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Pigment composition of PS II pigment protein complexes purified by anion exchange chromatography. identification of xanthophyll cycle pigment binding proteins
.........,......, ••••••AL.F.
Short Communication
© 1997 by Gustav Fischer Verlag, Jena
Pigment Composition of PS II Pigment Protein Complexes Purified by Anion Exchange Chromatography. Identification of Xanthophyll Cycle Pigment Binding Proteins REIMUND
Gossl, *,
MICHAEL RICHTER2,
and ALOYSIUS WILD 2
1
Institut fur Botanik, Universitlit Leipzig, Johannisallee 21, 04103 Leipzig, Germany
2
Institut fur Allgemeine Botanik, Johannes Gutenberg-Universitat Mainz, Saarsrra& 21,55099 Mainz, Germany
Received August 8, 1996 . Accepted November 6,1996
Summary
The pigment composition of the chlorophyll binding proteins of Photosystem II (PS II) of spinach
(Spinacea oleracea L.) has been determined using sucrose gradient ultracentrifugation, anion exchange
chromatography and HPLC based pigment analysis. The xanthophyll cycle pigments violaxanthin, antheraxanthin and zeaxanthin were exclusively found in the proteins of the outer PS II antenna, with the highest amounts being present in the minor chlorophyll alb binding proteins CP 29 and CP 26. PS II core particles containing the reaction centre proteins 01, 02, cytochrome b559 and the proteins of the inner antenna CP 47 and CP 43 bind ~carotene as the only carotenoid. The presence of the xanthophyll cycle pigments in the PS II antenna proteins gives further indication to their proposed role in the dissipation of excess excitation energy. The purification procedure presented in this study differs from previous isolation methods (i. e. isoelectric focusing) used to determine the pigment composition of the PS II proteins. The pigment stoichiometries shown here therefore provide further insight in the pigmentation of the PS II chlorophyll binding complexes.
Key words: Spinacea oieracea, anion txchange chromatography, LHC I/, CP 29, CP 26, PS II CO" particles, xanthophyU cycle pigments. AbbmJiations: chI = chlorophyll; CP = chlorophyll protein; OM = dodecyl maltoside; HPLC = high performance liquid chromatography; LHC II = light harvesting complex of photosystem II; PAGE = polyacrylamide gel electrophoresis; PAR= photosynthetically active radiation; PS II = photosystem II; SOS = sodium dodecyl sulphate. Introduction
In recent times many studies have underlined the important physiological role of the so-called xanthophyll cycle (for a review see Pfundel and Bilger, 1994). Zeaxanthin that is produced by deepoxidation of violaxanthin and antheraxanthin is thought to induce a protecting mechanism against * Correspondence. j. Plant PhysioL \>0/. 151. pp. 115-119 (1997)
an excess of absorbed excitation energy. Zeaxanthin increases non-radiative energy dissipation (Goss et al., 1995), possibly by a direct quenching of singlet excited chlorophyll a (Frank et al., 1994) or by the stimulation of LHC II aggregation (Horton and Ruban, 1992). Besides the informations resulting from fluorescence measurements only few studies were carried out to clarify the localization of the xanthophyll cycle pigments in the different pigment protein complexes of PS II. Early evidence for a
116
REIMUND Goss, MICHAEL RICHTER, and ALOYSIUS WILD
preferential binding of the xanthophyll cycle pigments to the PS II antenna proteins can be derived from a study by Siefermann-Harms (1984). Refined analysis by Bassi et al. (1993) and Ruban et al. (1994) showed that violaxanthin, antheraxanthin and zeaxanthin are mainly bound to the minor chlorophyll alb binding proteins of the PS II antenna. In contrast Lee and Thornber (1995) found the xanthophyll cycle pigments more equally distributed among the different PS II antenna polypeptides including the major LHC II. These conflicting results arose from different separation techniques and diversity in the plant material used in these studies. Bassi et al. (1993) and Ruban et al. (1994) separated PS II pigment protein complexes of maize and spinach, respectively by means of isoelectric focusing, whereas Lee and Thornber (1995) performed native gel electrophoresis on thylakoid membranes of ~arley. It is the aim of the present study to present pigment stoichiometries of purified PS II pigment proteins of spinach derived from an anion exchange chromatography separation. These results from another analytical approach provide further clarification of the localization of the xanthophyll cycle pigments. Materials and Methods
Preparation ofthylakoid membranes Spinach plants were cultivated in a growth chamber at the irradiance of 125 ~mol m -2 s-1 photosynthetically active radiation (PAR) with an 11 h photoperiod. Isolated thylakoids were prepared according to Richter et al. (1990). Grana membranes were obtained by solubilization of isolated thylakoids in Triton X-100 for 15 min on ice. The detergent per chlorophyll ratio was 12.5 mg Triton X-I00 per mg chlorophyll. The solubilized thylakoids were then centrifuged for 30 min at 40,000 g., (Beckman }2-21, rotor }A-20). The pellet containing the grana membranes was then used for further purification steps.
Sucrose density gradient centrifugation Grana membranes were solubilized in 1 % OM for 30 min on ice and then spun for 15 min at 40,000 g., at 4·C. The supernatant was then rapidly loaded Onto a continuous 0.1-0.8 mollL sucrose gradient containing 40 mmol/L HEPES pH 6,7 and 0.06 % OM. The ultracentrifugation was done using a swing out rotor (TST 28/38, Kontron) at 100,000 g., for 16 hat 4·C.
Preparation of CP 29 and CP 26 containing PS II core particles CP 29 and CP 26 containing core particles were isolated following strictly the procedure described by Mishra and Ghanotakis (1994).
Anion txchange chromatography Anion exchange chromatography was performed with both the fraction 2 of sucrose gradient centrifugation and the CP 29 and CP 26 containing core particles as starting material. 10 mmol/L MgC12 were added to band 2 obtained by sucrose gradient ultracentrifugation, that contained LHC II, CP 29 and CP 26. Complete precipitation of the respective proteins was achieved within 30 min incubation at room temperature. The precipitated
proteins were solubilized in 1 % OM for 15 min on ice and then spun for 15 min at 40,000 g., at 4·C. The resulting supernatant was immediately loaded onto a 2.5 X 10 cm anion exchange column (Q-Sepharose FF, Pharmacia). CP 29 and CP 26 containing core particles were solubilized in 1 % OM for 15 min on ice, centrifuged and loaded onto the anionexchange column. In both cases elution of the proteins was performed with a continuous KCl gradient (30 to 300 mmollL KCl in 90 min) in 20 mmol/L HEPES pH 7.5 and 0.025 % OM at a flow rate of 1 mLl min. The elution of the differen~ proteins was detected by their absorption at 280 nm.
Pigment analysis The pigment composition of the purified pigment protein complexes was determined using reversed phase high performance liquid chromatography (HPLC). The HPLC separation was based on a method described by Thayer and Bjorkman (1990) with a modified solvent A (acetonitrile: methanol 85: 15 was exchanged by acetonitrile: tris-HCl pH 7.8 98: 2). The calibration of the HPLC system was done by injection of defined amounts of purified pigments and by regression analysis of the integration areas over the molar amounts of standards. The concentrations of the purified standards •were calculated using extinction coefficients reported by Hager and Meyer-Bertenrath (1966). ChI alb contents were determined according to Ziegler and Egle (1965).
Absorption spectroscopy Absorption spectra of the isolated pigment proteins were taken with a Shimadzu MPS-2000 spectrophotometer.
SDS-PAGE The polypeptide composition and the state of purification was analyzed using denaturing polyacrylamide gel electrophoresis as described by Laemmli (1970) and silver staining according to Heukeshoven and Demick (1985).
Results
CP 29 and CP 26 from the different isolation methods were identical with regard to the polypeptide pattern, pigment stoichiometries and spectroscopy. We, therefore, do not differentiate between the two methods in the presentation of the respective data.
SDS-PAGE Fig. 1 shows the polypeptide composition of isolated PS II core complexes, LHC II, CP 29 and CP 26 after separation by anion exchange chromatography. PS II core complexes (Fig. 1 A) consisted of the proteins of the inner antenna CP 47 and CP 43 and the proteins Dl, D2 and Cyt b 559 of the PS II reaction centre. The (X- and ~-subunits of Cyt b559 with molecular masses of 9 and 4 kDa, respectively are not resolved as defined bands in the applied gel system (12 % acrylamide). Their presence in the purified PS II core particles was confirmed using gels with an acrylamide concentration of 15 or 18 % (data not shown). The purified LHC II of spinach (Fig. 1 B) showed two apoproteins with molecular masses of 27 and 25 kDa, respectively (see also Ryrie et al., 1980). The
Pigment composition of PS II pigment protein complexes
67
67
45
117
67 . . .
45 45 • • 45
29
29
i
I
29 29 21
I:' .....
r
27lcDa 25lcDa
_~
y
~,
:,~
'::,.CP26 :. .~~
...
21
21
..::
21
12.5
12.5 12.5 12.5 1
2
lA
1
2
IB
1
2
Ie
1
2
ID
Fig. 1: Polypeptide composition of the purified PS II pigment protein complexes as detected by SDS-polyacrylamide gel electrophoresis.
Fig.IA: Lane 1: Molecular weight markers, lane 2: PS II core particles. Fig.IB: Lane 1: Molecular weight markers, lane 2: LHC II. Fig. Ie: Lane 1: Molecular weight markers, lane 2: CP 29. Fig. 1 D: Lane 1: Molecular weight markers, lane 2: CP 26.
minor chlorophyll alb binding proteins CP 29 (Fig. 1 C) and CP 26 (Fig. 1 D) migrated according to their molecular weight, with CP 29 being visible just below the 29 kDa molecular weight marker.
Absorption spectroscopy and chlorophyll alb ratios LHC II contained high amounts of chlorophyll b (chI alb ratio 1.3), that were clearly visible in the absorption spectrum (Fig. 2 B) as peaks at 472 and 651 nm. The purified minor chlorophyll alb binding protein CP 29 showed a chlorophyll alb ratio of about 3.1. This value is slightly higher than the chlorophyll alb ratio of 2.9 mentioned by Bassi et aI. (1993), but significantly lower than the ratio of 4.0 proposed by Ruban et aI. (1994). The absorption spectrum of CP 29
(Fig. 2 A) was dominated by chlorophyll a absorption with maxima at 436 and 676 nm. Chlorophyll b and the xanthophylls bound to CP 29 became visible as a pronounced shoulder in the range 450 to 480 nm. Interestingly the absorption spectrum of CP 29 showed a characteristic shoulder at 636 nm, that is also described by Dainese et aI. (1990), Ruban et aI. (1994) and Machold and Meister (1979), who first resolved CP 29. CP 26, in comparison to CP 29, contained higher amounts of chlorophyll b (chlorophyll alb ratio 2.8). Dainese and Bassi (1991) reported lower chlorophyll alb ratios for CP 26 (2.2), whereas Ruban et aI. (1994) found higher values (2.9). The increased content of chlorophyll b became visible as a more pronounced chlorophyll b absorption berween 450 and 480 nm (Fig. 2 A). The absorption maxima of chlorophyll a were the same when compared to
118
REIMUND Goss, MICHAEL RICHTER, and ALoYSIUS WILD
Absorption (reI. units)
0,5
0,4
Table 1: Pigment contents (mol pigment) per 100 mol of chlorophyll a of rhe purified PS II pigment protein complexes. Pigment proteins were purified from dark adapted spinach leaves and light treated thylakoids (500 ~mol m- 2 S-l PAR for 30 min in the presence of 30 mM ascorbate), respectively.
neo
0,4
0,3
0,3 0,2 0,2 0,1
0,1
o
0
350 400 450 500 550 600 650 700 750 nm
VIO
ant lut
dark thylakoids 8.9 8.9 0.7 LHC II 13.4 3.7 CP 29 8.9 15.0 0.6 CP 26 8.6 9.3 0.6 PS II core light thylakoids 9.3 LHC II 16.5 CP 29 8.5 CP 26 9.2 PS II core
zea chi b chi a pheo
~-car
22.5 0.3 35.9 100 29.1 72.7 100 15.4 33.4 100 14.2 36.3 100 100 5.7
13.7
100 100 100 100 100 6.1
10.8
4.2 0.5 21.8 5.4 40.9 2.2 26.8 72.5 8.3 13.6 3.4 31.7 4.4 12.6 1.8 36.8
1.7 1.4 16.2
1.6 . 1.2 15.9
neo: neoxanrhin; vio: violaxanthin; ant: anrheraxanrhin; lut: lutein; zea: zeaxanrhin; chi b: chlorophyll b; chi a: chlorophyll a; pheo: pheophytin; ~-car: ~-carotene. Table 1 shows the mean values of pigment contents of rhree individual separations wirh a standard deviation < 8 %. 417 nm, ~-carotene could be identified by its absorption in the range 450 to 500 nm.
Absorption (rei. units)
0,6
0,15
0,1
0,4
0,05
0,2
o
0 350 400 450 500 550 600 650 700 750 nm
Hg.2A: Absorption spectra ofCP 29 (solid line) and CP 26 (dotted line) at room temperature. Fig. 2 8: Absorption spectra of PS II core particles (solid line) and LHC II (dotted line) at room temperature.
CP 29. Purified PS II core complexes did not contain chlorophyll b (see Table 1). The absorption spectrum was clearly dominated by chlorophyll a peaking at 435 and 673 nm (Fig. 2 B). Pheophytin became visible as a pronounced shoulder at
Pigment composition Table 1 (upper part) shows the pigment contents (mol) of the purified PS II pigment protein complexes isolated from dark adapted spinach leaves per 100 mol of chlorophyll a. The highest amounts of xanthophyll cycle pigments (i.e. violaxanthin) were found in the minor chlorophyll alb binding proteins CP 29 and CP 26 with values of 15.0 and 9.3 mol violaxanthin per 100 mol chlorophyll a, respectively. LHC II also contained violaxanthin, but with lower amounts being present (3.7 mol violaxanthin per 100 mol chlorophyll a). The entichment of xanthophyll cycle pigments in the minor chlorophyll alb binding proteins was also seen in pigment proteins purified from light treated isolated thylakoids. After illumination with 500 J.l.mol m- 2 s-1 (PAR) for 30 min in the presence of ascorbate and subsequent purification CP 29 bound 3.4 mol zeaxanthin and 8.3 mol violaxanthin per 100 mol chlorophyll a, CP 26 1.8 mol zeaxanthin and 4.4 mol violaxanthin, while 2.2 mol violaxanthin but no zeaxanthin could be detected in the peripheral LHC II (Table 1 lower part). Comparison with the pigment contents of light treated isolated thylakoids (4.2 mol violaxanthin and 5.4 mol zeaxanthin) reveals that after violaxanthin deepoxidation and subsequent purification part of the zeaxanthin is no longer bound to the antenna proteins. From Table 1 it becomes obvious that the pigments of the xanthophyll cycle were only present in the pigment protein complexes of the outer PS II antenna. PS II core complexes containing the proteins of the reaction centre and the inner antenna only bound chlorophyll a, pheophytin and ~-caro tene. Identical results were obtained for PS II core complexes prepared from illuminated thylakoids.
Pigment composition ofPS II pigment protein complexes
Discussion
The pigment stoichiometries of the purified PS II pigment protein complexes described in this study show that the xanthophyll cycle pigments are exclusively bound to the proteins of the outer PS II antenna. They are enriched in the minor chlorophyll alb binding proteins CP 29 and CP 26, whereas the peripheral LHC II only binds them at a smaller xanthophyll/chlorophyll ratio. This finding is in agreement with results published by Bassi et al. (1993) and Ruban et al. (1994), where the majority of the xanthophyll cycle pigments is also found in CP 29, CP 26 and CP 24. However one should consider that a significant part of the xanthophyll cycle pigments must be bound to LHC II, given the assumption that the latter contains four times the chlorophyll of the minor chlorophyll alb binding proteins (Dainese and Bassi, 1991). The complete absence of zeaxanthin in LHC II and the diminished contents in CP 29 and CP 26 purified from light treated isolated thylakoids could be due to a decreased stability in the pigment protein binding in the case of zeaxanthin. Our data would also be consistent with a recent suggestion by Havaux and Tardy (1995). These authors proposed that zeaxanthin formed in strong light might interact with the membrane lipid phase rather than the pigment proteins, ~hereby simultaneously providing protection against heat and light stress. Both explanations would predict higher amounts of zeaxanthin in the free pigment when compared to the concentrations of free violaxanthin in purifications using dark adapted plants as starting material. As Bassi et al. (1993) and Ruban et al. (1994) use identical isolation procedures (isoelectric focusing), the pigment compositions of the isolated pigment proteins derived from anion exchange chromatography provide an alternative approach to clarify the localization of the xanthophyll cycle pigments. Recent studies by Jahns (1995) and Lee and Thornber (1995) showed an association of the xanthophyll cycle pigments with the PS II core complex. It is, therefore, important to state that in our preparations PS II core particles without contaminating proteins of the outer antenna do not contain pigments of the xanthophyll cycle. They solely bind chlorophyll a, pheophytin and ~-carotene. This is in agreement with data published by Alfonso et al. (1994). Frank et al. (1994) proposed that zeaxanthin might act as a direct quencher of chlorophyll a in the singlet excited state. According to Dainese et al. (1992) the function of zeaxanthin in non-photochemical energy dissipation is favoured by its specific binding to the minor chlorophyll alb binding proteins which in turn are located in between the peripheral antenna (i.e. LHC II) and the PS II core. On the other hand, taking into account results that the PS II antenna is arranged as a very shallow funnel (Schatz et al., 1988; Jennings et al., 1993) and that the funnel organization does not playa major role in energy trapping the importance of the localization of the quencher might be questioned.
119
Acknowledgements
Our thanks are due to Prof. Dr. C. Wilhelm for critically reading the manuscript and to Ralf Nickel for his help regarding all aspects of anion exchange chromatography.
References ALFONSO, M., G. MONTOYA, R. CASES, R. RODRIGUEZ, and R. PI COREL: Biochemistry 33,10494-10500 (1994). BASSI, R., B. PINEAU, P. DAINESE, and J. MARQUARDT: Eur. J. Biochern. 212, 297-303 (1993). BERTHOLD, D. A., G. T. BABCOCK, and C. F. YOCUM: FEBS 13412, 231-234 (1981). DAINESE, P., G. HOYER-HANSEN, and R. BASSI: Photochem. PhotobioI. 51, 693-703 (1990). DAINESE, P. and R. BASSI: J. BioI. Chern. 266, 8136-8142 (1991). DAINESE, P., J. MARQUARDT, B. PINEAU, and R. BASSI: In: MURATA, N. (ed.): Research in Photosynthesis, Vol. IL pp. 287-290. Kluwer Academic Publishers, Netherlands (1992). FRANK, H. A., A. CUA, V. CHYUWAT, A. YOUNG, D. GOSZTOLA, and P. M. R. WASIELEWSKI: Photosynth. Res. 41,389-395 (1994). Goss, R., M. RiCHTER, and A. WILD: J. Photochem. Photobiol. B: Biology 27, 147-152 (1995). HAvAUX, M. and F. TARDY: In: MATHIS, P. (ed.): Photosynthesis: from Light to Biosphere, Vol. Iv, pp. m-782. Kluwer Academic Publishers, Netherlands (1995). HAGER, A. and T. MEYER-BERTENRATH: Planta 69, 128-217 (1966). HEUKESHOVEN, J. and R. DERNICK: Electrophoresis 6, 103-112 (1985). HORTON, P. and A. V. RUBAN: Photosynth. Res. 34, 375-385 (1992). JAHNS, P.: Plant Physiol. 108, 149-156 (1995). JENNINGS, R. c., R. BASSI, F. M. GARLASCHI, P. DAINESE, and G. ZUCCHELLI: Biochemistry 32,3203-3210 (1993). WMMLI, U. K: Nature 227, 680-685 (1970). LEE, A. L. C. and J. P. THORNBER: Plant Physiol. 107, 565-574 (1995). MACHOLD, o. and A. MEISTER: Biochim. Biophys. Acta 546, 472480 (1979). MISHRA, R. K and D. F. GHANOTAKIS: Photosynth. Res. 42, 37-42 (1994). PFUNDEL, E. and W. BILGER: Photosynth. Res. 42, 89-109 (1994). RiCHTER, M., w. RUHLE, and A. WILD: Photosynth. Res. 24, 229235 (1990). RYRIE, I. J., J. M. ANDERSSON, and D. J. GOODCHILD: Eur. J. Biochern. 107, 345-354 (1980). RUBAN, A. v., A. J. YOUNG, A. A. PASCAL, and P. HORTON: Plant Physiol. 104, 227-234 (1994). SCHATZ, G., H. BROCK, and A. R. HOLZWARTH: Biophys. J. 54, 397-405 (1988). SIEFERMANN-HARMS, D.: Photochem. Photobiol. 40, 507-512 (1984). THAYER, S. S. and O. BJORKMAN: Photosynth. Res. 23, 331-343 (1990). VAN LEEUWEN, P. J., M. C. NIEVEEN, and H. J. VAN GORKOM: Photosynth. Res. 28, 149-153 (1991). ZIEGLER, R. and K EGLE: Beitr. BioI. Pflanzen 41, 11-37 (1965).
Short Communication
© 1997 by Gustav Fischer Verlag, Jena
Pigment Composition of PS II Pigment Protein Complexes Purified by Anion Exchange Chromatography. Identification of Xanthophyll Cycle Pigment Binding Proteins REIMUND
Gossl, *,
MICHAEL RICHTER2,
and ALOYSIUS WILD 2
1
Institut fur Botanik, Universitlit Leipzig, Johannisallee 21, 04103 Leipzig, Germany
2
Institut fur Allgemeine Botanik, Johannes Gutenberg-Universitat Mainz, Saarsrra& 21,55099 Mainz, Germany
Received August 8, 1996 . Accepted November 6,1996
Summary
The pigment composition of the chlorophyll binding proteins of Photosystem II (PS II) of spinach
(Spinacea oleracea L.) has been determined using sucrose gradient ultracentrifugation, anion exchange
chromatography and HPLC based pigment analysis. The xanthophyll cycle pigments violaxanthin, antheraxanthin and zeaxanthin were exclusively found in the proteins of the outer PS II antenna, with the highest amounts being present in the minor chlorophyll alb binding proteins CP 29 and CP 26. PS II core particles containing the reaction centre proteins 01, 02, cytochrome b559 and the proteins of the inner antenna CP 47 and CP 43 bind ~carotene as the only carotenoid. The presence of the xanthophyll cycle pigments in the PS II antenna proteins gives further indication to their proposed role in the dissipation of excess excitation energy. The purification procedure presented in this study differs from previous isolation methods (i. e. isoelectric focusing) used to determine the pigment composition of the PS II proteins. The pigment stoichiometries shown here therefore provide further insight in the pigmentation of the PS II chlorophyll binding complexes.
Key words: Spinacea oieracea, anion txchange chromatography, LHC I/, CP 29, CP 26, PS II CO" particles, xanthophyU cycle pigments. AbbmJiations: chI = chlorophyll; CP = chlorophyll protein; OM = dodecyl maltoside; HPLC = high performance liquid chromatography; LHC II = light harvesting complex of photosystem II; PAGE = polyacrylamide gel electrophoresis; PAR= photosynthetically active radiation; PS II = photosystem II; SOS = sodium dodecyl sulphate. Introduction
In recent times many studies have underlined the important physiological role of the so-called xanthophyll cycle (for a review see Pfundel and Bilger, 1994). Zeaxanthin that is produced by deepoxidation of violaxanthin and antheraxanthin is thought to induce a protecting mechanism against * Correspondence. j. Plant PhysioL \>0/. 151. pp. 115-119 (1997)
an excess of absorbed excitation energy. Zeaxanthin increases non-radiative energy dissipation (Goss et al., 1995), possibly by a direct quenching of singlet excited chlorophyll a (Frank et al., 1994) or by the stimulation of LHC II aggregation (Horton and Ruban, 1992). Besides the informations resulting from fluorescence measurements only few studies were carried out to clarify the localization of the xanthophyll cycle pigments in the different pigment protein complexes of PS II. Early evidence for a
116
REIMUND Goss, MICHAEL RICHTER, and ALOYSIUS WILD
preferential binding of the xanthophyll cycle pigments to the PS II antenna proteins can be derived from a study by Siefermann-Harms (1984). Refined analysis by Bassi et al. (1993) and Ruban et al. (1994) showed that violaxanthin, antheraxanthin and zeaxanthin are mainly bound to the minor chlorophyll alb binding proteins of the PS II antenna. In contrast Lee and Thornber (1995) found the xanthophyll cycle pigments more equally distributed among the different PS II antenna polypeptides including the major LHC II. These conflicting results arose from different separation techniques and diversity in the plant material used in these studies. Bassi et al. (1993) and Ruban et al. (1994) separated PS II pigment protein complexes of maize and spinach, respectively by means of isoelectric focusing, whereas Lee and Thornber (1995) performed native gel electrophoresis on thylakoid membranes of ~arley. It is the aim of the present study to present pigment stoichiometries of purified PS II pigment proteins of spinach derived from an anion exchange chromatography separation. These results from another analytical approach provide further clarification of the localization of the xanthophyll cycle pigments. Materials and Methods
Preparation ofthylakoid membranes Spinach plants were cultivated in a growth chamber at the irradiance of 125 ~mol m -2 s-1 photosynthetically active radiation (PAR) with an 11 h photoperiod. Isolated thylakoids were prepared according to Richter et al. (1990). Grana membranes were obtained by solubilization of isolated thylakoids in Triton X-100 for 15 min on ice. The detergent per chlorophyll ratio was 12.5 mg Triton X-I00 per mg chlorophyll. The solubilized thylakoids were then centrifuged for 30 min at 40,000 g., (Beckman }2-21, rotor }A-20). The pellet containing the grana membranes was then used for further purification steps.
Sucrose density gradient centrifugation Grana membranes were solubilized in 1 % OM for 30 min on ice and then spun for 15 min at 40,000 g., at 4·C. The supernatant was then rapidly loaded Onto a continuous 0.1-0.8 mollL sucrose gradient containing 40 mmol/L HEPES pH 6,7 and 0.06 % OM. The ultracentrifugation was done using a swing out rotor (TST 28/38, Kontron) at 100,000 g., for 16 hat 4·C.
Preparation of CP 29 and CP 26 containing PS II core particles CP 29 and CP 26 containing core particles were isolated following strictly the procedure described by Mishra and Ghanotakis (1994).
Anion txchange chromatography Anion exchange chromatography was performed with both the fraction 2 of sucrose gradient centrifugation and the CP 29 and CP 26 containing core particles as starting material. 10 mmol/L MgC12 were added to band 2 obtained by sucrose gradient ultracentrifugation, that contained LHC II, CP 29 and CP 26. Complete precipitation of the respective proteins was achieved within 30 min incubation at room temperature. The precipitated
proteins were solubilized in 1 % OM for 15 min on ice and then spun for 15 min at 40,000 g., at 4·C. The resulting supernatant was immediately loaded onto a 2.5 X 10 cm anion exchange column (Q-Sepharose FF, Pharmacia). CP 29 and CP 26 containing core particles were solubilized in 1 % OM for 15 min on ice, centrifuged and loaded onto the anionexchange column. In both cases elution of the proteins was performed with a continuous KCl gradient (30 to 300 mmollL KCl in 90 min) in 20 mmol/L HEPES pH 7.5 and 0.025 % OM at a flow rate of 1 mLl min. The elution of the differen~ proteins was detected by their absorption at 280 nm.
Pigment analysis The pigment composition of the purified pigment protein complexes was determined using reversed phase high performance liquid chromatography (HPLC). The HPLC separation was based on a method described by Thayer and Bjorkman (1990) with a modified solvent A (acetonitrile: methanol 85: 15 was exchanged by acetonitrile: tris-HCl pH 7.8 98: 2). The calibration of the HPLC system was done by injection of defined amounts of purified pigments and by regression analysis of the integration areas over the molar amounts of standards. The concentrations of the purified standards •were calculated using extinction coefficients reported by Hager and Meyer-Bertenrath (1966). ChI alb contents were determined according to Ziegler and Egle (1965).
Absorption spectroscopy Absorption spectra of the isolated pigment proteins were taken with a Shimadzu MPS-2000 spectrophotometer.
SDS-PAGE The polypeptide composition and the state of purification was analyzed using denaturing polyacrylamide gel electrophoresis as described by Laemmli (1970) and silver staining according to Heukeshoven and Demick (1985).
Results
CP 29 and CP 26 from the different isolation methods were identical with regard to the polypeptide pattern, pigment stoichiometries and spectroscopy. We, therefore, do not differentiate between the two methods in the presentation of the respective data.
SDS-PAGE Fig. 1 shows the polypeptide composition of isolated PS II core complexes, LHC II, CP 29 and CP 26 after separation by anion exchange chromatography. PS II core complexes (Fig. 1 A) consisted of the proteins of the inner antenna CP 47 and CP 43 and the proteins Dl, D2 and Cyt b 559 of the PS II reaction centre. The (X- and ~-subunits of Cyt b559 with molecular masses of 9 and 4 kDa, respectively are not resolved as defined bands in the applied gel system (12 % acrylamide). Their presence in the purified PS II core particles was confirmed using gels with an acrylamide concentration of 15 or 18 % (data not shown). The purified LHC II of spinach (Fig. 1 B) showed two apoproteins with molecular masses of 27 and 25 kDa, respectively (see also Ryrie et al., 1980). The
Pigment composition of PS II pigment protein complexes
67
67
45
117
67 . . .
45 45 • • 45
29
29
i
I
29 29 21
I:' .....
r
27lcDa 25lcDa
_~
y
~,
:,~
'::,.CP26 :. .~~
...
21
21
..::
21
12.5
12.5 12.5 12.5 1
2
lA
1
2
IB
1
2
Ie
1
2
ID
Fig. 1: Polypeptide composition of the purified PS II pigment protein complexes as detected by SDS-polyacrylamide gel electrophoresis.
Fig.IA: Lane 1: Molecular weight markers, lane 2: PS II core particles. Fig.IB: Lane 1: Molecular weight markers, lane 2: LHC II. Fig. Ie: Lane 1: Molecular weight markers, lane 2: CP 29. Fig. 1 D: Lane 1: Molecular weight markers, lane 2: CP 26.
minor chlorophyll alb binding proteins CP 29 (Fig. 1 C) and CP 26 (Fig. 1 D) migrated according to their molecular weight, with CP 29 being visible just below the 29 kDa molecular weight marker.
Absorption spectroscopy and chlorophyll alb ratios LHC II contained high amounts of chlorophyll b (chI alb ratio 1.3), that were clearly visible in the absorption spectrum (Fig. 2 B) as peaks at 472 and 651 nm. The purified minor chlorophyll alb binding protein CP 29 showed a chlorophyll alb ratio of about 3.1. This value is slightly higher than the chlorophyll alb ratio of 2.9 mentioned by Bassi et aI. (1993), but significantly lower than the ratio of 4.0 proposed by Ruban et aI. (1994). The absorption spectrum of CP 29
(Fig. 2 A) was dominated by chlorophyll a absorption with maxima at 436 and 676 nm. Chlorophyll b and the xanthophylls bound to CP 29 became visible as a pronounced shoulder in the range 450 to 480 nm. Interestingly the absorption spectrum of CP 29 showed a characteristic shoulder at 636 nm, that is also described by Dainese et aI. (1990), Ruban et aI. (1994) and Machold and Meister (1979), who first resolved CP 29. CP 26, in comparison to CP 29, contained higher amounts of chlorophyll b (chlorophyll alb ratio 2.8). Dainese and Bassi (1991) reported lower chlorophyll alb ratios for CP 26 (2.2), whereas Ruban et aI. (1994) found higher values (2.9). The increased content of chlorophyll b became visible as a more pronounced chlorophyll b absorption berween 450 and 480 nm (Fig. 2 A). The absorption maxima of chlorophyll a were the same when compared to
118
REIMUND Goss, MICHAEL RICHTER, and ALoYSIUS WILD
Absorption (reI. units)
0,5
0,4
Table 1: Pigment contents (mol pigment) per 100 mol of chlorophyll a of rhe purified PS II pigment protein complexes. Pigment proteins were purified from dark adapted spinach leaves and light treated thylakoids (500 ~mol m- 2 S-l PAR for 30 min in the presence of 30 mM ascorbate), respectively.
neo
0,4
0,3
0,3 0,2 0,2 0,1
0,1
o
0
350 400 450 500 550 600 650 700 750 nm
VIO
ant lut
dark thylakoids 8.9 8.9 0.7 LHC II 13.4 3.7 CP 29 8.9 15.0 0.6 CP 26 8.6 9.3 0.6 PS II core light thylakoids 9.3 LHC II 16.5 CP 29 8.5 CP 26 9.2 PS II core
zea chi b chi a pheo
~-car
22.5 0.3 35.9 100 29.1 72.7 100 15.4 33.4 100 14.2 36.3 100 100 5.7
13.7
100 100 100 100 100 6.1
10.8
4.2 0.5 21.8 5.4 40.9 2.2 26.8 72.5 8.3 13.6 3.4 31.7 4.4 12.6 1.8 36.8
1.7 1.4 16.2
1.6 . 1.2 15.9
neo: neoxanrhin; vio: violaxanthin; ant: anrheraxanrhin; lut: lutein; zea: zeaxanrhin; chi b: chlorophyll b; chi a: chlorophyll a; pheo: pheophytin; ~-car: ~-carotene. Table 1 shows the mean values of pigment contents of rhree individual separations wirh a standard deviation < 8 %. 417 nm, ~-carotene could be identified by its absorption in the range 450 to 500 nm.
Absorption (rei. units)
0,6
0,15
0,1
0,4
0,05
0,2
o
0 350 400 450 500 550 600 650 700 750 nm
Hg.2A: Absorption spectra ofCP 29 (solid line) and CP 26 (dotted line) at room temperature. Fig. 2 8: Absorption spectra of PS II core particles (solid line) and LHC II (dotted line) at room temperature.
CP 29. Purified PS II core complexes did not contain chlorophyll b (see Table 1). The absorption spectrum was clearly dominated by chlorophyll a peaking at 435 and 673 nm (Fig. 2 B). Pheophytin became visible as a pronounced shoulder at
Pigment composition Table 1 (upper part) shows the pigment contents (mol) of the purified PS II pigment protein complexes isolated from dark adapted spinach leaves per 100 mol of chlorophyll a. The highest amounts of xanthophyll cycle pigments (i.e. violaxanthin) were found in the minor chlorophyll alb binding proteins CP 29 and CP 26 with values of 15.0 and 9.3 mol violaxanthin per 100 mol chlorophyll a, respectively. LHC II also contained violaxanthin, but with lower amounts being present (3.7 mol violaxanthin per 100 mol chlorophyll a). The entichment of xanthophyll cycle pigments in the minor chlorophyll alb binding proteins was also seen in pigment proteins purified from light treated isolated thylakoids. After illumination with 500 J.l.mol m- 2 s-1 (PAR) for 30 min in the presence of ascorbate and subsequent purification CP 29 bound 3.4 mol zeaxanthin and 8.3 mol violaxanthin per 100 mol chlorophyll a, CP 26 1.8 mol zeaxanthin and 4.4 mol violaxanthin, while 2.2 mol violaxanthin but no zeaxanthin could be detected in the peripheral LHC II (Table 1 lower part). Comparison with the pigment contents of light treated isolated thylakoids (4.2 mol violaxanthin and 5.4 mol zeaxanthin) reveals that after violaxanthin deepoxidation and subsequent purification part of the zeaxanthin is no longer bound to the antenna proteins. From Table 1 it becomes obvious that the pigments of the xanthophyll cycle were only present in the pigment protein complexes of the outer PS II antenna. PS II core complexes containing the proteins of the reaction centre and the inner antenna only bound chlorophyll a, pheophytin and ~-caro tene. Identical results were obtained for PS II core complexes prepared from illuminated thylakoids.
Pigment composition ofPS II pigment protein complexes
Discussion
The pigment stoichiometries of the purified PS II pigment protein complexes described in this study show that the xanthophyll cycle pigments are exclusively bound to the proteins of the outer PS II antenna. They are enriched in the minor chlorophyll alb binding proteins CP 29 and CP 26, whereas the peripheral LHC II only binds them at a smaller xanthophyll/chlorophyll ratio. This finding is in agreement with results published by Bassi et al. (1993) and Ruban et al. (1994), where the majority of the xanthophyll cycle pigments is also found in CP 29, CP 26 and CP 24. However one should consider that a significant part of the xanthophyll cycle pigments must be bound to LHC II, given the assumption that the latter contains four times the chlorophyll of the minor chlorophyll alb binding proteins (Dainese and Bassi, 1991). The complete absence of zeaxanthin in LHC II and the diminished contents in CP 29 and CP 26 purified from light treated isolated thylakoids could be due to a decreased stability in the pigment protein binding in the case of zeaxanthin. Our data would also be consistent with a recent suggestion by Havaux and Tardy (1995). These authors proposed that zeaxanthin formed in strong light might interact with the membrane lipid phase rather than the pigment proteins, ~hereby simultaneously providing protection against heat and light stress. Both explanations would predict higher amounts of zeaxanthin in the free pigment when compared to the concentrations of free violaxanthin in purifications using dark adapted plants as starting material. As Bassi et al. (1993) and Ruban et al. (1994) use identical isolation procedures (isoelectric focusing), the pigment compositions of the isolated pigment proteins derived from anion exchange chromatography provide an alternative approach to clarify the localization of the xanthophyll cycle pigments. Recent studies by Jahns (1995) and Lee and Thornber (1995) showed an association of the xanthophyll cycle pigments with the PS II core complex. It is, therefore, important to state that in our preparations PS II core particles without contaminating proteins of the outer antenna do not contain pigments of the xanthophyll cycle. They solely bind chlorophyll a, pheophytin and ~-carotene. This is in agreement with data published by Alfonso et al. (1994). Frank et al. (1994) proposed that zeaxanthin might act as a direct quencher of chlorophyll a in the singlet excited state. According to Dainese et al. (1992) the function of zeaxanthin in non-photochemical energy dissipation is favoured by its specific binding to the minor chlorophyll alb binding proteins which in turn are located in between the peripheral antenna (i.e. LHC II) and the PS II core. On the other hand, taking into account results that the PS II antenna is arranged as a very shallow funnel (Schatz et al., 1988; Jennings et al., 1993) and that the funnel organization does not playa major role in energy trapping the importance of the localization of the quencher might be questioned.
119
Acknowledgements
Our thanks are due to Prof. Dr. C. Wilhelm for critically reading the manuscript and to Ralf Nickel for his help regarding all aspects of anion exchange chromatography.
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