Involvement of hydrogen peroxide in the regulation of senescence in pear

Endogenous peroxide levels in pear fruit (Pyrus communis) were measured using a titanium assay method, and were found to increase during senescence in both Bartlett and Bosc varieties. Application of glycolic acid or xanthine, serving as substrates for the formation of H₂O₂, increased the peroxide content of the tissue and accelerated the onset of ripening, as measured by increased softening and ethylene evolution. Application of ethylene also induced increased peroxide levels. Ripening processes were similarly promoted when peroxides were conserved by inhibiting the activity of catalase with hydroxylamine or potassium cyanide. By comparison, the inhibition of glycolate oxidase with alphahydroxy-2-pyridinemethanesulfonic acid decreased the peroxide content of the tissue and delayed the onset of ripening. These results indicate that the onset of ripening correlates with the peroxide content of fruit tissues as occurring under normal conditions or as influenced by the treatments. Hydrogen peroxide may be involved in oxidative processes required in the initiation and the promotion of ripening.     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress

Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ‐SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild‐type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress‐related metabolites. Despite this, FA plants were more sensitive to short‐ and long‐term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non‐stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non‐stress and stress conditions is important for enabling the plants to cope with stress conditions.     

Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio‐ (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants

Angiosperm resurrection plants exhibit poikilo‐ or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit‐induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non‐enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO‐dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit‐induced poikilochlorophylly occurs via the well‐characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.     

The role of lager beer yeast in oxidative stability of model beer

Aims: In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Methods and Results: Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma‐mass spectrometry, and the slowest formation of radicals in the model beers. Conclusions: A more oxidative stable beer is not obtained by a more‐oxidative‐stress‐tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less‐oxidative‐stress‐tolerant strain, exhibiting a higher iron uptake. Significance and Impact of the Study: To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin.     

Changes in oxidative processes and components of the antioxidant system during tomato fruit ripening

Analysis of the oxidative processes taking place during fruit ripening in a salad tomato variety (Lycopersicon esculentum Mill. cv. Ailsa Craig) revealed changes in oxidative and antioxidative parameters. Hydrogen peroxide content, lipid peroxidation and protein oxidation were measured as indices of oxidative processes and all were found to increase at the breaker stage. The levels of the aqueous-phase antioxidants, glutathione and ascorbate, increased during the ripening process and these increases were associated with significant changes in their redox status, becoming more reduced as ripening progressed. Changes in the activities of superoxide dismutase, catalase and the enzymes involved in the ascorbate-glutathione cycle during ripening indicated that the antioxidative system plays a fundamental role in the ripening of tomato fruits.     

Jasmonic acid is involved in the water-stress-induced betaine accumulation in pear leaves

Jasmonic acid (JA) is known to be involved in the response of plants to environmental stresses such as drought, and betaine (glycinebetaine) is an osmopretectant accumulated in plants under environmental stresses including drought. However, it remains currently unclear whether JA is involved in the water‐stress‐induced betaine accumulation in plant leaves. The present experiment, performed with the whole pear plant (Pyrus bretschneideri Redh. cv. Suli), revealed that the exogenously applied JA induced a significant increase of the betaine level in the pear leaves when the plants were not yet stressed by drought, and when the plants were subjected to water stress, the ‘JA plus drought’ treatment induced a significant higher betaine level than did the drought treatment alone. Meanwhile, the ‘JA plus drought’ treatment induced higher levels of betaine aldehyde dehydrogenase (BADH, E C 1.2.1.8) and activities in the leaves than did the drought treatment alone. These results obtained in the whole plant experiments were supported by the results of detached leaf experiments. In detached leaves JA induced significant increases in betaine levels, BADH activities and BADH protein amounts in a time‐ and concentration‐dependent manner. These data demonstrate that JA is involved in the drought‐induced betaine accumulation in pear leaves.     

2D-DIGE analysis of mango (Mangifera indica L.) fruit reveals major proteomic changes associated with ripening

A comparative proteomic investigation between the pre-climacteric and climacteric mango fruits (cv. Keitt) was performed to identify protein species with variable abundance during ripening. Proteins were phenol-extracted from fruits, cyanine-dye-labeled, and separated on 2D gels at pH 4-7. Total spot count of about 373 proteins spots was detected in each gel and forty-seven were consistently different between pre-climacteric and climacteric fruits and were subjected to LC-MS/MS analysis. Functional classification revealed that protein species involved in carbon fixation and hormone biosynthesis decreased during ripening, whereas those related to catabolism and the stress-response, including oxidative stress and abiotic and pathogen defense factors, accumulated. In relation to fruit quality, protein species putatively involved in color development and pulp softening were also identified. This study on mango proteomics provides an overview of the biological processes that occur during ripening.     

Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120‐mediated connexin43 phosphorylation

The ω‐3 fatty acids exert as an antioxidant via the G protein‐coupled receptor 120 (GPR120). Icosapent ethyl, a purified eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connexin43 expression and whether the GPR120 signalling is involved in the protection. Male Wistar rats after ligating coronary artery were assigned to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative‐nitrosative stress. In concert, myocardial connexin43 levels revealed a significant decrease in vehicle‐treated infarcted rats compared with sham. These changes of oxidative‐nitrosative stress and connexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative‐nitrosative stress and connexin43 phosphorylation were abolished by administering AH‐7614, an inhibitor of GPR120. SIN‐1 abolished the Cx43 phosphorylation of icosapent ethyl without affecting GPR120 levels. Taken together, chronic use of icosapent ethyl after infarction is associated with up‐regulation of connexin43 phosphorylation through a GPR120‐dependent antioxidant pathway and thus plays a beneficial effect on arrhythmogenic response to programmed electrical stimulation.     

Chloroplast protection in plum pox virus‐infected peach plants by L‐2‐oxo‐4‐thiazolidine‐carboxylic acid treatments: effect in the proteome

Sharka, a disease caused by plum pox virus (PPV), has a significant economic impact on fruit tree production. In this work, we analysed the effect of (2,1,3)‐benzothiadiazole (BTH) and L‐2‐oxo‐4‐thiazolidine‐carboxylic acid (OTC) on plant growth and virus content. OTC reduced sharka symptom, stimulated plant growth and alleviated PPV‐induced oxidative stress, indicated by a lack of changes in some oxidative stress parameters. PPV infection reduced chloroplast electron transport efficiency. However, in the presence of BTH or OTC, no changes in the chlorophyll fluorescence parameters were observed. PPV produced an alteration in chloroplast ultrastructure, giving rise to a decrease in starch contents that was less dramatic in OTC‐treated plants. Furthermore, PPV reduced the abundance of proteins associated with photosynthesis, carbohydrate and amino acid metabolism and photorespiration. These changes did not take place in OTC‐treated plants, and increases in the expression of proteins related with the aforementioned processes, including ADP‐glucose pyrophosphorylase, were produced, which correlated with the lower decrease in starch contents observed in PPV‐infected plants treated with OTC. The results suggested that OTC treatment provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV‐infected peaches. Thus, OTC could have practical implications in agriculture in improving the vigour of different plant species as well as in immunizing plants against pathogens.