED) gene and downregulated the reduced glutathione synthetase (GSHS) gene. These inhibitors also decreased the stomatal aperture, levels of H 2 O 2 and reduced glutathione (GSH), but increased the ABA content under salt stress and Put treatment conditions. The order of influence is AG > 1, 8-DO > DPI > SHAM. However, Put-induced downregulation of ABA content and upregulation of GSH content under salt stress were effectively blocked by N, N′-dimethylthiourea (DMTU, H 2 O 2 scavenger) and 1-chloro-2,4-dinitrobenzene (CDNB, GSH scavenger) treatments. Taken together, these results suggest that Put induced the formation of H 2 O 2 signaling mediates the degradation of PAs by diamine oxidase (DAO), increasing GSH content and inhibiting the accumulation of ABA in leaves, thereby promoting stomatal opening in salt-stressed cucumber leaves. • Primarily salinity-induced photosynthesis inhibition caused by stomatal limitation. • Putrescine alleviated the photosynthetic inhibition of cucumber under salt stress. • Putrescine regulates stomatal changes through H 2 O 2 pathway. • Reduced glutathione can inhibit the synthesis of ABA. [ABSTRACT FROM AUTHOR]

Polyamine oxidases (PAOs) are flavin adenine dinucleotide-dependent enzymes that catalyze oxidation of polyamines (PAs), and play an important roles in growth and development and stress tolerance of plant. In this study, we identified four PAO genes (CsPAO1 - CsPAO4) in cucumber (Cucumis sativus L), and classified them into three clades (Ⅰ, Ⅱ and Ⅲ). CsPAO1 and CsPAO3 were located in cell membrane or nucleus while CsPAO2 and CsPAO4 were likely localized in peroxisome. CsPAO1 - 2 and CsPAO4 consisted of ten exons and nine introns, but no intron was observed in CsPAO3. The CsPAOs showed higher expression level in root, with the exception of CsPAO1, which was mainly expressed in flower, and they had various responses to hormone and stress treatments. Stress treatments induced more obvious change of gene expression, and CsPAO2–4 responded to NaCl stress earlier both in root and leaf. Overexpression of CsPAO3 in Arabidopsis improved seed germination and root growth on medium containing NaCl. Furthermore, under salt stress, electrolyte leakage (EL), malonaldehyde (MDA) and hydrogen peroxide (H 2 O 2) content were decreased, but peroxidase (POD) and catalase (CAT) activities were increased in transgenic Arabidopsis lines as compared with wild type (WT), contributing to alleviating the salt stress-induced growth suppression. The OE15 line, on the other hand, had the highest expression level of CsPAO3 , exhibited identical phenotype to WT, suggesting that excess H 2 O 2 produced by polyamine oxidation might be harmful to plants. Taken together, our results indicated that CsPAO family members are involved in plant development and stress responses, and a proper level of CsPAO3 overexpression enhances salt tolerance of plants. • There are four PAO family members in cucumber genome. • CsPAO3 was a candidate gene enhancing plants' tolerance to salt. • Higher overexpression level of CsPAO3 couldn't lead to stronger salt resistance of plants, and excess H 2 O 2 produced by polyamine oxidation may be harmful to plants. [ABSTRACT FROM AUTHOR]

Peptide: N -glycanase (PNGase; EC 3.5.1.52) is a deglycosylation enzyme that is responsible for deglycosylating misfolded glycoproteins in the endoplasmic reticulum. However, the role of PNGase in plants is largely unknown. Here, we cloned and characterized the function of peptide: N -glycanase (CsPNG1) from cucumber. The amino acid encoded by CsPNG1 gene contained a typical transglutaminase (TGase) catalytic triad domain and belonged to the "TGase superfamily". Subcellular localization showed that CsPNG1 was located in the cell membrane and nucleus. Promoter sequence analysis and qPCR tests showed that CsPNG1 could respond to a variety of abiotic stresses and hormone treatments. Yeast one-hybrid assays revealed the interaction between the transcription factor CsGT-3b and CsPNG1 promoter. Importantly, overexpression of CsPNG1 in tobacco increased the tolerance to salt stress of transgenic plants. In addition, CsPNG1 interacted with CsRAD23 family proteins and the C-terminal UBA domain of CsRAD23 protein was responsible for binding to CsPNG1, indicating that CsPNG1 was involved in the ER-associated degradation pathway (ERAD). Taken together, our study demonstrated that CsPNG1 plays a positive role in improving plant salt tolerance, and these findings might provide a basis for further functional analysis of CsPNG1 genes in abiotic stress and ERAD. • CsPNG1 positively responded to abiotic stresses, and its promoter can be bound by the transcription factor CsGT-3b. • The overexpression of CsPNG1 resulted in increased salt stress tolerance in transgenic tobacco. • There are physical interactions between CsPNG1 and CsRAD23 proteins. [ABSTRACT FROM AUTHOR]