Albert C. Purvis

Does the alternative pathway ameliorate chilling injury in sensitive plant tissues

Free radical processes have been observed in senescence and several membrane-associated disorders of plants including chilling, freezing, and desiccation injuries. The mitochondria of plant tissues exposed to low temperatures, and other abiotic and biotic stresses, produce superoxide and/or hydrogen peroxide when electron transport through the cytochrome pathway is impaired due to the energy state of the cell or to stress-induced physical changes in the membrane components. The superoxide and/or hydrogen peroxide produced can diffuse throughout the cell causing peroxidation of membrane lipids which results in membrane disruption, increased permeability and metabolic disturbances, and eventually the visible symptoms of chilling injury. The alternative pathway of electron transport in the mitochondria, which is induced by low temperatures in some plant tissues, can mediate these degradative processes by reducing the level of superoxide generated by the mitochondria.

Differences in chilling sensitivity of cucumber varieties depends on storage temperature and the physiological dysfunction evaluated

Abstract Fruit from eight Plant Introduction (PI) lines, 12 F 1 hybrids of crosses between chilling-sensitive and chilling-resistant lines and two commercial cultivars of cucumber ( Cucumis sativus L.) were stored for 7 days at 1 or 4°C followed by 2 days at 24°C and evaluated for chilling injury. The chilling-induced symptoms and physiological dysfunctions compared included visible pitting, decay, weight loss during storage at low temperature, electrolyte leakage, chlorophyll fluorescence ratios, respiration rates and pyruvate accumulation in the mesocarp tissue. Severity of chilling-induced injury among the lines, hybrids and cultivars depended on the particular symptom or physiological dysfunction examined. Fruit from all lines, crosses and cultivars exhibited greater injuries after storage at 1°C than after storage at 4°C. Other than visible pitting and decay, only small, sometimes indistinguishable, differences were observed among the various lines as to their sensitivity to low temperatures. In this study, chlorophyll fluorescence ratios proved to be of little value in distinguishing between chilling-sensitive and chilling-resistant lines of cucumber. Decay and weight loss were the only measurements that were significantly correlated with chilling-induced visible pitting. Respiration rates were correlated with weight loss, but not with visible pitting and decay, during storage at 1 and 4°C. It is concluded that low temperatures do not affect all biochemical and physiological processes of cucumber to the same extent. Furthermore, there is a continuum of sensitivity of each process to low temperature, and whether a cultivar is deemed to be chilling-sensitive or chilling-resistant depends on which particular biochemical or physiological process is evaluated.

Stress-induced changes in ubiquinone concentration and alternative oxidase in plant mitochondria.

We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.

Diphenylamine reduces chilling injury of green bell pepper fruit

Diphenylamine (DPA), an antioxidant widely used to control superficial scald in apples stored at low temperatures, reduced the chilling-induced pitting of green bell pepper (Capsicum annuum L.) fruit in a concentration-dependent manner up to 12 mM. Treatment was effective whether applied as a 2-min dip or injected into the seed cavity prior to storing peppers at 1° C for 6 or 8 days followed by storage at 20° C for an additional 2 days. Ethanol (5%), the solvent used to dissolve the diphenylamine, also reduced chilling injury of green bell peppers, but to a lesser extent than DPA. Changes in all of the chlorophyll fluorescence parameters were observed within 24 h following the exposure of green bell peppers to low temperatures, whereas visible manifestation of chilling injury was not observed until after the peppers had been exposed to several days of low temperatures. Diphenylamine reduced the chilling-induced decline in maximal chlorophyll fluorescence and increased the time taken for maximal chlorophyll fluorescence to occur and the size of the electron acceptor pool. If chilling injury results from oxidative stress induced by low storage temperatures, it is unlikely that chloroplasts were the site of production of active oxygen species in these green bell peppers since they were stored in the dark. The rapidity of chlorophyll fluorescence responses to low temperatures makes chlorophyll fluorescence analysis useful to distinguish between chilling-sensitive and chilling-resistant plant tissues, but does not measure the extent of chilling injury.

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Summary Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper ( Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers. Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production. Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.

HIGH-TEMPERATURE CONTINUOUS-FLOW CURING OF SWEET ONIONS

A study was undertaken to investigate the feasibility of heat treating sweet onions under controlled commercial conditions. Three test runs were conducted whereby approximately 4 m3 of onions for each test were passed through a continuous-flow drier. Set-point temperatures of 43, 43, and 460C and durations of heat treatment of 17, 24 and 24 h were used respectively during the three tests. Samples of heat treated onions were taken from the dryer at regular intervals and, after prescribed storage intervals, were inspected for the presence of Botrytis allii with the aid of a dye. The increase in disease was calculated. There was a significantly (P<0.01) less increase in disease during storage for those onions having received heat treatment compared with onions conventionally cured. In comparing the least square means, 24 h of heat treatment resulted in a lower incidence of disease than 17 h. Similarly, a set-point temperature of 460C resulted in a lower incidence of disease than 430C . Based upon the results of the study, a combination of heat treatment and conventional curing was recommended.