Photosynthesis limitations of grapevine after treatment with the fungicide fludioxonil

The aim of this work was to determine the major limitations to photosynthesis induced by the fungicide fludioxonil (fdx) on nontarget grapevines using cutting as a model. The fdx treatments (1.2, 6, and 30 mM) induced a net photosynthetic rate ( P n) decrease without changes in stomatal conductance, suggesting a nonstomatal limitation. Fdx effects on P n were related neither to photosynthetic capacity alteration in leaves nor to loss in PSII activity. The mechanism underlying photosynthesis reduction differed according to the concentration. Fdx at 6 mM led to an increase of light requirement for photosynthesis while 30 mM fdx induced an increase in the respiration rate in the light. P n decrease after 1.2 mM fdx could rather be related to wetness caused by the spraying than to fungicide toxicity. P n recovered 10 days after treatment, meaning that fdx had little deleterious effect on plant physiology or that grapevine has a great capacity to overcome this temporary stress.     

Is the application of carbendazim harmful to healthy plants? Evidence of weak phytotoxicity in tobacco.

To understand the phytotoxic effects that certain bezimidazole fungicides exert on plant growth, the aim of the present study was to determine the effect of the fungicide carbendazim, on foliar biomass, pigment content, and nutrient levels in tobacco plants (Nicotiana tabacum L. cv. Tennessee 86). The fungicide applied was carbendazim with a purity of 100%, at three different rates: 1.3 mM (carb1), 2.6 mM (the recommended concentration, carb2), and 5.2 mM (carb3). The control treatment was without carbendazim. The application of dosages of this fungicide lower than recommended (1.3 mM) resulted, on the one hand, in greater dry weight and, on the other, higher carotenoid concentrations, as well as higher N and K concentrations with respect to control. On the contrary, the application of the carbendazim dosage higher than recommended (5.2 mM) caused a decrease in dry weight and in all of the foliar pigments, as well as all of the nutrients, with respect to the other dosages and control. These results appear to indicate that besides its direct antibiotic action against pathogens, the effects of this fungicide in plants could be dangerous, especially at higher dosages. Nevertheless, the negative effects of carbendazim can be avoided by reducing the amount applied in current agriculture.     

Phenylboronic acid--a potent inhibitor of lipase from Oryza sativa

The kinetics of inhibition of rice bran lipase (RBL) by phenylboronic acid (PBA) was studied to elucidate the nature of inhibition and the effect of the inhibitor on the structure-function of RBL. The effectiveness of an inhibitor is normally expressed by the constant K(i), which is calculated from the Lineweaver-Burk plot and found to be 1.7 mM at pH 7.4. The kinetics of inhibition by PBA was competitive, indicating the presence of serine in the active site of the enzyme. The loss of activity of RBL was concentration dependent on the inhibitor (PBA), and the inactivation followed a pseudo-first-order kinetics. Fluorescence emission measurements indicated a decrease in the fluorescence emission intensity and a red shift in the emission maximum as the inhibitor concentration was increased. The inhibition of the enzyme by PBA was also confirmed by thermal denaturation measurements, which indicated a shift in the thermal denaturation temperature of the enzyme toward lower temperatures. The far-UV-CD data suggest that there were no significant changes in the conformation of the enzyme as a result of binding of PBA. These results indicate that PBA is a potential inhibitor of RBL and binds to the enzyme in bringing about inhibition without any structural alterations.     

Astaxanthin protects against oxidative stress and calcium-induced porcine lens protein degradation

Astaxanthin (ASTX), a carotenoid with potent antioxidant properties, exists naturally in various plants, algae, and seafoods. In this study, we investigated the in vitro ability of ASTX to protect porcine lens crystallins from oxidative damage by iron-mediated hydroxyl radicals or by calcium ion-activated protease (calpain), in addition to the possible underlying biochemical mechanisms. ASTX (1 mM) was capable of protecting lens crystallins from being oxidized, as measured by changes in tryptophan fluorescence, in the presence of a Fenton reaction solution containing 0.2 mM Fe2+ and 2 mM H2O2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that beta(high)-crystallin was the most vulnerable protein under these conditions of free radical exposure. The proteolysis of lens crystallins induced by calcium ion-activated calpain was also inhibited by ASTX (0.03-1 mM) as determined by daily measurement of the light-scattering intensity at 405 nm for five consecutive days. ASTX at 1 mM was as potent as a concentration of 0.1 mM calpain inhibitor E64 in protecting the oxidative damage/hydrolysis of porcine crystallins. At a concentration of 1 mM, ASTX provided better protection than the endogenous antioxidant glutathione in terms of suppressing calcium-induced turbidity of lens proteins. Thin-layer chromatography analysis indicated that ASTX interacted with calcium ions to form complexes, which we believe interfere with the hydrolysis of lens crystallins by calcium-activated calpain. This in vitro study shows that ASTX is capable of protecting porcine lens proteins from oxidative insults and degradation by calcium-induced calpain.     

Structure and stability of the potato cysteine protease inhibitor group (cv. Elkana)

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.     

Calcium activation of maize root phospholipase D

Calcium activation of plant lipases which occurs at relatively high concentrations is poorly understood. The effects of divalent cations on the activities of the soluble and membrane‐bound phospholipase D were compared. Both the soluble and membrane‐bound activities were stimulated by 1 mM Ca and to a lesser extent by 1 mM Ba, but not by 1 mM Mg, Mn, Zn, or Cd. The two enzyme forms differ in the concentration dependence of Ca activation. The membrane‐bound activity exhibited some activity in the absence of Ca, whereas no activity was observed with the soluble enzyme without Ca. Maximal activation was observed at micromolar concentration with the membrane‐bound enzyme compared to millimolar levels for the soluble enzyme. The effects of Ca on the structure of substrate vesicles for the soluble enzyme were determined by following changes in fluorescence of incorporated probes. Calcium had little effects on membrane structure at micromolar concentration, but alter the bilayer structure at millimolar concentrations. Thus, there was a strong association between the concentration dependence of the soluble phospholipase D activity and changes in membrane structure.     

Effect of phosphite–phosphate interaction on growth and quality of hydroponic lettuce (Lactuca sativa)

Although the fungicidal properties of phosphite have been recognized, its potential as a fertilizer is still being debated. The information on how phosphite affects the growth and quality of plants in relation to phosphate (P_i) also remains unknown. This study was conducted to investigate the effect of phosphite in relation to P_i on growth and quality parameters of lettuce (Lactuca sativa L.). The results showed that addition of phosphite to the nutrient solution at different rates ranging from 0.05 to 2 mM significantly increased total P, water‐extractable P_i, and phosphite in both shoots and roots, but did not improve plant growth under various P_i supplies (0.05, 0.1, 0.15, and 0.3 mM as P_i levels for approximately 50%, 80%, 90%, and 100% of the maximum plant growth, respectively), indicating that phosphite was well absorbed by roots and mobile inside the plants, but did not provide any P nutrition. Also, no stimulating effect of any P_i–phosphite combination was observed. The effect of phosphite on plant growth was strongly dependent on the level of P_i supply. In general, application of phosphite up to 2 mM did not influence the growth of P_i‐sufficient plants. However, plants fertilized with P_i for about 90% of maximum growth were still vulnerable to phosphite at 2 mM. The negative effect of phosphite was found even at concentrations as low as 0.2 mM, when plants were supplied with P_i adequate for about 80% of maximum growth or less. At 0.05 mM, phosphite had marginal effects on plant growth under all the P_i levels. Although phosphite itself had little influence on the ascorbate and mineral concentrations of lettuce, its application to P_i‐deficient plants may decrease the mineral concentrations of plants brought about by the inhibitory effect of phosphite on root growth and hence nutrient uptake. Since phosphite is an effective fungicide for lettuce, care should be taken on P_i supplies prior to application of phosphite products to minimize the harmful effects.     

Promotion of phytotoxic bacteria in rhizospheres of leatherleaf fern by benlate DF

Foliar damage symptoms to leatherleaf fern (Rumohra adiantiformis [Forst] Ching) in Florida during widespread use of the fungicide Benlate DF could not be attributed to nutrition, cultural practices, environmental conditions, or new pathogens developing on the crop. The objective of this study was to assess the involvement of rhizosphere bacteria in the damage symptoms using bioassays to detect phytotoxic activity. Rhizosphere bacteria were cultured from rhizospheres of leatherleaf ferns sampled from ferneries where Benlate DF was routinely applied and from check ferneries not receiving the fungicide. Using a lettuce seedling bioassay, the proportion of rhizosphere bacteria that was plant growth‐inhibitory ranged from 7.5% and 11% for isolates from two check ferneries to 70% for those from a fernery previously treated with Benlate DF. Rhizosphere bacteria originating from Benlate DF‐treated leatherleaf ferns caused most severe damage on seedlings with some isolates reducing root growth 70% compared to control seedlings. Other symptoms induced by these bacteria, which were mainly fluorescent and nonfluorescent pseudomonads, included necroses and inhibition of root hair development. Results suggested that Benlate DF affected the composition of bacteria in leatherleaf fern rhizospheres by promoting a bacterial component with phytotoxic properties toward plant growth.     

Do reactive oxygen species (ROS) induced by NaCl contribute to ammonium accumulation in Spartina alterniflora?

Growth, activity of antioxidant enzymes viz. glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX), and some metabolic processes related to ammonium metabolism were investigated in a salt‐tolerant Spatina alterniflora. In comparison to 0 mM–NaCl treatment, growth of S. alterniflora plant increased significantly at 200 mM NaCl, but was highly inhibited at 500 mM NaCl. Ammonium concentration in the leaves and roots increased 2.1–3.4 times when plants were treated with 500 mM NaCl. Under 200 mM NaCl, antioxidant‐enzyme activities increased, however, at 500 mM the antioxidant system was unable to compensate reactive oxygen species induced by NaCl. At this high level of salinity, ammonium production through nitrate reductase (NR) was inhibited, but no significant changes in the activities of glutamine synthetase (GS) or glutamate dehydrogenase (GDH) were found. We conclude that the accumulation of ammonium under high salt stress was not due to inhibition of the assimilatory activities of GS or GDH. Ammonia accumulation under high salinity may result from amino acid and protein catabolism activated by reactive oxygen species (ROS) and/or a lack of carbon skeletons to incorporate ammonium into organic molecules due to a decrease in photosynthetic activity in salt‐stressed plants.     

Long-Term Effect of Nitrate Application from Lower Part of Roots on Nodulation and N2 Fixation in Upper Part of Roots of Soybean (Glycine max (L.) Merr.) in Two-Layered Pot Experiment

The long-term effect of the concentration and duration of application of nitrate from the lower part of soybean roots on the nodulation and nitrogen fixation in the upper part of roots was investigated using a two-layered pot system separating the upper roots growing in a vermiculite medium and the lower roots growing in a nutrient solution. Continuous absence of nitrate (hereafter referred to as “0–0 treatment”), and continuous 1 mM (1–1 treatment) and 5 mM (5–5 treatment) nitrate treatments were imposed in the lower pot from transplanting to the beginning of the maturity stage. In addition, 5 mM nitrate was supplied partially from the beginning of the pod stage till the beginning of the maturity stage (0–5 treatment) or from transplanting till the beginning of the pod stage (5–0 treatment). The values of the total plant dry weight and seed dry weight were highest in the 5–5 treatment, intermediate in the 1–1, 5–0, 0–5 treatments, and lowest in the 0–0 treatment. The values of the nodule dry weight and nitrogen fixation activity (acetylene reduction activity) were lowest in the 5–5 treatment. The value of the nodule dry weight in the upper roots was highest in the plants subjected to the 1–1 treatment and exceeded that in the 0–0 treatment. Total nitrogen fixation activity of the upper nodules per plant at the beginning of the pod stage was also highest in the 1–1 treatment. These results indicated that long-term supply of a low level of nitrate from the lower roots could promote nodulation and nitrogen fixation in the upper part of roots. Withdrawal of 5 mM nitrate after the beginning of the pod stage (5–0 treatment) markedly enhanced nodule growth and ARA per plant in the upper roots at the beginning of the maturity stage when the values of both parameters decreased in the other treatments. The nitrate concentration in the nodules attached to the upper roots was low, including the 5–5 treatment regardless of the stages of growth. This indicated that the inhibitory effect of 5 mM nitrate or promotive effect of 1 mM nitrate supplied from the lower roots was not directly controlled by nitrate itself, but was mediated by some systemic regulation, possibly by the C or/and N requirement of the whole plant.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.     

Bacterial degradation of fungicide captan

The phthalimide fungicide captan has been widely used to control plant pathogenic fungi. A strain of Bacillus circulans utilized the fungicide captan as sole source of carbon and energy. The organism degraded captan by a pathway involving its initial hydrolysis to yield cis-1,2,3,6-tetrahydrophthalimide, a compound without fungicidal activity. The formation of this compound was confirmed by HPLC, IR, NMR, and mass spectral analysis. The results also revealed that cis-1,2,3,6-tetrahydrophthalimide was further degraded to o-phthalic acid by a protocatechuate pathway. These findings indicated that there was a complete mineralization of fungicide captan by B. circulans.     

Inhibition of diphenolase activity of tyrosinase by vitamin b(6) compounds

The vitamin B(6) compounds pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and pyridoxamine 5'-phosphate (PMP) inhibited the diphenolase activity of mushroom tyrosinase. PM showed the highest inhibition; the control activity was inhibited by 38% at 1.5 mM. Each PL, PN, and PMP showed about 30% inhibition at the same concentration. Lineweaver-Burk plots showed that PM and PN were mixed-type inhibitors with K(I) values of 4.3 and 5.2 mM, respectively. Because PM and PN cannot form a Schiff base with a primary amino group of the enzyme, their inhibition is not attributable to the formation of the Schiff base. Alternatively, their quenching function of reactive oxygen species (ROS) was postulated to be responsible for the inhibition. Thus, the inhibitory effect of ROS was examined. The representative singlet oxygen quenchers l-histidine, sodium azide, Trolox, and anthracene-9,10-dipropionic acid (AAP) inhibited the activity. The specific scavenger of superoxide, proxyl fluorescamine, also inhibited the activity. The scavengers of hydroxyl radical, d-mannitol and dimethyl sulfoxide, showed no inhibition. The fluorescence of AAP was decayed during the diphenolase reaction, and PM inhibited the decay. AAP was also a mixed-type inhibitor. The results showed that the vitamin B(6) compounds inhibited the diphenolase activity by quenching ROS (probably singlet oxygen) generated during some reaction step of the diphenolase reaction.     

New insights on glyphosate mode of action in nodular metabolism: Role of shikimate accumulation

The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.     

Effect of heat-induced aggregation on the IgE binding of patatin (Sol t 1) is dominated by other potato proteins

The interaction of the major potato allergen patatin, Sol t 1, with IgE was investigated on a quantitative level as a function of heat treatment at different temperatures. On the basis of a number of publications, potato is considered to be a heat-labile allergen, but the molecular explanation for this behavior was not given. In this work, heat treatment of patatin in the absence and presence of other potato proteins mimicking the proteinaceous environment of the potato was studied. Using far-UV circular dichrosim spectroscopy, tryptophan fluorescence spectroscopy, and differential scanning calorimetry, the molecular transitions during heating of patatin were investigated. It was found that as long as patatin is not aggregated, denaturation of patatin on a secondary or tertiairy folding level is reversible with only a minor effect on the IgE affinity. Aggregation of patatin results in a nonreversible unfolding and a concomitant important decrease in affinity for IgE (25-fold). Aggregation of patatin in the presence of other potato proteins results in a less condensed aggregate compared to the situation of isolated patatin, resulting in a more pronounced decrease of affinity for IgE (110-fold). It is concluded that the heat lability of patatin-IgE interaction is explained by aggregation of patatin with other potato proteins rather than by denaturation of patatin itself.     

Biotransformation of vinclozolin by the fungus Cunninghamella elegans

This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.     

Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera)

Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-cancer effects when consumed in the human diet. Glycosylation of stilbenes increases their solubility and may make them more easily absorbed by the intestine. We have found an activity in extracts of cultured cells of Vitis vinifera (cv. Gamay Freaux) that glucosylates the stilbene resveratrol to form piceid. The Km for UDP-Glucose was 1.2 mM, and the Km for resveratrol was 0.06 mM, values similar to those of other phenolic glucosyltransferases. We investigated the resveratrol glucosylating activity of the enzyme extracted from cells grown under different light treatments (dark, visible light, light + ultraviolet (UVC) radiation) and found the activity to be unaffected or slightly reduced. In contrast, UVC light strongly stimulated extractable quercetin glucosyltransferase activity. These results, combined with analysis of phenolic compounds extracted from the differently treated cells, suggest that the resveratrol glucosyltransferase is distinct from the glucosyltransferase(s) active on other phenolics.     

Extraction of isoflavone malonylglucosides from Trifolium pratense L

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.