Carotenoids are involved in a variety of biological functions, yet the underlying mechanisms are poorly understood, in part because of the long-standing difficulty in assigning the location of the first excited (S 1 ) state. Here, we present a method for determining the energy of the forbidden S 1 state, on the basis of ultrafast spectroscopy of the short lived level. Femtosecond transient absorption spectra and kinetics of the S 1 3 S 2 transition revealed the location of the intermediate level in two carotenoid species involved in the xanthophyll cycle, zeaxanthin and violaxanthin, and yielded surprising implications regarding the mechanism of photoregulation in photosynthesis.The diversity and abundance of carotenoids in nature reflect their importance in protecting and supporting the organisms in which they are found (1). In photosynthetic and physiological systems alike, carotenoids are antioxidants that prevent damage by quenching triplet states and scavenging singlet oxygen. Furthermore, they play an integral role in photoregulation in plants, assisting with light harvesting or dissipating excess energy as needed (2). The mechanisms that underlie their functions are poorly understood, in part because of the long standing difficulty in assigning the location of their first excited (S 1 ) states. In higher conjugated carotenoids, the S 1 state is spectroscopically ''dark,'' hence previous assignments of its energy have relied on extrapolation of data from shorter-chain carotenoids (3). Whereas application of the ''energy-gap law'' (4) to carotenoids of similar composition (i.e., with changes only in conjugation length) may yield reasonable estimates of S 1 energies (5), quantitative information for markedly different species is not reliable.The S 1 level of carotenoids is likely involved in numerous functions and may be particularly relevant to the xanthophyll cycle of higher plants. In this photoprotection function, carotenoids are involved in regulating the amount of light energy funneled into the reaction centers according to the given environmental conditions. The equilibrium within an interconversion reaction cycle involving three carotenoid species, zeaxanthin N antheraxanthin N violaxanthin, is shifted according to the amount of light available. In high light, the concentration of zeaxanthin increases, and correspondingly excess energy is dissipated out of the antenna complexes (as monitored by fluorescence quenching), whereas in low light violaxanthin is the predominant carotenoid and no quenching is observed. The S 1 states of both carotenoids have been implicated in the mechanism of this function, according to estimates of the energy levels via application of the energy gap law (3, 6). However, without precise knowledge of the S 1 energetics, this interpretation is highly speculative.Here we present a general method for measuring the S 1 -state energy of carotenoids directly, with specific application to zeaxanthin and violaxanthin. The key factor of this experiment lies in the ability to ...

1997. The xanthophyll cycle, its regulation and components. -Physiol. Plant. 100: 806-816.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 (K^ = 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.

Human obesity is a global epidemic, which causes a rapidly increased frequency of diabetes and cardiovascular disease. One reason for obesity is the ready availability of refined food products with high caloric density, an evolutionarily new event, which makes over-consumption of food inevitable. Fat is a food product with high caloric density. The mechanism for regulation of fat intake has therefore been studied to a great extent. Such studies have shown that, as long as fat stays in the intestine, satiety is promoted. This occurs through the fat-released peptide hormones, the best known being CCK (cholecystokinin), which is released by fatty acids. Hence, retarded fat digestion with prolonged time for delivery of fatty acids promotes satiety. Pancreatic lipase, together with its protein cofactor, co-lipase, is the main enzymatic system responsible for intestinal fat digestion. We found that biological membranes, isolated from plants, animals or bacteria, inhibit the lipase/co-lipase-catalysed hydrolysis of triacylglycerols even in the presence of bile salt. We propose that the inhibition is due to binding of lipase/co-lipase to the membranes and adsorption of the membranes to the aqueous/triacylglycerol interface, thereby hindering lipase/co-lipase from acting on its lipid substrate. We also found that chloroplast membranes (thylakoids), when added to refined food, suppressed food intake in rats, lowered blood lipids and raised the satiety hormones, CCK and enterostatin. Consequently, the mechanism for satiety seems to be retardation of fat digestion allowing the fat products to stay longer in the intestine.

The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.

The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.