Harry Y. Yamamoto

Resolution of lutein and zeaxanthin using a non-endcapped, lightly carbon-loaded C18 high-performance liquid chromatographic column

Abstract A new rapid and reproducible high-performance liquid chromtographic method using Spherisorb ODS-1, a non-endcapped, lightly carbon-loaded column material, for the separation of higher-plant chloroplast pigments is described. The method resolves lutein and zeaxanthin, as well as all other major and most minor pigments at or near baseline by either of two solvent programs. Program I is faster and more sensitive than program I while the latter resolves pheophytin a and β,e-carotene slightly better than program I. Both programs use an inital buffered aqueous mixture that apperas critical for this application of ODS-1. The method is well suited for analysis of xanthophyll-cycle pigment changes.

BIOCHEMISTRY OF THE VIOLAXANTHIN CYCLE IN HIGHER PLANTS

Abstract The biochemistry of the violaxanthin cycle in relationship to photosynthesis is reviewed. The cycle is a component of the thylakoid and consists of a reaction sequence in which violaxanthin is converted to zeaxanthin (de-epoxidation) and then regenerated (epoxidation) through separate reaction mechanisms. The arrangement of the cycle in the thylakoid is transmembranous with the de-epoxidation system situated on the loculus side and epoxidation on the outer side of the membrane. Photosynthetic activities affect turnover of the cycle but the cycle itself consists entirely of dark reactions. Light has at least two roles in de-epoxidation. It establishes through the proton pump the acidic pH in the loculus that is required for de-epoxidase activity and it induces a presumed conformational change in the inner membrane surface which determines the fraction of violaxanthin in the membrane that enters the cycle. De-epoxidation, which requires ascorbate, is presumed to proceed by a reductive-dehydration mechanism. Non-cyclic electron transport can provide the required reducing potential through the dehydroascorbate-ascorbate couple. Whether ascorbate reduces the de-epoxidase system directly or through an intermediate has not been settled. Epoxidation requires NADPH and O2 which suggests a reductive mechanism. In contrast with de-epoxidation, it has a pH optimum near neutrality. The coupling of photosynthetically generated NADPH to epoxidation has been shown. Turnover of the cycle under optimal conditions is estimated to be about two orders of magnitude below optimal electron transport rates. This low rate appears to exclude a direct role of the cycle in photosynthesis or a role in significantly affecting photosynthate levels in a back reaction. The fact that the cycle is sensitive to events both before and after Photosystem I suggests a regulatory role, possibly through effects on membrane properties. A model showing the various relationships of the cycle to photosynthesis is presented. The contrasting view that the cycle can participate directly in photosynthesis, such as in oxygen evolution, is discussed. Violaxanthin de-epoxidase has been purified. It is a lipoprotein which contains monogalactosyldiglyceride (MG) exclusively. The enzyme is a mono-de-epoxidase which is specific for 3-OH, 5–6-epoxy carotenoids that are in a 3R , 5S , 6R configuration. In addition, the polyene chain must be all- trans . A model has been presented which depicts enzymic MG in a receptor role and the stereospecific active center situated in a narrow well-like depression that can accommodate only the all- trans structure.

Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching

Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and ΔpH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.

The effects of dithiothreitol on violaxanthin de-epoxidation and absorbance changes in the 500-nm region☆

Abstract The effects of dithiothreitol on absorbance changes at 505 and 515 nm in isolated lettuce chloroplasts were investigated. Dithiothreitol inhibited the ascorbate-dependent 505-nm change that is due to the de-epoxidation of violaxanthin to zeaxanthin. Dithiothreitol was effective for both light-induced de-epoxidation at pH 7 and dark de-epoxidation at pH 5. Titration of de-epoxidase activity with dithiothreitol resulted in complete inhibition at about 5 μmoles dithiothreitol per mg chlorophyll. Removal of dithiothreitol restored de-epoxidase activity. These results are consistent with the view that dithiothreitol inhibits violaxanthin de-epoxidation and the corresponding 505-nm change by reducing a disulfide that is required for de-epoxidase activity. Dithiothreitol was effective in resolving absorbance changes due to violaxanthin de-epoxidation and other changes that were superimposed under some conditions. At 515 nm and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), phenazine methosulfate, and ascorbate, dithiothreitol inhibited the large, slow and irreversible change which was due to de-epoxidation but not the fast and reversible so-called 515-nm change. At 505 nm and under similar conditions, dithiothreitol revealed the presence of a slow reversible change in addition to the one from de-epoxidation. Results with dithiothreitol showed that the absorbance change at 505 nm in the presence of DCMU, 2,6-dichlorophenolindophenol and ascorbate was due entirely to de-epoxidation. Similarly, absorbance changes at 515 nm also appeared to be mainly from de-epoxidation but with the presence of a small transient change due to some other components. It is suggested that dithiothreitol may be useful in resolving complex light-induced absorbance changes in other photosynthetic systems as well as in enabling new studies on reversible absorbance changes in the 500-nm region.

Carotenoids: Localization and Function

Carotenoids are components of every pigment-protein complex in the photosynthetic apparatus of higher plants. These pigments, previously referred to as ‘accessory,’ are now recognized to fulfill indispensable functions in light harvesting, protection against photooxidation, and regulation of Photosystem II efficiency. The wealth of information accumulated in recent years dealing with the closely related questions of carotenoid organization and functions are summarized in this chapter. In the first section the distribution of carotenoids in the different pigment proteins is reported showing that each photosystem subunit has its characteristic composition. The organization of the different xanthophylls within the antenna complexes is discussed on the basis of recent structural and biochemical evidence. In the second section, advances in photophysical mechanisms through which carotenoids perform their classical light harvesting and protective functions are discussed. In addition, particular attention is given to discussion of the xanthophyll cycle which, in conjunction with the transthylakoid ΔpH1 down-regulates Photosystem II photochemical efficiency by non-radiative dissipation of energy in the light-harvesting complexes. Down-regulation helps to keep PS II traps open, thereby helping to maintain electron transport and to protect the reaction center from photoinhibition.

SHORT‐TERM RESPONSE OF THE DIADINOXANTHIN CYCLE AND FLUORESCENCE YIELD TO HIGH IRRADIANCE IN CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

The relationship between the diadinoxanthin cycle and changes in fluorescence yield in the diatom Chaetoceros muelleri Lemm. (clone CH10, Amorient Aquafarm, Inc., Hawaii) was investigated. High‐light‐induced changes in fluorescence yield and xanthophyll de‐epoxidation occurred very rapidly (first order rate constant 1.60 min−1). The observed light‐induced changes in diatoxanthin and diadinoxanthin concentration were consistent with a two‐pool scheme for diadinoxanthin, one of which does not undergo de‐epoxidation. Changes in xanthophyll concentration correlated with changes in in vivo absorbance indicating that diadinoxanthin cycle activity in vivo can be monitored spectrophotometrically. However, changes in cell absorbance were small relative to total optical absorption cross section. Increases in the concentration of diatoxanthin were linearly correlated with increases in the rate constant for thermal de‐excitation in the antenna of photosystem II (PSII). Antenna quenching produced or mediated by diatoxanthin may, thus, protect the PSII reaction center in diatoms. Changes in the maximum fluorescence yield suggested that changes in the reaction center also contributed to nonphotochemical quenching of fluorescence. Thus, reaction center quenching affected the relationship between antenna quenching and changes in photochemical efficiency producing the effect of a decrease in fluorescence yield without a decrease in photochemical efficiency.

Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase.

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.

Violaxanthin De-Epoxidase (Purification of a 43-Kilodalton Lumenal Protein from Lettuce by Lipid-Affinity Precipitation with Monogalactosyldiacylglyceride)

Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photo-chemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy in the antennae. Previously, violax-anthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a novellipid-affinity precipitation step with monogalactosyldiacylglyceride. The association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.

Membrane barriers and Mehler-peroxidase reaction limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts

The presence of an acidic lumen and the xanthophylls, zeaxanthin and antheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recently reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Mehler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 99: 1354–1361]. It is demonstrated that in non-CO2-fixing intact chloroplasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbate available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In intact chloroplasts, this results in biphasic kinetic behavior for light-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and the terminal low activity due to limiting ascorbate from the effects of chloroplast membranes barriers and a light-dependent process. A five-fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 120 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the Mehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H2O2 transiently inhibited de-epoxidase activity and concomitantly increased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the KM values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support this conclusion. These results indicate that regulation of xanthophyll-dependent non-radiative energy dissipation in the pigment bed of Photosystem II is modulated not only by lumen acidification but also by ascorbate availability.

Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of trans-thylakoid ΔpH

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide‐free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (ΔpH), epoxidation can proceed irrespective of a ΔpH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light‐induced thylakoid ΔpH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid ΔpH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.