High- but not low-intensity light leads to oxidative stress and quality loss of cold-stored baby leaf spinach

BACKGROUND: Quality management in the fresh produce industry is an important issue. Spinach is exposed to various adverse conditions (temperature, light, etc.) within the supply chain. The present experiments were conducted to investigate the effect of light conditions (dark, low-intensity light (LL) and high-intensity light (HL)) and photoperiod (6 h HL and 18 h dark) on the quality changes of cold-stored spinach. RESULTS: HL exposure resulted in oxidative stress, causing tissue damage and quality loss as evidenced by increased membrane damage and water loss. The content of total ascorbic acid was reduced under HL conditions. On the other hand, storage of spinach under LL conditions gave promising results, as nutritional quality was not reduced, while texture maintenance was improved. No significant differences, with the exception of nutritional quality, were found between spinach leaves stored under continuous (24 h) low-intensity light (30–35 µmol m−2 s−1) and their counterparts stored under the same light integral over 6 h (130–140 µmol m−2 s−1). CONCLUSION: LL extended the shelf-life of spinach. The amount of light received by the leaves was the key factor affecting produce quality. Light intensity, however, has to be low enough not to cause excess oxidative stress and lead to accelerated senescence

Impact of elevated CO2 concentration on dynamics of leaf photosynthesis in Fagus sylvatica is modulated by sky conditions

AbstractIt has been suggested that atmospheric CO2 concentration and frequency of cloud cover will increase in future. It remains unclear, however, how elevated CO2 influences photosynthesis under complex clear versus cloudy sky conditions. Accordingly, diurnal changes in photosynthetic responses among beech trees grown at ambient (AC) and doubled (EC) CO2 concentrations were studied under contrasting sky conditions. EC stimulated the daily sum of fixed CO2 and light use efficiency under clear sky. Meanwhile, both these parameters were reduced under cloudy sky as compared with AC treatment. Reduction in photosynthesis rate under cloudy sky was particularly associated with EC-stimulated, xanthophyll-dependent thermal dissipation of absorbed light energy. Under clear sky, a pronounced afternoon depression of CO2 assimilation rate was found in sun-adapted leaves under EC compared with AC conditions. This was caused in particular by stomata closure mediated by vapour pressure deficit

The xanthophyll cycle, its enzymes, pigments and regulation

The xanthophyll cycle involves the light-dependent and reversible conversion of violaxanthin to zeaxanthin. Zeaxanthin has been implicated in the protection of the photosynthetic machinery from over-excitation. The enzyme violaxanthin de-epoxidase catalyses the conversion of violaxanthin to zeaxanthin. The enzyme is located in the thylakoid lumen and its activity is strictly pH controlled. Apart from violaxanthin, ascorbate and monogalactosyldiacylglycerol are required for activity. The Km for ascorbate showed a pH dependence from which we conclude that the acid form of ascorbate is the substrate for the reaction. Release of the enzyme from the thylakoid membrane is strongly pH dependent, with a cooperativity of 4 with respect to protons. A new method for the irreversible inhibition of violaxanthin de-epoxidase was developed. From the pH dependence of this inhibition, we suggest that violaxanthin de-epoxidase undergoes a conformational change upon membrane binding. Purification of violaxanthin de-epoxidase by gel filtration, hydrophobic interaction chromatography and DEAE anion exchange chromatography resulted in a 5 000-fold enrichment and identification of a 43 kDa protein as violaxanthin de-epoxidase. The sequence of the enzyme is unique and show no apparent homology with other known proteins. Pigment analysis of sub-thylakoid membrane domains shows that violaxanthin is evenly distributed in the membrane. Addition of purified violaxanthin de-epoxidase to these domains converted violaxanthin to zeaxanthin from both sides of the membrane to the same maximum degree, independent of pigment-protein complex composition. It is therefore concluded that this conversion takes place in the lipid matrix and not in the pigment-protein complexes. When spinach plants are shifted from low to high light the quantities of the xanthophyll cycle pigments and ascorbate are increased, but the amount and activity of violaxanthin de-epoxidase are decreased. The increase in xanthophyll cycle pigments and ascorbate only partly explains the increased rate of conversion of violaxanthin to zeaxanthin. The most probable explanation of a faster conversion is an increased accessibility of violaxanthin in the membrane. Temperature has a strong influence on both the rate and degree of maximum conversion. It is proposed that diffusion of violaxanthin between pigment protein complexes and the lipid matrix regulates the degree of conversion

Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light

Zeaxanthin, a carotenoid in the xanthophyll cycle, has been suggested to play a role in the protection against photodestruction. We have studied the importance of the parameters involved in zeaxanthin formation by comparing spinach plants grown in low light (100 to 250 mol m-2 s-1) to plants transferred to high light (950 mol m-2 s-1). Different parameters were followed for a total of 11 days. Our experiments show that violaxanthin de-epoxidase decreased between 15 and 30%, the quantity of xanthophyll cycle pigments doubled to 100 mmol (mol Chl)-1, corresponding to 27 mol m-2, and the rate of violaxanthin to zeaxanthin conversion was doubled. Lutein and neoxanthin increased from 50 to 71 mol m-2 and from 16 to 23 mol m-2, respectively. On a leaf area basis, chlorophyll and -carotene levels first decreased and then after 4 days increased. The chlorophyll a/b ratio was unchanged. The quantity of ascorbate was doubled to 2 mmol m-2, corresponding to an estimated increase in the chloroplasts from 25 to 50 mM. In view of our data, we propose that the increase in xanthophyll cycle pigments and ascorbate only partly explain the increased rate of conversion of violaxanthin to zeaxanthin, but the most probable explanation of the faster conversion is an increased accessibility of violaxanthin in the membrane

Enzymes and mechanisms for violaxanthin-zeaxanthin conversion

The xanthophyll cycle is of great importance in relation to light stress. Particularly, interest has been focused on the possible photoprotective role of zeaxanthin. In higher plants under light stress, zeaxanthin is formed from violaxanthin in a reaction catalyzed by violaxanthin de-epoxidase (VDE). The reverse reaction is catalyzed by zeaxanthin epoxidase (ZE) under low light or in darkness. VDE has been purified from spinach and lettuce as a 43-kDa protein. The gene has been cloned and sequenced from several species, and a few mutants have been isolated. The gene is nuclear encoded and the transit peptide is characteristic for targeting to the thylakoid lumen. The activity of VDE is affected by factors such as a pH-dependent binding to the thylakoid membrane, concentration of ascorbic acid, temperature and availability of violaxanthin in relation to amount, type and distribution of pigment-protein complexes in the membrane. The information about ZE is more limited. The enzyme has not yet been isolated but its gene has been cloned and sequenced and a number of mutants have been isolated. The role of the xanthophyll cycle in the dissipation of excess light energy will be discussed particularly in relation to the recent progress in studies on various mutants. The possible role of the xanthophyll cycle in other processes, such as protection against oxidative stress of lipids, regulation of membrane fluidity, participation in blue light responses, and regulation of abscisic acid synthesis will also be presented

Chemical and mutational modification of histidines in violaxanthin de-epoxidase from Spinachcia oleracea.

The violaxanthin de-epoxidase (VDE) gene from spinach (Spinacia oleracea) was cloned, sequenced (GenBank AJ 250433), and expressed in Escherichia coli. The highest obtained conversion rate of violaxanthin was 86 nmol s-1 per litre of growth medium, corresponding to an amount of active enzyme of 0.4 mg l-1. Sequence comparison between VDE from different species were made and particular interest was focused on four highly conserved histidines (H121,124,167,173) and their possible involvement in enzymatic activity. Chemical modification of the histidines using DEPC or by site-directed mutations resulted in partial or total inactivation of the enzyme. The chemical modification could be reversed by hydroxylamine treatment, regenerating a large percentage of the original activity. The histidine residues, which are located in pairs close to each other, were pairwise substituted for either alanine or arginine. This resulted in one inactive mutant (H121,124R) and three mutants with very different activities and decreased binding of ascorbic acid, as reflected by an up to four-fold increase in Km. A substitution of all four histidines for either alanine or arginine resulted in inactive enzymes. Based on these results it is suggested that the histidine residues are important for the activity of VDE

The xanthophyll cycle, its regulation and components

During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de-epoxidase has recently been purified from spinach and lettuce as a 43-kDa protein. It was found as 1 molecule per 20-100 electron-transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear-encoded and lumen-localized proteins. The activity of violaxanthin de-epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pKa= 4.2) the true substrate (Km= 0.1 mM) for the violaxanthin de-epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de-epoxidase occurs