Proteomic and physiological analyses to elucidate nitric oxide-mediated adaptive responses of barley under cadmium stress.

Nitric oxide (NO) is known to induce plant resistance for several environmental stresses. The protective roles of NO in cadmium (Cd) toxicity have been well documented for various plant species; nevertheless, little information is available about its molecular regulation in improving Cd tolerance of barley plants. Therefore, we combined a comparative proteomics with physiological analyses to evaluate the potential roles of NO in alleviating Cd stress (50 µM) in barley (Hordeum vulgare L.) seedlings. Exogenous application of NO donor sodium nitroprusside (SNP, 100 µM) decreased the Cd-mediated seedling growth inhibition. This observation was supported by the reduction of lipid peroxidation as well as the improvement of chlorophyll content and inhibition of hydrogen peroxide accumulation. Activities of the superoxide dismutase and guaiacol peroxidase were reduced following the application of SNP, while ascorbate peroxidase activity was enhanced. In this study, a total of 34 proteins were significantly regulated by NO in the leaves under Cd stress using a gel-based proteomic approach. The proteomic analysis showed that several pathways were noticeably influenced by NO including photosynthesis and carbohydrate metabolism, protein metabolism, energy metabolism, stress defense, and signal transduction. These results provide new evidence that NO induce photosynthesis and energy metabolism which may enhance Cd tolerance in barley seedlings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01214-3.

Growth responses and essential oil profile of Salvia officinalis L. Influenced by water deficit and various nutrient sources in the greenhouse.

Salvia officinalis L. is a medicinal plant extensively used in foods, traditional medicine, and the pharmacological industry. In the current study, the effects of different irrigation regimes [irrigation after 70 ± 5 (regular), 105 ± 5 (moderate drought stress), and 140 ± 5 (severe drought stress) mm evaporation] and nutrient sources (control, NPK, farmyard manure, foliar fertilizer, and hydrogel) were investigated on the growth parameters and essential oil (EO) components of S. officinalis in the greenhouse. The plants were harvested two times. The regular irrigation treatment had the most significant effect on plant height (51 cm), fresh and dry herb weight (51.5 and 18.1 g plant-1), and fresh and dry leaf weight (40.1 and 13.1 g plant-1). The highest amount of EO was observed after moderate drought stress (1.48%). The NPK treatment had the greatest effect on plant height (40 cm), branch number (19 per plant), fresh and dry herb weight (53.4 and 18.9 g plant-1), fresh and dry leaf weight (41.2 and 13.6 g plant-1), and EO content (1.67%). The 1st cutting was superior in EO amount, while the 2nd cutting had a high agronomic yield. α-Thujone (from 21.6 to 34.2%) was identified as the predominant compound. Additionally, the content of α-thujone in the 2nd cutting was higher after moderate drought stress, NPK, and hydrogel treatments. Moreover, 1,8-cineole, ß-thujone, camphene, α-pinene, α-humulene, viridiflorol, borneol, and bornyl acetate were the other main compounds. As a general result, regular irrigation and NPK treatments improved the agronomic yield of S. officinalis. The plants under drought stress produced high amounts of EO. The farmyard manure also improved plant yield by providing a part of the plant's nutritional needs. Therefore, it could be concluded that it is crucial to determine the effects of limited water availability and various nutrient sources on yield and chemical compositions for medicinal and aromatic plant growth.

Proteomic analysis reveals the role of exogenous cysteine in alleviating chromium stress in maize seedlings.

Cysteine (Cys) is incorporated into several compounds which are involved in detoxification of heavy metals. It is evident from recent studies that Cys is effective in alleviating the toxicity of heavy metals. Nevertheless, little is known about the Cys-mediated alleviation of chromium (Cr) toxicity. In our study, the impacts of exogenous Cys on Cr-stressed maize (Zea mays L.) were examined by using physiological and proteomic analyses. The results showed that Cr (100 µM) increased the accumulation of hydrogen peroxide, decreased cell viability, enhanced lipid peroxidation and consequently inhibited plant growth. The application of Cys (500 µM) attenuated the adverse effects of Cr on seedling growth. Cys supplementation to Cr treated plants decreased Cr accumulation in the shoots and increased Cr accumulation in roots. Cys treatment also modulated the activities of antioxidant enzymes and increased endogenous Cys content. Sixty proteins in root tissue were significantly affected by exogenous Cys under Cr stress using two-dimensional electrophoresis. Forty-six differentially expressed proteins were successfully identified by MALDI-TOF/TOF mass spectrometry. These differentially expressed proteins were involved in various biological pathways such as stress response (41.3%), energy and carbohydrate metabolism (21.7%), protein metabolism (6.5%), amino acid metabolism (6.5%), and others of unknown functions. The defense response-related proteins including glutathione peroxidase, glutathione S-transferases, pathogenesis-related proteins, glyoxalases and superoxide dismutase were differently regulated by Cys suggesting their roles in the Cys-mediated Cr tolerance.

Proteomic analysis of the anticancer effect of various extracts of endemicThermopsisturcica in human cervical cancer cells

Background/aim: Thermopsisturcica is a perennial species endemic to Turkey and different extracts of T. turcica have an antiproliferative effect on cancer cells, but there has not been any report on HeLa (human cervical cancer) cells. Materials and methods: To get a better understanding of the molecular mechanism of anticancer activity of methanolic extracts of leaves (LE) and flowers (FE) of T. turcica, we employed 2-DE-based proteomics to explore the proteins involved in anticancer activity in HeLa cells. Results: T. turcica extracts showed a potent cytotoxic effect on HeLa cells with the IC50 values of 1.75 mg/mL for LE and 3.25 mg/mL for FE. The induction of apoptosis by LE and FE was also consistent with increased expression of caspase mRNAs and DNA fragmentation. In terms of the proteomic approach, 27 differentially expressed proteins were detected and identified through MALDI-TOF/TOF mass spectrometry. These altered proteins were involved in cytoskeleton organization and movement, protein folding, proteolysis and translation, cell cycle and proliferation, signal transduction, cell redox homeostasis, and metabolism. Conclusion: Up-regulation of protein disulfide isomerases and down-regulation of Rho GDP-dissociation inhibitor, heterogeneous nuclear ribonucleoproteins, and heat shock proteins may contribute to the induction of apoptosis and arresting of the cell cycle in HeLa cells.

Proteomic analysis of chromium stress and sulfur deficiency responses in leaves of two canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance.

Sulfur (S) is an essential macronutrient for plant growth and development, and it plays an essential role in response to environmental stresses. Plants suffer with combined stress of S deficiency and hexavalent chromium [Cr(VI)] in the rhizosphere. Little is known about the impact of S deficiency on leaf metabolism of canola (Brassica napus L.) under Cr(VI) stress. Therefore, this study is the first to examine the effects of Cr(VI) stress and S deficiency in canola at a molecular level. A comparative proteomic approach was used to investigate the differences in protein abundance between Cr-tolerant NK Petrol and Cr-sensitive Sary cultivars. The germinated seeds were grown hydroponically in S-sufficient (+S) nutrient solution for 7 days and then subjected to S-deficiency (-S) for 7 days. S-deficient and +S seedlings were then exposed to 100µM Cr(VI) for 3 days. Protein patterns analyzed by two-dimensional electrophoresis (2-DE) revealed that 58 protein spots were differentially regulated by Cr(VI) stress (+S/+Cr), S-deficiency (-S/-Cr) and combined stress (-S/+Cr). Of these, 39 protein spots were identified by MALDI-TOF/TOF mass spectrometry. Differentially regulated proteins predominantly had functions not only in photosynthesis, but also in energy metabolism, stress defense, protein folding and stabilization, signal transduction, redox regulation and sulfur metabolism. Six stress defense related proteins including 2-Cys peroxiredoxin BAS1, glutathione S-transferase, ferritin-1, l-ascorbate peroxidase, thiazole biosynthetic enzyme and myrosinase-binding protein-like At3g16470 exhibited a greater increase in NK Petrol. The stress-related proteins play an important role in the detoxification of Cr(VI) and maintaining cellular homeostasis under variable S nutrition.

Interactive effects of sulfur and chromium on antioxidative defense systems and BnMP1 gene expression in canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance.

Plants suffer with combined stress of sulfur (S) deficiency and hexavalent chromium [Cr(VI)] in soils. There are a few reports on the interactive effects of S-deficiency and Cr(VI) stress. Therefore, the interactions between S nutrition and Cr(VI) stress were investigated in hydroponically grown canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance. The relatively Cr(VI)-tolerant (NK Petrol) and Cr(VI)-susceptible (Sary) cultivars were grown in S-sufficient nutrient solution and then exposed to variable S concentrations [deficient (0 mM S, -S) and sufficient (1 mM S, +S)]. The seedlings were then exposed to 100 µM Cr(VI) for 3 days. S-deficiency (-S/-Cr) and combined stress (-S/+Cr) caused a significant decrease in growth parameters of Sary than NK Petrol (P < 0.05). In -S/+Cr treatment, Cr accumulation in Sary was significantly higher than NK Petrol. The higher level of Cr in Sary increased lipid peroxidation and decreased chlorophyll content. The activities of antioxidant enzymes and cysteine content were significantly higher in NK Petrol than in Sary under combined stress. The levels of ascorbate (AsA) and glutathione (GSH) were significantly decreased by S deficiency. The expression level of metallothionein gene (BnMP1) in the tolerant NK Petrol was increased by -S/+Cr treatment. However, expression level of BnMP1 gene in the susceptible Sary was enhanced by +S/+Cr treatment. This result suggests metallothionein (MT) may be involved in Cr(VI) tolerance under S-deficient condition. In conclusion, S nutrition influenced Cr accumulation and enhanced tolerance caused by a positive effect on defense systems and gene expression.

Proteomic and biochemical responses of canola (Brassica napus L.) exposed to salinity stress and exogenous lipoic acid.

To evaluate the mitigating effects of exogenous lipoic acid (LA) on NaCl toxicity, proteomic, biochemical and physiological changes were investigated in the leaves of canola (Brassica napus L.) seedlings. Salinity stress decreased the growth parameters and contents of ascorbate (AsA) and glutathione (GSH), and increased the contents of malondialdehyde (MDA), proline, cysteine and the activities of antioxidant enzymes such as superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX). The foliar application of LA alleviated the toxic effects of salinity stress on canola seedlings and notably decreased MDA content and increased growth parameters, cysteine content, and activities of CAT and POD. In the proteomic analyses, total proteins from the leaves of control, LA, NaCl and NaCl+LA treated-seedlings were separated using two-dimensional gel electrophoresis (2-DE). A total of 28 proteins were differentially expressed. Of these, 21 proteins were successfully identified by MALDI-TOF/TOF MS. These proteins had functions related to photosynthesis, stress defense, energy metabolism, signal transduction, protein folding and stabilization indicating that LA might play important roles in salinity through the regulation of photosynthesis, stress defense and signal transduction related proteins. The proteomic findings have provided new insight to reveal the effect of LA on salinity stress for the first time.

Variations in chromium tolerance and accumulation among canola (Brassica napus L.) cultivars.

Phytoremediation is a green technology for the remediation of contaminated ecosystems by using plants. In the present study, a hydroponic experiment was conducted to investigate the phytoremediation potential of eight canola (Brassica napus L.) cultivars for hexavalent chromium [Cr(VI)]. Chromium significantly affected dry weight, lipid peroxidation, chlorophylls, non-protein thiol and antioxidant enzymes. Based on the dry weight, the tolerance index was found maximum in cultivar (cv.) NK Petrol and minimum in cv. Sary. The cv. Sary accumulated the maximum amount of Cr (705.8 µg g(-1) DW), which was correlated with the lowest levels of chlorophyll content and highest levels of lipid peroxidation. However, Cr accumulation was lowest (255.0 µg g(-1) DW) in NK Petrol. Although cv. NK Petrol may be a Cr(VI) excluder relative to cv. Sary, it may have the potential for the phytoremediation of Cr-contaminated sites as it possesses higher resistance to Cr(VI) by producing higher biomasses.

Protective role of hydrogen peroxide pretreatment on defense systems and BnMP1 gene expression in Cr(VI)-stressed canola seedlings.

To evaluate the ameliorating effects of hydrogen peroxide (H2O2, 200 µM) on hexavalent chromium [Cr(VI)] toxicity in canola (Brassica napus L.), we focused on the plant growth, chlorophyll content, thiol contents, lipid peroxidation, antioxidant enzymes, and the expression of metallothionein protein (BnMP1) mRNA. Cr(VI) at 50 µM significantly decreased the plant growth (fresh and dry weights). The decrease in growth was accompanied by increased lipid peroxidation and decreased chlorophyll content in leaves. Hydrogen peroxide pretreatment, however, enhanced plant growth parameters and led to the reduced levels of lipid peroxidation and higher levels of pigment. In addition, H2O2 pretreatment increased Cr accumulation in aerial parts of seedlings. The tendency of increase in thiol content under Cr(VI) stress was further increased with H2O2 pretreatment. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) were differentially altered. SOD and POD activities increased under Cr(VI) stress, whereas APX and CAT activities decreased. The SOD and CAT activities remained unaffected in both durations due to H2O2 pretreatment, but activities of APX and POD were promoted in the Cr(VI)-stressed seedlings. Metallothioneins are a family of low-molecular-weight Cys-rich proteins and are thought to play a possible role in metal metabolism or detoxification. In real-time quantitative PCR analysis, the expression level of BnMP1 mRNA was increased at 1 day after treatment (DAT), whereas it was decreased at 7 DAT in Cr(VI)-stressed seedlings. At 1 DAT, pretreatment of H2O2 before Cr(VI) stress reduced the expression of BnMP1 mRNA as compared to Cr(VI) stress alone, but this effect was not significant. At 7 DAT, H2O2 pretreatment alleviated the Cr(VI) stress-mediated decrease in the expression of BnMP1 mRNA. These results suggest that H2O2 may act as a signal that triggers defense mechanisms which in turn protects canola seedlings from Cr(VI)-induced oxidative damage.

Cytotoxic effects of nasal buserelin on nasal mucosal tissue in rabbits.

To investigate the cytotoxic effects of nasal buserelin on rabbit nasal mucosal tissue, twenty-four female rabbits were studied prospectively. The rabbits were divided into 4 groups including 6 rabbits. The rabbits' left noses were included in the all study groups: 150 µg/puff/day of buserelin acetate was administered topically twice daily during 21, 42 and 63 days. Saline was administered topically twice daily to the left nasal cavity in the control group. The nasal septal mucosal stripe tissue was carefully removed from underlaying cartilage after sedation. HE staining, Masson's trichrome, toluidine blue and TUNEL staining were used to evaluate mucosal changes. Each preparation was investigated via apoptotic cells, and they were accounted. Kruskal-Wallis test was used to evaluate nonparametric comparison of apoptotic cells. Mononuclear cells have been raised in the sub-epithelial connective tissue, nucleuses of epithelial cells in the apical region were pyknotic, and apoptotic cells were determined on 21-day group. In the 42-day group, nasal epithelial tissue was similar to 21-day group and epithelial cells including pyknotic nucleus were present in this group, too. In the 63-day group, epithelial cells were light colored. Venous sinuses in the sub-epithelial connective tissue were wide but not congested and not raised collagen filaments. In the intra-epithelial tissue, some of cells were TUNEL (+). Apoptotic cells were fewer in the control group according to 21-day group. In 42- and 63-day groups, these cells were fewer than in 21-day group. Numerical difference was present between the groups, but statistical significance was not found between the groups. We concluded that nasal buserelin cytotoxicity was not potent in the nasal cavity in rabbits. We use nasal buserelin in all indications with confidence.

Evaluation of 2,4-D and Dicamba genotoxicity in bean seedlings using comet and RAPD assays.

The present study was undertaken to evaluate genotoxic potential of two auxinic herbicides [2,4-dicholorophenoxy acetic acid (2,4-D) and 3,6-dichloro-2-methoxybenzoic acid (Dicamba)] in the roots of common bean (Phaseolus vulgaris L.) seedlings. Two-day-old etiolated seedlings were treated with 10 ppm methyl methanesulfonate (MMS, positive control) or 0.1, 0.2, or 0.3 ppm of either 2,4-D or Dicamba. At the end of a 96 h growth period, root growth, total soluble protein content, DNA damage in individual cells (comet assay scores) and randomly amplified polymorphic DNA (RAPD) profiles were used as endpoints of genotoxicity. 2,4-D and Dicamba were clearly dose-dependent root growth inhibitors. Total soluble protein content was significantly decreased in the positive control and at high concentrations (0.2 and 0.3 ppm) of Dicamba. Soluble protein content increased significantly only at 0.3 ppm 2,4-D (P<0.05). In the comet assay, DNA fragmentation increased in a dose-dependent manner. The diagnostic and phenetic analyzes of appeared and/or disappeared RAPD bands indicated that dose-dependent DNA polymorphism was induced by both herbicides. Genomic template stability was significantly affected at all 2,4-D and Dicamba doses tested. Overall 2,4-D and Dicamba have similar effects on DNA damage detected by comet and RAPD assays.

Montelukast protects against testes ischemia/reperfusion injury in rats.

INTRODUCTION: In this study, we investigate the effect of montelukast on histologic damage induced by testicular torsion-detorsion in rats. METHODS: Twenty-one male Sprague-Dawley rats were separated into 3 groups, each containing 7 rats. A sham operation was performed in group 1 (control). In group 2 (ischemia-reperfusion [IR]/untreated), 1-hour detorsion of the testis was performed after 6 hours of unilateral testicular torsion. In group 3 (I-R/dextroamphetamine), after performing the same surgical procedures as in group 2, montelukast was given intraperitoneally. In all experimental rats, ipsilateral orchiectomies were performed for histological examination and tissue malondialdehyde (MDA), glutathione and myeloperoxidase assays. RESULTS: Montelukast treatment significantly decreased the I-R-induced elevation in testes tissue MDA and glutathione levels were found to be preserved. The level of myeloperoxidase (MPO) activity was significantly increased in the testes tissue of the IR/untreated group. However, in I-R/montelukast treatment group significantly decreased testes tissue MPO level. Histopathologically, the in the group 2 rats, edema, congestion, hemorrhage between seminiferous tubules and necrosis of the germinal cells were predominant features in sections. However, most of the specimens in the montelukast treated group 3 showed grades-I and II injury. Additionally, the testicular injury score was lower in group 3 rats compared with group 2. CONCLUSION: The current findings demonstrate that the montelukast decreased the severity of testicular injury by reversing the oxidative effects of testes I-R.

Exogenous melatonin treatment reduces hepatocyte damage in rats with experimental acute pancreatitis.

BACKGROUND/PURPOSE: The hormone melatonin affects cellular immunity in particular and the immune system in general both directly and indirectly. We report our evaluation of the effects of decreasing and increasing serum melatonin levels on hepatocyte damage in rats with experimental acute pancreatitis. METHODS: Wistar Albino rats with experimentally induced acute pancreatitis were divided into three groups of ten rats each: (1) control (induced acute pancreatitis only); (2) rats with induced acute pancreatitis plus surgical pinealectomy (no melatonin injections); (3) rats with induced acute pancreatitis plus injections of exogenous melatonin. The effects of melatonin levels were evaluated using biochemical and histopathological parameters. RESULTS: Rats undergoing the pinealectomy had increased amylase and lactate dehydrogenase (LDH) levels, while those receiving injections of exogenous melatonin had decreased amylase, aspartate transaminase, LDH, and bilirubin levels but increased levels of alanine transferase levels. CONCLUSION: Melatonin may have a therapeutic or protective effect on acute pancreatitis and obstructive jaundice.

Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays.

We used the anaphase-telophase chromosome aberration and comet (Single Cell Gel Electrophoresis, SCGE) assays to evaluate the genotoxic effects of copper sulphate (CS) and cobalt chloride (CC) chemicals prepared in two concentrations (EC(50), 2xEC(50)), using methyl methanesulfonate (MMS) as a positive control and untreated cells as a negative control. In Allium root growth inhibition test, EC(50) values for CS and CC are 1.5 and 5.5 ppm, respectively. Mitotic index (MI) decreased in all concentrations tested of CS and CC compared to the control at each exposure time. The bridge, stickiness, vagrant chromosomes, fragments, c-anaphase and multipolarity chromosome aberrations were observed in anaphase-telophase cells. The total chromosome aberrations were more frequent with an increasing in the exposure time and the concentrations of both chemicals. The genotoxicity of CS and CC in Allium cepa root cells was analyzed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. In all the concentrations, CS and CC induced a significant increase (P<0.05) in DNA damage. No significant difference was found between positive control (300+/-5.81) and 3 ppm CS (280+/-4.61). The methods used are applicable for biological monitoring of environmental pollutants.

The effect of pinealectomy and leptin hormone on the proliferation and apoptosis activation in Syrian hamster testis in different photoperiods.

The effects of pinealectomy and leptin hormone on proliferative and apoptotic processes in the epithelia of testicular seminiferous tubules of Syrian hamsters have been investigated. Proliferative and apoptotic processes were assessed semi-quantitatively by proliferating cell nuclear antigen (PCNA) and caspase-3 immune stainings. Animals used in the study were divided into four groups; control, pinealectomy (PinX), leptin-treated (10 microg/mL/day/kg body weight, intraperitoneally) and pinealectomy + leptin groups. Half of the hamsters in each group were exposed to short and the other half to long photoperiods for 8 weeks. In short photoperiod, PCNA activity especially in spermatogonia was significantly higher in the pinealectomy and leptin-treated groups compared with the control group. Histological score (HSCORE) value of PCNA in the PinX + leptin group was lower than those of PinX and leptin-treated groups. HSCORE value of caspase-3 in PinX and PinX + leptin groups was increased. In the long photoperiod, PCNA activation in the PinX group was significantly lower than the control group while the differences between the controls and other groups were not significant. The difference between the increases in caspase-3 activity in the PinX and control groups was significant. Thus, it was observed that photoperiods had no effect on the proliferation activity in the control groups. The inhibiting effect of short photoperiod on testis was not observed throughout 8 weeks. PinX eliminated the inhibiting effect of short photoperiod but did not alter the stimulating effect of long photoperiod. Leptin did not show any effect in long photoperiod but decreased proliferation by stimulating melatonin in short photoperiod.