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.     

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 (pK_a= 4.2) the true substrate (K_m= 0.1 m M ) 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.     

Chemical and mutational modification of histidines in violaxanthin de-epoxidase from Spinacia 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 K _m. 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.