Exogenous Melatonin Modulates Endogenous H2S Homeostasis and L-Cysteine Desulfhydrase Activity in Salt-Stressed Tomato (Solanum lycopersicum L. var. cherry) Seedling Cotyledons

Although melatonin has been reported to function as a stress signaling molecule, not much information is available on the biochemical and molecular events associated with probable melatonin-hydrogen sulfide crosstalk in plants. Present work provides evidence on the role of melatonin in the modulation of H₂S homoeostasis during NaCl stress in dark-grown tomato (Solanum lycopersicum L. var. cherry) seedlings. NaCl stress (120 mM) inhibits hypocotyl elongation, promotes primary root growth and enhances electrolytic leakage from tomato seedlings. Treatment with H₂S donor (100 µM; NaHS) tends to reverse these effects, all the more so (additive effect) in the presence of melatonin. NaCl stress and exogenous melatonin (30 µM) treatments modulate endogenous H₂S accumulation and positively upregulate the activity of L-cysteine desulfhydrase (L-DES; EC 4.4.1.15; cytosolic). Melatonin has been observed to temporally modulate the activity of specific isoforms of H₂S biosynthesizing enzyme, L-DES in seedling cotyledons. Zymographic analysis of L-DES isoforms in tomato seedling cotyledons has provided novel findings in plant system. Melatonin treatment decreases H₂S accumulation in NaCl-stressed seedling cotyledons which is accompanied by a contrasting increase in L-DES activity. Melatonin, therefore, regulates endogenous H₂S concentration in seedling cotyledons (NaCl treated), thus indicating the role of H₂S catabolism pathways in H₂S homoeostasis. Present findings thus reveal that exogenous melatonin modulates early H₂S signaling in cotyledons of tomato seedlings subjected to NaCl stress. Furthermore, exogenous melatonin and H₂S in combination (additive effect) ameliorate NaCl stress-induced growth changes in tomato seedlings.

     

Melatonin mitigates cadmium and aluminium toxicity through modulation of antioxidant potential in Brassica napus L

• Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth.
• In this study, we applied different doses of melatonin (50 and 100 µm ) to alleviate Al (25 µm ) and Cd (25 µm ) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity.
• Melatonin increased the expression of melatonin biosynthesis‐related Brassica napus genes for caffeic acid O‐methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT‐1, BnCOMT‐5 and BnCOMT‐8 showed up‐regulated expression, while BnCOMT‐4 and BnCOMT‐6 were down‐regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al‐ and Cd‐treated seedlings.
• These results suggest that melatonin‐induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.

     

Arsenic-induced oxidative stress and thiol metabolism in two cultivars of rice and its possible reversal by phosphate

Groundwater arsenic contamination, a grave threat in Bangladesh and parts of West Bengal (India), causes biochemical and physiological disorders in plants. Arsenic and phosphorus (plant macronutrient) have similar electronic configurations, resulting in their competitive interaction for the same uptake system in plant roots. Arsenic exposure initiates production of reactive oxygen species. Hence, the contents of proline, hydrogen peroxide, glutathione, ascorbate, and activities of ascorbate peroxidase, catalase were investigated in 21-day-old rice seedlings (cv. Khitish and cv. Nayanmani). Additionally, impact of arsenate together with phosphate on growth, total glutathione contents and activity of its regulatory enzymes were altered in the test cultivars to varying extents. Inductively coupled plasma-optical emission spectroscopic study of arsenic content in the root and shoot also showed variable uptake of arsenic by the two cultivars. Arsenate reductase enzyme activity primarily observed in the root, also differed from one cultivar to the other. Different phytochelatin (PCs) levels were recorded in the shoot and root of the cultivars under arsenate and phosphate treatment by reverse phase-high performance liquid chromatography. PC content increased with increasing arsenate concentrations, whereas phosphate and arsenate co-application resulted in reduced PC levels. The degree of elevation in PC contents varied significantly in the cultivars. Based on the above-mentioned parameters, cv. Khitish appeared to be more susceptible to arsenic toxicity than cv. Nayanmani which showed selective tolerance to the said metal stress.     

Differential plant growth and osmotic effects of two maize (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) cultivars to exogenous glycinebetaine application under drought stress

We investigated the effects of exogenous glycinebetaine (GB) and drought stress (DS) on grain yield (GY) and production of dry matter (DM) and osmolytes in two maize (Zea mays L.) cultivars i.e. Shaandan 9 (S₉) and Shaandan 911 (S₉₁₁) during the entire growing period. Drought stress substantially reduced DM and GY but increased free proline, endogenous GB, soluble sugar and K⁺ concentrations in leaves of both cultivars. The DM production, GY, drought index (DI) and concentrations of these osmolytes were greater for S₉ than those for S₉₁₁ under DS. The significant differences in these parameters suggested that S₉ was more drought-tolerant as compared to S₉₁₁. Additionally, foliar application of GB increased the concentrations of all osmolytes measured, DM and GY of both cultivars under DS. These positive responses of exogenous GB spray were more pronounced in S₉₁₁ as compared to those in S₉. Further correlation analysis involving a number of parameters indicated that maize production was tighterly correlated with accumulation of the osmolytes measured during DS rather than well-watered controls. Accordingly, this study demonstrated the notion of an anti-drought role of exogenous GB by osmoregulation under DS, particularly in this drought sensitive cultivar. Thus, exogenous GB application might be firstly used with drought sensitive species/cultivars when exposed to DS.     

Melatonin supplementation can ameliorate the detrimental effects of heat stress on performance and carcass traits of Japanese quail

Melatonin, the major pineal hormone, modulates growth in poultry by influencing hormones involved in growth. We investigated the effects of dietary melatonin supplementation on performance, carcass characteristics, and excretion of nitrogen and some minerals in broiler Japanese quails (Coturnix coturnix japonica) exposed to high-ambient-temperature stress (34°C). One hundred twenty Japanese quails (10 d old) were randomly assigned to 4 treatment groups, 3 replicates of 10 birds each. The birds were kept in either an environment-controlled room at a constant 22°C or were kept at 22°C for 16 h/d and at 34°C for 8 h/d (9:00 am to 5:00 pm). At both temperatures birds were fed either a basal diet or the basal diet supplemented with 40 mg of melatonin per kilogram of diet. The experiment lasted for 32 d. Melatonin improved feed efficiency in both temperatures groups compared with their corresponding controls. Although feed intake was similar in all groups, the improvement in feed efficiency was more noticable in melatonin-fed quails kept at high temperature (p<0.01). Supplemental melatonin significantly increased live weight gain and carcass characteristics under stress conditions (p<0.01) but did not show the same effect at thermoneutral conditions (p>0.05). Heat exposure increased excretion of N, Ca, P, Zn, Fe, and Cr and decreased retention rates for them. Dietary melatonin supplementation returned these values to normal (p<0.01). No interactions between melatonin and temperature were found in the parameters measured. The results of the study show that melatonin supplementation attenuated the retardation in performance as well as the excretion of minerals caused by heat stress in broiler quails. Our data suggest that melatonin might offer protection against heat-stress-related depression in the performance of broiler quails.     

Effects of Melatonin on Growth,Physiology and Gene Expression in Rice Seedlings Under Cadmium Stress

Melatonin (MLT) is a hormonal substance found in many organisms and can improve plant stress resistance. In this study, the japonica rice variety Y32 and indica rice variety NJ6 were cultivated in hydroponics under different concentrations of CdCl₂ at the two-leaf stage. The growth, physiological and biochemical responses of the seedlings and the expression of cadmium (Cd)-related genes under exogenous melatonin (MLT) treatment were assessed. The results indicated that Cd stress destroyed the dynamic balance between reactive oxygen species (ROS) production and removal, resulting in ROS accumulation, membrane lipid peroxidation, and impaired growth and development. Following the application of exogenous MLT to rice seedlings, increases in plant biomass including both underground and above-ground areas were observed. MLT also scavenged the inhibition of superoxide dismutase (SOD) and peroxidase (POD) in a concentration dependent manner in response to Cd stress. Catalase (CAT) activity and malondialdehyde (MDA) expression also decreased following MLT treatment. Amongst the six Cd-related genes assessed, five genes were down-regulated and one was up-regulated in response to MLT treatment. Taken together, these data demonstrate that MLT improves the resilience of rice seedlings at the biochemical, physiological, and molecular levels, and diminishes the damage caused by Cd stress.     

Melatonin protects against myocardial ischemia–reperfusion injury by elevating Sirtuin3 expression and manganese superoxide dismutase activity

Myocardial ischemia–reperfusion (MI/R) injury is a crucial cause for mortality throughout the world. Recent studies indicated that melatonin might exert profound cardio-protective effect in MI/R injury. However, the underlying mechanisms are not completely understood. In the current study, we aimed to explore the potential effect of melatonin in the pathological process of MI/R. Both in vivo MI/R model and in vitro H9c2 cell line simulated I/R (SIR) model were applied with or without melatonin supplementation. We found that Sirtuin3 (Sirt3) expression and activity were markedly decreased under MI/R and SIR conditions. Melatonin treatment significantly increased myocardial Sirt3 expression, and alleviated MI/R-induced cardiac morphology changes and cardiac dysfunction, as well as myocardial apoptosis level. In addition, DHE and JC-1 staining results demonstrated that melatonin reduced mitochondrial reactive oxygen species (ROS) generation and restored ATP production after SIR injury via elevating Sirt3 expression. By using siRNA targeting Sirt3, we confirmed that the beneficial effects of melatonin were dependent on Sirt3, which in turn deacetylated and activated manganese superoxide dismutase (MnSOD). Collectively, the current study demonstrated the protective effect of melatonin against MI/R injury via alleviating myocardial oxidative stress. Moreover, these beneficial effects were associated with the deacetylation modification of Sirt3 on MnSOD.     

Physiological and Biochemical Mechanisms of Exogenous Calcium Chloride on Alleviating Salt Stress in Two Tartary Buckwheat (<i>Fagopyrum tataricum</i>) Varieties Differing in Salinity Tolerance

Salt stress is one of the most serious abiotic stresses limiting plant growth and development. Calcium as an essential nutrient element and important signaling molecule plays an important role in ameliorating the adverse effect of salinity on plants. This study aimed to investigate the impact of exogenous calcium on improving salt tolerance in Tartary buckwheat cultivars, cv. Xinong9920 (salt-tolerant) and cv. Xinong9909 (salt-sensitive). Four-week-old Tartary buckwheat seedlings under 100 mM NaCl stress were treated with and without exogenous calcium chloride (CaCl₂), Ca²⁺ chelator ethylene glycol tetraacetic acid (EGTA) and Ca²⁺-channel blocker lanthanum chloride (LaCl₃) for 10 days. Then, some important physiological and biochemical indexes were determined. The results showed that salt stress significantly reduced seedling growth, decreased photosynthetic pigments, inhibited antioxidants and antioxidant enzyme activities. However, it increased the reactive oxygen species (ROS) levels in the two Tartary buckwheat cultivars. Exogenous 10 mM CaCl₂ application on salt-stressed Tartary buckwheat seedlings obviously mitigated the negative effects of NaCl stress and partially restored seedlings growth. Ca²⁺-treated salt-stressed seedlings diplayed a suppressed accumulation of ROS, increased the contents of total chlorophyll, soluble protein, proline and antioxidants, and elevated the activities of antioxidant enzymes compared with salt stress alone. On the contrary, the addition of 0.5 mM LaCl₃ and 5 mM EGTA on salt-stressed Tartary buckwheat seedlings exhibited the opposite effects to those with CaCl₂ treatment. These results indicate that exogenous Ca²⁺ can enhance salt stress tolerance and Ca²⁺ supplementation may be an effective practice to cultivate Tartary buckwheat in saline soils.     

Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in spermidine-treated maize roots

Inhibition of root growth and accumulation of putrescine caused by exogenous spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H₂O₂ produced from spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing spermidine-generated H₂O₂ by DMTU. Over-expression of APX was associated with increased APX activity in spermidine-treated seedlings whereas the addition of DMTU to spermidine completely prevented spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H₂O₂, a spermidine catabolic product.     

Melatonin-induced phase and dose responses in a diurnal mammal,Funambulus pennantii

ABSTRACT

Melatonin, an essential pineal hormone, acts as a marker of the circadian clock that regulates biological rhythms in animals. The effects of exogenous melatonin on the circadian system of nocturnal rodents have been extensively studied; however, there is a paucity of studies on the phase-resetting characteristics of melatonin in diurnal rodents. We studied the phase shifting effects of exogenous melatonin as a single melatonin injection (1 mg/kg) at various phases of the circadian cycle on the circadian locomotor activity rhythm in the palm squirrel, Funambulus pennantii. A phase response curve (PRC) was constructed. Adult male squirrels (N = 10) were entrained to a 12:12 h light-dark cycle (LD) in a climate-controlled chronocubicle with food and water provided ad libitum. After stable entrainment, squirrels were transferred to constant dark condition (DD) for free-running. Following stable free run, animals were administered a single dose of melatonin (1 mg/kg in 2% ethanol-phosphate buffered saline (PBS) solution) or vehicle (2% ethanol-PBS solution) at circadian times (CTs) 3 h apart to evoke phase shifts. The phase shifts elicited at various CTs were plotted to generate the PRC. A dose response curve was generated using four doses (0.5, 1, 2 and 4 mg/kg) administered at the CT of maximum phase advance. Melatonin evoked maximum phase advances at CT0 (1.23 ± 0.28 h) and maximum phase delays at CT15 (0.31 ± 0.09 h). In the dose response experiment, maximal phase shifts were evoked with 1 mg/kg. In contrast, no significant shifts were observed in control groups. Our study demonstrates that the precise timing and appropriate dose of melatonin administration is essential to maximize the amelioration of circadian rhythm–related disorders in a diurnal model.     

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease

Background

Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice.

Methods

Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase β, GRP78 and CHOP) and metabolic dysfunction (RPB4, β-catenin) and cellular longevity (SIRT1) were analyzed.

Results

Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase β were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and β-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD.

Conclusions

Melatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.     

Melatonin Antagonizes Cytokinin Responses to Stimulate Root Growth in Arabidopsis

Melatonin functions as the key growth regulator in various plant species. The mechanisms of the interactions between melatonin and cytokinins remain largely unknown. In this study, the kinetic effects of melatonin over a range of concentrations were investigated. The results showed that melatonin functioned as a positive regulator of root growth ranged from 0.1 to 100 nM. In contrast, exogenous cytokinin at 0.5–1 nM and overexpression of cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE 8 (IPT8) inhibited primary root growth. Combined treatments with melatonin and cytokinin indicated that melatonin antagonized the inhibitory effect of cytokinin 6-benzylaminopurine (6-BA) on primary root elongation. Further analysis revealed that melatonin promotes primary root growth by modulating expression and distribution of auxin efflux transporters PIN2/3 and influx transporter AUX1. Moreover, the cytokinin signaling components AHK4, AHP2/3/5, and type-A ARR15 were down-regulated after melatonin treatment. The polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) impaired the promotive effect of melatonin on primary root growth, indicating that auxin transport is essential in melatonin-mediated root growth. Taken together, our data provided evidence to show that melatonin regulates primary root growth in coordination with cytokinin partially through auxin-dependent pathway.

     

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia

Endothelial dysfunction is associated with K_Ca3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial K_Ca3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial K_Ca3.1 dysfunction occurs in vessels from women with preeclampsia. We compared K_Ca3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on K_Ca3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial K_Ca3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in K_Ca3.1 current. Plasma from PE induced K_Ca3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced K_Ca3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced K_Ca3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1–NOX2-mediated pathway and downregulates endothelial K_Ca3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests K_Ca3.1as a novel target for patients with preeclampsia.     

Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

Daily variations of immunoreactive melatonin in the visual system of crayfish

In crustaceans, melatonin has been detected in the central nervous system and some other organs. The aim of this study was to analyze the melatonin content in the visual system of Procambarus clarkii, by means of radioimmunoassay, at different day-night phases. We have also studied the action of exogenous melatonin on the main properties of the electroretinogram (ERG) circadian rhythm. Experiments were conducted with 25 specimens maintained under controlled conditions of 16°C and 12 h of light alternating with 12 h of darkness. Eyes where cut in dim red light and shock frozen with liquid nitrogen and pulverized in a mortar until a homogenous powder was obtained. Melatonin was extracted with acetone, followed by centrifugation, diluted with an equal volume of equa bidest to ensure freezing at ?80°C for at least 90 min and lyophilization at the same temperature. Lyophilizates, after having been dissolved in RIA buffer, were used for determinations of melatonin. Long-term recordings of electrical responses to light (ERG) were obtained for 10 or more consecutive days. At the 5th day, a single dose of melatonin was injected and its effects on amplitude and period of the ERG circadian rhythm were measured. Melatonin concentrations differed considerably depending on the circadian time and attained a maximum during dark phase. Among the crustaceans, Procambarus clarkii represents the first case in which melatonin peaks during the night following the typical pattern known in the majority of organisms. After melatonin injection, period and amplitude of the ERG circadian rhythm were increased. This effect suggests the involvement of melatonin in the oscillators underlying the generation and expression of circadian rhythms in crayfish.