RESUMO
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
Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4-5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.
Assuntos
Germinação , Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Subtilisinas/metabolismo , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas , Hordeum/enzimologia , Hordeum/metabolismo , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Plântula/enzimologia , Plântula/metabolismoRESUMO
Isoflavonoid compositions, phenylalanine ammonia lyase (PAL) activity and antioxidant capacity were evaluated in chickpea (Cicer arietinum L.) sprouts germinated after soaking with different sodium selenite (Na2SeO3) concentrations (0, 1 and 2mg/100g seeds). Chickpea seeds were germinated during four days at 24°C and the isoflavonoid profiles and concentrations evaluated by HPLC-UV daily during four days of germination. Eleven isoflavones and two pterocarpan phytoalexins forms were identified in sprouts, being malonylated formononetin glycoside, formononetin, isoformononetin glycoside and malonylated biochanin A glycoside the major compounds. Compared to untreated sprouts, total isoflavonoid, PAL activity and antioxidant capacity showed a remarkable increase of 83%, 56%, and 33%, respectively in chickpea sprouts that were treated with a high sodium selenite content (2mg/100g seeds). Results suggest that Se-enriched chickpea sprouts could represent a good source of dietary Se and as an upgraded source of isoflavonoids.
Assuntos
Antioxidantes , Cicer/efeitos dos fármacos , Isoflavonas , Fenilalanina Amônia-Liase/genética , Selenito de Sódio/farmacologia , Cicer/química , Cicer/enzimologia , Regulação da Expressão Gênica de Plantas , Plântula/química , Plântula/efeitos dos fármacos , Plântula/enzimologia , Sesquiterpenos , FitoalexinasRESUMO
The mobilization of palm seed reserves is a complex process because of the abundance and diversity of stored compounds and results from the development of a highly specialized haustorium. This work focused on the important Neotropical oleaginous palm Acrocomia aculeata, with the aim of defining phases of seedling development associated with mobilization of reserves and elucidating the role of haustorium and endosperm in this process. Standard methods were performed, including biometric, anatomical, and histochemical analyses, as well as the evaluation of the activities of the enzymes endo-ß-mannanase and lipase, throughout the reserve mobilization in seeds during germination and in seedlings. Seeds of A. aculeata stored large quantities of proteins, lipids, and polysaccharides in the embryo and endosperm. The mobilization of reserves initiated in the haustorium during germination and subsequently occurred in the endosperm adjacent to the haustorium, forming a gradually increasing zone of digestion. Proteins and polysaccharides were the first to be mobilized, followed by lipids and cell wall constituents. The haustorium activates and controls the mobilization, forming transitory reserves and translocating them to the vegetative axis, while the endosperm, which also has an active role, serves as a site of intense enzymatic activity associated with protein bodies. Seedling development can be described as occurring in six phases over a long period (approximately 150 days) due to the large amount of seed reserves. This process exhibits an alternation between stages of accumulation and translocation of protein, lipid, and carbohydrate reserves in the haustorium, which favors the seedling establishment and the reproductive success of the species.
Assuntos
Arecaceae/crescimento & desenvolvimento , Endosperma/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Ácido Abscísico , Arecaceae/citologia , Arecaceae/enzimologia , Endosperma/citologia , Endosperma/enzimologia , Metabolismo Energético , Germinação , Manosidases/metabolismo , Proteínas de Plantas/metabolismo , Plântula/citologia , Plântula/enzimologiaRESUMO
Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa.
Assuntos
Agaricales/fisiologia , Cacau/enzimologia , Cisteína Endopeptidases/genética , Genoma de Planta/genética , Doenças das Plantas/imunologia , Sequência de Aminoácidos , Cacau/genética , Cacau/crescimento & desenvolvimento , Cacau/imunologia , Análise por Conglomerados , Cotilédone/enzimologia , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Cotilédone/imunologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Modelos Estruturais , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/imunologia , Alinhamento de SequênciaRESUMO
Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC50) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg(-1) of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg(-1) day(-1)) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.
Assuntos
Acacia/efeitos dos fármacos , Acacia/metabolismo , Arseniatos/toxicidade , Poluentes do Solo/toxicidade , Acacia/enzimologia , Acacia/genética , Arseniatos/metabolismo , Arsênio/metabolismo , Arsênio/toxicidade , Biodegradação Ambiental , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Poluentes do Solo/metabolismoRESUMO
INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH-box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron-containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2-silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing-regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell-cell communication in plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Plasmodesmos/metabolismo , RNA Helicases/genética , Splicing de RNA , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Cloroplastos/enzimologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Genes Reporter , Íntrons/genética , Mutação , Fotossíntese , Plantas Geneticamente Modificadas , RNA Helicases/metabolismo , RNA de Cloroplastos/genética , RNA de Cloroplastos/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/metabolismoRESUMO
Corn DNA was introduced into dry seeds of rice (cv. 'YuJing-6') by ion beam irradiation. Proteinase activities in rice seedling roots were subsequently analyzed by renaturation electrophoresis at pH 4.5, 7.0, and 8.5. Proteinase activity was more pronounced on gels at higher pH. Irradiation of rice seedling roots caused the loss of some proteinase bands at all pH conditions although a novel 50-kDa band was found at both pH 7.0 and 8.5. No new proteinase activity was detected at pH 4.5. However, novel bands and bands showing stronger activity were observed at pH 7.0 and 8.5. The data indicate that the expression of proteinases in rice seedling roots was altered following low energy ion beam mediated transformation with corn DNA.
Assuntos
DNA de Plantas/genética , Oryza/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Sementes/genética , Transformação Genética , Zea mays/química , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Expressão Gênica , Concentração de Íons de Hidrogênio , Íons , Cinética , Nitrogênio/química , Oryza/enzimologia , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Radiação não Ionizante , Plântula/enzimologia , Plântula/genética , Sementes/enzimologiaRESUMO
We evaluated the salt tolerance of hybrids of pepper (Capsicum annuum L.) during germination. Treatments were applied at 0, 25, and 50 mM NaCl with preparations of supplemental extracts of the microalgae Dunaliella salina and Phaeodactylum tricornutum to determine the percentage germination rate as well as measured indicators of oxidative stress caused by the salt treatments during seed germination. We found that root growth was favorably influenced by the microalgae leading to increased germination rate. Tissues were analyzed in terms of superoxide radical production, lipid peroxidation, and activity of antioxidant enzymes viz. superoxide dismutase, catalase, and glutathione peroxidase. Our results suggest that application of microalgae extracts significantly reduced (p < 0.05) superoxide radical production, as well as lower lipid peroxidation in comparison to plants without extracts of microalgae. The antioxidant enzymes increased in the presence of microalgae showing a significant difference (p < 0.05). The results suggest differences in oxidative metabolism in response to the magnitude of salt stress and concentrations of microalgae help mitigate salt stress in plants during the germination process.
Assuntos
Antioxidantes/farmacologia , Capsicum/crescimento & desenvolvimento , Microalgas/química , Superóxidos/metabolismo , Antioxidantes/metabolismo , Capsicum/efeitos dos fármacos , Capsicum/enzimologia , Capsicum/metabolismo , Germinação , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Cloreto de Sódio/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
To elucidate how physiological and biochemical mechanisms of chilling stress are regulated by abscisic acid (ABA) pretreatment, pepper variety (cv. 'P70') seedlings were pretreated with 0.57 mM ABA for 72 h and then subjected to chilling stress at 10°/6°C (day/night). Chilling stress caused severe necrotic lesions on the leaves and increased malondialdehyde and H(2)O(2) levels. Activities of monodehydroascorbate reductase (DHAR), dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, ascorbate peroxidase, ascorbate, and glutathione increased due to chilling stress during the 72 h, while superoxide dismutase and catalase activities decreased during 24 h, suggesting that chilling stress activates the AsA-GSH cycle under catalase deactivation in pepper leaves. ABA pretreatment induced significant increases in the above-mentioned enzyme activities and progressive decreases in ascorbate and glutathione levels. On the other hand, ABA-pretreated seedlings under chilling stress increased superoxide dismutase and guaiacol peroxidase activities and lowered concentrations of other antioxidants compared with untreated chilling-stressed plants. These seedlings showed concomitant decreases in foliage damage symptoms, and levels of malondialdehyde and H(2)O(2). Induction of Mn-SOD and POD was observed in chilling-stressed plants treated with ABA. The expression of DHAR1 and DHAR2 was altered by chilling stress, but it was higher in the presence than in the absence of ABA at 24 h. Overall, the results indicate that exogenous application of ABA increases tolerance of plants to chilling-induced oxidative damage, mainly by enhancing superoxide dismutase and guaiacol peroxidase activities and related gene expression.
Assuntos
Ácido Abscísico/farmacologia , Antioxidantes/metabolismo , Capsicum/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Folhas de Planta/enzimologia , Estresse Fisiológico/genética , Capsicum/efeitos dos fármacos , Capsicum/enzimologia , Catalase/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Oxirredutases/metabolismo , Peroxidase/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Estresse Fisiológico/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Most of the elements involved in the integration of signals of low external K(+)-supply into a physiological response pathway remain essentially unknown. The aim of this work was to study the influence exerted by DELLA proteins, which are known to be key components for the control of growth, on plant responses during K(+) deprivation in wheat (Triticum aestivum) by using two sets of near-isogenic lines (NILs) in the Maringa and April Bearded cultivars. After K(+) shortage, the NILs of both cultivars containing the Rht-B1b,Rht-D1b alleles, which encode altered function DELLA proteins, displayed either a slight or no decrease in chlorophyll content, in contrast to the sharp decrease observed in the NILs having the wild type alleles (Rht-B1a,Rht-D1a). That difference was accompanied by a lower relative decrease of biomass accumulation only in the Maringa cultivar. In both cultivars, high chlorophyll retention was coupled with K(+) starvation-induced differences in superoxide dismutase and ascorbate peroxidase activities, which were enhanced in K(+)-starved Rht-B1b,Rht-D1b NILs. In addition, Rht-B1b,Rht-D1b and Rht-B1a,Rht-D1a NILs markedly differed in the accumulation of the major cations Ca(2+), Na(+) and K(+). These results suggest a major role of the Rht-1 genes in the control of physiological responses during K(+) deprivation.
Assuntos
Proteínas de Plantas/genética , Potássio/metabolismo , Estresse Fisiológico/fisiologia , Triticum/fisiologia , Alelos , Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Biomassa , Cruzamento , Cálcio/metabolismo , Clorofila/metabolismo , Genes de Plantas/genética , Genótipo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sódio/metabolismo , Superóxido Dismutase/metabolismo , Triticum/enzimologia , Triticum/genética , Triticum/crescimento & desenvolvimentoRESUMO
Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.
Assuntos
Fosfatase Ácida/metabolismo , Ascomicetos/enzimologia , Basidiomycota/enzimologia , Magnoliopsida/enzimologia , Micorrizas/enzimologia , Fósforo/metabolismo , Ascomicetos/fisiologia , Basidiomycota/fisiologia , Transporte Biológico , Modelos Lineares , Magnoliopsida/microbiologia , Magnoliopsida/fisiologia , Micélio/enzimologia , Micélio/fisiologia , Micorrizas/fisiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Brotos de Planta/metabolismo , Plântula/enzimologia , Plântula/microbiologia , Plântula/fisiologia , Solo/química , SimbioseRESUMO
The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H(2)O(2)) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H(2)O(2) content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H+G+S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.
Assuntos
Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Glycine max/efeitos dos fármacos , Lignina/metabolismo , Benzoatos/farmacologia , Parede Celular/enzimologia , Parede Celular/metabolismo , Cinamatos/farmacologia , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Ácidos Cumáricos/farmacologia , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Lignina/análise , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/efeitos dos fármacos , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Glycine max/enzimologia , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Transcinamato 4-Mono-Oxigenase/antagonistas & inibidores , Transcinamato 4-Mono-Oxigenase/metabolismoRESUMO
Aluminum (Al) is one of the most abundant elements of the planet and exposure to this metal can cause oxidative stress and lead to various signs of toxicity in plants. Plants are essential organisms for the environment as well as food for humans and animals. The toxic effect of aluminum is the major cause of decreased crop productivity. Thus, the objective of the present study was to analyze the effects of aluminum on the activity of antioxidant enzymes such as catalase (CAT - E.C. 1.11.1.6), superoxide dismutase (SOD - E.C.1.15.1.1) and ascorbate peroxidase (APX - E.C. 1.11.1.11), and on lipid peroxidation, electrolyte leakage percentage (ELP) and chlorophyll and protein oxidation levels in Cucumis sativus L. (cv. Aodai). Seedlings were grown at different concentrations of aluminum ranging from 1 to 2000 microM for 10 days. The increase in ELP and H(2)O(2) production observed in the seedlings may be related to the decreased efficiency of the antioxidant system at higher aluminum concentrations. The antioxidant system was unable to overcome toxicity resulting in negative effects such as lipid peroxidation, protein oxidation and a decrease in the growth of Cucumis seedlings. Aluminum toxicity triggered alterations in the antioxidant and physiological status of growing cucumber seedlings.
Assuntos
Alumínio/toxicidade , Cucumis sativus/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plântula/efeitos dos fármacos , Ascorbato Peroxidases , Catalase/metabolismo , Clorofila/metabolismo , Cucumis sativus/enzimologia , Cucumis sativus/metabolismo , Relação Dose-Resposta a Droga , Eletrólitos/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/enzimologia , Brotos de Planta/metabolismo , Plântula/enzimologia , Plântula/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.
Assuntos
Desidratação/metabolismo , Magnoliopsida/microbiologia , Micorrizas/fisiologia , Nitrogênio/metabolismo , Fósforo/metabolismo , Secas , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/metabolismo , Nodulação , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , SimbioseRESUMO
We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified approximately 40 and approximately 80kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.
Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Proteínas de Domínio MADS/metabolismo , Medicago sativa/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Núcleo Celular/enzimologia , Citosol/enzimologia , Glicólise , Dados de Sequência Molecular , Plântula/enzimologia , Sementes/enzimologiaRESUMO
Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH4)2SO4 precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (Km=9.86mM, Vmax=24.66 EU [DeltaAmin(-1)]) and catechol (Km=3.44mM, Vmax=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.
Assuntos
Catecol Oxidase/metabolismo , Fabaceae/enzimologia , Doenças das Plantas , Plântula/enzimologia , Catecol Oxidase/biossíntese , Catecol Oxidase/isolamento & purificação , Cinética , Extratos Vegetais/metabolismoRESUMO
Triasulfuron (TS) is a widely used sulfonylurea herbicide which inhibits the acetolactate synthase in broad-leaf weeds and in some wheat crop grasses (Triticum aestivum L.). Residues can be found in soil and superficial water with high toxicity to primary producers. In cereals, TS metabolism depends on cytochromes P450 (CYPs), the age of seedlings and the interaction with compounds. The genotoxicity of TS was demonstrated in the wing spot test of Drosophila melanogaster, an in vivo assay based on the loss of heterozygosity of the mwh and flr markers in the wing imaginal disk cells of larvae fed with chemical agents. Chronic treatments with analytical grade TS, commercial formulation TS (Amber) 75WG) (0.5mg/mL) and commercial formulation bentazon (Basagran) 480) (0.24mg/mL) were performed with three-day-old larvae of the standard (ST) and the high bioactivation (HB) crosses with regulated and high constitutive levels of CYPs, respectively. To demonstrate the effect of winter wheat metabolism on TS genotoxicity, T. aestivum L. seedlings were immersed for 4h in these herbicides, and aqueous extracts (AEs) of the roots were prepared to expose the larvae. TS and Amber 75WG produced similar genotoxic effects in both crosses. Wheat metabolism modulated the genotoxicity because the AEs yielded statistically significant lower spot frequencies in the HB cross than in the ST cross. Differences between the two crosses of the wing spot test in D. melanogaster must be related to CYPs levels. Basagran 480 was genotoxic only in the HB cross, and wheat metabolism did not modulate its genotoxicity.
Assuntos
Perda de Heterozigosidade/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/análise , Plântula , Compostos de Sulfonilureia/análise , Triticum , Asas de Animais , Animais , Benzotiadiazinas/análise , Benzotiadiazinas/metabolismo , Cruzamentos Genéticos , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Marcadores Genéticos , Herbicidas/análise , Herbicidas/metabolismo , Larva/genética , Larva/metabolismo , Testes de Mutagenicidade/métodos , Mutagênicos/metabolismo , Proteínas de Plantas/metabolismo , Plântula/enzimologia , Compostos de Sulfonilureia/metabolismo , Fatores de Tempo , Triticum/enzimologiaRESUMO
Catalase (CAT) dismutates the reactive oxygen species H2O2 into water and dioxygen and in plants; it is located in peroxisomes and glyoxysomes. In the present study, we investigated the effect of cadmium (a well-known oxidative stress inducer) on catalase in roots and cotyledons of developing sunflower seedlings, at 10 microM and 100 microM. Although germination was unaltered after 48 h of exposure to 100 microM Cd2+, root length was significantly reduced. CAT activity was also significantly reduced, but this activity was completely restored (10 microM treatment) or even enhanced (100 microM treatment) 24 h later. Although CAT protein abundance remained similar to control in roots and cotyledons of Cd-treated seedlings, cadmium produced CAT protein oxidation, indicating that the mechanism of CAT inactivation by Cd2+ involves oxidation of the protein structure. The transcripts of the four genes described for sunflower (CATA1 to CATA4) increased after cadmium treatment; CATA1 and CATA2 were the most overexpressed in cotyledon and root, respectively. The differential expression of catalase genes in sunflower seedlings under Cd stress might be related to the synthesis of CAT isoforms less sensitive to oxidation, which would prevent enzyme inactivation and H2O2 accumulation.
Assuntos
Cádmio/farmacologia , Catalase/metabolismo , Helianthus/enzimologia , Plântula/enzimologia , Catalase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Germinação/efeitos dos fármacos , Germinação/fisiologia , Helianthus/efeitos dos fármacos , Helianthus/crescimento & desenvolvimento , Peróxido de Hidrogênio/metabolismo , Cinética , Plântula/efeitos dos fármacosRESUMO
Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu(2+), Hg(2+), and Zn(2+) ions, stimulated by MgCl(2), and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.