Age-Dependent Effects of Jasmonic Acid Treatment and Wind Exposure on Foliar Oxidase Activity and Insect Resistance in Tomato

Jasmonic acid (JA) treatment of tomato plants induces several defense-related oxidative enzymes and increases pest resistance in a manner thought to simulate natural insect wounding. In a full-factorial greenhouse experiment, we examined the independent and interactive effects of plant age and exposure to wind-induced mechanical stress (MS), on the ability of JA to induce defense in tomato. In general, treatment of 4-, 6-, and 8-week-old tomato plants with 1 mM JA resulted in the induction of peroxidase and polyphenol oxidase activity and reduced the relative growth rate of first-instar Manduca sexta larvae fed treated leaves, in accordance with other studies. Peroxidase activity increased with plant age and was induced by JA most strongly in older plants. In contrast, polyphenol oxidase activity did not change with plant age and was induced by JA most strongly in young plants. While relative growth rates of M. sexta were lower on older plants overall, JA reduced growth rates most strongly in young plants, in which JA treatment enhanced polyphenol oxidase activity by more than 70%. MS enhanced the activity of peroxidase, but substantially reduced the activity of polyphenol oxidase; the latter most intensely on older plants. M. sexta tended to grow more slowly on MS-treated plants, although this effect was not significant. Thus, reduced polyphenol oxidase activity in MS-treated plants did not lead to an increase in growth rate of M. sexta, possibly because peroxidase activity was still elevated in MS-treated plants. Significant interactions between JA and MS and three-way interactions were not detected for any variable, although the inductive effects of both JA and MS interacted in complex ways with plant age. Our results indicate that resistance traits in tomato are differentially affected by JA and wind exposure and differ in their relative contribution to defense as plants age.     

The molecular mechanism of membrane proteins probed by evanescent infrared waves

The catalytic action of membrane proteins is vital to many cellular processes. Yet the molecular mechanisms remain poorly understood. We describe here the technique of evanescent infrared difference spectroscopy as a tool to decipher the structural changes associated with the enzymatic action of membrane proteins. Functional changes as minute as the protonation state of individual amino acid side chains can be observed and linked to interactions with a ligand, agonist, effector, or redox partner.     

Reassessment of treatments to retard browning of fresh‐cut Russet potato with emphasis on controlled atmospheres and low concentrations of bisulphite

The cultivar Pacific Russet with high browning susceptibility was used for most testing. Controlled atmospheres (0.3%, 3% and 21% O₂ in combination with 0%, 6% or 12% CO₂) and anti‐browning chemicals were studied in relation to quality retention and wound‐induced phenolic metabolism of fresh‐cut slices for up to 16 days at 5 °C. The 3% O₂+ 12% CO₂ atmosphere was most effective among those tested, and retarded increases in phenolics and phenylalanine ammonia lyase activity, but had only slight benefit on visual quality. A 1.25% ascorbic acid +1.25% citric acid treatment was ineffective, but when combined with 3% O₂+ 12% CO₂, it was comparable with 0.025% sodium bisulphite. Bisulphite concentrations from 0.05% to 0.25% provided similar effective control of discolouration. Bisulphite as low as 0.025% with 3% O₂+ 12% CO₂ resulted in a visual quality score at the limit of marketability after 8 days at 5 °C. Chemical treatments did not retard increases in phenolic concentrations or phenolic enzyme activities.     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

Evaluation of amperometric glucose biosensors based on glucose oxidase encapsulated within enzymatically synthesized polyaniline and polypyrrole

In this article potential and suitability of enzymatically synthesized conducting polymers polyaniline (PANI) and polypyrrole (PPY) for fabrication of enzymatic amperometric glucose biosensors were evaluated. The polymerisation of these polymers was induced by catalytic activity of glucose oxidase (GOx) from Penicillium vitale cross-linked by glutaraldehyde (GA) on the graphite rod electrode (GOx-electrode) surface. The main precursors for initiation of polymerisation reactions were hydrogen peroxide as an initiator of polymerisation reaction and β-d-gluconic acid as a medium, which reduced the pH towards acidic one is the most suitable for the formation of PANI and PPY. During the polymerisation reactions the immobilized GOx was self-encapsulated within formed PANI or PPY layers in order to form GOx/PANI- and GOx/PPY-modified electrodes (GOx/PANI-electrode and GOx/PPY-electrode, respectively). Kinetic properties of GOx, which is acting as a biocatalyst in GOx/PANI- and GOx/PPY-electrodes, were studied and results were compared with GOx-electrode. The results show that in both GOx/PANI- and GOx/PPY-electrodes self-encapsulated GOx exhibited different parameters of catalysed reaction kinetics due to increasing diffusion limitations if compared with that of the GOx-electrode and it allowed the detection of glucose in a wider concentration interval. Moreover, both GOx/PANI- and GOx/PPY-electrodes exhibited good operational stability and reproducibility of analytical signal. The electrochemical characteristics of formed PANI and PPY in the GOx/PANI- and GOx/PPY-electrodes were also determined. In addition, the influence of temperature, pH and common interfering compounds on the steady-state current response of modified electrodes were investigated and discussed.     

Reagentless recycling of pyruvate oxidase on a conducting polymer-modified electrode

The enzyme pyruvate oxidase (PyOD) covalently immobilized on an original conducting copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic-1,4-naphthoquinone acid) can be recycled under anaerobic conditions, at +0.1 V versus SCE. It is first demonstrated that the quinone group is an efficient co-substrate for PyOD in homogeneous conditions, then this efficiency is preserved when the quinone group is embedded in the polymer structure. The copolymer remains efficient even in aerated media. The low working potential avoids side-oxidations of interfering species as ascorbic acid or salycilate.     

GLUCOSE OXIDASE REACTION FOR ESTIMATION OF GLUCOSE WITHOUT HORSERADISH PEROXIDASE. SOME MICROBIOLOGICAL AND FERMENTATION APPLICATIONS

An enzymic method has been developed for analysis of glucose. Glucose oxidase acts on glucose to produce hydrogen peroxide which acts to directly reduce the green Cu(II) 2–2′-bicinchoninate complex to a violet complex without horseradish peroxidase. A concentration range of 20–200 μM glucose was used but the reaction shows a linear range of 20–800 μM glucose. Interference is controlled by using a blank determination which has not been treated with glucose oxidase. The reaction has been used to estimate glucose levels in Saccharomyces cerevisiae fermentation and α-amylase, invertase and β-galactosidase reactions and coloured corn-steep fermentation media.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

外源乙烯对桃果实采后贮藏过程中线粒体内细胞色素氧化酶的影响

以硬溶质型桃果实‘加纳岩’为试材,研究外源乙烯处理对桃果实采后贮藏过程中呼吸速率、乙烯释放量、细胞色素氧化酶（cytochrome C oxidase,COX）活性以及线粒体编码的COX 3 种亚基（COXⅠ 、COXⅡ 、COXⅢ ）基因表达量的影响。结果表明：外源乙烯处理加速了硬溶质型桃果实‘加纳岩’在采后贮藏过程中硬度的下降,促进了呼吸作用和内源乙烯释放,使呼吸高峰和乙烯释放高峰提前出现。同时外源乙烯抑制了COX酶活性,抑制了贮藏后期COXⅠ 和COXⅡ 亚基基因的表达,抑制了整个贮藏过程中COXⅢ 亚基基因的表达。外源乙烯处理抑制了COX途径的呼吸,加速了果实的软化衰老。