Blind Detection of Broadband Signal Based on Weighted Bi-Directional Feature Pyramid Network.

With the development of wireless technology, signals propagating in space are easy to mix, so blind detection of communication signals has become a very practical and challenging problem. In this paper, we propose a blind detection method for broadband signals based on a weighted bi-directional feature pyramid network (BiFPN). The method can quickly perform detection and automatic modulation identification (AMC) on time-domain aliased signals in broadband data. Firstly, the method performs a time-frequency analysis on the received signals and extracts the normalized time-frequency images and the corresponding labels by short-time Fourier transform (STFT). Secondly, we build a target detection model based on YOLOv5 for time-domain mixed signals in broadband data and learn the features of the time-frequency distribution image dataset of broadband signals, which achieves the purpose of training the model. The main improvements of the algorithm are as follows: (1) a weighted bi-directional feature pyramid network is used to achieve a simple and fast multi-scale feature fusion approach to improve the detection probability; (2) the Efficient-Intersection over Union (EIOU) loss function is introduced to achieve high accuracy signal detection in a low Signal-Noise Ratio (SNR) environment. Finally, the time-frequency images are detected by an improved deep network model to complete the blind detection of time-domain mixed signals. The simulation results show that the method can effectively detect the continuous and burst signals in the broadband communication signal data and identify their modulation types.

Physiological mechanism of strigolactone enhancing tolerance to low light stress in cucumber seedlings.

Strigolactone is a newly discovered type of plant hormone that has multiple roles in modulating plant responses to abiotic stress. Herein, we aimed to investigate the effects of exogenous GR24 (a synthetic analogue of strigolactone) on plant growth, photosynthetic characteristics, carbohydrate levels, endogenous strigolactone content and antioxidant metabolism in cucumber seedlings under low light stress. The results showed that the application of 10 µM GR24 can increase the photosynthetic efficiency and plant biomass of low light-stressed cucumber seedlings. GR24 increased the accumulation of carbohydrates and the synthesis of sucrose-related enzyme activities, enhanced antioxidant enzyme activities and antioxidant substance contents, and reduced the levels of H2O2 and MDA in cucumber seedlings under low light stress. These results indicate that exogenous GR24 might alleviate low light stress-induced growth inhibition by regulating the assimilation of carbon and antioxidants and endogenous strigolactone contents, thereby enhancing the tolerance of cucumber seedlings to low light stress.

CsPAO2 Improves Salt Tolerance of Cucumber through the Interaction with CsPSA3 by Affecting Photosynthesis and Polyamine Conversion.

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.

A Comprehensive Evaluation of Salt Tolerance in Tomato (Var. Ailsa Craig): Responses of Physiological and Transcriptional Changes in RBOH's and ABA Biosynthesis and Signalling Genes.

Salinity is a ubiquitous stressor, depleting osmotic potential and affecting the tomato seedlings' development and productivity. Considering this critical concern, we explored the salinity response in tomato seedlings by evaluating them under progressive salt stress duration (0, 3, 6, and 12 days). Intriguingly, besides the adverse effect of salt stress on tomato growth the findings exhibited a significant role of tomato antioxidative system, RBOH genes, ABA biosynthesis, and signaling transcription factor for establishing tolerance to salinity stress. For instance, the activities of enzymatic and non-enzymatic antioxidants continued to incline positively with the increased levels of reactive oxygen species (O2•-, H2O2), MDA, and cellular damage, suggesting the scavenging capacity of tomato seedlings against salt stress. Notably, the RBOH transcription factors activated the hydrogen peroxide-mediated signalling pathway that induced the detoxification mechanisms in tomato seedlings. Consequently, the increased gene expression of antioxidant enzymes and the corresponding ratio of non-enzymatic antioxidants AsA-GSH suggested the modulation of antioxidants to survive the salt-induced oxidative stress. In addition, the endogenous ABA level was enhanced under salinity stress, indicating higher ABA biosynthesis and signalling gene expression. Subsequently, the upregulated transcript abundance of ABA biosynthesis and signalling-related genes suggested the ABA-mediated capacity of tomato seedlings to regulate homeostasis under salt stress. The current findings have revealed fascinating responses of the tomato to survive the salt stress periods, in order to improve the abiotic stress tolerance in tomato.

Auxin is involved in arbuscular mycorrhizal fungi-promoted tomato growth and NADP-malic enzymes expression in continuous cropping substrates.

BACKGROUND: Despite significant limitations of growth medium reuse, a large amount of organic substrate is reused in soilless cultivation of horticultural crops in China. Arbuscular mycorrhizal fungi (AMF) can promote nutrient absorption and improve plant tolerance to biotic and abiotic stresses. However, the mechanisms governing the effects of AMF on crop growth in organic continuous cropping substrates have not been elucidated. RESULTS: In this study, we showed that the inoculation of AMF in continuous cropping substrates promoted growth and root development, and increased the root and NADP-malic enzyme (NADP-ME) activity of tomato seedlings. Root transcriptome analysis demonstrated that the plant hormone signal transduction pathway was highly enriched, and 109 genes that positively correlated with the AMF-inoculated plant phenotype were obtained by gene set enrichment analysis (GSEA), which identified 9 genes related to indole acetic acid (IAA). Importantly, the levels of endogenous IAA in tomato seedlings significantly increased after AMF inoculation. Furthermore, the application of AMF significantly increased the expression levels of NADP-ME1 and NADP-ME2, as well as the activity of NADP-ME, and enhanced the root activity of tomato seedlings in comparison to that observed without inoculation of AMF. However, these effects were blocked in plants treated with 2,3,5-triiodobenzoic acid (TIBA), a polar transport inhibitor of IAA. CONCLUSIONS: These results suggest that IAA mediates the AMF-promoted tomato growth and expression of NADP-MEs in continuous cropping substrates. The study provides convincing evidence for the reuse of continuous cropping substrates by adding AMF as an amendment.

CsCDPK6, a CsSAMS1-Interacting Protein, Affects Polyamine/Ethylene Biosynthesis in Cucumber and Enhances Salt Tolerance by Overexpression in Tobacco.

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.

Systematic identification and analysis of heat-stress-responsive lncRNAs, circRNAs and miRNAs with associated co-expression and ceRNA networks in cucumber (Cucumis sativus L.).

Researchers have shown that long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) act as competitive endogenous RNAs (ceRNAs) and are mutually regulated by competition for binding to common microRNA response elements (MREs). However, a comprehensive identification and analysis of lncRNAs and circRNAs as ceRNAs have not yet been completed in cucumber (Cucumis sativus L.) exposed to high-temperature stress. In our study, 32 663 coding transcripts, 2085 lncRNAs, 2477 circRNAs and 348 differentially expressed miRNAs were identified using RNA sequencing. In addition, six heat-stress-responsive miRNAs (five known and one novel miRNAs) and eight lncRNAs were selected for qPCR to confirm their expression profiles. By analyzing the cis effects of lncRNAs, we constructed a lncRNA-mRNA co-expression network. Based on the results, the corresponding lncRNAs play a regulatory role in the stress response in cucumber plants. In our study, the PatMatch software was used to predict the potential function of lncRNAs and circRNAs as ceRNAs. A total of 18 lncRNAs and seven circRNAs were predicted to bind to 114 differentially expressed miRNAs and compete with 359 mRNAs for miRNA binding sites. These mRNAs are predicted to be involved in various pathways, such as plant hormone signal transduction, plant-pathogen interaction and glutathione metabolism. Among them, TCONS_00031790, TCONS_00014332, TCONS_00014717, TCONS_00005674, novel_circ_001543 and novel_circ_000876 may interact with miR9748 by plant hormone signal transduction pathways in response to high-temperature stress. Moreover, indole-3-acetic acid (IAA) and 1-aminocyclopropane-l-carboxylic acid (ACC) levels decreased in the high-temperature treatment group, indicating that IAA and ethylene signaling might be involved in response to high-temperature stress. In this study, we conducted a full transcriptomic analysis in response to high-temperature stress in cucumber and, for the first time, integrated the potential ceRNA functions of lncRNAs/circRNAs. The results provide a basis for studying the potential functions of lncRNAs/circRNAs in response to high-temperature stress.

Melatonin alleviates nickel phytotoxicity by improving photosynthesis, secondary metabolism and oxidative stress tolerance in tomato seedlings.

Arable land contamination with nickel (Ni) has become a major threat to worldwide crop production. Recently, melatonin has appeared as a promising stress-relief substance that can alleviate heavy metal-induced phytotoxicity in plants. However, the plausible underlying mechanism of melatonin function under Ni stress has not been fully substantiated in plants. Herein, we conducted an experiment that unveiled critical mechanisms in favor of melatonin-mediated Ni-stress tolerance in tomato. Ni stress markedly inhibited growth and biomass by impairing the photosynthesis, photosystem function, mineral homeostasis, root activity, and osmotic balance. In contrast, melatonin application notably reinforced the plant growth traits, increased photosynthesis efficiency in terms of chlorophyll content, upregulation of chlorophyll synthesis genes, i.e. POR, CAO, CHL G, gas exchange parameters, and PSII maximum efficiency (Fv/Fm), decreased Ni accumulation and increased mineral nutrient homeostasis. Moreover, melatonin efficiently restricted the hydrogen peroxide (H2O2) and superoxide radical production and increased RBOH expression and restored cellular integrity (less malondialdehyde and electrolyte leakage) through triggering the antioxidant enzyme activities and modulating AsA-GSH pools. Notably, oxidative stress was effectively mitigated by upregulation of several defense genes (SOD, CAT, APX, GR, GST, MDHAR, DHAR) and melatonin biosynthesis-related genes (TDC, T5S, SNAT, ASMT). Besides, melatonin treatment enhanced secondary metabolites (phenols, flavonoids, and anthocyanin) contents along with their encoding genes (PAL, CHS) expression, and these metabolites potentially restricted excess H2O2 accumulation. In conclusion, our findings deciphered the potential functions of melatonin in alleviating Ni-induced phytotoxicity in tomato through boosting the biomass production, photosynthesis, nutrient uptake, redox balance, and secondary metabolism.

Melatonin alleviates heat-induced damage of tomato seedlings by balancing redox homeostasis and modulating polyamine and nitric oxide biosynthesis.

BACKGROUND: Melatonin is a pleiotropic signaling molecule that plays multifarious roles in plants stress tolerance. The polyamine (PAs) metabolic pathway has been suggested to eliminate the effects of environmental stresses. However, the underlying mechanism of how melatonin and PAs function together under heat stress largely remains unknown. In this study, we investigated the potential role of melatonin in regulating PAs and nitric oxide (NO) biosynthesis, and counterbalancing oxidative damage induced by heat stress in tomato seedlings. RESULTS: Heat stress enhanced the overproduction of reactive oxygen species (ROS) and damaged inherent defense system, thus reduced plant growth. However, pretreatment with 100 µM melatonin (7 days) followed by exposure to heat stress (24 h) effectively reduced the oxidative stress by controlling the overaccumulation of superoxide (O2•-) and hydrogen peroxide (H2O2), lowering the lipid peroxidation content (as inferred based on malondialdehyde content) and less membrane injury index (MII). This was associated with increased the enzymatic and non-enzymatic antioxidants activities by regulating their related gene expression and modulating the ascorbate-glutathione cycle. The presence of melatonin induced respiratory burst oxidase (RBOH), heat shock transcription factors A2 (HsfA2), heat shock protein 90 (HSP90), and delta 1-pyrroline-5-carboxylate synthetase (P5CS) gene expression, which helped detoxify excess ROS via the hydrogen peroxide-mediated signaling pathway. In addition, heat stress boosted the endogenous levels of putrescine, spermidine and spermine, and increased the PAs contents, indicating higher metabolic gene expression. Moreover, melatonin-pretreated seedlings had further increased PAs levels and upregulated transcript abundance, which coincided with suppression of catabolic-related genes expression. Under heat stress, exogenous melatonin increased endogenous NO content along with nitrate reductase- and NO synthase-related activities, and expression of their related genes were also elevated. CONCLUSIONS: Melatonin pretreatment positively increased the heat tolerance of tomato seedlings by improving their antioxidant defense mechanism, inducing ascorbate-glutathione cycle, and reprogramming the PAs metabolic and NO biosynthesis pathways. These attributes facilitated the scavenging of excess ROS and increased stability of the cellular membrane, which mitigated heat-induced oxidative stress.

Exogenous putrescine alleviates photoinhibition caused by salt stress through cooperation with cyclic electron flow in cucumber.

When plants suffer from abiotic stresses, cyclic electron flow (CEF) is induced for photo-protection. Putrescine (Put), a primary polyamine in chloroplasts, plays a critical role in stress tolerance. However, the relationship between CEF and Put in chloroplasts for photo-protection is unknown. In this study, we investigated the role of Put-induced CEF for salt tolerance in cucumber plants (Cucumis sativus L). Treatment with 90 mM NaCl and/or Put did not influence the maximum photochemical efficiency of PSII (Fv/Fm), but the photoactivity of PSI was severely inhibited by NaCl. Salt stress induced a high level of CEF; moreover, plants treated with both NaCl and Put exhibited much higher CEF activity and ATP accumulation than those exhibited by single-salt-treated plants to provide an adequate ATP/NADPH ratio for plant growth. Furthermore, Put decreased the trans-membrane proton gradient (ΔpH), which was accompanied by reduced pH-dependent non-photochemical quenching (NPQ) and an increased the effective quantum yield of PSII (Y(II)). The ratio of NADP+/NADPH increased significantly with Put in salt-stressed leaves compared with the ratio in leaves treated with NaCl, indicating that Put relieved over-reduction pressure at the PSI acceptor side caused by salt stress. Collectively, our results suggest that exogenous Put creates an excellent condition for CEF promotion: a large amount of pmf is predominantly stored as Δψ, resulting in moderate lumen pH and low NPQ, while maintaining high rates of ATP synthesis (high pmf).

Overexpression of Transglutaminase from Cucumber in Tobacco Increases Salt Tolerance through Regulation of Photosynthesis.

Transglutaminase (TGase) is a regulator of posttranslational modification of protein that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms of TGase-mediated salt tolerance remain largely unknown. Here, we found that the transcription of cucumber TGase (CsTGase) was induced in response to light and during leaf development, and the CsTGase protein was expressed in the chloroplast and the cell wall. The overexpression of the CsTGase gene effectively ameliorated salt-induced photoinhibition in tobacco plants, increased the levels of chloroplast polyamines (PAs) and enhanced the abundance of D1 and D2 proteins. TGase also induced the expression of photosynthesis related genes and remodeling of thylakoids under normal conditions. However, salt stress treatment reduced the photosynthesis rate, PSII and PSI related genes expression, D1 and D2 proteins in wild-type (WT) plants, while these effects were alleviated in CsTGase overexpression plants. Taken together, our results indicate that TGase-dependent PA signaling protects the proteins of thylakoids, which plays a critical role in plant response to salt stress. Thus, overexpression of TGase may be an effective strategy for enhancing resistance to salt stress of salt-sensitive crops in agricultural production.

Identification of microRNAs associated with the exogenous spermidine-mediated improvement of high-temperature tolerance in cucumber seedlings (Cucumis sativus L.).

BACKGROUND: High-temperature stress inhibited the growth of cucumber seedlings. Foliar spraying of 1.0 mmol·L- 1 exogenous spermidine (Spd) to the sensitive cucumber cultivar 'Jinchun No. 2' grown at high-temperature (42 °C/32 °C) in an artificial climate box improved the high-temperature tolerance. Although there have been many reports on the response of microRNAs (miRNAs) to high-temperature stress, the mechanism by which exogenous Spd may mitigate the damage of high-temperature stress through miRNA-mediated regulation has not been studied. RESULTS: To elucidate the regulation of miRNAs in response to exogenous Spd-mediated improvement of high-temperature tolerance, four small RNA libraries were constructed from cucumber leaves and sequenced: untreated-control (CW), Spd-treated (CS), high-temperature stress (HW), and Spd-treated and high-temperature stress (HS). As a result, 107 known miRNAs and 79 novel miRNAs were identified. Eight common differentially expressed miRNAs (miR156d-3p, miR170-5p, miR2275-5p, miR394a, miR479b, miR5077, miR5222 and miR6475) were observed in CS/CW, HW/CW, HS/CW and HS/HW comparison pairs, which were the first set of miRNAs that responded to not only high-temperature stress but also exogenous Spd in cucumber seedlings. Five of the eight miRNAs were predicted to target 107 potential genes. Gene function and pathway analyses highlighted the integral role that these miRNAs and target genes probably play in the improvement of the high-temperature tolerance of cucumber seedlings through exogenous Spd application. CONCLUSIONS: Our study identified the first set of miRNAs associated with the exogenous Spd-mediated improvement of high-temperature tolerance in cucumber seedlings. The results could help to promote further studies on the complex molecular mechanisms underlying high-temperature tolerance in cucumber and provide a theoretical basis for the high-quality and efficient cultivation of cucumber with high-temperature resistance.

Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

KEY MESSAGE: The mechanism of exogenous Spd-induced Ca(NO3)2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO3)2 stress resistance. Ca(NO3)2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO3)2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO3)2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO3)2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO3)2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H2O2 and MDA, were detected after exogenous Spd application during Ca(NO3)2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO3)2 stress tolerance mediated by exogenous Spd.

The effect of exogenous calcium on cucumber fruit quality, photosynthesis, chlorophyll fluorescence, and fast chlorophyll fluorescence during the fruiting period under hypoxic stress.

BACKGROUND: Plants often suffer from hypoxic stress during waterlogging and hydroponic culturing. This study investigated the response of cucumber (Cucumis sativus L.) plant growth parameters, leaf photosynthesis, chlorophyll fluorescence, fast chlorophyll a fluorescence transient (OJIP), and fruit quality parameters to hypoxic stress alleviated by exogenous calcium. During the fruiting period, cucumber plants were exposed to hypoxia and hypoxia + Ca2+ treatment (4 mM Ca2+) for 9 d. RESULT: Exogenous calcium application enhanced the biomass and fruit quality of hypoxic stressed cucumber and also increased the net photosynthesis rate, stomatal conductance, intercellular CO2 concentration, maximum quantum efficiency of photosystem II photochemistry, actual photochemical efficiency of PSII, photochemical quenching coefficient, and non-photochemical quenching coefficient. Additionally, measurement of chlorophyll a fluorescence transients showed the positive K- and L-bands were more pronounced in leaves treated with hypoxia compared with those with hypoxia + Ca2+, indicating that hypoxic treatment induced uncoupling of the oxygen-evolving complex and inhibited electron transport beyond plastoquinone pool (Qa, Qb) including possible constraints on the reduction of end electron acceptors of photosystem I. Exogenous calcium can reduce these stress-induced damages in cucumber. CONCLUSION: This research focused the effect of exogenous calcium on cucumber photosynthesis during the fruiting period under hypoxic stress. Hypoxic stress might impair the photosynthetic electron-transport chain from the donor side of PSII up to the reduction of end acceptors of PSI, and exogenous calcium enhanced electron transport capacity and reduced hypoxic damage of cucumber leaves.

NaCl stress induces CsSAMs gene expression in Cucumis sativus by mediating the binding of CsGT-3b to the GT-1 element within the CsSAMs promoter.

MAIN CONCLUSION: The CsSAMs promoter is a salt-stress-inducible promoter containing three GT-1 elements that are sufficient for the salt-stress response. The transcription factor CsGT-3b was found to bind to the GT-1 element. The S-adenosyl-L-methionine synthase (SAMs) gene is among the functional genes induced during environmental stress. However, little is known about the regulatory mechanism and upstream regulators of this salt-inducible gene in cucumber plants. Thus, it is necessary to understand the characteristics of the SAMs gene by analyzing its promoter and transcription factors. In this study, we isolated and functionally analyzed a 1743-bp flanking fragment of the CsSAMs gene from Cucumis sativus. To examine promoter activity, the full-length promoter, as well as different promoter fragments, were fused to the ß-glucuronidase (GUS) reporter gene and introduced into the tobacco genome. The full-length promoter displayed maximal promoter activity, whereas the P4 promoter, containing 321 bp of upstream sequence, showed no basal promoter activity. In addition, the CsSAMs promoter exhibited stress-inducible regulation rather than tissue-specific activity in transgenic tobacco. Histochemical analysis revealed strong GUS staining in leaves, stems, and roots, especially in the veins of leaves, the vascular bundle of stems, and root tip zones following NaCl stress. A transient expression assay confirmed that the 242-bp region (-1743 to -1500) was sufficient for the NaCl-stress response. Yeast one-hybrid assays further revealed interaction between the NaCl-response protein CsGT-3b and the GT-1 (GAAAAA) element within the 242-bp region. Taken together, we revealed the presence of four salt-stress-responsive elements (GT-1 cis-elements) in the CsSAMs promoter and identified a transcription factor, CsGT-3b, that specifically binds to this sequence. These results might help us better understand the intricate regulatory network of the cucumber SAMs gene.

NO accumulation alleviates H2 O2 -dependent oxidative damage induced by Ca(NO3 )2 stress in the leaves of pumpkin-grafted cucumber seedlings.

Nitric oxide (NO) and hydrogen peroxide (H2 O2 ), two important signaling molecules, are stimulated in plants by abiotic stresses. In this study, we investigated the role of NO and its interplay with H2 O2 in the response of self-grafted (S-G) and salt-tolerant pumpkin-grafted (Cucurbita maxima × C. moschata) cucumber seedlings to 80 mM Ca(NO3 )2 stress. Endogenous NO and H2 O2 production in S-G seedlings increased in a time-dependent manner, reaching maximum levels after 24 h of Ca(NO3 )2 stress. In contrast, a transient increase in NO production, accompanied by H2 O2 accumulation, was observed at 2 h in rootstock-grafted plants. Nw -Nitro-l-Arg methyl ester hydrochloride (l-NAME), an inhibitor of nitric oxide synthase (NOS), tungstate, an inhibitor of nitrate reductase (NR), and 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (cPTIO), a scavenger of NO, were found to significantly inhibit NO accumulation induced by salt stress in rootstock-grafted seedlings. H2 O2 production was unaffected by these stress conditions. Ca(NO3 )2 stress-induced NO accumulation was blocked by pretreatment with an H2 O2 scavenger (dimethylthiourea, DMTU) and an inhibitor of NADPH oxidase (diphenyleneiodonium, DPI). In addition, maximum quantum yield of PSII (Fv/Fm), as well as the activities and transcript levels of antioxidant enzymes, were significantly decreased by salt stress in rootstock grafted seedlings after pretreatment with these above inhibitors; antioxidant enzyme transcript levels and activities were higher in rootstock-grafted seedlings compared with S-G seedlings. These results suggest that rootstock grafting could alleviate the oxidative damage induced by Ca(NO3 )2 stress in cucumber seedlings, an effect that may be attributable to the involvement of NO in H2 O2 -dependent antioxidative metabolism.

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings.

KEY MESSAGE: Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings. Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

Root proteomics reveals cucumber 24-epibrassinolide responses under Ca(NO3)2 stress.

KEY MESSAGE: The application of exogenous 24-epibrassinolide promotes Brassinosteroids intracellular signalling in cucumber, which leads to differentially expressed proteins that participate in different life process to relieve Ca(NO 3 ) 2 damage. NO3 (-) and Ca(2+) are the main anion and cation of soil secondary salinization during greenhouse cultivation. Brassinosteroids (BRs), steroidal phytohormones, regulate various important physiological and developmental processes and are used against abiotic stress. A two-dimensional electrophoresis gel coupled with MALDI-TOF/TOF MS was performed to investigate the effects of exogenous 24-epibrassinolide (EBL) on proteomic changes in cucumber seedling roots under Ca(NO3)2 stress. A total of 80 differentially accumulated protein spots in response to stress and/or exogenous EBL were identified and grouped into different categories of biological processes according to Gene Ontology. Under Ca(NO3)2 stress, proteins related to nitrogen metabolism and lignin biosynthesis were induced, while those related to cytoskeleton organization and cell-wall neutral sugar metabolism were inhibited. However, the accumulation of abundant proteins involved in protein modification and degradation, defence mechanisms against antioxidation and detoxification and lignin biosynthesis by exogenous EBL might play important roles in salt tolerance. Real-time quantitative PCR was performed to investigate BR signalling. BR signalling was induced intracellularly under Ca(NO3)2 stress. Exogenous EBL can alleviate the root indices, effectively reduce the Ca(2+) content and increase the K(+) content in cucumber roots under Ca(NO3)2 stress. This study revealed the differentially expressed proteins and BR signalling-associated mRNAs induced by EBL in cucumber seedling roots under Ca(NO3)2 stress, providing a better understanding of EBL-induced salt resistance in cucumber seedlings. The mechanism for alleviation provides valuable insight into improving Ca(NO3)2 stress tolerance of other horticultural plants.

Mitigative effects of spermidine on photosynthesis and carbon-nitrogen balance of cucumber seedlings under Ca(NO3)2 stress.

Ca(NO3)2 stress is one of the most serious constraints to plants production and limits the plants growth and development. Application of polyamines is a convenient and effective approach for enhancing plant salinity tolerance. The present investigation aimed to discover the photosynthetic carbon-nitrogen (C-N) mechanism underlying Ca(NO3)2 stress tolerance by spermidine (Spd) of cucumber (Cucumis sativus L. cv. Jinyou No. 4). Seedling growth and photosynthetic capacity [including net photosynthetic rate (P N), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr)] were significantly inhibited by Ca(NO3)2 stress (80 mM). However, a leaf-applied Spd (1 mM) treatment alleviated the reduction in growth and photosynthesis in cucumber caused by Ca(NO3)2 stress. Furthermore, the application of exogenous Spd significantly decreased the accumulation of NO3 (-) and NH4 (+) caused by Ca(NO3)2 stress and remarkably increased the activities of N metabolism enzymes simultaneously. In addition, photosynthesis N-use efficiency (PNUE) and free amino acids were significantly enhanced by exogenous Spd in response to Ca(NO3)2 stress, thus promoting the biosynthesis of N containing compounds and soluble protein. Also, the amounts of several carbohydrates (including sucrose, fructose and glucose), total C content and the C/N radio increased significantly in the presence of Spd. Based on our results, we suggest that exogenous Spd could effectively accelerate nitrate transformation into amino acids and improve cucumber plant photosynthesis and C assimilation, thereby enhancing the ability of the plants to maintain their C/N balance, and eventually promote the growth of cucumber plants under Ca(NO3)2 stress.

Effects of exogenous spermidine on photosynthetic capacity and expression of Calvin cycle genes in salt-stressed cucumber seedlings.

We investigated the effects of exogenous spermidine (Spd) on growth, photosynthesis and expression of the Calvin cycle-related genes in cucumber seedlings (Cucumis sativus L.) exposed to NaCl stress. Salt stress reduced net photosynthetic rates (PN), actual photochemical efficiency of PSII (ΦPSII) and inhibited plant growth. Application of exogenous Spd to salinized nutrient solution alleviated salinity-induced the inhibition of plant growth, together with an increase in PN and ΦPSII. Salinity markedly reduced the maximum carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Vcmax), the maximal velocity of RuBP regeneration (Jmax), triose-phosphate utilization capacity (TPU) and carboxylation efficiency (CE). Spd alleviated the negative effects on CO2 assimilation induced by salt stress. Moreover, Spd significantly increased the activities and contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and fructose-1,6-biphosphate aldolase (ALD; aldolase) in the salt-stressed cucumber leaves. On the other hand, salinity up-regulated the transcriptional levels of ribulose-1,5-bisphosphate (RCA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribrokinase (PRK) and down-regulated the transcriptional levels of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS), ALD, triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate phosphatase (FBPase) and 3-phosphoglyceric acid kinase (PGK). However, Spd application to salt-stressed plant roots counteracted salinity-induced mRNA expression changes in most of the above-mentioned genes. These results suggest that Spd could improve photosynthetic capacity through regulating gene expression and activity of key enzymes for CO2 fixation, thus confers tolerance to salinity on cucumber plants.