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Nitric oxide (NO) plays an important role in plant growth, development, and stress responses. The exogenous NO donor sodium nitroprusside (SNP) increased the plant height, root length, and biomass of tomato seedlings under nitrate stress, especially 100 μM SNP. The reactive oxygen species (ROS) and malondialdehyde (MDA) contents were decreased, while the antioxidant enzyme activities, ascorbate and glutathione contents, and expression of SlTrxh, SlNTRB, SlTpx were increased in tomato after SNP and nitrate stress treatment, compared with nitrate stress. The tomato plants showed shorter root length, more MDA and ROS contents after the NADPH-dependent thioredoxin reductase (NTR) inhibitor Auranofin (ANF) was added, suggesting that ANF inhibited the alleviating effect of NO under nitrate stress. The NO and S-nitrosothiol (SNO) contents and the SlNR transcript levels were decreased and the expression of SlGLB, SlGSNOR was increased after NO and nitrate stress treatment. The S-nitrosylated level in tomato roots was increased after nitrate stress. The SlMDHAR, SlTrxh, and SlNTRB protein were S-nitrosylated after NO and nitrate stress treatment. These results suggested that key enzymes in ascorbate-glutathione cycle and Trx system played an important role in alleviating the effect of NO in tomato seedlings under nitrate stress involving the S-nitrosylation modification.

H2O2 is an important signaling molecule that involved in multiple physiological metabolic activities in plants. Excess H2O2 can destroy biological macromolecules to poison the cell. Thioredoxin peroxidase (Tpx) plays an important role in protecting plants from oxidative damage by clearing the H2O2. In this study, tomato Tpx (SlTpx) gene was cloned and bioinformatic analysis was done. The mRNA transcript level of SlTpx in tomato roots and leaves were increased significantly after NaCl stress treatment. SlTpx overexpression transgenic tobacco plants were obtained to study its function under NaCl stress. The seed germination rate of SlTpx overexpression plants was higher than that in wild plants (WT) under NaCl treatment. The ROS and NO accumulation in transgenic tobacco root tips were all less than in WT under NaCl stress. Meanwhile,compared with salt stress, transgenic plants had low ROS accumulation, high antioxidant enzyme activity and high proline and soluble sugar content. The Na+ metabolism genes expression was higher in transgenic plants than the WT. The SlTpx transgenic plants showed higher tolerance to H2O2 and MV treatment, compared with the WT. Besides, the growth of prokaryotic strain of SlTpx was better than the pET28a strain with H2O2 treatment. The above results indicate that the SlTpx gene improves the plant salt tolerance by scaveging H2O2.

S-nitrosylation, the addition of nitric oxide (NO) moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. S-nitrosoglutathione reductase (GSNOR) is thought to be the major regulator of total cellular SNO levels in plants. However, its role in excess nitrate stress has not been investigated in spinach. In this study, a spinach GSNOR gene (GenBank Accession No. KR381778) was amplified and designated as SoGSNOR. The transcript and protein level of SoGSNOR were reduced after excess nitrate treatment for 24 h. Addition of NO donor to the nitrate solution decreased the SoGSNOR expression, while supplementation inhibitor of nitrate reductase and nitric oxide synthase increased its expression. Overexpression of SoGSNOR in tobacco increased the germination rate of transgenic seeds, compared to the wild type (WT) under nitrate stress. Higher photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency and expression level of some stress-related genes were detected in the transgenic seedlings than the WT under nitrate stress. The transgenic tobacco seedlings have lower malondialdehyde content, reactive oxygen species (ROS) fluorescence, and higher activities and transcript level of superoxide dismutase, catalase, peroxidase under nitrate stress. SoGSNOR transgenic tobacco plants have lower NR activity and protein level, higher GSNOR and non-symbiotic class 1 hemoglobin (nsHb) protein level than the WT plants, leading to lower NO accumulation and SNOs contents under nitrate stress. These results suggested that overexpression of SoGSNOR increased nitrate stress tolerance of tobacco by regulating ROS and RNS metabolism. A spinach S-nitrosoglutathione reductase (SoGSNOR) gene was isolated from spinach root. The expression level and activities of SoGSNOR were reduced by excess nitrate treatment. Overexpression of SoGSNOR in tobacco increased nitrate stress tolerance by regulating ROS and RNS metabolism.

Background Transcranial direct current stimulation (tDCS) has been explored in epilepsy with limited samples, varied parameters, and inconclusive results. We aimed to study the efficacy of tDCS for patients with refractory focal epilepsy. Method We conducted a randomized, double-blind, sham-controlled, and three-arm (Group 1 (sham), Group 2 (20-min), and Group 3 (2 × 20-min)) tDCS parallel multicenter study. The primary outcome measurement was seizure frequencies (SFs). The study consisted of 28-days baseline, 14-days treatment, and 56-days follow-up. The cathode was placed over the epileptogenic focus, and the current intensity was 2 mA. The generalized estimating equations model, one-way analysis of variance, chi-square and Kruskal-Wallis test were used for analysis. Results Of the 82 enrolled patients, 70 patients were included for final analysis (Group 1, n = 21; Group 2, n = 24; and Group 3, n = 25). There was a significant reduction in SFs for both active tDCS groups compared with the sham group. Patients in Group 2 showed a significantly 50.73–21.91% greater reduction in SFs that lasted for 4 weeks (p = 0.008–0.060). Patients in Group 3 showed a significantly 63.19–49.79% greater reduction in SFs compared with the sham group that lasted for 5 weeks (p = 0.011–0.045). Patients in Group 3 had a 64.98–66.32% greater reduction in SFs at W9–W10, when compared with Group 2 (p = 0.021–0.022). Conclusion Fourteen consecutive days tDCS significantly decreased SFs in patients with refractory focal epilepsy, with 2 × 20-min daily stimulation protocol being superior to 20-min daily stimulation protocol.

Phytotoxicity reduction is one of the main aims of reusing walnut green husk (WGH) for cultivation purposes. This study was conducted to evaluate the efficiency of bio-detoxification by selected Trametes and Schizophyllum strains under solid-state fermentation. Ninety-two isolates of filamentous fungi were obtained from forest litter, and nine strains belonging to Trametes and Schizophyllum genus were selected due to their efficient laccase production and rapid growth using WGH and corncob (w/w, 1:1) as the carbon source under solid-state fermentation. Further, enzyme activities and phytotoxicity assays showed that water extract of WGH treated by Trametes strains 120301, 10288, and 10306 showed a vast lignocellulose enzyme system, higher laccase activity (about 620 U/g), and maximal germination rate (100%), compared with 20% germination rate of the untreated extract of WGH. Our results indicated that Trametes strains are suitable for the phytotoxicity reduction of WGH and laccase production, and showed the possible role of laccase in the detoxification of agricultural wastes rich in phenols such as WGH.

Objectives: to evaluate the effect of sodium reduction based on a modified DASH diet on blood pressure in hypertensive patients with type 2 diabetes. Material and methods: sixty-one hypertensive patients with type 2 diabetes were selected from the community and randomly allocated to a common salt group and low sodium salt group receiving the 8-week dietary intervention, in which weeks 1-2 was the dietary guidance phase, weeks 3-4 was the centralized feeding phase, and weeks 5-8 was the home medical care phase. Participants were followed up in the hospital once a week to collect information on outpatient blood pressure, salt, and drug use. Physical examinations were conducted at 4 weeks and the end of the intervention, as well as at baseline. Results: after the intervention, the blood pressure of both the low sodium group (SBP: -14.32 mmHg, p0.001; DBP: -6.32mmHg, p0.001) and the common salt group (SBP: -10.98 mmHg, p0.001; DBP: -5.24 mmHg, p = 0.001) decreased significantly with a more pronounced decrease in the low sodium group but no statistically significant differences between the two groups (SBP: -0.28 mmHg, p = 0.929; DBP: -3.32 mmHg, p = 0.093). At the end of the intervention, sodium intake was significantly decreased, but potassium intake was increased in the low sodium group (p0.05); however, the common salt group had no significant change. Conclusion: reducing sodium intake based on the modified DASH diet had a good effect on systolic and diastolic blood pressure in hypertensive patients with type 2 diabetes. Sodium reduction based on the modified DASH diet is safe and effective, and can be used as a guide for healthy living in hypertensive patients.Objetivos: evaluar el efecto de la reducción de sodio basada en una dieta DASH modificada sobre la presión arterial en pacientes hipertensos con diabetes de tipo 2. Material y métodos: sesenta y un pacientes hipertensos con diabetes de tipo 2 fueron seleccionados de la comunidad y asignados aleatoriamente a un grupo de sal común y un grupo de sal baja en sodio, que recibieron una intervención dietética de 8 semanas en la que las semanas 1-2 fueron la fase de orientación dietética, las semanas 3-4 fueron la fase de alimentación centralizada, y las semanas 5-8 fueron la fase de atención médica domiciliaria. Los participantes fueron seguidos en el hospital una vez por semana para recopilar información sobre la presión arterial, la sal y el uso de drogas en pacientes ambulatorios. Los exámenes físicos se realizaron a las 4 semanas y al final de la intervención, así como al inicio del estudio. Resultados: después de la intervención, la presión arterial tanto del grupo bajo en sodio (PAS: -14,32 mmHg, p0,001; PAD: -6,32 mmHg, p0,001) como del grupo de sal común (PAS: -10,98 mmHg, p0,001; PAD: -5,24 mmHg, p = 0,001) disminuyó significativamente con una disminución más pronunciada en el grupo bajo en sodio pero sin diferencias estadísticamente significativas entre los dos grupos (PAS: -0,28 mmHg, p = 0,929; PAD: -3,32 mmHg, p = 0,093). Al final de la intervención, la ingesta de sodio disminuyó significativamente, pero la ingesta de potasio aumentó en el grupo bajo en sodio (p0,05); sin embargo, el grupo de la sal común no tuvo cambios significativos. Conclusión: la reducción de la ingesta de sodio basada en la dieta DASH modificada tuvo un buen efecto sobre la presión arterial sistólica y diastólica en pacientes hipertensos con diabetes de tipo 2. La reducción de sodio basada en la dieta DASH modificada es segura y eficaz, por lo que puede utilizarse como guía para una vida saludable en pacientes hipertensos.

Introduction: hypertension and diabetes are chronic disorders associated with an increased risk of cardiovascular disease. Objectives: to evaluate the effect of 52 % low-sodium salt applied to the Chinese-modified DASH (CM-DASH) diet on risk of atherosclerotic cardiovascular disease (ASCVD) in patients with hypertension and type-2 diabetes. Methods: the low-sodium salt group (LSSG) took 5 g/day of 52 % low-sodium salt plus CM-DASH diet for 8 weeks, while the normal-sodium salt group (NSSG) took the same dose of normal-sodium salt plus CM-DASH diet for 8 weeks. Blood tests, 24-hour urine tests, anthropometric measurements, and 10-year risk of ASCVD prediction were assessed. Results: compared with baseline, both LSSG and NSSG showed a significant reduction in 10-year risk of ASCVD, but we did not find any statistically significant differences in 10-year risk of ASCVD between LSSG and NSSG. Conclusions: our study shows that salt limits and DASH diets reduce the risk of cardiovascular disease whereas low-sodium salt containing 52 % sodium chloride did not significantly lower the risk of cardiovascular disease when compared to regular salt. Due to the limitations of the research, additional studies will be necessary to confirm our findings.Introducción: la hipertensión y la diabetes son trastornos crónicos asociados a un mayor riesgo de enfermedad cardiovascular. Objetivos: evaluar el efecto de la sal baja en sodio (52 %) aplicada a la dieta DASH modificada en China (CM-DASH) sobre los riesgos de enfermedad cardiovascular aterosclerótica (ECA) en pacientes con hipertensión arterial y diabetes de tipo 2. Métodos: el grupo de sal baja en sodio (LSSG) tomó 5 g/día de sal baja en sodio al 52 % más dieta CM-DASH durante 8 semanas, mientras que el grupo de sal normal en sodio (NSSG) tomó la misma dosis de sal normal en sodio más dieta CM-DASH durante 8 semanas. Se evaluaron los análisis de sangre, los análisis de orina de 24 horas, las mediciones antropométricas y los riesgos de predicción de ECVA a 10 años. Resultados: en comparación con el valor basal, tanto el LSSG como el NSSG mostraron una reducción significativa de los riesgos a 10 años de ECVA, mientras que no se encontraron diferencias estadísticamente significativas en los riesgos a 10 años de ECVA entre el LSSG y el NSSG. Conclusiones: nuestro estudio muestra que los límites de sal y las dietas DASH reducen el riesgo de enfermedad cardiovascular mientras que la sal baja en sodio con un 52 % de cloruro sódico no redujo significativamente el riesgo de enfermedad cardiovascular en comparación con la sal normal. Debido a las limitaciones de la investigación, serán necesarios estudios adicionales para confirmar nuestros hallazgos.

Phenolic glycosides are the important bioactive molecules, and their bioavailability can be influenced by enzyme hydrolysis, such as β-glucosidases (EC3.2.1.21) and other glycosyl hydrolases (GHs). Wood rotting fungi possess a superfamily of GHs, but little attention has been paid to the GHs and their potential applications in biotransformation of phenolic glycosides. In this study, two GH3 gene family members of

To elucidate the mechanism of soybean resistance to Al, physiological and biochemical indices and antioxidant enzyme expression and activities were systematically analyzed in Al-sensitive (Glycine max Merr., Yunnan Province of China, SB) and Al-resistant Dambo (Glycine max Merr., Kyoto of Japan, RB) black soybean plants. According to the results, the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in RB root tips were significantly lower than those in SB root tips, though the opposite results occurred for soluble protein contents. Moreover, the expression and activities of superoxide dismutase (SOD, EC1.15.1.1.1.1.1.1), peroxidase (POD, EC1.11.1.7) and catalase (CAT, EC1.11.1.6) under 0–400 μM Al for 0–96 h were greater in RB than in SB. However, below 100 μM Al, the activities of those enzymes in SB increased with increasing Al concentration and treatment duration, with SOD activity being lowest and CAT activity exceeding that of POD with increasing Al concentration. Overall, enzyme activity in SB treated with Al at concentrations greater than 200 μM was lower than that in the SB control (CK; not treated with Al) and decreased with treatment duration. Additionally, at Al concentrations lower than 200 μM, enzyme activities in RB were significantly greater than those in RB CK and increased with both Al concentration and treatment duration. Moreover, enzyme activity in RB treated with 400 μM Al was slightly greater than that in RB CK. Thus, CAT activity determines soybean resistance to Al. These results indicate that soybean resistance to Al can be enhanced by regulating the expression and activity of antioxidant enzymes to remove H2O2 under Al stress.

White-rot fungi, especially Trametes strains, are the primary source of industrial laccases in bioenergy and bioremediation. Trametes strains express members of the laccase gene family with different physicochemical properties and expression patterns. However, the literature on the expression pattern of the laccase gene family in Trametes trogii S0301 and the response mechanism to Cu(2+), a key laccase inducer, in white-rot fungal strains is scarce. In the present study, we found that Cu(2+) could induce the mRNAs and proteins of the two alternative splicing variants of heat shock transcription factor 2 (TtHSF2). Furthermore, the overexpression of alternative splicing variants TtHSF2α and TtHSF2β-I in the homokaryotic T. trogii S0301 strain showed opposite effects on the extracellular total laccase activity, with maximum laccase activities of approximately 0.6 and 3.0 U ml(−1), respectively, on day 8, which are 0.4 and 2.3 times that of the wild-type strain. Similarly, TtHSF2α and TtHSF2β-I play opposite roles in the oxidation tolerance to H(2)O(2). In addition, the direct binding of TtHSF2α to the promoter regions of the representative laccase isoenzymes (TtLac1 and TtLac13) and protein-protein interactions between TtHSF2α and TtHSF2β-I were detected. Our results demonstrate the crucial roles of TtHSF2 and its alternative splicing variants in response to Cu(2+). We believe that these findings will deepen our understanding of alternative splicing of heat shock transcription factors (HSFs) and their regulatory mechanism of the laccase gene family in white-rot fungi. IMPORTANCE The members of laccase gene family in Trametes strains are the primary source of industrial laccase and have gained widespread attention. Increasing the yield and enzymatic properties of laccase through various methods has always been a topic worthy of attention, and there is no report on the regulation of laccase expression through HSF transcription factor engineering. Here, we found that two alternative splicing variants of TtHSF2 functioned oppositely in regulating the expression of laccase genes, and copper can induce the expression of almost all members of the laccase gene family. Most importantly, our study suggested that TtHSF2 and its alternative splicing variants are vital for copper-induced production of laccases in T. trogii S0301.

Nitrogen (N) is one of the essential macronutrients that plays important roles in plant growth and development. To better understand the response of antioxidant system and N metabolism under N starvation and re-supply condition, physiological and transcriptomic analysis were performed in tomato roots. The malondialdehyde (MDA) and reactive oxygen species (ROS) contents increased significantly in tomato seedlings after N starvation for 24 h. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR), the ratio of ASA/DHA and GSH/GSSG, the NO3- contents, nitrate reductase (NR) activity were decreased after N starvation treatment and increased after N re-supply for 24 h. Compared with the control, 1766 genes were up-regulated and 2244 genes were down-regulated after N starvation in tomato. These differentially expressed genes (DEGs) are mainly enriched in functional items such as cellular process, metabolic process and catalytic activity. The KEGG pathways revealed that the DEGs were mainly involved in phenpropane biosynthesis, amino sugar and nucleotide sugar metabolism, and N metabolism. The expression patterns of tomato SlSOD, SlCAT, SlAPX, SlMDHAR, thioredoxin (SlTrxh), peroxiredoxin (SlPrx) and glutaredoxin (SlGrx) genes, and nitrate transporter SlNRT2.4, SlNR, glutamine synthetase (SlGS2), nitrite reductase (SlNiR) decreased after N starvation and increased after N re-supply, which were validated by qRT-PCR. Our results provide a basis for understanding the response of tomato to N deficiency and re-supply and a theoretical reference for cultivation regulation.

Aim to compare the home blood pressure monitoring (HBPM) and visit blood pressure monitoring in a clinical phase I single-arm pilot trial. The 18% sodium substitute salt was used in 43 hypertensives for 8 weeks, and visited once a week, while weekly visit blood (VBP) pressure, daily home blood pressure (HBP) and urine test results before and after intervention were collected. 43 hypertensive patients were recruited, 4 were lost. And enrolled 39 patients for analysis. The VBP were lower than morning HBP and night HBP (P P P P P P P = 0.003) and night home SBP (− 10.2 mmHg, 95% CI: − 15.8 to − 4.6, P = 0.001) decreased significantly, after intervention. Both HBP and VBP showed that 18% substitute salt intervention could decrease the blood pressure of hypertensives. Medication led to VBP lower than HBP, but the two still had a good correlation.Trial registration: NCT03226327. Registered 21 July 2017—Retrospectively registered, http://www.clinicaltrials.gov.

Sunlight enhanced peel color and significantly up-regulated the expression of PyMYB10 and PybHLH genes. MYB-bHLH-WD40 transcriptional complex forms in the light and is involved in regulating anthocyanin accumulation in the peel. Anthocyanin is the major pigment in the peel of Yunnan red pear (Pyrus pyrifolia (Burm.) Nak.). A transcriptional activation protein complex, involving members of the transcription factor classes of MYB, bHLH and WD40, regulates anthocyanin biosynthesis. This complex was examined in the peel of red pear. In order to clarify the interaction of PyMYB10, PybHLH and PyWD40, fruit were bagged then peel samples collected 0, 3, 5, and 7 days after bag removal. Samples were used for Western blotting and protein interaction analysis. The results showed that sunlight enhanced peel color and significantly up-regulated the expression of both PyMYB10 and PybHLH genes. Co-immunoprecipitation (Co-IP) analysis showed that PybHLH interacted with PyMYB10 or PyWD40, and PyMYB10 interacted with PyWD40. Using onion cells as a model system, bimolecular fluorescence complementation (BiFC) confirmed these interactions and showed that the interaction localized to the nuclei. GST Pull down and Far-Western blotting assays demonstrated that PybHLH interacted with PyMYB10 or PyWD40, respectively, and PyMYB10 interacted with PyWD40 in vitro. In addition, EMSA assay showed that PyMYB10 can directly bind to the promoter of the gene encoding the anthocyanin biosynthesis enzyme anthocyanidin synthase (PyANS). Taken together, these results showed that the ternary complex of PyMYB10, PybHLH and PyWD40 transcription factors forms to regulate anthocyanin biosynthesis and accumulation in Yunnan red pear.

A thermo-activation and thermostable laccase isoenzyme (Lac 37 II) produced by Trametes trogii S0301 at 37°C was purified to apparent homogeneity by anionic exchange chromatography and sephadex G-75 chromatography, with 12.3% of yeiled and a specific activity of 343.1 U mg-1. The molecular weight of the purified Lac 37 II was estimated to be approximately 56 kDa in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the protein was 2.7 and 60°C, respectively. The purified Lac 37 II showed higher resistance to all tested metal ions and organic solvents except for Fe2+ and Cd2+ at 37°C and the activity of the purified Lac 37 was significantly enhanced by Cu2+ at 50 mM. The K cat , K m , and K cat /K m of Lac 37 II were 2.977 s-1, 16.1 μM, and 184.9 s-1 μM-1, respecively, in the condition of pH 2.7 and 60°C using ABTS as a substrate. Peptide-mass fingerprinting analysis showed that the Lac 37 II matched to the gene-deduced sequences of lcc3 in T. trogii BAFC 463, other than Lcc1, Lcc 2, and Lcc 4. Compared with laccase prepared at 28°C, the onset of thermo-activation of Lac 37 II activity occurred at 30°C with an increase of 10%, and reached its maximum at the temperatures range of 40-60°C with an increase of about 40% of their original activity. Furthermore, Lac 37 II showed the efficient decolorization ability toward triphenylmethane dyes at 60°C, with decolorization rates of 100 and 99.1% for 25 mg L-1 malachite and crystal violet in 5 h, respectively, when hydroxybenzotriazole (HBT) was used as a mediator. In conclusion, it is the first time to report a thermo-activation laccase from a thermophilic T. trogii strain, which has a better enzyme property and higher decolorization ability among fungal laccases, and it also has a further application prospective in the field of biotechnology.

To survive, sessile plants must adapt to grow and develop when facing water-deficit stress. However, the molecular mechanisms underlying fine-tuning of the antagonistic action between stress response and growth remain to be determined. Here, plants overexpressing Lateral Organ Boundaries Domain 15 (LBD15) showed abscisic acid (ABA) hypersensitivity and tolerance of water-deficit stress, whereas the loss-of-function mutant lbd15 presented decreased sensitivity to ABA and increased sensitivity to water-deficit stress. Further analysis revealed that LBD15 directly binds to the promoter of the ABA signaling pathway gene ABSCISIC ACID INSENSITIVE4 (ABI4) to activate its expression, thereby forming an LBD15-ABI4 cascade to optimally regulate ABA signaling-mediated plant growth and tolerance of water-deficit stress. In addition, drought stress-induced ABA signaling promoted LBD15 expression, which directly activates expression of ABI4 to close stomata. As a result, water loss is reduced, and then water-deficit stress tolerance is increased. The results of this study reveal a molecular mechanism by which LBD15 coordinates and balances plant growth and resistance to water-deficit stress.

The thioredoxin (Trx) system plays a vital function in cellular antioxidative defense. However, little is known about Trx in tomato under excess nitrate. In this study, we isolated the tomato gene encoding h-type Trx gene (SlTrxh). The mRNA transcript of SlTrxh in roots and leaves of tomato was induced incrementally under excess nitrate for 24 h. Subcellular localization showed that SlTrxh might localize in the cytoplasm, nucleus and plasma membrane. Enzymatic activity characterization revealed that SlTrxh protein possesses the disulfide reductase function and Cysteine (Cys) 54 is important for its activity. Overexpressing SlTrxh in tobacco resulted in increasing seed germination rate, root length and decreasing H2O2 and O2− accumulation, compared with the wild type (WT) tobacco under nitrate stress. While overexpressing SlTrxhC54S (Cysteine 54 mutated to Serine) in tobacco showed decreased germination rate and root length compared with the WT after nitrate treatment. After nitrate stress treatment, SlTrxh overexpressing transgenic tobacco plants have lower malonaldehyde (MDA), H2O2 contents and Reactive Oxygen Species (ROS) accumulation, and higher mRNA transcript level of NtP5CS, NtDREB2, higher ratio of ASA/DHA and GSH/GSSG, higher activities of ascorbate peroxidase and NADP thioredoxin reductase. Besides, SlTrxh overexpressing plants showed higher tolerance to Methyl Viologen (MV) in the seed germination and seedling stage. The yeast two-hybrid, pull-down, Co-immunoprecipitation and Bimolecular luciferase complementation assay confirmed that SlTrxh physically interacted with tomato peroxiredoxin (SlPrx). These results suggest that SlTrxh contributes to maintaining ROS homeostasis under excess nitrate stress interacting with SlPrx and Cys54 is important for its enzyme activity.

The inducible vectors pER8-Gateway-3Flag and pER8-Gateway-3Flag-SRDX have been subjected to considerable research in terms of the function of transcription factors (TFs) via transcription activity and repression, respectively. Approximately 1500 TFs have been identified in Arabidopsis thaliana genome. To identify their functions, loss-of-function and gain-of-function mutants were generated to analyze the phenotype. However, many loss-of-function mutants did not show any evident phenotype because of the functional redundancy within the TF family. The constitutive misexpression of some TFs may result in lethality or sterility. To overcome these problems, we produced a Gateway-compatible inducible binary vector system that facilitates fast and reliable DNA cloning and biological function identification. The vector can be used for the inducible expression of protein fusions to a polypeptide protein tag named 3xFLAG tag. This vector system can also be used to generate an inducible transcription inhibition of protein fusion to an Ethylene-Responsive Factor-associated amphiphilic repression (EAR) motif. The EAR motif makes it possible to get rid of redundancy within a TF family, thereby facilitating studies on loss of function. With these Gateway vectors, conventional subcloning technology that depends on restriction digestion and ligation is avoided. Thus, these Gateway vectors should be useful not only for the rapid analysis of the functions of redundant plant TFs, but also for the manipulation of TF overexpression, resulting in plant lethality or sterility, via an inducible promoter.

Soil secondary salinization caused by excess nitrate addition is one of the major obstacles in greenhouse vegetable production. Excess nitrate inhibited the growth of tomato plants, while application of 100 μM H2S donor NaHS efficiently increased the plant height, fresh and dry weight of shoot and root, root length, endogenous H2S contents and L-cysteine desulfhydrases activities. NaHS altered the oxidative status of nitrate-stressed plants as inferred by changes in reactive oxygen species (ROS) accumulation and lipid peroxidation accompanied by regulation of the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX). Besides, NaHS increased the nitric oxide (NO) and total S-nitrosothiols (SNOs) contents, nitrate reductase (NR) activities and decreased the S-nitrosoglutathione reductase (GSNOR) activities under nitrate stress. Furthermore, microarray analysis using the Affymetrix Tomato GeneChip showed that 5349 transcripts were up-regulated and 5536 transcripts were down-regulated under NaHS and excess nitrate stress treatment, compared to the excess nitrate stress alone. The differentially expressed genes (log2 fold change >2 or

Hydrogen sulfide (H2S) has been proposed as the third gasotransmitter after nitric oxide (NO) and carbon monoxide in animals and plants. In this study, the effect of H2S under excess nitrate stress in tomato root was studied. The results showed that excess nitrate inhibited the growth of tomato seedlings, while application of the H2S donor NaHS efficiently alleviated the growth inhibition, and decreased the lipid peroxidation, hydrogen peroxide (H2O2), and protein carbonyl contents, as well as the reactive oxygen species accumulation, especially 100 μM NaHS. Treatment with NaHS under nitrate stress increased the endogenous H2S and l -cysteine desulfhydrases (LCD) activity. Moreover, treatment with NaHS induced NO production and the synthesis of NO was mediated through nitrate reductase, but not through nitric oxide synthase in the presence or absence of nitrate. Besides, exogenous NaHS increased antioxidant enzyme genes (SOD, POD, CAT, and APX) expression, reducing the oxidative stress caused by excess nitrate. The alleviating effects of lipid peroxidation, H2O2 contents, and enhanced antioxidant enzyme activities by NaHS and NO donor (sodium nitroprusside, SNP), were reversed by 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the specific scavenger of NO. Together, above results suggested that NR-dependent NO production by NaHS was involved in the alleviation of nitrate stress by enhancing antioxidant enzyme activities in tomato root.

As a signal molecule, hydrogen sulfide (H2S) plays an important role in plant growth. In this study, the effect of different concentration of H2S donor, sodium hydrosulfide (NaHS), on growth of tomato seedlings was tested in hydroponic culture. The effects of 50 μM NaHS on tomato seedlings were not significant, compared with the control. After 100 μM NaHS treatment, plant height, shoot fresh weight and dry weight increased significantly. However, 300, 500 and 1000 μM NaHS had inhibitory effect on tomato seedlings, especially 1000 μM NaHS. 100 μM NaHS could increase chlorophyll a, chlorophyll b, and total chlorophyll content, stomatal conductance, net photosynthetic rate, transpiration rate and intercellular CO2 concentration. The activities of nitrate reductase, glutamate synthase and glutamine synthase, and L-cysteine demethylase (LCD), and nitrate content, were increased significantly after 100 μM NaHS treatment. The microarray results showed that 9122 genes changed after treated with 100 μM NaHS for 3 h, and 430 genes changed more than 2 times. These genes were grouped into metabolism, signal transduction, stress resistance, transcription factors, transport, protein synthesis and degradation, hormone response, and cell wall related genes in tomato seedlings. RT-PCR analysis showed that the expression of some MAPK family genes changed significantly when exogenous NaHS was added, especially SlMAPK3 and SlMAPK13. The MAPK inhibitor PD98059 reduce the LCD activity and H2S content in tomato seedlings, which indicated that MAPK signaling pathway was involved in the process of H2S promoting tomato seedling growth.

AtPrx64 is one of the peroxidases gene up-regulated in Al stress and has some functions in the formation of plant second cell wall. Its overexpression may improve plant tolerance to Al by some ways. Studies on its function under Al stress may help us to understand the mechanism of plant tolerance to Al stress. In Arabidopsis thaliana, the expressions of some genes (AtPrxs) encoding class III plant peroxidases have been found to be either up-regulated or down-regulated under aluminum (Al) stress. Among 73 genes that encode AtPrxs in Arabidopsis, AtPrx64 is always up-regulated by Al stress, suggesting this gene plays protective roles in response to such stress. In this study, transgenic tobacco plants were generated to examine the effects of overexpressing of AtPrx64 gene on the tolerance to Al stress. The results showed that overexpression of AtPrx64 gene increased the root growth and reduced the accumulation of Al and ROS in the roots. Compared with wild type controls, transgenic tobaccos had much less soluble proteins and malondialdehyde in roots and much more root citrate exudation. The activity of plasma membrane (PM) H+-ATPase, the phosphorylation of PM H+-ATPase and its interaction with 14-3-3 proteins increased in transgenic tobaccos; moreover, the content of lignin in root tips also increased. Taken together, these results showed that overexpression of AtPrx64 gene might enhance the tolerance of tobacco to Al stress.

In this study, the role of nitric oxide (NO) in response to excess nitrate stress in spinach was investigated. The results showed that excess nitrate stress provoked a significant reduction in the seedling growth and an increase in lipid peroxidation and H 2 O 2 content in spinach. The gene and protein expression of nitrate reductase and non-symbiotic hemoglobin were up-regulated, while S-nitrosoglutathione reductase (GSNOR) was down-regulated with the accumulation of NO and total S -nitrosothiols (SNOs) after 160 mM nitrate treatment for 24 h. Nitrate stress treatment increased the S -nitrosylation level of spinach. Using biotin-switch coupled with LC–MS/MS method, several proteins were identified as targets of S -nitrosylation including ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, phosphoglycerate kinase, 23 kDa oxygen-evolving protein (OEC), 16 kDa protein of the photosynthetic OEC, and ribulose-1,5-bisphosphate carboxylase oxygenase. We further demonstrated that exogenous NO donor (SNP) increased the superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and GSNOR activities, which would reduce reactive oxygen species (ROS) and reactive nitrogen species (RNS) contents and thereby enhance spinach tolerance to nitrate stress. Suppressing NO accumulation by NR inhibitor, NOS inhibitor, and NO scavenger aggravated the damage to spinach under nitrate stress. These results suggest that NO acts as an essential signal to enhance spinach tolerance to nitrate stress via reducing ROS and RNS toxicity.

A thermo-activation and thermostable laccase isoenzyme (Lac 37 II) produced by Trametes trogii S0301 at 37°C was purified to apparent homogeneity by anionic exchange chromatography and sephadex G-75 chromatography, with 12.3% of yeiled and a specific activity of 343.1 U mg–1. The molecular weight of the purified Lac 37 II was estimated to be approximately 56 kDa in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the protein was 2.7 and 60°C, respectively. The purified Lac 37 II showed higher resistance to all tested metal ions and organic solvents except for Fe2+ and Cd2+ at 37°C and the activity of the purified Lac 37 was significantly enhanced by Cu2+ at 50 mM. The Kcat, Km, and Kcat/Km of Lac 37 II were 2.977 s–1, 16.1 μM, and 184.9 s–1 μM–1, respecively, in the condition of pH 2.7 and 60°C using ABTS as a substrate. Peptide-mass fingerprinting analysis showed that the Lac 37 II matched to the gene-deduced sequences of lcc3 in T. trogii BAFC 463, other than Lcc1, Lcc 2, and Lcc 4. Compared with laccase prepared at 28°C, the onset of thermo-activation of Lac 37 II activity occurred at 30°C with an increase of 10%, and reached its maximum at the temperatures range of 40–60°C with an increase of about 40% of their original activity. Furthermore, Lac 37 II showed the efficient decolorization ability toward triphenylmethane dyes at 60°C, with decolorization rates of 100 and 99.1% for 25 mg L–1 malachite and crystal violet in 5 h, respectively, when hydroxybenzotriazole (HBT) was used as a mediator. In conclusion, it is the first time to report a thermo-activation laccase from a thermophilic T. trogii strain, which has a better enzyme property and higher decolorization ability among fungal laccases, and it also has a further application prospective in the field of biotechnology.

Additional file 6. BUSCO validation of the T. trogii S0301 genome annotation result.

As a major antioxidant in plants, ascorbic acid (AsA) plays a very important role in the response to aluminum (Al) stress. However, the effect of AsA on the mitigation of Al toxicity and the mechanism of nitrate nitrogen (NO3 −–N) uptake by plants under Al stress are unclear. In this study, a hydroponic experiment was conducted using peak 1 A rice (sterile line, Indica) with weaker resistance to Al and peak 1 superior 5 rice (F1 hybrid, Indica) with stronger resistance to Al to study the effects of exogenous AsA on the physiological and biochemical responses to NO3 −–N uptake by rice roots exposed to 50 μmol L−1 Al. Al stress induced increases in the concentrations of H2O2 and malondialdehyde (MDA) and in the activities of antioxidant enzymes [such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)]. Plasma membrane (PM) H+-ATPase and H+-pump activities, endogenous AsA content and NO3 −–N uptake in rice roots decreased under Al stress. After treatment with 2 mmol L−1 exogenous AsA combined with Al, concentrations of H2O2 and MDA in roots notably decreased, and endogenous AsA content and activities of SOD, POD, CAT, and APX in rice roots increased significantly; furthermore, the interaction of PM H+-ATPase and the 14-3-3 protein was also enhanced significantly compared with that in control plants without AsA treatment, which clearly increased NO3 −–N uptake. Based on all of these data, the application of AsA significantly reduced the accumulation of H2O2 and MDA and increased the activities of PM H+-ATPase and the H+-pump by increasing the endogenous AsA content, the antioxidant enzyme activities, and the interaction of PM H+-ATPase and the 14-3-3 protein in the roots of the two rice cultivars under Al stress, thereby improving the uptake of NO3 −–N in rice.

Hydrogen sulfide (H2S) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. Mitogen-activated protein kinase (MAPK) and nitric oxide (NO) are essential for abiotic stress signaling. This work investigated the effects of H2S and the crosstalk between H2S, MAPK and NO in cucumber roots under nitrate stress. The inhibitory effect of 140 mM nitrate on the growth of shoot and root was substantially alleviated by treatment with H2S donor sodium hydrosulfide (NaHS), especially 100 μM NaHS. Treatment with 100 μM NaHS reduced malondialdehyde (MDA) and H2O2 contents, ROS accumulation and increased the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). CsNMAPK transcript level was up-regulated by NaHS treatment, while significantly decreased by propargylglycine (PAG, specific inhibitor of H2S biosynthesis) and hypotaurine (HT, H2S scavenger) in cucumber roots under nitrate stress. NO accumulation was increased by NaHS treatment under nitrate stress, but reduced by HT, PAG and PD98059, indicating that NO might function downstream of MAPK and H2S. MAPK inhibitor PD98059 and NO scavenger (cPTIO) reversed the alleviating effect of H2S by increasing MDA and H2O2 contents, and decreasing antioxidant enzyme activities of SOD, CAT, POD, APX, and the endogenous H2S contents and LCD activities under nitrate stress. In conclusion, H2S played a protective role in cucumber seedlings under nitrate stress and MAPK/NO signaling were involved in the process by regulating antioxidant enzyme activities.

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Sterol C24 methyltransferase2 (SMT2) governs the pattern of phytosterols synthesized in higher plants. In this study, the spinach sterol C24 methyltransferase2 ( SoSMT2 ) cDNA was cloned by RT-PCR and RACE-PCR. The full length of SoSMT2 consists of 1614 bp, including a 278 bp 5′-untranslated region (UTR), a 1086 bp open reading frame (ORF), and a 250 bp 3′-UTR. The SoSMT2 gene encodes a polypeptide of 361 amino acid residues with a predicted molecular mass of 40.47 kDa. RT-PCR analysis indicated that the expression of SoSMT2 was induced by excess nitrate in the root and shoot of spinach. The transcript level of SoSMT2 was decreased by NaCl treatment. In contrast, dehydration, and H 2 O 2 treatment increased the transcript levels of SoSMT2 . To examine the biological roles of SoSMT2 in stress responses, transgenic Arabidopsis plants that constitutively overexpress SoSMT2 under the control of cauliflower mosaic virus 35S promoter were generated. The germination rate of transgenic seeds was higher than wild type (WT) in MS medium supplemented with 160 mM nitrate. When seedlings were transferred to MS liquid medium with 160 mM nitrate for 0, 8, 24 and 48 h, the transgenic plants showed lower levels of MDA and H 2 O 2 contents, compared with WT plants. Additionally, transgenic plants had relatively higher SOD, CAT and POD activities and soluble sugar contents than WT plants under nitrate stress with the increasing of treatment time. These data suggest that SoSMT2 is involved in nitrate tolerance in spinach.

SummaryIn this study, the effects of the exogenous H2S donor, NaHS, on the germination of tomato seeds under nitrate (NO3 -) ion stress were investigated. The results showed that under increased nitrate ion stress (range 0 - 200 mM), the germination percentage of tomato seeds decreased from 92.3% to 1.0%. The application of NaHS, up to 100 ?M, alleviated the inhibitory effect of nitrate stress by enhancing seed germination up to 1.3-fold compared to the nitrate stress treatment alone. Further study showed that malondialdehyde concentrations and the accumulation of reactive oxygen species declined significantly after all NaHS treatments. Amylase, superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase activities increased significantly after 100 ?M NaHS treatment. In addition, nitric oxide (NO) fluorescence increased when NaHS was added to the nitrate solution. Our results suggest that exogenous H2S can alleviate the damage caused by nitrate stress, possibly through increasing anti-oxidant enz...

Soil secondary salinization caused by excess nitrate addition is one of the major obstacles in greenhouse vegetable production. Excess nitrate inhibited the growth of tomato plants, while application of 100 μM H

SummaryGermin-like proteins (GLPs) are ubiquitous plant glycoproteins that have been implicated in various plant physiological and developmental processes. In this study, a cDNA clone, designated SoGLP, encoding a germin-like protein from spinach (Spinacia oleracia L.) was isolated and characterised. SoGLP encodes a 208-amino-acid polypeptide with a predicted molecular mass of 22.54 kDa and a pI of 5.95. Phylogenetic analysis indicated that SoGLP belonged to sub-family 3 of the GLP family. Sub-cellular localisation of an SoGLP-GFP fusion protein appeared to detect the protein on the cell walls in transgenic tobacco plants. Real-time quantitative RT-PCR analysis showed that the level of expression of SoGLP in the salt-resistant spinach cultivar (‘Chaoji’) was generally higher than in the salt-sensitive spinach cultivar (‘Daye’) grown in 160 mM nitrate ions for 0.5, 3.0, or 6.0 h. Expression of the SoGLP gene was also induced by other abiotic stresses including polyethylene glycol (PEG), NaCl, salicylic aci...

SummaryAluminium (Al) toxicity is the major factor-limiting crop productivity in acid soils. In the present study, physiological and transcriptional responses of broad bean leaves to Al stress were investigated. Malondialdehyde (MDA) content, H2O2 content and protein carbonyls (PC) level in leaves were increased after 100 μm AlCl3 stress treatment, whereas the total protein content was decreased, compared with the plants without Al treatment. Stomatal closure in leaves of broad bean was increased after Al stress, suggesting that the photosynthesis rate might be affected by Al stress. The relative citrate secretion in leaves was decreased after Al treatment for 24 h according to the 13C-NMR analysis, indicating that citrate in leaves might be transported to the root to chelate Al3+. To investigate the molecular mechanisms of Al toxicity in leaves of broad bean, a suppression subtractive hybridization (SSH) library was constructed to identify up-regulated genes: cDNA from leaves subjected to 12, 24, 48 and 72 h of 50 and 100 μm AlCl3 stress were used as testers and cDNA from leaves subjected to 0 μm AlCl3 treatment for the same lengths of time as above were used as a driver. The SSH analysis identified 156 non-redundant putative Al stress-responsive expressed sequence tags (ESTs) out of 960 clones. The ESTs were categorized into ten functional groups, which were involved in metabolism (0·21), protein synthesis and protein fate (0·10), photosynthesis and chloroplast structure (0·09), transporter (0·08), cell wall related (0·06), signal transduction (0·05), defence, stress and cell death (0·05), energy (0·03), transcription factor (0·03) and unknown proteins (0·30). The effect of Al treatment on expression of 15 selected genes was investigated by reverse transcription polymerase chain reaction (RT–PCR), confirming induction by Al stress. The results indicated that genes involved in organic acid metabolism, transport, photosynthesis and chloroplast structure, defence, stress and cell death might play important roles under Al stress.

SummarySalinity negatively impacts plant growth and productivity, but little is known about nitrate stress responses in celery (Apium graveolens L.). To improve our understanding of the adaptation of celery in nitrate-rich environments, we constructed a suppression-subtractive hybridisation (SSH) library. cDNAs from celery root tissue exposed to 100 mM total nitrate for 2, 4, 8, 24, 48, or 72 h were used as testers, and cDNAs of roots exposed to 0 mM nitrate for the same treatment times were used as drivers. Sequence analysis of the SSH cDNA library generated 202 high-quality expression sequence tags (ESTs), of which 112 were non-redundant. Eighty ESTs showed similarities to proteins of known function, and could be categorised into 11 functional groups, including metabolism, protein synthesis, cell defense and rescue, signal transduction, transport facilitation, genetic information processing, energy, cell structure, transcription, unknown, and no hits. Ten of the ESTs were selected by reverse transcripti...

A class 1 non-symbiotic hemoglobin family gene, SoHb, was isolated from spinach. qRT-PCR showed that SoHb was induced by excess nitrate, polyethylene glycol, NaCl, H2O2, and salicylic acid. Besides, SoHb was strongly induced by application of nitric oxide (NO) donor, while was suppressed by NO scavenger, nitrate reductase inhibitor, and nitric oxide synthase inhibitor. Overexpression of SoHb in Arabidopsis resulted in decreased NO level and sensitivity to nitrate stress, as shown by reduced root length, fresh weight, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm), and higher malondialdehyde contents. The activities and gene transcription of superoxide dioxidase, and catalase decreased under nitrate stress. Expression levels of RD22, RD29A, DREB2A, and P5CS1 decreased after nitrate treatment in SoHb-overexpressing plants, while increased in the WT plants. Moreover, SoHb-overexpressing plants showed decreased tolerance to NaCl and osmotic stress. In addition, the SoHb-overexpression lines showed earlier flower by regulating the expression of SOC, GI and FLC genes. Our results indicated that the decreasing NO content in Arabidopsis by overexpressing SoHb might be responsible for lowered tolerance to nitrate and other abiotic stresses.

To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-e group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR.

The objective of this study was to develop an efficient regeneration and transformation protocol for cucumber (Cucumis sativus L.). Cotyledonary node explants showed a very high regeneration frequency (94.3 %) when incubated on Murashige and Skoog medium (MS) supplemented with 1.5 mg l−1 6-benzyladenine (BA). An Agrobacterium-mediated transformation methodology for cucumber was developed using this protocol. A construct containing a sense mitogen-activated protein kinase gene (CsNMAPK) was used to transform cucumber cotyledonary nodes. Adventitious shoots were obtained in selection media containing 150 mg l−1 kanamycin and 250 mg l−1 carbenicillin 4 weeks after bacterial inoculation. A total of twelve transgenic plants were confirmed using PCR-specific analysis. The transgene copy number of four transgenic plants (random selection) was found to be a single gene copy using Southern blot analysis and the expression level of the CsNMAPK gene in the transgenic plants was significantly higher than in the wild-type plants. The transgenic rate was approximately 4.8 %.