Dariusz Latowski

Mechanism and regulation of the violaxanthin cycle: the role of antenna proteins and membrane lipids.

The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.

Heavy metal-induced oxidative damage, defense reactions, and detoxification mechanisms in plants

Heavy metal (HMs) contamination is widespread globally due to anthropogenic, technogenic, and geogenic activities. The HMs exposure could lead to multiple toxic effects in plants by inducing reactive oxygen species (ROS), which inhibit most cellular processes at various levels of metabolism. ROS being highly unstable could play dual role (1) damaging cellular components and (2) act as an important secondary messenger for inducing plant defense system. Cells are equipped with enzymatic and non-enzymatic defense mechanisms to counteract this damage. Some are constitutive and others that are activated only when a stress-specific signal is perceived. Enzymatic scavengers of ROS include superoxide dismutase, catalase, glutathione reductase, and peroxidase, while non-enzymatic antioxidants are glutathione, ascorbic acid, α-tocopherol, flavonoids, anthocyanins, carotenoids, and organic acids. The intracellular and extracellular chelation mechanisms of HMs are associated with organic acids such as citric, malic and oxalic acid, etc. The important mechanism of detoxification includes metal complexation with glutathione, amino acids, synthesis of phytochelatins and sequestration into the vacuoles. Excessive stresses induce a cascade, MAPK (mitogen-activated protein kinase) pathway and synthesis of metal-detoxifying ligands. Metal detoxification through MAPK cascade and synthesis of metal-detoxifying ligands will be of considerable interest in the field of plant biotechnology. Further, the photoprotective roles of pigments of xanthophylls cycle under HMs stress were also discussed.

Carotenoids and Environmental Stress in Plants: Significance of Carotenoid-Mediated Modulation of Membrane Physical Properties

Carotenoids, apart of their antenna function in photosynthesis, play an important role in the mechanisms protecting the photosynthetic apparatus against various harmful environmental factors. They protect plants against overexcitation in strong light and dissipate the excess of absorbed energy, they scavenge reactive oxygen species formed during photooxidative stress and moderate the effect of extreme temperatures. One of the important factors involved in the protective action of carotenoids is their influence on the molecular dynamics of membranes. To obtain complex information about interactions between carotenoids and lipids in a membrane, different techniques were used. In this review, the data resulting from EPR–spin label spectrometry, 31P- and 13C-NMR, differential scanning calorimetry, and computer simulation of the membrane molecular dynamics are presented. The effects of selected, structurally different carotenoid species on various physical parameters of model and natural membranes are described and their relevance to protection against some environmental stresses are discussed.

The xanthophyll cycle - molecular mechanism and physiological significance

The light-dependent, cyclic changes of xanthophyll pigments: violaxanthin, antheraxanthin and zeaxanthin, called the xanthophyll cycle, have been known for about fifty years. This process was characterised for higher plants, several fern and moss species and in some algal groups. Two enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE), belonging to the lipocalin protein family, are engaged in the xanthophyll cycle. VDE requires for its activity ascorbic acid and reversed hexagonal structure formed by monogalactosyldiacylglycerol. ZE, postulated to be a flavoprotein, has not been purified yet and it is known from its gene sequence only. Zeaxanthin epoxidation is dependent on the reducing power of NADPH and presence of additional proteins.The xanthophyll cycle is postulated to play a role in many important physiological processes. Zeaxanthin, formed from violaxanthin under high light conditions, is thought to be a main photoprotector in autotrophic cells due to its ability to dissipate excess of absorbed light energy that can be measured as a non-photochemical quenching. In addition the zeaxanthin formation is important in protection of the thylakoid membranes against lipid peroxidation. Other postulated functions of the xanthophyll cycle, which include regulation of membrane physical properties, blue light reception and regulation of abscisic acid synthesis, are also discussed.

Thermotropic phase behaviour of α-dipalmitoylphosphatidylcholine multibilayers is influenced to various extents by carotenoids containing different structural features- evidence from differential scanning calorimetry

Carotenoids are the effective modulators of physical properties of model and natural membranes. To demonstrate the relationship between the structure of carotenoids and their effect on the molecular dynamics of membranes, we have investigated the influence of five structurally different carotenoids: beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin and additionally carotane--a fully saturated derivative of beta-carotene, on thermotropic phase behaviour of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles by means of differential scanning calorimetry (DSC). The results obtained indicate that the carotenoids used modulated the thermotropic properties of multibilayers to various extents, broadening the pretransition and the main phase transition peaks and shifting them to lower temperatures. Pronounced decrease of pretransition enthalpy (DeltaH(p)) proves that carotenoids very strongly alter the membrane properties in its gel phase. Comparison of the influence of several carotenoids shows that a rigid, polyisoprenoid chain plays a basic role in altering the thermotropic properties of such membranes and the presence of rings without oxygen-containing groups has a minor significance for the observed interactions. Carotenoids containing epoxy and/or hydroxy groups attached to their rings modify the thermotropic phase behaviour of DPPC multilamellar vesicles stronger than carotenes--a result of their orientation in the DPPC bilayer.

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII).

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.

Xanthophyll cycle – a mechanism protecting plants against oxidative stress

Abstract Six different xanthophyll cycles have been described in photosynthetic organisms. All of them protect the photosynthetic apparatus from photodamage caused by light-induced oxidative stress. Overexcitation conditions lead, in the chloroplast, to the over-reduction of the NADP pool and production of superoxide, which can subsequently be metabolized to hydrogen peroxide or a hydroxyl radical, other reactive oxygen species (ROS). On the other hand, overexcitation of photosystems leads to an increased lifetime of the chlorophyll excited state, increasing the probability of chlorophyll triplet formation which reacts with triplet oxygen forming single oxygen, another ROS. The products of the light-dependent phase of xanthophyll cycles play an important role in the protection against oxidative stress generated not only by an excess of light but also by other ROS-generating factors such as drought, chilling, heat, senescence, or salinity stress. Four, mainly hypothetical, mechanisms explaining the protective role of xanthophyll cycles in oxidative stress are presented. One of them is the direct quenching of overexcitation by products of the light phase of xanthophyll cycles and three others are based on the indirect participation of xanthophyll cycle carotenoids in the process of photoprotection. They include: (1) indirect quenching of overexcitation by aggregation-dependent light-harvesting complexes (LHCII) quenching; (2) light-driven mechanisms in LHCII; and (3) a model based on charge transfer quenching between Chl a and Zx. Moreover, results of the studies on the antioxidant properties of xanthophyll cycle pigments in model systems are also presented.

Regulation of LHCII aggregation by different thylakoid membrane lipids.

In the present study the influence of the lipid environment on the organization of the main light-harvesting complex of photosystem II (LHCII) was investigated by 77K fluorescence spectroscopy. Measurements were carried out with a lipid-depleted and highly aggregated LHCII which was supplemented with the different thylakoid membrane lipids. The results show that the thylakoid lipids are able to modulate the spectroscopic properties of the LHCII aggregates and that the extent of the lipid effect depends on both the lipid species and the lipid concentration. Addition of the neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) seems to induce a modification of the disorganized structures of the lipid-depleted LHCII and to support the aggregated state of the complex. In contrast, we found that the anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) exert a strong disaggregating effect on the isolated LHCII. LHCII disaggregation was partly suppressed under a high proton concentration and in the presence of cations. The strongest suppression was visible at the lowest pH value (pH 5) and the highest Mg(2+) concentration (40 mM) used in the present study. This suggests that the negative charge of the anionic lipids in conjunction with negatively charged domains of the LHCII proteins is responsible for the disaggregation. Additional measurements by photon correlation spectroscopy and sucrose gradient centrifugation, which were used to gain information about the size and molecular mass of the LHCII aggregates, confirmed the results of the fluorescence spectroscopy. LHCII treated with MGDG and DGDG formed an increased number of aggregates with large particle sizes in the micromm-range, whereas the incubation with anionic lipids led to much smaller LHCII particles (around 40 nm in the case of PG) with a homogeneous distribution.

The xanthophyll cycle activity in kidney bean and cabbage leaves under salinity stress

Seven-day-old kidney bean and cabbage seedlings were treated with 0.1–0.3 M NaCl solutions for 3 days. Chlorophyll content decreased in NaCl-treated Phaseolus seedlings, but did not significantly decrease in Brassica seedlings. Photochemical efficiency of photosystem II at dark-adapted state was similar in both Phaseolus and Brassica. The de-epoxidation state of violaxanthin increased more than sixfold in Phaseolus but showed no significant change in Brassica seedlings during NaCl treatment under low light. Maximum de-epoxidation state of violaxanthin in vivo tested in high light (2000 μmol quanta/(m2 s) increased in salt-stressed Phaseolus but decreased in Brassica seedlings. The nonphotochemical quenching (NPQ) also increased in Phaseolus but decreased in Brassica. This suggests that xanthophyll cycle pigments influence the NPQ in both Phaseolus and Brassica, but in an opposite way. The increase in the de-epoxidation state of violaxanthin in salt-stressed Phaseolus even under low light may be considered an early light signal to protect the pigment-protein complexes from salt-stress induced photodamage. It is proposed that in salt-stressed Brassica, the de-epoxidation is retarded and/or the epoxidation is accelerated leading to the accumulation of violaxanthin and a lower de-epoxidation state. Thus, light-induced violoxanthin cycle operation largely controls the photoprotection of photosynthetic apparatus in kidney bean leaves.

Incorporation of plastoquinone and ubiquinone into liposome membranes studied by HPLC analysis.: The effect of side chain length and redox state of quinone

The efficiency of incorporation of plastoquinones and ubiquinones into phospholipid liposomes has been studied. The representatives of short (PQ1 and UQ1) middle (PQ4 and UQ4) and long (PQ9, UQ9 and UQ10) prenylquinones have been used to investigate the effect of quinone side chain length. The properties of hydroquinones have been also thoroughly examined in relation to the quinone forms. The extraction procedure was modified and further developed which enables removing of nonincorporated quinone by pentane washing and then determination of quinone content inside the lipid bilayer. The quantitatively evaluation of the amount of prenylquinone was assayed by means of HPLC analysis which offers much greater sensitivity and could be easily applied in case of hydroquinones. It has been found that PQ1 and UQ1 as well as their reduced forms were present mainly (about 80%) in the aqueous phase, when attempting to introduce them into phospholipid bilayer. In case of quinones having four and more isoprenyl units in side chain, a high level of quinone incorporation, ranging about 95%, was observed. The results pointed out that when comparing the effects of different exogenous quinones on membrane related processes, one has to consider the effectiveness of their incorporation within lipid bilayer.