Russian Journal of Plant Physiology, Vol. 50, No. 2, 2003, pp. 173–177. From Fiziologiya Rastenii, Vol. 50, No. 2, 2003, pp. 194–199. Original English Text Copyright © 2003 by Latowski, Kostecka-Guga l a, Strza l ka.
Effect of the Temperature on Violaxanthin De-Epoxidation: Comparison of the In Vivo and Model Systems1 D. Latowski, A. Kostecka-Guga l a, and K. Strza l ka Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland; fax: (4812) 633-6907; e-mail:
[email protected] Received October 8, 2001
Abstract—The xanthophyll cycle is a photoprotective mechanism operating in the thylakoid membranes of all higher plants, ferns, mosses and several algal groups. The occurrence of inverted hexagonal domains of monogalactosyldiacylglycerol (MGDG) in the membrane is postulated as an essential factor involved in violaxanthin de-epoxidation. The violaxanthin de-epoxidation was investigated in high-light illuminated Lemna trisulca at three temperatures (4, 12, and 25°C). The temperature dependence of this reaction was compared with kinetics of violaxanthin de-epoxidation at the same temperatures in MGDG micelles and in phosphatidylcholine (PC)–MGDG unilamellar liposomes. In both model systems and in the illuminated plants, a decrease in temperature resulted in lower zeaxanthin production. We found that the presence of MGDG in PC liposomes was necessary for the de-epoxidation reaction. With the increase in MGDG proportion in liposomes, the percentage of transformed violaxanthin was also increasing. We suggest that the violaxanthin de-epoxidation takes place within lipid matrix of the thylakoid membranes inside the MGDG-rich domains. Presence of the reversed hexagonal phase in the thylakoid membranes has been already reported in our previous papers and by other authors using 31P-NMR and freeze-fracturing techniques. Key words: Lemna trisulca - xanthophyll cycle - monogalactosyldiacylglycerol
INTRODUCTION Violaxanthin de-epoxidation is one of two reactions (de-epoxidation of violaxanthin and epoxidation of zeaxanthin) involved in the xanthophyll cycle (Fig. 1). This process was discovered by Sapozhnikov et al. in 1957 [1] and characterized in the 1960s and 1970s by pioneering studies of the groups headed by Yamamoto and Hager [2–4]. Very little progress was made in understanding the role of the xanthophyll cycle until the finding by Deming et al. [5] of the possible relationship between zeaxanthin formation and dissipation of excess light energy. To-day this process is known to play an important role in the protection against photodestruction. Other possible functions suggested for xanthophyll cycle are protection against oxidative stress of lipids, regulation of membrane fluidity, participation in a blue light response, and regulation of ABA synthesis. Unfortunately, molecular mechanism of the xanthophyll cycle reactions remains unclear. The activity of the enzyme responsible for de-epoxidation, vio1The
article was submitted by the authors in English. It was reported at the International Conference “Ecological Physiology of Plants: Problems and Possible Solutions in the XXI Century” (Syktyvkar, Russia, October 2001).
Abbreviations: MGDG—monogalactosyldiacylglycerol; PC— egg-yolk phosphatidylcholine; PCS—photon correlation spectroscopy; VDE—violaxanthin de-epoxidase; ZE—zeaxanthin epoxidase.
laxanthin de-epoxidase (VDE), is strongly pH-dependent. At high lumen pH (above 7.0), VDE is a soluble enzyme, but upon illumination the electron transport chain generates a proton gradient across the thylakoid membrane and the enzyme becomes attached to the membrane. At pH values below 6.0, VDE is fully bound to the membrane [6]. The pH optima of 4.8 for VDE from isolated chloroplasts and 5.2 for the isolated enzyme were reported. VDE was found to be specific for xanthophylls that have the 3-hydroxy, 5,6-epoxy group in a 3S, 5R, 6S configuration and are all trans in the polyene chain [7]. To convert violaxanthin, VDE requires ascorbate as a reductant [3, 4] as well as the major thylakoid lipid monogalactosyldiacylglycerol (MGDG) [7]. This lipid, because of its small head group area and critical packing parameter value superior to one, is a nonbilayer-prone lipid which forms reversed hexagonal phase in water instead of bilayer structures [8]. It is known that MGDG forms hexagonal phases over a wide temperature range from –15°C to 80°C at concentrations higher than 50% lipid in water, and this process also depends on the degree of unsaturation of the acyl chains. Until now, in vitro studies on the VDE activity have been carried out in the system described by Yamamoto [9] which contained violaxanthin, ascorbic acid, enzyme and MGDG. According to Yamamoto [7], violaxanthin might be closed within MGDG micelles, thus being available for VDE. In this article, the results of studies on influence of tempera-
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ture on violaxanthin de-epoxidation in plants (Lemna trisulca) and in the model systems were presented. MATERIALS AND METHODS Violaxanthin was isolated from dark-stored leaves of alfalfa (Medicago sativa) by extraction with acetone and saponification followed by column chromatography on Silica Gel F254 (Merck, Germany) in petroleum ether : acetone (4 : 1, v/v). VDE was isolated and purified from the leaves of 7-day-old wheat seedlings grown at 28°C according to the method described by Hager and Holocher [10]. Its activity was determined according to Yamamoto [9] by dual-wavelength measurements (502, 540 nm) at 25°C using a DW-2000 SLM Aminco spectrophotometer. Lemna trisulca plants were grown for three weeks at low light intensity (approximately 80 µmol/(m2 s)) in Hoagland nutrient solution diluted with source water. The growth temperature was about 25°C. De-epoxidation of violaxanthin was induced by exposure of plants to high light (2000 µmol/(m2 s)) for 3 h at 4, 12, and 25°C. De-epoxidation of violaxanthin in a MGDG reversed hexagonal structures was also performed at 4, 12, and 25°C. The composition of the reaction mixture was the same as for the enzyme activity determination. When the effect of MGDG on kinetics of violaxanthin de-epoxidation reaction was investigated, liposomes with the fixed concentrations of MGDG (12.9 µM) and violaxanthin (0.33 µM) were used, and egg yolk phosphatidylcholine (PC) amount was changed in order to obtain the following MGDG proportions: 5, 15, and 30 mol %. The PC–MGDG liposomes were prepared as follows. The mixture of lipids with violaxanthin in chloroform was evaporated under stream of nitrogen to form
a thin film and dried under vacuum for one hour. The dried lipids were dissolved in ethanol and the solution was injected slowly with a Hamilton syringe into 0.1 M sodium citrate buffer, pH 5.1, under continuous bubbling with nitrogen. The final ethanol concentration did not exceed 1.25%. Subsequently, the liposome suspension was extruded through a polycarbonate membrane with a pore diameter of 100 nm. For electron microscopy analyses, one drop of PC– MGDG liposomes (350 µM lipid concentration) or MGDG structures (12.9 µM lipid concentration) in citrate buffer, pH 5.2, was placed on a Formvar-coated grid and, after 30 s, one drop of staining solution was added. Negative staining was performed with uranyl acetate at room temperature [11]. After 30 s, excess of the solution was drained off with filter paper and the grid was allowed to dry in air. The grids were examined under a JEM 100SX electron microscope (Japan) operating at 80 kV. The diameter of PC–MGDG liposomes and MGDG reversed hexagonal micelle was measured by photon correlation spectroscopy (PCS). A 10 mW He–Ne laser (633 nm) was used as a light source. The selected angle was 90°, the viscosity was 0.890 centipoise, and refractive index was equal to 1.333. All analyses were performed at 25°C and equilibration time of 2 min. Total lipid concentration in the case of PC–MGDG liposomes was 43 µM (30.1 µM PC, 12.9 µM MGDG) and 12.9 µM for MGDG structures. Both liposomes and MGDG reversed hexagonal micelles were suspended in 0.1 M sodium citrate buffer, pH 5.1. PC was purchased from Sigma (United States) and plant MGDG was obtained from Lipid Products (United Kingdom). Pigment separation was performed by reverse phase HPLC using a RP-18 column (5 µm particle size)
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Fig. 2. Time-courses of violaxanthin to zeaxanthin conversion at various temperatures in Lemna trisulca. (a) at 4°C, (b) at 12°C, (c) at 25°C. 䊏—violaxanthin, 䊉—antheraxanthin, 䉱—zeaxanthin.
Fig. 3. Time-courses of violaxanthin to zeaxanthin conversion at various temperatures in the MGDG system. (a) at 4°C, (b) at 12°C, (c) at 25°C. 䊏—violaxanthin, 䊉—antheraxanthin, 䉱—zeaxanthin.
according to the modified method of Gilmore and Yamamoto [12] at the flow rate of 0.7 ml/min. The eluted pigments were monitored at 440 nm and measured according to [12].
cycle studies in vitro, MGDG produces large aggregates. Therefore, we defined a new system, better corresponding to natural conditions of violaxanthin deepoxidation reaction [13], namely the liposome membranes composed of PC and supplemented with MGDG which was found to be necessary for VDE activity. With the rise in MGDG proportion in PC liposomes to a certain level, the percentage of transformed violaxanthin also increased (Fig. 4). However, at 35 mol % of MGDG, the de-epoxidation rate became significantly lower due to liposome aggregation, and the suspension became turbid. The increase in turbidity was followed by sedimentation of the lipid aggregates formed. These changes were caused probably by the fusion of liposomes or the appearance of MGDG aggregates in its high proportion to PC in the lipid mixture [8, 14]. The liposome suspension with the MGDG content up to 30 mol % was transparent and showed no tendency to aggregate. The presence of liposomes and absence of aggregates from such suspension was confirmed by electron microscopy and PCS (data not shown). Violax-
RESULTS The comparison of the temperature effect on violaxanthin de-epoxidation in vivo (Lemna trisulca) with the model system described by Yamamoto [9] showed similarity in the kinetics of the violaxanthin de-epoxidation (Figs. 2, 3) although the rates of the corresponding reactions at the same temperatures in the both systems were not exactly identical. These minor variations result from the structural differences between the systems. In the case of Lemna trisulca, the violaxanthin de-epoxidation proceeds in the thylakoid membrane, whereas in Yamamoto system it takes place inside the MGDG reversed hexagonal structures. As follows from our electron microscopic and PCS measurements, in the system which is commonly used in the xanthophyll RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 4. Effect of MGDG proportion in PC–MGDG liposomes on the level of xanthophylls after 20 min of the violaxanthin de-epoxidation reaction at 25°C. 䊏—Violaxanthin, 䊉—antheraxanthin, 䉱—zeaxanthin.
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anthin de-epoxidation was found to be strongly dependent not only on the concentration of MGDG, but also on the ratio of MGDG to PC in liposomes even if the absolute amount of MGDG in the reaction mixture and its proportion to violaxanthin and VDE were constant (Fig. 5). When the effect of temperature on violaxanthin deepoxidation was measured in such liposomes, the general course of the kinetics was like that in vivo and in Yamamoto system (data not shown). The characteristic feature of the kinetics in all systems is a more pronounced effect of the temperature on the first stage of the de-epoxidation, i.e., the conversion of the violaxanthin to antheraxanthin, whereas the second stage (the conversion of the antheraxanthin to zeaxanthin) seems to be less sensitive to temperature. DISCUSSION The results presented show that effects of the temperature on violaxanthin de-epoxidation kinetics both in vivo and in the model system are very similar (Figs. 2, 3). Under temperature changes, it is the first stage of violaxanthin de-epoxidation (transformation of violaxanthin to antheraxanthin) that determines the reaction progress. Like in [15], we also demonstrated that the flip-flop of antheraxanthin, which is probably a necessary step in the antheraxanthin to zeaxanthin conversion, is not the rate-limiting factor in the zeaxanthin formation. On the other hand, the presence of MGDG in PC-liposomes was found to be indispensable for the violaxanthin de-epoxidation reaction, and the rate of violaxanthin conversion was strongly dependent on concentration and proportion of the MGDG in the liposome membrane (Figs. 4, 5). As we have demonstrated,
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Fig. 5. Time-courses of violaxanthin to zeaxanthin conversion in PC–MGDG liposomes at 25°C. PC and MGDG concentrations were the following: (a) 245.1 µM and 12.9 µM (5 mol % of MGDG); (b) 73.1 µM and 12.9 µM (15 mol % of MGDG); (c) 30.1 µM and 12.9 µM (30 mol % of MGDG) and violaxanthin concentration was 0.33 µM in all cases. 䊏—Violaxanthin, 䊉—antheraxanthin, 䉱—zeaxanthin.
the rate of violaxanthin to antheraxanthin conversion depends on MGDG/PC ratio in the liposome membrane even if the absolute amount of MGDG in the reaction mixture and its proportion to violaxanthin and VDE remains constant (Fig. 5). These results may suggest that VDE binds only to certain membrane domains which are rich in MGDG and the de-epoxidation reactions take place in these domains. This concept is supported by recent study, in which MGDG was found to be four-fold more efficient in precipitating VDE as compared to another most common lipid in thylakoids, digalactosyldiacylglycerol, and up to 38 times more efficient than other lipids [16]. It is well known that nonbilayer prone lipids (e.g., MGDG) may form domains of reversed micelles in model lipid membranes and it has been reported that such structures exist in biological membranes [17, 18]. The presence of MGDG reversed hexagonal phase was detected by 31PNMR in PC–MGDG liposome membranes used in our experiments [19]. It is possible that the MGDG
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reversed micelles existing in the membrane may facilitate flip-flop of the antheraxanthin. The important role in this process may play the fact that the MGDG-containing inverted micelles contain the most unsaturated fatty acids, so the fluidity of this part of the membrane is higher than in other regions of the bilayer. Violaxanthin to be converted to antheraxanthin probably has to enter the MGDG-enriched domains by lateral diffusion, and the higher the temperature the easier the access of the violaxanthin to the MGDG rich domains. On the other hand, the higher the MGDG/PC ratio the greater the amount of such domains in the liposomal membrane. This is the way to shorten the diffusion path of violaxanthin molecules to these domains that results in higher rate of violaxanthin de-epoxidation (Figs. 4, 5). Apart from VDE, no other proteins or pigment–protein complexes were present in the model lipid membrane or required for this reaction. These findings confirm that violaxanthin to zeaxanthin transformation can take place within the lipid matrix of thylakoid membranes and not within pigment–protein complexes as suggested by Thayer and Björkman [20]. ACKNOWLEDGMENTS This work was supported by a grant no. 6 P04A 07321 from the Committee for Scientific Research (KBN) of Poland. REFERENCES 1. Sapozhnikov, D.I., Krasovskaya, T.A., and Maevskaya, A.N., Change in the Interrelationship of the Basic Carotenoids of the Plastids of Green Leaves under the Action of Light, Dokl. Akad. Nauk SSSR, 1957, vol. 113, pp. 465–467. 2. Yamamoto, H.Y., Nakayama, T.O.M., and Chichester, C.O., Studies on the Light and Dark Interconversions of Leaf Xanthophylls, Arch. Biochem. Biophys., 1962, vol. 97, pp. 168–173. 3. Yamamoto, H.Y., Biochemistry of the Violaxanthin Cycle in Higher Plants, Pure Appl. Chem., 1979, vol. 51, pp. 639–648. 4. Hager, A., Lichtbedingte pH-Erniedrigung in einem Chloroplasten Kompartiment als Ursache der enzymatischen Violaxanthin zu Zeaxanthin Umwandlung; Beziehungen zur Photophosphorilierung, Planta, 1969, vol. 89, pp. 224–243. 5. Deming, B., Winter, K., Kruger, A., and Czygan, F.-C., Photoinhibition and Zeaxanthin Formation in Intact Leaves, Plant Physiol., 1987, vol. 84, pp. 218–224. 6. Bratt, C.E., Arvidsson, P.-O., Carlsson, M., and Akerlund, H.-E., Regulation of Violaxanthin De-Epoxidase Activity by pH and Ascorbate Concentration, Photosynth. Res., 1995, vol. 45, pp. 169–175.
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7. Yamamoto, H.Y. and Higashi, R.M., Violaxanthin Deepoxidase, Lipid Composition and Substrate Specificity, Arch. Biochem. Biophys., 1978, vol. 190, pp. 514–522. 8. Israelaschvili, J.N. and Mitchell, D.J., A Model for the Packing of Lipids in Bilayer Membranes, Biochim. Biophys. Acta, 1975, vol. 389, pp. 13–19. 9. Yamamoto, H.Y., Xanthophyll Cycle, Methods Enzymol., 1995, vol. 110, pp. 303–312. 10. Hager, A. and Holocher, K., Localization of the Xanthophyll Cycle Enzyme Violaxanthin Deepoxidase within the Thylakoid Lumen and Abolition of Its Mobility by a (Light-Dependent) pH Decrease, Planta, 1994, vol. 192, pp. 581–589. 11. Enoch, H.G. and Stritmatter, Ph., Formation and Properties of 1000 Å-Diameter, Single-Bilayer Phospholipid Vesicles, Proc. Natl. Acad. Sci. USA, 1979, vol. 76, pp. 145–149. 12. Gilmore, A.M. and Yamamoto, H.Y., Resolution of Lutein and Zeaxanthin Using a Nonendcapped, Lightly Carbon-Loaded C18 High-Performance Liquid Chromatographic Column, J. Chromatogr., 1991, vol. 543, pp. 137–145. 13. Latowski, D., Kostecka, A., and Strza l ka, K., Effect of Monogalactosyldiacylglycerol and Other Thylakoid Lipids on Violaxanthin De-Epoxidation in Liposomes, Biochem. Soc. Trans., 2000, vol. 28, part 6, pp. 812–814. 14. Sprague, G.S. and Staehelin, L.A., Effects of Reconstitution Method on the Structural Organization of Isolated Chloroplast Membrane Lipids, Biochim. Biophys. Acta, 1984, vol. 777, pp. 306–322. 15. Arvidsson, P.-O., Carlsson, H., Stefansson, H., Albertsson, P.-A., and Åkerlund, H.-E., Violaxanthin Accessibility and Temperature Dependency for De-Epoxidation in Spinach Thylakoid Membranes, Photosynth. Res., 1997, vol. 52, pp. 39–48. 16. Rockholm, D.C. and Yamamoto, H.Y., Purification of a 43-Kilodalton Lumen Protein from Lettuce by LipidAffinity Precipitation with Monogalactosyldiacylglyceride, Plant Physiol., 1996, vol. 110, pp. 697–703. 17. De Kruijff, B., Verkleij, A.J., van Echteld, C.J.A., Gerritsen, W.J., Mombers, C., Noordam, P.C., and Gier, J., The Occurrence of Lipid Particles in Lipid Bilayers as Seen by 31P-NMR and Freeze-Fracture Electron Microscopy, Biochim. Biophys. Acta, 1979, vol. 555, pp. 200–209. 18. Walde, P., Giuliani, A.M., Boicelli, C.A., and Luisi, P.L., Phospholipid-Based Reversed Micelles, Chem. Phys. Lipids, 1990, vol. 53, pp. 265–288. 19. Latowski, D., Kruk, J., Burda, K., Skrzynecka-Jaskier, M., Kostecka-Guga l a, A., and Strza l ka, K., Kinetics of Violaxanthin De-epoxidation by Violaxanthin De-epoxidase, a Xanthophyll Cycle Enzyme, Is Regulated by Membrane Fluidity in Model Lipid Bilayers, Eur. J. Biochem., 2002, vol. 269, pp. 4656–4665. 20. Thayer, S.S. and Björkman, O., Carotenoid Distribution and Deepoxidation in Thylakoid Pigment–Protein Complexes from Cotton Leaves and Bundle-Sheath Cells of Maize, Photosynth. Res., 1992, vol. 33, pp. 213–225.
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