RESUMO
Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.
Assuntos
Apocynaceae/enzimologia , Leite/química , Peptídeo Hidrolases/química , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteólise , Sementes/enzimologia , Animais , Concentração de Íons de HidrogênioRESUMO
Considering the importance of blast caused by Pyricularia oryzae in the decrease of rice yield worldwide, this study aimed to assess the photosynthetic performance [leaf gas exchange and chlorophyll (Chl) a fluorescence parameters as well as the photosynthetic pigments concentration], the activities of antioxidant enzymes [ascorbate peroxidase, catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase] and concentrations of hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) in the leaves of rice plants non-supplied (-Glu) or supplied (+Glu) with glutamate (Glu) and non-infected or infected by P. oryzae. Blast severity was reduced in the leaves of +Glu plants. On the infected leaves of +Glu plants, the values for internal CO2 concentration were lower while the values for net carbon assimilation rate, stomatal conductance as well as for the concentrations of Chl a, Chl b and carotenoids were higher in comparison to infected leaves of -Glu plants. The functionality of the photosynthetic apparatus was preserved in the infected leaves of +Glu plants. The activities of CAT, GPX, GR, POX and SOD increased in the infected leaves of both -Glu and +Glu plants compared to their non-inoculated counterparts, but their activities were lower for +Glu plants. The lower activity of these antioxidative enzymes was triggered by the reduced hydrogen peroxide concentration in the infected leaves of +Glu plants resulting in lower MDA concentration. It can be concluded that photosynthesis was less impaired in infected plants supplied with glutamate due to the lower biochemical constraints for CO2 fixation. Moreover, there was a need for lower activity of reactive oxygen species scavenging enzymes in infected leaves of plants supplied with glutamate due to the lower oxidative stress imposed by P. oryzae infection.
Assuntos
Antioxidantes/metabolismo , Ácido Glutâmico/farmacologia , Oryza/microbiologia , Oryza/fisiologia , Fotossíntese , Doenças das Plantas/microbiologia , Clorofila/análise , Peróxido de Hidrogênio/análise , Malondialdeído/análise , Estresse Oxidativo , Folhas de Planta/enzimologiaRESUMO
Leaf hyperspectral reflectance has been used to estimate nutrient concentrations in plants in narrow bands of the electromagnetic spectrum. The aim of this study was to estimate leaf nutrient concentrations using leaf hyperspectral reflectance and verify the variable selection methods using the partial least squares regression (PLSR). Two studies were carried out using stands with Eucalyptus clones. Study I was established in Eucalyptus stands with three clones, classifying leaves into five colour patterns using the Munsell chart for plant tissues. Immediately after leaf collection, leaf reflectance was read and the chemical analysis was performed. Study II was carried out in commercial clonal stands of Eucalyptus performing the same leaf sampling and chemical analysis as used in Study I. All leaf reflectance spectra were smoothed and three more pre-processing procedures were applied. In addition, three methods of PLSR were tested. The first derivative was more accurate for predicting nitrogen ( Rcv2=0.95 ), phosphorous ( Rcv2=0.93 ), and sulphur concentration ( Rcv2=0.85 ). The estimates for concentrations of calcium ( Rcv2=0.81 ), magnesium ( Rcv2=0.22 ), and potassium ( Rcv2=0.76 ) were more accurate using the logarithm transformation. Only the estimates for iron concentrations were performed with higher accuracy ( Rcv2=0.35 ) using the smoothed reflectance. The copper concentrations were more accurate ( Rcv2=0.78 ) using the logarithm transformation. Concentrations of boron ( Rcv2=0.68 ) and manganese ( Rcv2=0.79 ) were more accurate using the first derivative, while zinc ( Rcv2=0.31 ) concentration was most accurate using the second derivative.(AU)
Assuntos
Folhas de Planta/química , Folhas de Planta/enzimologia , Eucalyptus/enzimologiaRESUMO
Leaf hyperspectral reflectance has been used to estimate nutrient concentrations in plants in narrow bands of the electromagnetic spectrum. The aim of this study was to estimate leaf nutrient concentrations using leaf hyperspectral reflectance and verify the variable selection methods using the partial least squares regression (PLSR). Two studies were carried out using stands with Eucalyptus clones. Study I was established in Eucalyptus stands with three clones, classifying leaves into five colour patterns using the Munsell chart for plant tissues. Immediately after leaf collection, leaf reflectance was read and the chemical analysis was performed. Study II was carried out in commercial clonal stands of Eucalyptus performing the same leaf sampling and chemical analysis as used in Study I. All leaf reflectance spectra were smoothed and three more pre-processing procedures were applied. In addition, three methods of PLSR were tested. The first derivative was more accurate for predicting nitrogen ( Rcv2=0.95 ), phosphorous ( Rcv2=0.93 ), and sulphur concentration ( Rcv2=0.85 ). The estimates for concentrations of calcium ( Rcv2=0.81 ), magnesium ( Rcv2=0.22 ), and potassium ( Rcv2=0.76 ) were more accurate using the logarithm transformation. Only the estimates for iron concentrations were performed with higher accuracy ( Rcv2=0.35 ) using the smoothed reflectance. The copper concentrations were more accurate ( Rcv2=0.78 ) using the logarithm transformation. Concentrations of boron ( Rcv2=0.68 ) and manganese ( Rcv2=0.79 ) were more accurate using the first derivative, while zinc ( Rcv2=0.31 ) concentration was most accurate using the second derivative.
Assuntos
Eucalyptus/enzimologia , Folhas de Planta/enzimologia , Folhas de Planta/químicaRESUMO
KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.
Assuntos
Arabidopsis/enzimologia , Carbono/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/enzimologia , Mutagênese Insercional , Nitratos/metabolismo , Fenótipo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas , Subunidades Proteicas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
Leaf blast is the main rice disease in the world causing significant losses in productivity. Blast integrate management (BIM) requires the use of genetic resistance, cultural practices, and chemical control, although for sustainable BIM, the insertion of biological agents may be the fourth component for. The objective of this work was to test three formulations of Burkholderia pyrrocinia (BRM32113) previously selected and to verify the effectiveness in resistance induction and blast control in rice. Two experiments were carried out, in a completely randomized design with three replications, in the greenhouse (E1 and E2). E1 aimed to select the best treatment for suppressing leaf blast severity and activating plant defense mechanisms. It was composed of 8 treatments: (1) formulated 11+ B. pyrrocina × Magnaporthe oryzae; (2) formulated 17+ B. pyrrocina × M. oryzae; (3) formulated 32+ B. pyrrocina × M. oryzae; (4) formulated 11 × M. oryzae; (5) B. pyrrocinia 17 × M. oryzae; (6) formulated 32 × M. oryzae; (7) B. pyrrocina × M. oryzae; (8) M. oryzae; (9) control (water). E2 aimed to investigate the effect of the best treatments, for the promotion of plant growth and suppression of leaf blast by calculating AUDPC. It was composed of 6 treatments: (1) formulated 11+ B. pyrrocina × M. oryzae; (2) formulated 32+ B. pyrrocina × M. oryzae; (3) formulated 11 × M. oryzae; (4) formulated 32 × M. oryzae; (5) B. pyrrocina × M. oryzae; (6) water. And after, we did two assays aimed to localize this biological agent after application at seed, soil, and rice plant. In E1, formulated 11+ B. pyrrocinia and 32+ formulated and B. pyrrocina were the best, suppressing leaf blast by up to 97% and providing the significant increase of the enzymes ß-1,3-glucanase, chitinase, phenylalanine ammonia lyase, lipoxygenase, and salicylic acid at 24 h and 48 h after inoculation with M. oryzae. In E2, treatments formulated 11+ B. pyrrocinia, formulated 32+ B. pyrrocinia, and B. pyrrocina provided more significant increases in growth promotion and reduced area under disease progress curve. B. pyrrocinia was detected in the rice plant for 18 days, predominantly in the root system (internal and external). The use of B. pyrrocinia formulations based on sugarcane molasses and glycerol can be an essential strategy for sustainable management. Although all the benefits come from these sustainable formulations, the adoption by commercial biological segment depends on an established formulation process. It seems that all the results showed here by this research will be readily assimilated by startups of the organic segment.
Assuntos
Agentes de Controle Biológico/farmacologia , Burkholderia , Resistência à Doença/efeitos dos fármacos , Magnaporthe , Oryza/microbiologia , Doenças das Plantas/prevenção & controle , Agentes de Controle Biológico/isolamento & purificação , Burkholderia/metabolismo , Magnaporthe/crescimento & desenvolvimento , Oryza/enzimologia , Doenças das Plantas/microbiologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Distribuição Aleatória , Ácido Salicílico/metabolismoRESUMO
We investigated the effects of Azospirillum brasilense strains Ab-V5 and Ab-V6 in the induction of mechanisms of systemic acquired resistance (SAR) and induced system resistance (ISR) on maize (Zea mays L.) plants. Under normal growth conditions, the treatments consisted of the standard inoculation of cells at sowing, and leaf spray of cells or their metabolites at the V2.5 growth stage; under saline stress (170 mM NaCl), the treatment consisted of standard single and co-inoculation of A. brasilense and Rhizobium tropici. The main compounds in the Azospirillum metabolites were identified as indole-3-acetic acid (IAA) and salicylic acid (SA). Under normal conditions, A. brasilense cells applied at sowing or by leaf spray increased the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in leaves, and of ascorbate peroxidase (APX) in roots; however, interestingly, in general the highest activities were observed by leaf spray of metabolites. Under normal conditions, the highest levels of salicylic acid (SA) and jasmonic acid (JA) were achieved in leaves by leaf spray of metabolites, of SA in roots by leaf spray of cells, and of JA in roots by standard inoculation and leaf spray of metabolites. Under saline stress, plant protection occurred via SA and abscisic acid (ABA), but not JA. In general, inoculation resulted in further increases in SA in leaves and roots, and ABA in leaves. We hypothesize that A. brasilense confers protection to maize plants by simultaneous induction of JA and SA pathways, and, under saline stressing conditions, by SA and ABA pathways.
Assuntos
Antioxidantes/metabolismo , Azospirillum brasilense/metabolismo , Zea mays/metabolismo , Ácido Abscísico/metabolismo , Catalase/metabolismo , Ciclopentanos/metabolismo , Ácidos Indolacéticos/metabolismo , Malondialdeído/metabolismo , Oxilipinas/metabolismo , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Ácido Salicílico/metabolismo , Estresse Fisiológico , Superóxido Dismutase/metabolismo , Zea mays/enzimologia , Zea mays/microbiologiaRESUMO
Cladosporium herbarum is a plant pathogen associated with passion fruit scab and mild diseases in pea and soybean. In this study, a peptidogalactomannan (pGM) of C. herbarum mycelium was isolated and structurally characterized, and its role in plant-fungus interactions was evaluated. C. herbarum pGM is composed of carbohydrates (76%) and contains mannose, galactose and glucose as its main monosaccharides (molar ratio, 52:36:12). Methylation and 13C-nuclear magnetic resonance (13C-NMR) spectroscopy analysis have shown the presence of a main chain containing (1â¯ââ¯6)-linked α-D-Manp residues, and ß-D-Galf residues are present as (1â¯ââ¯5)-interlinked side chains. ß-Galactofuranose containing similar structures were characterized by our group in A. fumigatus, A. versicolor, A. flavus and C. resinae. Tobacco BY-2â¯cells were used as a model system to address the question of the role of C. herbarum pGM in cell viability and induction of the expression of plant defense-related genes. Native and partially acid hydrolyzed pGMs (lacking galactofuranosyl side-chain residues) were incubated with BY-2â¯cell suspensions at different concentrations. Cell viability drastically decreased after exposure to more than 400⯵gâ¯ml-1 pGM; however no cell viability effect was observed after exposure to a partially acid hydrolyzed pGM. BY-2â¯cell contact with pGM strongly induce the expression of plant defense-related genes, such as phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX), as well as the pathogen-related PR-1a, PR-2 and PR-3 genes, suggesting that pGM activates defense responses in tobacco cells. Interestingly, contact with partially hydrolyzed pGM also induced defense-related gene expression at earlier times than native pGM. These results show that the side chains of the (1â¯ââ¯5)-linked ß-D-galactofuranosyl units from pGM play an important role in the first line fungus-plant interactions mediating plant responses against C. herbarum. In addition, it was observed that pGM and/or C. herbarum conidia are able to induced HR when in contact with tobacco leaves and in vitro plantlets roots, producing necrotic lesions and peroxidase and NO burst, respectively.
Assuntos
Cladosporium , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Nicotiana , Doenças das Plantas/microbiologia , Folhas de Planta , Raízes de Plantas , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Raízes de Plantas/microbiologia , Nicotiana/citologia , Nicotiana/enzimologia , Nicotiana/microbiologiaRESUMO
The present study was designed to detect the effect of heavy metals in two zones of the Metropolitan Area of Mexico City (MAMC), the Centro de Ciencias de la Atmósfera (CCA), and the Altzomoni station in the Iztaccíhuatl-Popocatépetl National Park. Taraxacum officinale was selected as the indicator organism of responses to atmospheric contamination by heavy metals. Determinations of heavy metals were performed, and total mRNA was extracted to quantify the expression of microRNA398 (miR398), superoxide dismutase 2 (CSD2), and the amounts of free radicals using the bromide of 3-(4,5-dimethylthiazole-2-ilo)-2,5-diphenyltetrazole (MTT) salts reduction assay. Results from the Altzomoni station showed high concentrations of five heavy metals, especially Aluminum, while three heavy metals were identified in the CCA-UNAM zone, most importantly, Vanadium, both in the dry season; miR398 expression presented subtle changes but was greater in the leaves from the stations with higher concentrations of heavy metals. Observations included a significant expression of CSD2, mainly in the dry season in both study zones, where levels were significant with respect to controls (p < 0.05). Reduced MTT was also higher in the dry season than in the rainy season (p < 0.05). In conclusion, the increase in heavy metals on the leaves of Taraxacum officinale induces increased expression of the CSD2 gene and reduced MTT; thus, they can be used as indicators for biomonitoring heavy metal concentrations.
Assuntos
Poluentes Atmosféricos/análise , Monitoramento Ambiental/métodos , Metais Pesados/análise , Folhas de Planta/química , Taraxacum/química , Cidades , Expressão Gênica/efeitos dos fármacos , México , Parques Recreativos , Folhas de Planta/enzimologia , Chuva , Estações do Ano , Superóxido Dismutase/genética , Taraxacum/enzimologiaRESUMO
Natural rubber (polyisoprene) from the rubber tree Hevea brasiliensis is synthesized by specialized cells called laticifers. It is not clear how rubber particles arise, although one hypothesis is that they derive from the endoplasmic reticulum (ER) membrane. Here we cloned the genes encoding four key proteins found in association with rubber particles and studied their intracellular localization by transient expression in Nicotiana benthamiana leaves. We show that, while the cis-prenyltransferase (CPT), responsible for the synthesis of long polyisoprene chains, is a soluble, cytosolic protein, other rubber particle proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and Hevea rubber transferase 1-REF bridging protein (HRBP) are associated with the endoplasmic reticulum (ER). We also show that SRPP can recruit CPT to the ER and that interaction of CPT with HRBP leads to both proteins relocating to the plasma membrane. We discuss these results in the context of the biogenesis of rubber particles.
Assuntos
Antígenos de Plantas/metabolismo , Hevea/enzimologia , Proteínas de Plantas/metabolismo , Borracha/metabolismo , Transferases/metabolismo , Sequência de Aminoácidos , Antígenos de Plantas/genética , Citosol/enzimologia , Retículo Endoplasmático/metabolismo , Genes Reporter , Hevea/citologia , Hevea/genética , Modelos Biológicos , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Alinhamento de Sequência , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismo , Transferases/genéticaRESUMO
Background: Inferior Tieguanyin oolong tea leaves were treated with tannase. The content and bioactivity of catechins in extracts from the treated tea leaves were investigated to assess the improvement in the quality of inferior Tieguanyin oolong tea. Results: Analysis showed that after treatment, the esterified catechin content decreased by 23.5%, whereas non-galloylated catechin and gallic acid contents increased by 15.3% and 182%, respectively. The extracts from tannase-treated tea leaves showed reduced ability to bind to BSA and decreased tea cream levels. The extracts also exhibited increased antioxidant ability to scavenge OH and DPPH radicals, increased ferric reducing power, and decreased inhibitory effects on pancreatic α-amylase and lipase activities. Conclusions: These results suggested that tannase treatment could improve the quality of inferior Tieguanyin oolong tea leaves.
Assuntos
Chá/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Chá/metabolismo , Chá/química , Temperatura , Catálise , Catequina/análise , Folhas de Planta/enzimologia , Fermentação , Hidrólise , Lipase/antagonistas & inibidores , Lipase/metabolismo , AntioxidantesRESUMO
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
A new plant peroxidase was isolated from the leaves of guinea grass (Panicum maximum) and partially purified using a biphasic polymer system (poly(ethylene glycol) - ammonium sulfate) followed by size-exclusion chromatography and ultracentrifugation until obtaining a homogeneous extract containing a high peroxidase activity. The novel peroxidase was characterized as having a specific activity of 408U/mg and a molecular weight of 30kDa. The pH for its optimum activity was 8.0 and exhibited a high thermostability at 66°C with a kinact of 8.0×10-3min-1. The best substrates for peroxidase from guinea grass are o-dianisidine and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). POD from guinea grass was directly immobilized on the surface of a graphene screen printed electrode and cyclic voltammograms in the presence of potassium ferrocyanide ([Fe(CN)6]3-/4-) as a redox species demonstrated an increase in the electron transfer process. The graphene- modified electrode exhibits excellent electrocatalytic activity to the reduction of H2O2, with a linear response in the 100µM to 3.5mM concentration range and a detection limit of 150µM. The new peroxidase from guinea grass allowed the modification of a graphene electrode providing a potential sensor detection system for determination of H2O2 in real samples with some biomedical or environmental importance.
Assuntos
Técnicas Biossensoriais/métodos , Panicum/enzimologia , Peroxidase/metabolismo , Folhas de Planta/enzimologia , Eletroquímica , Estabilidade Enzimática , Grafite/química , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/química , Limite de Detecção , Peroxidase/química , Especificidade por SubstratoRESUMO
9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.
Assuntos
Dioxigenases/biossíntese , Nicotiana/fisiologia , Proteínas de Plantas/biossíntese , Ácido Abscísico/biossíntese , Adaptação Fisiológica , Citrus/enzimologia , Citrus/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Secas , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Estresse Fisiológico , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2 , and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc- ) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc- S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2 . On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc- strains from C4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and overexpressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C4 compounds and redox shuttling in plant cells.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Malato Desidrogenase (NADP+)/genética , Malatos/metabolismo , NADP/metabolismo , NAD/metabolismo , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clonagem Molecular , Expressão Gênica , Teste de Complementação Genética , Engenharia Genética , Glucose/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Malato Desidrogenase (NADP+)/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Transformação Genética , TransgenesRESUMO
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Assuntos
Aldeído Redutase/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Prunus domestica/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Frutosedifosfatos/metabolismo , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Dissulfeto de Glutationa/metabolismo , Hexosefosfatos/metabolismo , Hexosefosfatos/farmacologia , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismoRESUMO
Lipids are among the major chemical compounds present in coffee beans, and they affect the flavor and aroma of the coffee beverage. Coffee oil is rich in kaurene diterpene compounds, mainly cafestol (CAF) and kahweol (KAH), which are related to plant defense mechanisms and to nutraceutical and sensorial beverage characteristics. Despite their importance, the final steps of coffee diterpenes biosynthesis remain unknown. To understand the molecular basis of coffee diterpenes biosynthesis, we report the content dynamics of CAF and KAH in several Coffea arabica tissues and the transcriptional analysis of cytochrome P450 genes (P450). We measured CAF and KAH concentrations in leaves, roots, flower buds, flowers and fruit tissues at seven developmental stages (30-240 days after flowering - DAF) using HPLC. Higher CAF levels were detected in flower buds and flowers when compared to fruits. In contrast, KAH concentration increased along fruit development, peaking at 120 DAF. We did not detect CAF or KAH in leaves, and higher amounts of KAH than CAF were detected in roots. Using P450 candidate genes from a coffee EST database, we performed RT-qPCR transcriptional analysis of leaves, flowers and fruits at three developmental stages (90, 120 and 150 DAF). Three P450 genes (CaCYP76C4, CaCYP82C2 and CaCYP74A1) had transcriptional patterns similar to CAF concentration and two P450 genes (CaCYP71A25 and CaCYP701A3) have transcript accumulation similar to KAH concentration. These data warrant further investigation of these P450s as potential candidate genes involved in the final stages of the CAF and KAH biosynthetic pathways.
Assuntos
Coffea/genética , Sistema Enzimático do Citocromo P-450/genética , Diterpenos/metabolismo , Flores/enzimologia , Frutas/crescimento & desenvolvimento , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Transcrição Gênica , Cromatografia Líquida de Alta Pressão , Coffea/crescimento & desenvolvimento , Diterpenos/análise , Flores/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudos de Associação Genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genéticaRESUMO
Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (ß-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.
Assuntos
Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Proteínas Quinases/metabolismo , Solanum tuberosum/enzimologia , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras , Especificidade de Órgãos , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Tubérculos/citologia , Tubérculos/enzimologia , Tubérculos/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Solanum tuberosum/citologia , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimentoRESUMO
KEY MESSAGE: The seed treatment of a CPSMV-susceptible cowpea genotype with the mutagenic agent EMS generated mutagenized resistant plantlets that respond to the virus challenge by activating biochemical and physiological defense mechanisms. Cowpea is an important crop that makes major nutritional contributions particularly to the diet of the poor population worldwide. However, its production is low, because cowpea is naturally exposed to several abiotic and biotic stresses, including viral agents. Cowpea severe mosaic virus (CPSMV) drastically affects cowpea grain production. This study was conducted to compare photosynthetic and biochemical parameters of a CPSMV-susceptible cowpea (CE-31 genotype) and its derived ethyl methanesulfonate-mutagenized resistant plantlets, both challenged with CPSMV, to shed light on the mechanisms of virus resistance. CPSMV inoculation was done in the fully expanded secondary leaves, 15 days after planting. At 7 days post-inoculation, in vivo photosynthetic parameters were measured and leaves collected for biochemical analysis. CPSMV-inoculated mutagenized-resistant cowpea plantlets (MCPI) maintained higher photosynthesis index, chlorophyll, and carotenoid contents in relation to the susceptible (CE-31) CPSMV-inoculated cowpea (CPI). Visually, the MCPI leaves did not exhibit any viral symptoms neither the presence of the virus as examined by RT-PCR. In addition, MCPI showed higher SOD, GPOX, chitinase, and phenylalanine ammonia lyase activities, H2O2, phenolic contents, and cell wall lignifications, but lower CAT and APX activities in comparison to CPI. All together, these photosynthetic and biochemical changes might have contributed for the CPSMS resistance of MCPI. Contrarily, CPI plantlets showed CPSMV accumulation, severe disease symptoms, reduction in the photosynthesis-related parameters, chlorophyll, carotenoid, phenolic compound, and H2O2 contents, in addition to increased ß-1,3-glucanase, and catalase activities that might have favored viral infection.
Assuntos
Comovirus/fisiologia , Resistência à Doença , Mutagênese/genética , Fotossíntese , Doenças das Plantas/virologia , Vigna/fisiologia , Vigna/virologia , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Metanossulfonato de Etila , Homeostase , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Oxirredução , Fenóis/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/virologia , Proteínas de Plantas/metabolismo , SolubilidadeRESUMO
Different rice (Oryza sativa L.) genotypes were subjected to high salinity and low temperature (150 mM NaCl and 13°C, respectively) for 0, 6, 24, 48, or 72 h. We evaluated the simultaneous expression of the genes OsCATA, OsCATB, and OsCATC, correlated gene expression with enzyme activity, and verified the regulation of these genes through identification of cis-elements in the promoter region. The hydrogen peroxide content increased in a tolerant genotype and decreased in a sensitive genotype under both stress conditions. Lipid peroxidation increased in the tolerant genotype when exposed to cold, and in the sensitive genotype when exposed to high salinity. Catalase activity significantly increased in both genotypes when subjected to 13°C. In the tolerant genotype, OsCATA and OsCATB were the most responsive to high salinity and cold, while in the sensitive genotype, OsCATA and OsCATC responded positively to saline stress, as did OsCATA and OsCATB to low temperature. Cis-element analysis identified different regulatory sequences in the catalase promoter region of each genotype. The sensitive genotype maintained a better balance between hydrogen oxyacid levels, catalase activity, and lipid peroxidation under low temperature than the resistant genotype. OsCATA and OsCATB were the most responsive in the salt-tolerant genotype to cold, OsCATA and OsCATC were the most responsive to saline stress, and OsCATA and OsCATB were the most responsive to chilling stress in the sensitive genotype. There were positive correlations between catalase activity and OsCATB expression in the tolerant genotype under saline stress and in the sensitive genotype under cold stress.