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1.
Science ; 363(6422)2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30606819

RESUMO

Photorespiration is required in C3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity by >40% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C3 crop yield under agricultural field conditions.


Assuntos
Cloroplastos/metabolismo , Glicolatos/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo , Biomassa , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/enzimologia , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Cucurbita/enzimologia , Escherichia coli/enzimologia , Regulação da Expressão Gênica de Plantas , Genes Bacterianos , Redes e Vias Metabólicas/genética , Fotossíntese , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Ribulosefosfatos/metabolismo , Estresse Fisiológico , Biologia Sintética , Temperatura Ambiente
2.
Appl Biochem Biotechnol ; 186(4): 1061-1073, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29862444

RESUMO

A cantilever nanobiosensor functionalized with vegetable source of peroxidase was developed as an innovative way for glyphosate herbicide detection over a wide concentration range (0.01 to 10 mg L-1) using atomic force microscopy (AFM) technique. The extract obtained from zucchini (Cucurbita pepo source of peroxidase), with high enzymatic activity and stability has been used as bio-recognition element to develop a nanobiosensor. The polarization-modulated reflection absorption infrared spectroscopy (PM-RAIRS) demonstrated the deposition of enzyme on cantilever surface using self-assembled monolayers (SAM) by the presence of the amide I and II bands. The detection mechanism of glyphosate was based on the changes in surface tension caused by the analyte adsorption, resulting in a conformational change in the enzyme structure. In this way, the results of nanobiosensor demonstrate the potential of the sensing device for detecting glyphosate with a detection limit of 0.028 mg L-1.


Assuntos
Técnicas Biossensoriais/métodos , Cucurbita/enzimologia , Glicina/análogos & derivados , Peroxidase/química , Proteínas de Plantas/química , Glicina/análise
3.
Ecotoxicol Environ Saf ; 147: 151-156, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28841531

RESUMO

Phytoremediation is a low cost technology based on the use of plants to remove a wide range of pollutants from the environment, including the insecticide DDT. However, some pollutants are known to enhance generation of reactive oxygen species (ROS), which can generate toxic effects on plants affecting the phytoremediation efficiency. This study aims to analyze the potential use of antioxidant responses as a measure of tolerance to select plants for phytoremediation purposes. Tomato and zucchini plants were grown for 15 days in soils contaminated with DDTs (DDT + DDE + DDD). Protein content, glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) activities were measured in plant tissues. Exposure to DDTs did not affect protein content or CAT activity in any of the species. GST, GR and GPx activity showed different responses in exposed and control tomato plants. After DDTs exposure, tomato showed increased GR and GPX activity in stems and leaves, respectively, and a decrease in the GST activity in roots. As no effects were observed in zucchini, results suggest different susceptibility and/or defense mechanisms involved after pesticide exposure. Finally, both species differed also in terms of DDTs uptake and translocation. The knowledge about antioxidant responses induced by pesticides exposure could be helpful for planning phytoremediation strategies and for the selection of tolerant species according to particular scenarios.


Assuntos
Antioxidantes/metabolismo , Cucurbita/efeitos dos fármacos , DDT/toxicidade , Lycopersicon esculentum/efeitos dos fármacos , Poluentes do Solo/toxicidade , Biodegradação Ambiental , Cucurbita/enzimologia , DDT/metabolismo , Lycopersicon esculentum/enzimologia , Solo/química , Poluentes do Solo/metabolismo
4.
Development ; 143(23): 4425-4429, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27789625

RESUMO

Gibberellins (GAs) are hormones that control many aspects of plant development, including flowering. It is well known that stamen is the source of GAs that regulate male and bisexual flower development. However, little is known about the role of GAs in female flower development. In cucumber, high levels of GA precursors are present in ovaries and high levels of bioactive GA4 are identified in sepals/petals, reflecting the expression of GA 20-oxidase and 3-oxidase in these organs, respectively. Here, we show that the biologically inactive precursor GA9 moves from ovaries to sepal/petal tissues where it is converted to the bioactive GA4 necessary for female flower development. Transient expression of a catabolic GA 2-oxidase from pumpkin in cucumber ovaries decreases GA9 and GA4 levels and arrests the development of female flowers, and this can be restored by application of GA9 to petals thus confirming its function. Given that bioactive GAs can promote sex reversion of female flowers, movement of biologically inactive precursors, instead of the hormone itself, might help to maintain floral organ identity, ensuring fruit and seed production.


Assuntos
Cucumis sativus/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Flores/metabolismo , Giberelinas/metabolismo , Oxigenases de Função Mista/metabolismo , Cucurbita/enzimologia , Cucurbita/genética , Giberelinas/genética , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Plantas Geneticamente Modificadas/genética , Transdução de Sinais
5.
Nanotoxicology ; 10(9): 1243-53, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27308847

RESUMO

The effect of dissolved organic matter (DOM) on nanoparticle toxicity to plants is poorly understood. In this study, tannic acid (TA) was selected as a DOM surrogate to explore the mechanisms of neodymium oxide NPs (Nd2O3 NPs) phytotoxicity to pumpkin (Cucurbita maxima). The results from the tested concentrations showed that 100 mg L(-1) Nd2O3 NPs were significantly toxic to pumpkin in term of fresh biomass, and the similar results from the bulk particles and the ionic treatments were also evident. Exposure to 100 mg L(-1) of Nd2O3 NPs and BPs in 1/5 strength Hoagland's solution not only significantly inhibited pumpkin growth, but also decreased the S, Ca, K and Mg levels in plant tissues. However, 60 mg L(-1) TA significantly moderated the observed phytotoxicity, decreased Nd accumulation in the roots, and notably restored S, Ca, K and Mg levels in NPs and BPs treated pumpkin. TA at 60 mg L(-1) increased superoxide dismutase (SOD) activity in both roots (17.5%) and leaves (42.9%), and catalase (CAT) activity (243.1%) in the roots exposed to Nd2O3 NPs. This finding was confirmed by the observed up-regulation of transcript levels of SOD and CAT in Nd2O3 NPs treated pumpkin analyzed by quantitative reverse transcription polymerase chain reaction. These results suggest that TA alleviates Nd2O3 BPs/NPs toxicity through alteration of the particle surface charge, thus reducing the contact and uptake of NPs by pumpkin. In addition, TA promotes antioxidant enzymatic activity by elevating the transcript levels of genes involved in ROS scavenging. Our results shed light on the mechanisms underlying the influence of DOM on the bioavailability and toxicity of NPs to terrestrial plants.


Assuntos
Cucurbita/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Nanopartículas/toxicidade , Neodímio/toxicidade , Óxidos/toxicidade , Taninos/farmacologia , Antioxidantes/metabolismo , Cucurbita/enzimologia , Cucurbita/genética , Cucurbita/crescimento & desenvolvimento , Expressão Gênica/efeitos dos fármacos , Nanopartículas/química , Neodímio/química , Óxidos/química , Tamanho da Partícula , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Propriedades de Superfície
6.
Acta Biol Hung ; 67(1): 53-63, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26960356

RESUMO

This study is aimed to investigate the activity of urease (EC 3.5.1.5, urea amidohydrolase) that catalyzes the hydrolysis of urea in 5-day-old Cucurbita pepo cotyledons subjected to various concentrations of different growth regulators. The treatment of C. pepo cotyledons with different concentrations (100-600 µmol) of different auxins [indole-3-acetic acid (IAA), indole butyric acid (IBA), indole propionic acid (IPA) and naphthalene acetic acid (NAA)]; or with different concentrations (100-300 µmol) of different cytokinins [kinetin, zeatin and benzyladenine (6-BA)] resulted in a significant increase of urease activity, compared to control. The optimal effects were recorded for each of 500 µmol of IAA and 300 µmol of zeatin treatments. A gradual increase in urease activity was detected in cotyledons treated with various concentrations (0.2-1.0 mM) of 28-homobrassinolide (HBL), in relative to control. A substantial increase in urease activity was observed in cotyledons subjected to different concentrations of triazole (10-60 mg L(-1)), containing either triadimefon (TDM) or hexaconazole (HEX), compared to control. The combination of 300 µmol zeatin with any of protein inhibitors, namely 5-fluorouridine (FUrd), cordycepin and α-amanitin, resulted in the alleviation of their inhibitory effect on the urease activity.


Assuntos
Cucurbita/enzimologia , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Triazóis/metabolismo , Urease/metabolismo , Cotilédone/enzimologia
7.
Amino Acids ; 47(11): 2335-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26036686

RESUMO

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 µg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.


Assuntos
Antígenos/química , Caseínas/química , Cucurbita/enzimologia , Proteínas de Plantas/química , Serina Proteases/química , Proteínas do Soro do Leite/química , Animais , Bovinos , Hidrólise , Imunoglobulina E/química , Imunoglobulina G/química
8.
Analyst ; 140(15): 5039-47, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26051011

RESUMO

This study demonstrates a new strategy to develop online electrochemical systems (OECSs) for continuously monitoring neurochemicals by efficiently integrating in vivo microdialysis with an oxidase-based electrochemical biosensor with low-potential electron mediators to shuttle the electron transfer of the oxidases. By using thionine and xanthine oxidase (XOD) as examples of low-potential mediators and oxidases, respectively, we demonstrate that the use of low-potential mediators to shuttle the electron transfer of oxidases would offer a new approach to the development of oxidase-based biosensors with theoretical and technical simplicity. To construct the XOD-based biosensor, thionine was adsorbed onto carbon nanotubes and used to shuttle the electron transfer of XOD. The XOD-based biosensor was positioned into an electrochemical cell that was directly coupled with in vivo microdialysis to form an online electrochemical system (OECS) for continuous and selective measurements of the substrate of XOD (with hypoxanthine as an example). The OECS based on the low-potential mediators is highly selective against the species endogenously existing in the brain system, which is attributed to the low operation potential benefited from the low redox potentials of the mediators. Moreover, the OECS demonstrated here is stable and reproducible and could thus be envisaged to find some interesting applications in physiological and pathological investigations. This study essentially offers a new strategy to develop online electrochemical systems, which is of great importance in understanding the molecular basis of physiological and pathological events.


Assuntos
Técnicas Biossensoriais/instrumentação , Química Encefálica , Enzimas Imobilizadas/metabolismo , Hipoxantina/análise , Dispositivos Lab-On-A-Chip , Xantina Oxidase/metabolismo , Adsorção , Animais , Ascorbato Oxidase/metabolismo , Cucurbita/enzimologia , Desenho de Equipamento , Microdiálise/instrumentação , Nanotubos de Carbono/química , Sistemas On-Line , Oxirredução , Fenotiazinas/metabolismo , Ratos
9.
Molecules ; 20(6): 11184-201, 2015 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-26091076

RESUMO

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.


Assuntos
Cucurbita/química , Lipase/química , Lipase/isolamento & purificação , Extração Líquido-Líquido , Sementes/química , Tensoativos , Xilitol , Cucurbita/enzimologia , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Tensoativos/química , Temperatura Ambiente , Xilitol/química
10.
Carbohydr Polym ; 113: 314-24, 2014 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-25256490

RESUMO

An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical.


Assuntos
Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cucurbita/enzimologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Som , Depuradores de Radicais Livres/isolamento & purificação , Depuradores de Radicais Livres/farmacologia
11.
J Proteomics ; 110: 107-16, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25138009

RESUMO

UNLABELLED: In the present study angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from egg-yolk protein preparation (YP). Enzymatic hydrolysis conducted using unconventional enzyme from Cucurbita ficifolia (dose: 1000 U/mg of hydrolyzed YP (E/S (w/w)=1:7.52)) was employed to obtain protein hydrolysates. The 4-h hydrolysate exhibited a significant (IC50=482.5 µg/mL) ACE inhibitory activity. Moreover, hydrolysate showed no cytotoxic activity on human and animal cell lines which makes it a very useful multifunctional method for peptide preparation. The compiled isolation procedure (ultrafiltration, size-exclusion chromatography and RP-HPLC) of bioactive peptides from YP hydrolysate resulted in obtaining peptides with the strong ACE inhibitory activity. One homogeneous and three heterogeneous peptide fractions were identified. The peptides were composed of 9-18 amino-acid residues, including mainly arginine and leucine at the N-terminal positions. To confirm the selected bioactive peptide sequences their analogs were chemically synthesized and tested. Peptide LAPSLPGKPKPD showed the strongest ACE inhibitory activity, with IC50 value of 1.97 µmol/L. BIOLOGICAL SIGNIFICANCE: Peptides with specific biological activity can be used in pharmaceutical, cosmetic or food industries. Because of their potential role as physiological modulators, as well as theirhigh safety profile, they can be used as natural pharmacological compounds or functional food ingredients. The development of biotechnological solutions to obtain peptides with desired biological activity is already in progress. Studies in this area are focused on using unconventional highly specific enzymes and more efficient methods developed to conduct food process technologies. Natural peptides have many advantages. They are mainly toxicologically safe, have wide spectra of therapeutic actions, exhibit less side effects compared to synthetic drugs and are more efficiently absorbed in the intestinal tract. The complexity of operation of large scale technologies and high cost of purification techniques are limiting factors to the commercialization of food-derived bioactive peptides. Research on the isolation of bioactive peptides in order to reduce the processing time and costs is continuously developing. Bioactive peptides can also be released from protein by-products of the food industry, which reduce the substrate expense and production cost as well as provide the added advantage of an efficient waste disposal. Moreover, proteins as precursors of food-derived peptides are well-tolerated by the human body and therefore their application in drug development may reduce costs and duration of toxicological studies during research, development and clinical trials.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Cucurbita/enzimologia , Proteínas do Ovo/química , Peptídeo Hidrolases/química , Peptídeos/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Cucurbita/classificação , Proteínas do Ovo/isolamento & purificação , Ativação Enzimática , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Especificidade da Espécie
12.
Plant Physiol Biochem ; 82: 161-71, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24960300

RESUMO

In order to investigate the contribution of polyamines and related amino acids in the maintenance of zucchini fruit quality during cold storage, two varieties of Cucurbita pepo with different degrees of chilling tolerance were used, Natura (more tolerant) and Sinatra (moresensitive). After harvest, free putrescine levels decreased during storage at 20 °C, whereas in fruit kept at 4 °C this polyamine accumulated in both varieties, but with higher levels in the sensitive variety (Sinatra). This behavior suggests that putrescine is accumulated as a response to low temperature in zucchini fruit by stress-induced chilling injury, and not due to the postharvest storage itself. ADC activity responds quickly to chilling but sharply decreases after 14 days, whereas its expression remains high in both varieties. ODC activity takes over when the cold stress is relatively severe, as this activity was found to be much higher in Sinatra. ODCexpression also correlated with ODC activity. DAO activity increased in Natura fruit, and conversely decreased in Sinatra fruit during storage at 4 °C, whereas the proline content was higher in Natura and lower in Sinatra. Therefore, we suggest that putrescine degradation and proline accumulation contribute to the acquisition of chilling tolerance in zucchini fruit. GABA content decreased in both varieties, with a greater reduction in Natura fruit and less in Sinatra fruit. In addition, GABA transaminase showed a higher activity in Natura fruit than in Sinatra fruit during cold storage, suggesting that GABA catabolism could be involved in the tolerance to postharvest cold storage in zucchini fruit.


Assuntos
Cucurbita/enzimologia , Cucurbita/metabolismo , Frutas/metabolismo , Poliaminas/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Temperatura Baixa , Prolina/metabolismo
13.
PLoS One ; 9(3): e92335, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24637721

RESUMO

Protein isolates of pumpkin (Cucurbita pepo L) seeds were hydrolyzed by acid protease to prepare antioxidative peptides. The hydrolysis conditions were optimized through Box-Behnken experimental design combined with response surface method (RSM). The second-order model, developed for the DPPH radical scavenging activity of pumpkin seed hydrolysates, showed good fit with the experiment data with a high value of coefficient of determination (0.9918). The optimal hydrolysis conditions were determined as follows: hydrolyzing temperature 50°C, pH 2.5, enzyme amount 6000 U/g, substrate concentration 0.05 g/ml and hydrolyzing time 5 h. Under the above conditions, the scavenging activity of DPPH radical was as high as 92.82%.


Assuntos
Antioxidantes/metabolismo , Biotecnologia/métodos , Cucurbita/metabolismo , Peptídeos/isolamento & purificação , Sementes/metabolismo , Análise de Variância , Compostos de Bifenilo/metabolismo , Cucurbita/enzimologia , Depuradores de Radicais Livres/farmacologia , Hidrólise/efeitos dos fármacos , Modelos Biológicos , Picratos/metabolismo , Análise de Regressão , Reprodutibilidade dos Testes , Sementes/enzimologia , Especificidade por Substrato/efeitos dos fármacos , Fatores de Tempo
14.
Plant Biol (Stuttg) ; 16(2): 457-66, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23879260

RESUMO

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo.


Assuntos
Cucurbita/genética , Genes de Plantas , Proteínas de Plantas/genética , Pólen/enzimologia , Poligalacturonase/genética , Sequência de Aminoácidos , Clonagem Molecular , Cucurbita/enzimologia , Cucurbita/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Pólen/crescimento & desenvolvimento , Polinização , Poligalacturonase/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência
15.
Int J Mol Sci ; 14(12): 24008-28, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24336062

RESUMO

The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant "C. moschata", thermolabile "C. maxima" and moderately heat-tolerant interspecific inbred line "Maxchata" genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. "C. moschata" exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2(-)) and malondialdehyde (MDA) contents in the roots compared to stems, followed by "Maxchata". The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among "C. maxima" and "Maxchata", most of these genes were highly induced under heat stress in "Maxchata", which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.


Assuntos
Antioxidantes/metabolismo , Cucurbita/enzimologia , Cucurbita/genética , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Oxirredutases/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Catalase/genética , Catalase/metabolismo , Cucurbita/química , Genótipo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Oxirredutases/genética , Peroxidases/genética , Peroxidases/metabolismo , Raízes de Plantas/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Caules de Planta/química , Caules de Planta/enzimologia , Caules de Planta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo
16.
J Environ Sci (China) ; 25(6): 1235-44, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24191614

RESUMO

Enzymatic decolourization of the azo dye, Direct Yellow (DY106) by Cucurbita pepo (courgette) peroxidase (CP) is a complex process, which is greatly affected by pH, temperature, enzyme activity and the concentrations of H2O2 and dye. Courgette peroxidase was extracted and its performance was evaluated by using the free-CP (FCP) and immobilized-CP (ICP) forms in the decolourization of DY106. Immobilization of peroxidase in calcium alginate beads was performed according to a strategy aiming to minimize enzyme leakage and keep its activity at a maximum value by optimizing sodium alginate content, enzyme loading and calcium chloride concentration. The initial conditions at which the highest DY106 decolourization yield was obtained were found at pH 2, temperature 20 degrees C, H2O2 dose 1 mmol/L (FCP) and 100 mmol/L (ICP). The highest decolourization rates were obtained for dye concentrations 50 mg/L (FCP) and 80 mg/L (ICP). Under optimal conditions, the FCP was able to decolorize more than 87% of the dye within 2 min. While with ICP, the decolourization yield was 75% within 15 min. The decolourization and removal of DY106 was proved by UV-Vis analysis. Fourier transform infrared (FT-IR) spectroscopy analysis was also performed on DY106 and enzymatic treatment precipitated byproduct.


Assuntos
Corantes/metabolismo , Cucurbita/enzimologia , Enzimas Imobilizadas/metabolismo , Peroxidases/metabolismo , Compostos Azo/metabolismo , Biodegradação Ambiental , Naftalenos/metabolismo
17.
J Agric Food Chem ; 61(26): 6393-403, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23773001

RESUMO

The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf-, and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms, could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that, in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.


Assuntos
Antioxidantes/metabolismo , Carotenoides/biossíntese , Cucurbita/metabolismo , Proteínas na Dieta/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Antioxidantes/análise , Carotenoides/análise , Cucurbita/enzimologia , Cucurbita/crescimento & desenvolvimento , Frutas/enzimologia , Frutas/crescimento & desenvolvimento , Liases Intramoleculares/biossíntese , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espanha
18.
Acta Biochim Pol ; 60(1): 117-22, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23520577

RESUMO

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (µmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.


Assuntos
Caseínas/metabolismo , Cucurbita/química , Cucurbita/enzimologia , Serina Proteases/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Hidrólise , Peptídeos/metabolismo
19.
Appl Biochem Biotechnol ; 167(7): 2023-38, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22644641

RESUMO

Ascorbate oxidase (AO), a biologically active macromolecule, was successfully immobilized into a biocompatible sandwich-type composite film for developing the vitamin C (VC) biosensor, and the content of VC in commercial juices was amperometrically determined. The biocompatible and conducting poly(3,4-ethylenedioxythiophene) composite film and highly stable and selective multiwalled carbon nanotubes -Nafion composite film were prepared as inner and outer films of biosensor. AO molecules were immobilized between these two composite films. The as-fabricated biosensor displayed an excellent bioelectrocatalytic performance towards the oxidation of VC, a fast current response, a low working potential, a high sensitivity, a wide linear range, and a low detection limit. Moreover, the working mechanism of the biosensor was proposed, and its kinetics was also discussed. In addition, the specificity, reproducibility, and feasibility of the as-fabricated biosensor were also evaluated. Good results of the VC determination in commercial juices indicated that the as-fabricated biosensor was a potential candidate for the electrochemical determination of VC in agricultural crops. Inner and outer films provided a promising platform for the immobilization of biologically active species.


Assuntos
Ascorbato Oxidase/metabolismo , Ácido Ascórbico/análise , Materiais Biocompatíveis/química , Técnicas Biossensoriais/métodos , Cucurbita/enzimologia , Eletroquímica/métodos , Enzimas Imobilizadas/metabolismo , Bebidas/análise , Biocatálise , Eletricidade , Eletrodos , Cinética , Microscopia Eletrônica de Varredura , Oxirredução , Propriedades de Superfície
20.
J Exp Bot ; 63(7): 2681-91, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22268154

RESUMO

Gibberellin (GA) signalling during pumpkin male flower development is highly regulated, including biosynthetic, perception, and transduction pathways. GA 20-oxidases, 3-oxidases, and 2-oxidases catalyse the final part of GA synthesis. Additionally, 7-oxidase initiates this part of the pathway in some cucurbits including Cucurbita maxima L. (pumpkin). Expression patterns for these GA-oxidase-encoding genes were examined by competitive reverse transcription-PCR (RT-PCR) and endogenous GA levels were determined during pumpkin male flower development. In young flowers, GA20ox3 transcript levels are high in stamens, followed by high levels of the GA precursor GA(9). Later, just before flower opening, transcript levels for GA3ox3 and GA3ox4 increase in the hypanthium and stamens, respectively. In the stamen, following GA3ox4 expression, bioactive GA(4) levels rise dramatically. Accordingly, catabolic GA2ox2 and GA2ox3 transcript levels are low in developing flowers, and increase in mature flowers. Putative GA receptor GID1b and DELLA repressor GAIPb transcript levels do not change in developing flowers, but increase sharply in mature flowers. Emasculation arrests floral development completely and leads to abscission of premature flowers. Application of GA(4) (but not of its precursors GA(12)-aldehyde or GA(9)) restores normal growth of emasculated flowers. These results indicate that de novo GA(4) synthesis in the stamen is under control of GA20ox3 and GA3ox4 genes just before the rapid flower growth phase. Stamen-derived bioactive GA is essential and sufficient for male flower development, including the petal and the pedicel growth.


Assuntos
Cucurbita/metabolismo , Flores/crescimento & desenvolvimento , Giberelinas/biossíntese , Cucurbita/enzimologia , Cucurbita/genética , Cucurbita/crescimento & desenvolvimento , Flores/enzimologia , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade da Espécie
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