Tannase application in secondary enzymatic processing of inferior Tieguanyin oolong tea

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.

Seasonal characterization of antioxidant responses in plants of Ipomoea nil cv. Scarlet O'Hara / Caracterização sazonal de respostas antioxidativas em plantas de Ipomoea nil cv. Scarlet O'Hara

Reactive oxygen species can be produced in leaf cells during normal aerobic metabolism or in a variety of exogenous factors, which may cause oxidative damage to plants, unless they have an efficient antioxidant defense system, consisting of enzymatic and non-enzymatic substances. This work raised the hypothesis that plants of Ipomoea nil cv. Scarlet O'Hara, a native species and ornamental vine of the tropics, might tolerate oxidative stress factors imposed by natural fluctuations in weather conditions through changes in the antioxidant profile.The objective of this study was to determine the variations in three leaf antioxidants in plants growing inside a greenhouse without air pollutants and exposed to varying meteorological conditions throughout the four seasons of the year and to observe if such variations are related to the oscillations in meteorological factors. Four experimental campaigns were carried out, one in each season of 2006. Each campaign lasted 28 days and started with 45 plants. Ascorbic acid (AA) concentrations and superoxide dismutase (SOD) and peroxidase (POD) activities were determined in leaves of five plants in nine sampling days of each campaign. The antioxidant responses oscillated throughout the year. The highest values were found during the spring. This seasonal antioxidant profile was associated to variations in temperature, relative humidity and global radiation. Plants of this cultivar may then tolerate oxidative stress naturally imposed by meteorological conditions.

As espécies reativas de oxigênio podem ser produzidas em células de folhas durante o metabolismo aeróbico normal ou o sob uma diversidade de fatores exógenos que, por sua vez, podem causar danos oxidativos às plantas, a menos que estas tenham um eficiente sistema de defesa antioxidativo, formado por substâncias enzimáticas e não enzimáticas. Neste trabalho, levantou-se a hipótese de que as plantas de Ipomoea nil cv. Scarlet O'Hara, uma espécie trepadeira ornamental e nativa dos trópicos, podem tolerar fatores de estresse oxidativo imposto por oscilações naturais nas condições meteorológicas por meio de mudanças no perfil antioxidativo. Assim, este trabalho objetivou determinar as variações em três espécies antioxidantes foliares em plantas crescidas em casa de vegetação sob ar filtrado e expostas a condições meteorológicas variáveis ao longo das quatro estações do ano de 2006, bem como verificar se tais variações estão relacionadas às oscilações de fatores meteorológicos. Para tanto, realizaram-se quatro campanhas experimentais. Cada campanha durou 28 dias e começou com 45 plantas. Concentrações de ácido ascórbico (AA) e as atividades de superóxido dismutase (SOD) e peroxidase (POD) foram determinadas em folhas de cinco plantas distintas e retiradas da casa de vegetação em nove dias de amostragem de cada campanha. As respostas antioxidativas oscilaram durante todo o ano, sendo os maiores valores encontrados durante a primavera. Este perfil sazonal de antioxidantes foi associado às variações de temperatura, umidade relativa e radiação global. As plantas desta cultivar podem, então, tolerar o estresse oxidativo naturalmente imposto pelas condições meteorológicas.

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

Pre-treatment of seeds with static magnetic field ameliorates soil water stress in seedlings of maize (Zea mays L.).

The effect of magnetic field (MF) treatments of maize (Zea mays L.) var. Ganga Safed 2 seeds on the growth, leaf water status, photosynthesis and antioxidant enzyme system under soil water stress was investigated under greenhouse conditions. The seeds were exposed to static MFs of 100 and 200 mT for 2 and 1 h, respectively. The treated seeds were sown in sand beds for seven days and transplanted in pots that were maintained at -0.03, -0.2 and -0.4 MPa soil water potentials under greenhouse conditions. MF exposure of seeds significantly enhanced all growth parameters, compared to the control seedlings. The significant increase in root parameters in seedlings from magnetically-exposed seeds resulted in maintenance of better leaf water status in terms of increase in leaf water potential, turgor potential and relative water content. Photosynthesis, stomatal conductance and chlorophyll content increased in plants from treated seeds, compared to control under irrigated and mild stress condition. Leaves from plants of magnetically-treated seeds showed decreased levels of hydrogen peroxide and antioxidant defense system enzymes (peroxidases, catalase and superoxide dismutase) under moisture stress conditions, when compared with untreated controls. Mild stress of -0.2 MPa induced a stimulating effect on functional root parameters, especially in 200 mT treated seedlings which can be exploited profitably for rain fed conditions. Our results suggested that MF treatment (100 mT for 2 h and 200 for 1 h) of maize seeds enhanced the seedling growth, leaf water status, photosynthesis rate and lowered the antioxidant defense system of seedlings under soil water stress. Thus, pre sowing static magnetic field treatment of seeds can be effectively used for improving growth under water stress.

Kinetic properties of cell wall bound superoxide dismutase in leaves of wheat (Triticum aestivum L.) following stripe rust (Puccinia striiformis) infection.

Stripe rust (Puccinia striiformis f.sp. tritici) is the most devastating disease of wheat (Triticum aestivum L.) accounting huge economical losses to the industry worldwide. HD 2329 was a widely grown wheat cultivar which had become highly susceptible to stripe rust and was used to understand the biochemical aspects of the host pathogen interaction through characterization of superoxide dismutase (SOD). In the present study, two types of SOD, ionically or covalently bound to the particulate fraction were found in the stripe rust infected and uninfected wheat leaves of susceptible cultivar HD 2329. Cell walls of leaves contained a high level of SOD, of which 41-44% was extractable by 2 M NaCl and 10-13% by 0.5% EDTA in infected and uninfected leaves. The NaCl-released SOD constituted the predominant fraction. It exhibited maximum activity at pH 9.0, had a Km value of 1.82-2.51 for uninfected and 1.77-2.37 mM for infected, respectively with pyrogallol as the substrate, and a Vmax of 9.55-21.4 and 12.4-24.1 A min-1g-1FW. A temperature optimum of 20oC was observed for SOD of both uninfected and infected leaves. SOD showed differential response to metal ions, suggesting their distinctive nature. Inhibition of wall bound SOD by iodine and its partial regeneration of activity by mercaptoethanol suggested the involvement of cysteine in active site of the enzyme. These two forms showed greater differences with respect to thermodynamic properties like energy of activation (Ea) and enthalpy change (H), while entropy change (S) and free energy change (G) were similar. The results further showed that pathogen infection of the leaves of susceptible wheat cultivar induced a decrease in the SOD activity and kinetics which might be critical during the response of plant cells to the infection.

The Relationship between Leaf Rolling and Ascorbate-Glutathione Cycle Enzymes in Apoplastic and Symplastic Areas of Ctenanthe setosa Subjected to Drought Stress

The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities ...

Changes in antioxidant enzyme activities in Eichhornia crassipes (Pontederiaceae) and Pistia stratiotes (Araceae) under heavy metal stress

Whole plants of Eichhornia crassipes and Pistia stratiotes were exposed to various concentrations (0, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 mM) of 8 heavy metals (Ag, Cd, Cr, Cu, Hg, Ni, Pb and Zn) hydroponically for 21 days. Spectrometric assays for the total activity of catalase, peroxidase, and superoxide dismutase in the leaves were studied. At the end of the experimental period, data referred to metal treated plants were compared to data of untreated ones (control). Heavy metals increased the activity of catalase, peroxidase and superoxide dismutase in both species and there was differential inducement among metals. Overall, Zn had the least inducement of antioxidant enzymes in both species while Hg had the highest inducement. The increase in antioxidant enzymes in relation to the control plants was more in E. crassipes than P. stratiotes. The results showed that E. crassipes tolerated higher metal concentrations in a greater number of metals than P. stratiotes. Rev. Biol. Trop. 55 (3-4): 815-823. Epub 2007 December, 28.

Plantas completas de Eichhornia crassipes y Pistia stratiotes fueron expuestas a varias concentraciones (0, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 mM) de metales pesados (Ag, Cd, Cr, Cu, Hg, Ni, Pb and Zn) utilizando hidroponía, por 21 días. Se realizaron análisis espectrométricos en las hojas para determinar la actividad total de la catalasa, peroxidasa y dismutasa superóxida. Al final del periodo experimental, se comparó con plantas no tratadas (control). Los metales pesados incrementan la actividad de la catalasa, peroxidasa y la dismutasa superóxida para ambas especies y hay diferencias entre los metales. El Zn produce el menor estímulo para enzimas antioxidantes en ambas especies; Hg produce el mayor estímulo. El incremento de las enzimas antioxidantes en relación con las plantas control fue mayor en E. crassipes que P. stratiotes. E. crassipes tolera altas concentraciones de metal en un gran número de ellos, mientras que la tolerancia en P. stratiotes es menor.

Changes in PEP Carboxylase, Rubisco and Rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Induction of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion (Allium cepa var aggregatum).

The Pseudomonas fluorescens isolate Pfl was found to inhibit the growth of pathogen Alternaria palandui, in vitro. In the present study, foliar application of a talc-based formulation of Pfl significantly reduced the incidence of leaf blight of onion, caused by A. palandui. Induction of defense-related proteins viz., chitinase, beta-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by application of Pfl, was studied against A. palandui infection in resistant (IHR 56) and susceptible (MDUI) onion cultivars. Chitinase in both cultivars, with or without challenge-inoculation of A. palandui revealed changes in the isoform pattern. The Native-PAGE of PO showed induction of PO2 isoform in both the cultivars, in response to inoculation of pathogen. Isoform analysis of PPO also exhibited induction in the Pfl-treated plants challenged with pathogen. Similarly, the activity of beta-1,3-glucanase was greatly induced in Pfl-treated plants, challenged with pathogen as compared to controls. Thus, the P. fluorescens-treated plants showed significant increase in the levels of the defense enzymes, in comparison to the plants challenged with the pathogen.

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments.

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.

Nitric oxide: a common antipathogenic factor of plants.

In the present study, nitric oxide synthase/nitric oxide (NOS/NO) status was tested in the host plants infected with fungi, bacteria and virus. In each case cytosolic nitric oxide synthase (Cyt-NOS) of diseased plants was inhibited and inhibition was competitive in nature in respect to l-arginine, the substrate for the enzymic activity. Elevation of host nitric oxide (NO) level before infection using nitric oxide (NO) donor protected disease initiation significantly. The nature of enzyme kinetics and the manner of disease protection by nitric oxide donor (NO-donor) was similar in all the three cases of infection. It was concluded that nitric oxide was a common antipathogenic factor of plants.

Effect of zinc on antioxidant response in maize (Zea mays L.) leaves.

Maize (Zea mays L. cv kanaujia) plants grown with Zn [10 (control), 0.1 (low) and 20 microM (high)], were investigated for concentration of antioxidants and activities of antioxidative enzymes in leaves. Young leaves of low Zn plants developed whitish-necrotic spots. Leaves of both low and high Zn plants showed decrease in chlorophyll concentration and accumulation of lipid peroxides, ascorbate and dehydroascorbate, associated with a decrease in the activity of ascorbate peroxidase and superoxide dismutase. Low and high Zn, however, showed diverse effect on glutathione reductase. While low Zn increased the activity of glutathione reductase, high Zn decreased its activity. Zinc effect on antioxidative constituents suggested Zn involvement in sustaining the antioxidative defense system in maize leaves.

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants.

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.

H+-ATPase as a biochemical marker for early detection of root (wilt) disease in coconut palms (Cocos nucifera L).

H+-ATPase activity in leaves and roots of coconut palms growing in 'root wilt disease-prevalent areas' was compared with that of coconut palms growing in 'disease-free areas'. The activity was found to be significantly less in the leaves and roots of palms in the disease-prevalent zone as compared to that in disease-free zone. Histochemical examination of the leaves showed results that corroborated the biochemical findings. The possible application of H+-ATPase activity as a marker for the early detection of wilt disease in coconut palms is suggested.

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves.

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.

Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass.

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.

Aldolase, carbonic anhydrase and catalase activity in faba bean leaves as affected by iron and zinc

The effect of zinc and iron withhold from the nutrient medium on the activities of aldolase, carbonic anhydrase and catalase enzymes was investigated using faba bean plants grown in nutrient solution [0.2 M strength]. A reduction in catalase activity was observed when iron alone or/and with zinc withhold from the growth medium. In the case of carbonic anhydrase, the absence of zinc and iron from the nutrient medium caused high depressive effect [91%] followed by the absence of zinc [75%] and iron [30%]. Aldolase activity showed no response to both elements. The results showed that the aldolase activity after the first spray [20 days after sowing] was increased with more zinc and less iron applied concentrations. The maximum aldolase activity was reached when plants were sprayed with 100 ppm zinc and 50 ppm iron. It was noted that the different successive levels of zinc or iron alone did not show any promotive effect on the activity of aldolase or carbonic anhydrase enzymes