RESUMO
Water deficit is a major environmental constraint on crop productivity and performance and nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. This study aims to test the hypothesis that leaf spraying of S-nitrosoglutathione (GSNO), an NO donor, improves the antioxidant defense in both roots and leaves of sugarcane plants under water deficit, with positive consequences for photosynthesis. In addition, the roles of key photosynthetic enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in maintaining CO2 assimilation of GSNO-sprayed plants under water deficit were evaluated. Sugarcane plants were sprayed with water or GSNO 100 µM and subjected to water deficit, by adding polyethylene glycol (PEG-8000) to the nutrient solution. Sugarcane plants supplied with GSNO presented increases in the activity of antioxidant enzymes such as superoxide dismutase in leaves and catalase in roots, indicating higher antioxidant capacity under water deficit. Such adjustments induced by GSNO were sufficient to prevent oxidative damage in both organs and were associated with better leaf water status. As a consequence, GSNO spraying alleviated the negative impact of water deficit on stomatal conductance and photosynthetic rates, with plants also showing increases in Rubisco activity under water deficit.
Assuntos
Doadores de Óxido Nítrico/farmacologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Saccharum/efeitos dos fármacos , Antioxidantes/metabolismo , Catalase/metabolismo , Desidratação , Oxirredução , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/enzimologia , Estômatos de Plantas/fisiologia , Transpiração Vegetal/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Saccharum/enzimologia , Saccharum/fisiologia , Superóxido Dismutase/metabolismo , Água/fisiologiaRESUMO
Exogenous supply of nitric oxide (NO) increases drought tolerance in sugarcane plants. However, little is known about the role of NO produced by plants under water deficit. The aim of this study was to test the hypothesis that drought-tolerance in sugarcane is associated with NO production and metabolism, with the more drought-tolerant genotype presenting higher NO accumulation in plant tissues. The sugarcane genotypes IACSP95-5000 (drought-tolerant) and IACSP97-7065 (drought-sensitive) were submitted to water deficit by adding polyethylene glycol (PEG-8000) in nutrient solution to reduce the osmotic potential to -0.4 MPa. To evaluate short-time responses to water deficit, leaf and root samples were taken after 24 h under water deficit. The drought-tolerant genotype presented higher root extracellular NO content, which was accompanied by higher root nitrate reductase (NR) activity as compared to the drought-sensitive genotype under water deficit. In addition, the drought-tolerant genotype had higher leaf intracellular NO content than the drought-sensitive one. IACSP95-5000 exhibited decreases in root S-nitrosoglutathione reductase (GSNOR) activity under water deficit, suggesting that S-nitrosoglutathione (GSNO) is less degraded and that the drought-tolerant genotype has a higher natural reservoir of NO than the drought-sensitive one. Those differences in intracellular and extracellular NO contents and enzymatic activities were associated with higher leaf hydration in the drought-tolerant genotype as compared to the sensitive one under water deficit.
Assuntos
Secas , Óxido Nítrico/metabolismo , Saccharum/metabolismo , Saccharum/fisiologia , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , S-Nitrosoglutationa/metabolismoRESUMO
MAIN CONCLUSION: Nitric oxide (NO)-mediated redox signaling plays a role in alleviating the negative impact of water stress in sugarcane plants by improving root growth and photosynthesis. Drought is an environmental limitation affecting sugarcane growth and yield. The redox-active molecule nitric oxide (NO) is known to modulate plant responses to stressful conditions. NO may react with glutathione (GSH) to form S-nitrosoglutathione (GSNO), which is considered the main reservoir of NO in cells. Here, we investigate the role of NO in alleviating the effects of water deficit on growth and photosynthesis of sugarcane plants. Well-hydrated plants were compared to plants under drought and sprayed with mock (water) or GSNO at concentrations ranging from 10 to 1000 µM. Leaf GSNO sprayed plants showed significant improvement of relative water content and leaf and root dry matter under drought compared to mock-sprayed plants. Additionally, plants sprayed with GSNO (≥ 100 µM) showed higher leaf gas exchange and photochemical activity as compared to mock-sprayed plants under water deficit and after rehydration. Surprisingly, a raise in the total S-nitrosothiols content was observed in leaves sprayed with GSH or GSNO, suggesting a long-term role of NO-mediated responses to water deficit. Experiments with leaf discs fumigated with NO gas also suggested a role of NO in drought tolerance of sugarcane plants. Overall, our data indicate that the NO-mediated redox signaling plays a role in alleviating the negative effects of water stress in sugarcane plants by protecting the photosynthetic apparatus and improving shoot and root growth.
Assuntos
Secas , Óxido Nítrico/farmacologia , Fotossíntese/efeitos dos fármacos , Saccharum/efeitos dos fármacos , Análise de Variância , Dióxido de Carbono/metabolismo , Desidratação , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Oxirredução/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , S-Nitrosoglutationa/metabolismo , S-Nitrosoglutationa/farmacologia , Saccharum/crescimento & desenvolvimento , Saccharum/metabolismo , Água/metabolismo , Água/farmacologiaRESUMO
Nitrogen assimilation plays a vital role in plant metabolism. Assimilation of nitrate, the primary source of nitrogen in soil, is linked to the generation of the redox signal nitric oxide (NO). An important mechanism by which NO regulates plant development and stress responses is through S-nitrosylation, that is, covalent attachment of NO to cysteine residues to form S-nitrosothiols (SNO). Despite the importance of nitrogen assimilation and NO signalling, it remains largely unknown how these pathways are interconnected. Here we show that SNO signalling suppresses both nitrate uptake and reduction by transporters and reductases, respectively, to fine tune nitrate homeostasis. Moreover, NO derived from nitrate assimilation suppresses the redox enzyme S-nitrosoglutathione Reductase 1 (GSNOR1) by S-nitrosylation, preventing scavenging of S-nitrosoglutathione, a major cellular bio-reservoir of NO. Hence, our data demonstrates that (S)NO controls its own generation and scavenging by modulating nitrate assimilation and GSNOR1 activity.
Assuntos
Arabidopsis/metabolismo , Óxido Nítrico/metabolismo , Nitrogênio/metabolismo , S-Nitrosotióis/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Arabidopsis/metabolismo , Glutationa Redutase/metabolismo , Homeostase/fisiologia , Modelos Biológicos , Nitratos/metabolismo , OxirreduçãoRESUMO
Although soybean isoflavones naturally accumulate in their conjugated forms, the beneficial effects on human health of soybean-containing foods have been credited to their aglycone forms. In the present study we analyzed the effects of a chemical agent, sodium nitroprusside (SNP), in eliciting the exudation of non-conjugated isoflavones from intact soybean seeds, embrionary axes and cotyledons. The isoflavones in the exudates were determined by high performance liquid chromatography and mass spectrometry. The effect of the exudates on the emission of nitric oxide (NO) and on the proliferation of breast carcinoma cells (MCF-7) was also evaluated. SNP elicitation increased the production of the aglycone forms dose- and time-dependently. Exudates of embrionary axes and cotyledons stimulated NO emission and showed biphasic effects on viability of MCF-7 cells. At lower concentrations both extracts presented proliferative effects (10-25%), and at higher concentrations inhibited (15%) cell proliferation. The biphasic effect might be due to the action of isoflavone aglycones in activating estrogen receptors which in turn stimulate the production of NO. Overall, the results suggest that soybean extracts enriched in isoflavone aglycones by elicitation with SNP could be exploited as a functional ingredient in the food industry.
RESUMO
The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.
Assuntos
Aldeído Oxirredutases/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/metabolismo , Óxido Nítrico/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , S-Nitrosotióis/análiseRESUMO
An atisane diterpene was isolated from Xylopia langsdorfiana St. Hilaire & Tulasne, Annonaceae, leaves, ent-atisane-7α,16α-diol (xylodiol). Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ Ψm). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ Ψm occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ Ψm is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ Ψm and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ Ψm leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.
RESUMO
It has been demonstrated that tumoral cells have a higher uptake of ascorbic acid compared to normal cells. This differential characteristic can be used as a way to improve the specificity of antitumoral compounds if combined with polymeric drug delivery systems. The aim of this study was to prepare, characterize and evaluate the antitumoral activity of poly- D,L-(lactide-co-glycolide) 50:50 loading the antitumoral compound violacein and capped with L-ascorbic acid. Nanoparticles were prepared using the nanoprecipitation method and morphologically characterized by scanning electron microscopy (SEM). The average diameter and Zeta potential were determined by photon correlation spectroscopy method (PCS), and assays were carried out to determine the content of ascorbic acid and in vitro drug release kinetics. The antitumoral activity of this system was also evaluated against HL-60 cells by tetrazolium reduction assay. Nanoparticles with size distribution between 300-400 nm and strong negative outer surface (-40 mV) were obtained by this method. Analysis of ascorbic acid content showed that this compound was mainly localized on the external surface of nanoparticles. Violacein loading efficiency was determined as 32% +/- 1% and this drug was gradually released from nanoparticles at different rates depending on the composition of the release media. In addition, this system was observed to be 2 x more efficient as an antitumoral compared with free violacein.
Assuntos
Ácido Ascórbico/química , Sobrevivência Celular/efeitos dos fármacos , Indóis/administração & dosagem , Indóis/química , Ácido Láctico/química , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Ácido Poliglicólico/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Difusão , Composição de Medicamentos/métodos , Células HL-60 , Humanos , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Diterpenos/farmacologia , Leucemia/patologia , Xylopia/química , Diterpenos/isolamento & purificação , HumanosRESUMO
Tumoral cells are known to have a higher ascorbic acid uptake than normal cells. Therefore, the aim of this study was to obtain polymeric nanoparticles containing the antitumoral compound trans-dehydrocrotonin (DHC) functionalized with L-ascorbic acid 6-stearate (AAS) to specifically target this system tumoral cells. Nanoparticle suspensions (NP-AAS-DHC) were prepared by the nanoprecipitation method. The systems were characterized for AAS presence by thin-layer chromatography and for drug loading (81-88%) by UV-Vis spectroscopy. To further characterize these systems, in vitro release kinetics, size distribution (100-140 nm) and Zeta potential by photon-correlation spectroscopic method were used. In vitro toxicity against HL60 cells was evaluated by tetrazolium reduction and Trypan blue exclusion assays. Cell death by apoptosis was quantified and characterized by flow cytometry and caspase activity. Zeta potential analyses showed that the system has a negatively charged outer surface and also indicate that AAS is incorporated on the external surface of the nanoparticles. In vitro release kinetics assay showed that DHC loaded in nanoparticles had sustained release behavior. In vitro toxicity assays showed that NP-AAS-DHC suspension was more effective as an antitumoral than free DHC or NP-DHC and increased apoptosis induction by receptor-mediated pathway.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Ácido Ascórbico/análogos & derivados , Diterpenos Clerodânicos/administração & dosagem , Antineoplásicos Fitogênicos/síntese química , Apoptose/efeitos dos fármacos , Ácido Ascórbico/química , Caspases/metabolismo , Linhagem Celular Tumoral , Cromatografia em Camada Fina , Corantes , Diterpenos Clerodânicos/síntese química , Sistemas de Liberação de Medicamentos , Eletroquímica , Excipientes , Citometria de Fluxo , Células HL-60 , Humanos , Cinética , Nanopartículas , Tamanho da Partícula , Espectrofotometria Ultravioleta , Suspensões , Sais de Tetrazólio , Tiazóis , Azul TripanoRESUMO
Violacein is a compound obtained from Chromobacterium violaceum, a bacterium found in the Amazonian region. Violacein-loaded poly (D, L-lactide-co-glycolide) nanoparticles has a similar inhibitory effect evaluated by trypan blue assay on leukemic HL60 cells than the free form. However, the cytotoxic effects evaluated by phosphatase activity and MTT reduction assays were lower for the encapsulated form than for free violacein. Based on morphological changes, violacein and violacein entrapped in nanoparticles were found to induce terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells. Thus, both formulations inhibit HL60 cell growth in vitro, partly by inducing cytotoxic effects and cell differentiation. Flow cytometric analysis of HL60 cells after treatment for 12 h showed that violacein-loaded PLGA induced apoptosis, with maximum cell death at a concentration of 2 microM. Violacein and violacein/PLGA induced opposite effects on the mitochondrial swelling which indicates altered mitochondrial function. The mitochondrial activity was also checked by flow cytometry studies. Labelled cells with the probe JC1 displayed a basal hypopolarized status of the mitochondria in treated cells. Based on morphological changes, alterations in phospholipid asymmetry and changes in mitochondrial polarization, violacein and nanoparticles containing violacein were found to trigger cell death by apoptosis. These methodologies are promising as diagnostic and mechanistic effects of nanoparticles in cell cultures.