Relationship between the organization of the PSII supercomplex and the functions of the photosynthetic apparatus

Journal of Photochemistry and Photobiology B: Biology 83 (2006) 114–122 www.elsevier.com/locate/jphotobiol

Relationship between the organization of the PSII supercomplex and the functions of the photosynthetic apparatus Emilia L. Apostolova a,*, Anelia G. Dobrikova a, Pavlina I. Ivanova a, Ivana B. Petkanchin b, Stefka G. Taneva a a

Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.21, Sofia 1113, Bulgaria b Institute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 21 September 2005; received in revised form 20 December 2005; accepted 20 December 2005 Available online 7 February 2006

Abstract The chlorophyll fluorescence and the photosynthetic oxygen evolution (flash-induced oxygen yield patterns and oxygen bursts under continuous irradiation) were investigated in the thylakoid membranes with different stoichiometry and organization of the chlorophyllprotein complexes. Data show that the alteration in the organization of the photosystem II (PSII) supercomplex, i.e. the amount and the organization of the light-harvesting chlorophyll a/b protein complex (LHCII), which strongly modifies the electric properties of the membranes, influences both the energy redistribution between the two photosystems and the oxygen production reaction. The decrease of surface electric parameters (charge density and dipole moments), associated with increased degree of LHCII oligomerization, correlates with the strong reduction of the energy transfer from PSII to PSI. In the studied pea thylakoid membranes (wild types Borec, Auralia and their mutants Coeruleovireus 2/16, Costata2/133, Chlorotica XV/1422) with enhanced degree of oligomerization of LHCII was observed: (i) an increase of the S0 populations of PSII in darkness; (ii) an increase of the misses; (iii) an alteration of the decay kinetics of the oxygen bursts under continuous irradiation. There is a strict correlation between the degree of LHCII oligomerization in the investigated pea mutants and the ratio of functionally active PSIIa to PSIIb centers, while in thylakoid membranes without oligomeric structure of LHCII (Chlorina f2 barley mutant) the PSIIa centers are not registered.  2006 Elsevier B.V. All rights reserved. Keywords: Light-harvesting chlorophyll a/b protein complex of photosystem II; Chlorophyll fluorescence; Photosynthetic oxygen evolution; Pea mutant thylakoid membranes

1. Introduction The light reaction of photosynthesis in higher plants is driven by the cooperation of two photosystems, PSI and PSII, which are laterally and functionally segregated mainly in non-appressed (stroma) and appressed (grana) domains of thylakoid membranes, respectively [1–4]. This creates regions with different charge density in the thylakoid membranes, which carry a net negative surface charge [5,6]. The physical separation of PSII and PSI is a dynamic process and allows the regulation of the excitation energy distribution between the two photosystems [7]. *

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1011-1344/$ - see front matter  2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.12.012

Functionally and structurally distinct populations of the PSII centers (PSIIa and PSIIb) in grana and stroma lamellae have been proposed (see [3] and references therein). They differ in the organization of PSII, in the size of Chlantenna system and in their ability to reduce the plastoquinone molecule [8]. The PSIIa centers in the appressed grana domains form dimers with large antenna (four LHCII trimers per monomer) whereas PSIIb centers in stroma lamellae have small Chl-antenna size [3]. The main function of PSII is to drive an electron transfer from the water-oxidizing manganese center at the lumenal side to plastoquinone (PQ) at the stromal side of thylakoid membrane and a formation of a transmembrane proton gradient [9]. The manganese-containing active sites of PSII, which involve four Mn atoms,

E.L. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 83 (2006) 114–122

catalyze the oxygen evolution at the donor side [9,10]. According to Kok et al. [11] the cooperation of five oxidizing equivalents in the individual reaction centers, generated by four successive photoreactions in the same PSII reaction center, is required for production of each oxygen molecule (non-cooperative mechanism). The intermediate redox states are termed S0–S4. S0 is the most reduced state, while S1, S2 and S3 represent higher oxidation states and the O2 being evolved at the transition of S4–S0 state [12,13]. In darkness the S0 and S1 states are stable, while S2 and S3 revert to S1 in a few minutes. The main photosynthetic antenna complex in higher plants is the light-harvesting Chl a/b protein complex of PSII (LHCII) that binds about half of the thylakoid chlorophyll molecules [14–16]. Its main physiological role is absorption of light and transfer of excitation energy towards the reaction center of PSII, as well as regulation of the excitation energy transfer between the two photosystems [17]. The light-harvesting proteins, that are associated with PSII, form either trimers (major peripheral LHCII) or monomers (the minor/inner antenna members CP24, CP26 and CP29) [14,18]. LHCII in the granal thylakoid membranes forms large chiral-aggregated structures [19,20]. This high order oligomeric structural organization of the major LHCII stabilizes the membrane ultrastructure and is important for the dynamics and functioning of the photosynthetic membranes [21–24]. The thylakoid membrane structure has been proposed to be maintained by an asymmetric distribution of electrical charges on the grana and stroma membrane surfaces [5]. The role of LHCII in the electrostatic control of thylakoid organization [25], in the orientation of thylakoid membranes in electric and magnetic fields [26–28] and its contribution to the overall electrical properties of thylakoid membranes [29] has been demonstrated. The structure of the thylakoid membrane is not rigid and the organization of the antenna complexes in the membranes undergoes dynamic changes responding to changes in the environmental conditions [30–32]. The relationship between the stoichiometry and organization of pigmentprotein complexes and the function of photosynthetic apparatus as well as the role of the different aggregation states of LHCII are still poorly understood. Our recent investigations were focused on characterization of thylakoid membranes from pea mutants with altered pigment-protein composition. We have found that the alteration of the extent of LHCII oligomerization (a multiple of heterotrimers) is associated with strong changes in the surface electric properties: surface charge density and electric dipole moments (permanent and induced) of the membranes, determined by means of microelectrophoresis and electric light scattering techniques [33,34]. In the present study we used a low-temperature chlorophyll fluorescence and polarographic oxygen rate electrode to study the energy transfer and photosynthetic oxygen production in thylakoid mem-

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branes from pigment mutants of pea and barley in order to get a greater insight into the role of PSII supercomplex organization on the functions of the photosynthetic apparatus. The functional changes are discussed in terms of the variations the surface electric charges of these membranes caused by different organization of LHCIIPSII complex. 2. Materials and methods 2.1. Preparation of thylakoid membranes Thylakoid membranes were isolated from Pisum sativum L. cv. Borec and its mutants Coeruleovireus 2/16 and Costata 2/133, and Pisum sativum L. cv. Auralia and its mutant Chlorotica XV/1422 as described in [35]. Barley thylakoids (Hordeum vulgare L.) were isolated by the same protocol, except that five times higher concentration of MgCl2 was used for Chlorina f2 mutant than for the wild type like in Bassi et al. [36]. The total chlorophyll concentration was determined by the method of Lichtentaler et al. [37]. 2.2. Low temperature (77 K) chlorophyll fluorescence Low temperature (77 K) chlorophyll fluorescence measurements were performed in cylindrical quartz cuvette in a medium containing 40 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2, 330 mM sucrose at chlorophyll concentration of 20 lg/ml. Fluorescence spectra were recorded from 600 to 780 nm using Jobin Yvon JY3 spectrofluorimeter equipped with a red sensitive photomultiplier (Hamamatsu R928) and a liquid nitrogen device. Chlorophyll fluorescence was excited either at 436 nm (Chl a) or at 472 nm (Chl b). The width of exciting and measuring slit was 4 nm. 2.3. Oxygen evolution measurements Oxygen flash yields and initial oxygen burst were measured using a home-built polarographic oxygen rate electrode described in [38]. Thylakoid membranes, in a medium without artificial electron acceptor containing: 40 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose, were illuminated at chlorophyll concentration of 150 lg/ml in 2 mm suspension layer (100 ll sample volume). Samples were preilluminated with 25 flashes and then dark adapted for 5 min before measurements. Oxygen flash yields were induced by saturating (4 J) and short (t1/2 = 10 ls) periodic flash sequences with 650 ms dark spacing between the flashes. The initial oxygen burst was recorded after irradiation with continuous white light (450 lmol photons m2 s1). The Kok’s model parameters (initial S0 and S1 state distribution, misses and double hits) were determined by the least square deviations fitting of the theoretically calculated yields according to the model of Kok et al. [11] with the experimentally obtained oxygen flash yields.

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3. Results

1.4

-0.070

A -0.065

1.0 -0.060 0.8 -0.055

σ [Cm-2]

24

p⊥ x 10 [Cm]

γ /S x 10 16 [F] ||

The energy transfer between chlorophyll-protein complexes of thylakoid membranes from several pea species (Borec wild type and its mutant forms: Coeruleovireus 2/16 and Costata 2/133, and Auralia wild type and its mutant Chlorotica XV/1422) with different stoichiometry of pigment-protein complexes and LHCII organization [33,34] was followed by the low temperature chlorophyll fluorescence (77 K). The emission spectra of thylakoid membranes at 77 K are characterized by two overlapping bands with emission maxima at 685 and 695 nm, attributed to PSII supercomplex and a band at 735 nm attributed to PSI [39–43]. Our data show that the low temperature chlorophyll fluorescence ratios F735/F685 and F695/F685 strongly depend on the oligomeric to monomeric LHCII ratio. A strict correlation was previously found between the ratio of the oligomeric (heterotrimers) to monomeric forms of LHCII (LHCIIo/LHCIIm) and the surface electric parameters: surface charge density (r), transmembrane charge asymmetry (i.e. permanent dipole moment, p^) and electric polarizability per unit area (ci/S, connected to the ions of the diffuse electric double layer and their mobility) of the thylakoid membranes [33,34]. Based on these data for the quantitative correlation between the LHCII oligomerization state and the electric parameters (given in Fig. 1A), the present results are presented as a function of the membrane surface charge density, r (Fig. 1B). The variation of F735/F685 and F695/F685 as a function of either the permanent dipole moment (p^) or electric polarizability (ci/S) shows the same tendency as the surface charge density (data not shown). The emission ratios F735/F685 upon excitation of both Chl a (436 nm) and Chl b (472 nm) increase with the increase of the negative surface charge density related to reduced ratio of oligomeric to monomeric LHCII proteins (Fig. 1 and Table 1). This indicates a strong reduction of the energy transfer from PSII to PSI with decrease in the membrane surface electric charge and charge asymmetry. An opposite tendency is observed for the low temperature chlorophyll fluorescence ratio F695/F685 (i.e., for the energy transfer between pigment-protein complexes in PSII) when the membranes were excited at 472 nm (Chl b). Thus, F695/F685 ratio decreases with the increase of the surface negative charge density, associated with decrease of the ratio LHCIIo/LHCIIm (Fig. 1 and Table 1). Therefore, the alteration of the energy distribution between the two photosystems (determined for excitation at both wavelengths 472 and 436 nm), as well as within the LHCII-PSII complex (for excitation only at 472 nm), correlate with the changes in the electric moments and the negative surface charge density of the membranes

1.2

0.6 -0.050

0.4

-0.045

0.2

3

2

4

5

6

7

LHCIIo/LHCIIm 1.4

B 1.3 1.2

F735 / F685 F695/ F685

3.1. Energy transfer between pigment-protein complexes of thylakoid membranes

1.1 1.0 0.9 0.8 0.7 -0.045

-0.050

-0.055

-0.060

-0.065

σ [Cm-2] Fig. 1. (A) Effect of LHCII organization (LHCIIo/LHCIIm) on the surface electric properties of thylakoid membranes from different pea species: permanent dipole moment, p^ (¤); electric polarizability per unit surface area, ci/S (m) and negative surface charge density, r (.). (B) Low temperature (77 K) chlorophyll fluorescence emission ratios: F735/F685 (for excitation at 436 nm (d) and 472 nm ( )) and F695/F685 (for excitation at 436 nm (j) and 472 nm (h)) as a function of the surface charge density, r.



(caused by the variation of LHCIIo/LHCIIm ratio in mutants), whereas the energy transfer within the LHCIIPSII complex does not change when chlorophyll a was excited 436 nm (Fig. 1B). This reveals a significant role of the stoichiometry of LHCII in the organization of the proteins in LHCII-PSII complex and in the energy transfer between molecules of the chlorophyll b and chlorophyll a. Rearrangement and/or destabilization of LHCII-PSII molecular assembly might lead to alteration of the energy transfer. 3.2. Photosynthetic oxygen evolution The oxygen induction curves registered under continuous irradiation of thylakoid membranes from the different pea species are shown in Fig. 2. The amplitude of the initial oxygen burst and the area under the curve (which is a measure of the oxygen volume evolved) depend on the number of functionally active PSII centers [44]. The amplitudes of the oxygen burst and the decay kinetics under continuous

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Table 1 Characteristics of pigment-protein complexes and photosynthetic oxygen evolution of thylakoid membranes from wild types of pea: Borec and Auralia, and the mutants: Chlorotica XV/1422, Costata 2/133, Coeruleovireus 2/16 Plant type

LHCIIo/LHCIIma

LHCII/PSIIa

A1/A2

k1 (s1)

k2 (s1)

S0 (%)

Misses (%)

Double hits (%)

Chlorotica XV/1422 Auralia wild type Costata 2/133 Borec wild type Coeruleovireus 2/16

2.45 2.82 3.34 4.57 6.62

1.97 2.60 3.70 3.37 3.21

0.61 1.38 2.28 2.50 2.71

1.59 2.67 4.30 4.71 4.05

0.24 0.35 0.56 0.68 0.41

16 17 20 21 25

17 19 17 20 24

5.7 5.8 4.3 4.5 5.0

Kinetic parameters of the initial oxygen burst decay under continuous irradiation with white light (450 lmol photons m2 s1). A1 and A2 are the amplitudes, and k1 and k2 are the rate constants of the fast and slow components of the oxygen burst decay, respectively. Initial dark distribution of the S0 and S1 states (S1 = 100  S0), values of misses and double hits according to the Kok’s model. Average data are from 4 to 5 independent experiments. a Data published in Refs. [33,34].

4

1000

5

Oxygen evolution rate [mV]

2

1 500

0

-2

0

2

4

6

8

10

12

14

16

18

Time [s] Fig. 2. Time course of the initial oxygen bursts (induction curves) at continuous irradiation of pea thylakoid membranes: (1) Chlorotica XV/ 1422; (2) Auralia wild type; (3) Borec wild type; (4) Costata 2/133; (5) Coeruleovireus 2/16.

irradiation differ in thylakoids from the investigated species (Fig. 2 and Table 1). The lowest oxygen burst is observed for the mutant Chlorotica XV/1422, characterized by the smallest LHCII/PSII and LHCIIo/LHCIIm ratios (Table 1). The induction curves after oxygen burst exhibit biphasic exponential decay (Fig. 2), the kinetic parameters are given in Table 1. The observed biphasic kinetics can be explained by the existence of two different mechanisms for oxygen production [44,45]. It is supposed that the cooperative mechanism is realized by diffusion of oxygen precursors within the different oxygen evolving centers (mainly PSIIb centers) and is characterized by a rate constant (k2) lower than that of the non-cooperative Kok’s mechanism (with a rate constant k1) realized by PSIIa centers [44]. The ratio between the amplitudes of the fast and slow components (A1/A2) most probably corresponds to the proportion of the functionally active PSIIa and PSIIb centers in thylakoid membranes. This ratio is found to decrease with the reduction of the LHCII oligomeric state (Fig. 5 and Table 1). This can be explained by the cooperative mechanism, which can have a dominant role with the

2000

1600

Oxygen flash yields [mV]

3

decrease of LHCIIo/LHCIIm ratio. The kinetic constants, k1 and k2, for thylakoid membranes from Auralia wild type and Chlorotica XV/1422 are smaller than those for Borec wild type and its mutants. This fact indicates slower damping of both O2-evolving mechanisms (slower blocking of active PSII centers or PQ depletion) for thylakoid membrane with smaller amount of LHCII per unit reaction center of PSII. It could be supposed that the ratio LHCII/PSII affects the interaction between QB and PQ pool molecules (i.e., the reduction of PQ molecules by QB). The oscillation patterns of the oxygen yields induced by a flash sequence in dark-adapted thylakoid membranes are shown in Fig. 3. Characteristic oscillations with a periodicity of 4, which are damped due to misses (centers not converted, i.e., zero-step advances), double hits (double step advances) and reduction of the number of functionally active PSII centers after each flash of a sequence, are observed. Analysis of the flash-induced oxygen yield patterns reveals higher average oxygen yields for Borec wild type and its mutants (Coeruleovireus 2/16, Costata 2/133)

1200

800

400

0 0

2

4

6

8

10

12

Flash numbers Fig. 3. Oscillation patterns of the oxygen flash yields as a function of the flash number in dark-adapted thylakoid membranes from different pea species: (e) Chlorotica XV/1422; (¤) Auralia wild type; (d) Borec wild type; ( ) Costata 2/133; (h) Coeruleovireus 2/16.



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than that for Auralia wild type and its mutant Chlorotica XV/1422 (Fig. 3). Therefore, the observed decrease of the flash-induced oxygen production correlates with the decrease in the amount LHCII per PSII reaction center in thylakoid membranes (Table 1). The evaluated values of the initial population of the redox states S0 in percent (S1 = 100  S0) in OEC before the flash train, misses and double hits, using the Kok’s model [11], are given in Table 1. The results show that the S0 (in the dark) population increases with increase of LHCIIo/LHCIIm ratio in thylakoid membranes (Fig. 5, Table 1). The highest percent of the centers in S0 states in darkness is observed for the mutant Coeruleovireus 2/16 (Table 1). This mutant is characterized with the highest degree of LHCII oligomerization among all pea species under study. The oxygen production was also studied in thylakoids from Chlorina f2 mutant of barley with strongly reduced antenna size and lack of oligomeric structure of LHCII [46,47]. The results reveal a dramatic reduction of the oxygen flash yields and smoothing of the initial burst in Chlorina f2 compared with wild type barley thylakoids (Fig. 4). The drastic decline of the amplitudes of the flash-induced oxygen yields, the loss of the characteristic oscillations (Fig. 4) and the reduction of the oxygen burst (Fig. 4 inset) indicate a dominant operation of the cooperative mechanism of oxygen evolution attributed mainly to stroma situated PSIIb centers. The calculated kinetic constants for thylakoid membranes from barley wild type are k1 = 2.0 s1, k2 = 0.45 s1. A mono-exponential decay (lacking of the fast A1 component) of the induction curve (k2 = 0.41 s1) is observed for Chlorina f2 thylakoids (Fig. 4 inset), indicating that the fast kinetic phase, corresponding to PSIIa centers, is lost in the mutant. 2000 Oxygen evolution rate [mV]

3600

Oxygen flash yields [mV]

1600

1

3000 2400 1800 1200

2

600

1200 0 0

2

4

6

8

10

12

14

Time [s]

800

1

400

2

0 0

2

4

6

8

10

12

Flash numbers Fig. 4. Oxygen flash yield patterns and initial oxygen bursts (inset) of thylakoid membranes from barley wild type (1) and mutant Chlorina f2 (2).

4. Discussion It is known that most of the LHCII complex and PSIIa subpopulation of reaction centers are located in grana thylakoid domains, whereas PSI and PSIIb subpopulation are predominantly distributed in stroma lamellae [3,6]. The stroma lamellae are characterized by a greater magnitude of negative electric charge density than the grana membrane domains [48,49]. This difference in the electric charge density between stroma and grana domains might be needed for efficient energy transfer. The energy distribution between the two photosystems is regulated by the distance between the two supramolecular complexes, which is mediated via changes in the membrane electric charge density and the electric double layer [50]. Changes in the organization of thylakoid membranes and the membrane stacking, in which LHCII proteins have a significant contribution, are connected with changes in the ratio of grana-stroma domains and in the distribution of PSIIa and PSIIb centers, respectively [3]. Our previous data suggested an important role of the LHCII and its organization in determining the surface electric properties of thylakoid membranes and the interfacial charge dynamics [33,34]. The present results show a correlation between the variation of negative charge density on the membrane surface and the energy distribution between the two photosystems (followed by the fluorescence ratio F735/F685) as well as the energy transfer between antenna pigments of PSII when Chl b is excited (Fig. 1B). Data suggest that higher degree of oligomerization of LHCII, which correlates with lower values of the membrane electric moments (Fig. 1A), causes reduction of the energy spillover between PSII and PSI (Fig. 1B). The ratio LHCIIo/LHCIIm has a determining role on the energy transfer within the supramolecular complex LHCII-PSII only in the case when the Chl b is excited, whereas no detectable effect on the energy transfer upon excitation of Chl a (Fig. 1B). Therefore the protein conformational changes in the LHCII-PSII complex, related to oligomerization of the major LHCII and the variation of the electric properties, might regulate the energy transfer between chlorophyll-protein complexes. While Gilmore et al. [51] have shown that the deexcitation of absorbed light energy is independent of the peripheral antenna size of PSII. The oxygen production and its alterations with the changes in organization of the LHCII-PSII complex is the second aspect of this work. An operation of two parallel mechanisms is proposed to explain the oxygen-evolution process [44,45]: the Kok’s non-cooperative mechanism in grana regions (PSIIa centers), registered by flash-induced oxygen production, and a cooperative mechanism involving cooperation between different oxygen evolving centers (stroma situated PSIIb centers). The initial oxygen burst under continuous irradiation involves production of both PSIIa and PSIIb centers, and therefore reflects the two mechanisms.

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We observed that the ratio A1/A2, that should reflect the ratio of PSIIa to PSIIb centers in thylakoid membranes, diminishes with decrease of LHCII oligomerization state (Fig. 5 and Table 1). This suggests a decrease in the proportion of functionally active PSIIa centers in thylakoids with low degree of LHCII oligomerization. Moreover, the lack of a fast component of the oxygen burst (A1) for Chlorina f2 mutant thylakoid membranes (Fig. 4 inset) could be attributed to the reduced grana formation [52], which is evidenced by the strongly diminished O2 flash yields (Fig. 4) attributed to PSIIa centers. Earlier observation of Ghirardi et al. [53], that thylakoid membranes from Chlorina f2 lack differentiation of PSII into PSIIa and PSIIb, is in support of this suggestion. Our data suggest that the reduced LHCIIo/LHCIIm ratio correlates with an increased proportion of PSIIb in investigated thylakoid membranes, that in turn leads to reduction of the oxygen-evolving production (Figs. 2–5 and Table 1). On the other hand, higher ratio of LHCII per unit PSII reaction center in pea thylakoid membranes (Table 1), that corresponds to enhanced O2 flash yields (Fig. 3), might be due to altered interaction between QB and PQ pool molecules. The results show slower damping of both O2-evolving mechanisms (i.e., lower ki values) in membranes with smaller ratio LHCII/PSII (Table 1). A lower structural stability of the OEC has been previously shown to accompany the reduced content of LHCII, which is assumed to form a hydrophobic shell around the OEC [54]. We have previously found that the steady-state PSII electron transport rate in presence of the artificial electron acceptor p-benzoquinone (p-BQ) depends on the amount of LHCII in thylakoid mutants [55,56]. The electron transport rate for thylakoid membranes with higher amount of LHCII and higher ratio of LHCII/PSII (Borec

3

28

24 2

22

A1/A2

S0 , α [%]

26

20 1

18 16 2

3

4

5

6

7

LHCIIo/LHCIIm Fig. 5. Effect of LHCII organization (LHCIIo/LHCIIm) in the pea thylakoid membranes on the S0 populations of PSII in darkness (j), the misses, a (h) and the ratio of functionally active PSIIa to PSIIb centers, A1/A2 (d).

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wild type and its mutants) is bigger than that for membranes with lower LHCII content (Auralia wild type and Chlorotica XV/1422) (Table 1). In support of these data is the observation that the photochemical activity (in the presence of p-BQ) of Chlorina f2 mutant is considerably smaller (about 50%) than that of wild type barley (data not shown). Thus the structural changes in the LHCII-PSII complex, induced by varied amount of LHCII, might modify the environment and/or the conformation of the oxygen-evolving complex and consequently its interaction with p-BQ. It has been also shown that the protein arrangement influences the plastoquinone diffusion [57]. Having in mind that the oxygen production in photosynthetic membranes, in a medium without artificial electron acceptor, depends on the accessibility of plastoquinone pool and the features of the quinone acceptor QB [58], one could expect that changes in the organization of LHCII and in the LHCII/ PSII ratio can somewhat modify the interaction between QB and PQ. The oxygen-evolution rate, corresponding to an electron flow from the water-oxidizing complex to the plastoquinon pool, and the kinetics of Pþ 680 reduction depend on the Si state transitions [9,59]. It is well known that the oxygenevolving complex accumulates successively four positive charges in the Mn-active site (S0–S4 states reflect different oxidation states of the Mn atoms) before the evolution of molecule oxygen [9,11–13]. Our results indicate that the different content of LHCII proteins, that surround PSII core, affects the S0–S1 state distribution in darkness (Table 1). This might be caused by structural reorganization of the oxygen-evolving complex governed by the modified LHCII. In darkness S1 is formed from S0 by reduction of Tyr-D+(from D2 protein), which oxidizes Mn2+ to Mn3+ and stabilizes the Mn cluster [60]. Thereby it can be expected that the kinetics of the reduction and O2 release should be sensitive to minor structural perturbations. Analysis of the flash induced oxygen yield patterns (Fig. 3) indicates that the stoichiometry and organization of the antenna pigment-protein complex of PSII strongly affect the oxygen-evolving activity. This can be accounted for changes in the conformation of the OEC and/or of the proteins surrounding the complex induced by altered surface electric parameters of the membranes. Structural changes in the Mn cluster could be responsible for different S0–S1 state distribution occurring during dark adaptation of thylakoid membranes (Table 1). Nugent et al. [9] also have shown that the oxygen-evolution process is particularly sensitive to structural changes, which may alter the redox potentials of the Si state intermediates, hydrogen bonding and pKa values of amino acids surrounding the complex. The enhanced population of centers in the S0 state (in darkness), observed in membranes with higher degree of LHCII oligomerization (Fig. 5 and Table 1), indicates a reduction of Mn3+ to Mn2+. Having in mind that there is a strict correlation between the LHCIIo/LHCIIm ratio

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and the electric properties of the membrane, it can be supposed that changes in the electric state of the membrane interface (namely in the ions along the membrane surface creating the diffuse double layer and the transmembrane charge asymmetry distribution) may induce changes in proximity of the Mn cluster and in the Si state transitions in darkness. The increase of S0 population is accompanied also with higher percent of misses (Fig. 5 and Table 1). Lower values of misses have been proposed [61] to be due to a faster Q A reoxidation. Therefore, the possibility that a significant fraction of Q A in membranes with smaller values of electric dipole moments (higher oligomeric degree of LHCII) cannot be reoxidized during the dark time between the flashes has to be considered. It has also been suggested that the reoxidation rate of Q A determines the values of the double hits (double step oxidation) at saturating flash intensities [61]. Data show an increase of double hits with lowering of LCHII/PSII ratio (Table 1), for which a slower damping of flash patterns is observed (Fig. 3), i.e. slower blocking of PSII active centers or reoxidation of PQ pool (respectively, QA). The oxygen-evolution capacity is known to depend strongly on pH [62,63]. Previous investigations have shown that pH induces structural changes around OEC and influences the S0–S1 distribution. It has been also found that S0 population increases at alkaline and decreases at low pH [64]. Our experiments at different pHs show that the initial distribution of S0–S1 states (in the dark) follow similar tendency (data not shown). In addition, the variation of pH affects the kinetics of the decay after the initial oxygen burst (data not shown). Lowering the pH from 7.6 to 5.6 is suggested to cause structural changes in the donor side of PSII [64], which might lead to decrease in the rate of generation of active centers, destabilization of the intermediate and modification of Mn-binding sites [65]. Our results show that the increase of the membrane surface electric parameters has a similar effect on the kinetics of the exponential decay as lowering pH. Data at pH 5.6 correspond well to those for the mutant Chlorotica XV/1422 characterized with the smallest ratio of LHCIIo/LHCIIm and the highest surface negative charge density (r). This might reflect similar changes in thylakoid membranes, and their surface properties, induced by low pH and low degree of LHCII oligomerization. The amount of LHCII per PSII reaction center also alters the environment around OEC at the donor side and reduces flash-induced O2 production (Fig. 3 and Table 1). In summary, the experimental results presented in this study suggest that the organization of LHCII-PSII complex (the amount and oligomerization of LHCII), which are strongly related to the membrane surface electric properties: (i) regulate the quantum distribution between two photosystems; (ii) affect the excitation energy transfer from chlorophyll b to chlorophyll a (iii) change the flash-induced oxygen production and the initial S0–S1 state distribution, misses and double hits; (iv) alter the kinetic parameters of the initial oxygen burst (evolution) under continuous

irradiation; (v) change the ratio of functionally active PSIIa and PSIIb centers. 5. Abbreviations Chl chlorophyll LHCII light-harvesting complex of photosystem II LHCIIm LHCII monomer LHCIIo LHCII oligomer OEC oxygen-evolving complex PQ plastoquinone PSI photosystem I PSII photosystem II primary plastoquinone acceptor of PSII QA QB secondary plastoquinone acceptor of PSII Si redox state i of the water oxidase

Acknowledgements The author would like to thank the laboratory of Prof. Huner from the University of Western Ontario, Canada, for their collaboration in the determination of the pigment-protein content of pea mutant thylakoid membranes. We would like to thank Prof. Y. Zeinalov from the Institute of Biophysics, Bulgarian Academy of Sciences, for helpful comments and critical reading of the manuscript. The work was supported by contract K-1301/03 with National Science Fund. The authors are grateful to Dr. N. Naydenova from the Institute of Genetics, Bulgarian Academy of Sciences, for the donation of seeds of pea chlorophyll mutants. References [1] J. Whitmarsh, Govindjee, The photosynthetic process, in: G.S. Singhal, G. Renger, K.-D. Irrgang, S. Sopory, Govindjee (Eds.), Concepts in Photobiology: Photosynthesis and Photomorphogenesis, Narosa Publishers, New Delhi, Kluwer Academic Publishers, Dordrecht, 1999, pp. 11–51. [2] J.M. Anderson, Changing concepts about the distribution of photosystems I and II between grana-appressed and stroma-exposed thylakoid membranes, Photosynth. Res. 73 (2002) 157–164. [3] R. Danielsson, P.-A. Albertsson, F. Mamedov, S. Styring, Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach, Biochim. Biophys. Acta 1608 (2004) 53–61. [4] J.P. Dekker, E.J. Boekema, Supramolecular organization of thylakoid membrane proteins in green plants, Biochim. Biophys. Acta 1706 (2005) 12–39. [5] J. Barber, Membrane surface changes and potential in relation to photosynthesis, Biochim. Biophys. Acta 594 (1980) 253–308. [6] D. Stys, Staking and separation of photosystem I and photosystem II in plant thylakoid membranes: a physico-chemical view, Physiol. Plant. 95 (1995) 651–657. [7] H.-W. Trissl, C. Wilhelm, Why do thylakoid membranes from higher plants form grana stacks? Trends. Biochem. Sci. 18 (1993) 415–419. [8] A. Melis, Functional properties of photosystem IIb in spinach chloroplasts, Biochim. Biophys. Acta 808 (1985) 334–342. [9] J.H.A. Nugent, A.M. Rich, M.C.W. Evans, Photosynthetic water oxidation: towards a mechanism, Biochim. Biophys. Acta 1503 (2001) 138–146.

E.L. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 83 (2006) 114–122 [10] B. Andersson, S. Styring, Photosystem II; molecular organization, function and acclimation, in: C.P. Lee (Ed.), Current Topics in Photosynthesis, vol. 16, Academic Press, San Diego, 1991, pp. 1–18. [11] B. Kok, B. Forbush, M. McGloin, Co-operation of charges in photosynthetic O2 evolution. I. A linear four step mechanism, Photochem. Photobiol. 11 (1970) 457–475. [12] J.E. Penner-Hahn, C.F. Yocum, The photosynthesis ‘‘oxygen clock’’ gets a new number, Science 310 (2005) 982–983. [13] M. Haumann, P. Liebisch, C. Mu¨ller, M. Barra, M. Grabolle, H. Dau, Photosynthetic O2 formation tracked by time-resolved X-ray experiments, Science 310 (2005) 1019–1021. [14] S. Jansson, The light-harvesting chlorophyll a/b-binding proteins, Biochim. Biophys. Acta 1184 (1994) 1–19. [15] H. Paulsen, Chlorophyll a/b binding proteins, Photochem. Photobiol. 62 (1995) 367–382. [16] Z. Liu, H. Yan, K. Wang, T. Kuang, J. Zhang, L. Gui, X. An, W. Chang, Crystal structure of spinach major light-harvesting complex at ˚ resolution, Nature 428 (2004) 287–291. 2.72 A [17] J.F. Allen, Thylakoid protein phosphorylation, state 1–state 2 transitions, and photosystem stoichiometry adjustment: redox control at multiple levels of gene expression, Physiol. Plant. 93 (1995) 196– 205. [18] J. Barber, Photosystem II: a multisubunit membrane protein that oxidizes water, Curr. Opin. Struct. Biol. 12 (2002) 523–530. [19] V. Barzda, A. Istokovics, I. Simidjiev, G. Garab, Structural flexibility of chiral macroaggregates of light-harvesting chlorophyll a/b pigment-protein complexes. Light-induced reversible structural changes associated with energy dissipation, Biochemistry 35 (1996) 8981–8985. [20] I. Simidjiev, V. Barzda, L. Musta´rdy, G. Garab, Role of thylakoid lipids in the structural flexibility of lamellar aggregates of the isolated light-harvesting chlorophyll a/b complex of photosystem II, Biochemistry 37 (1998) 4169–4173. [21] G. Garab, A. Faludi-Daniel, J.C. Sutherland, G. Hind, Macroorganization of chlorophyll a/b light-harvesting complex in thylakoids and aggregates: information from circular differential scattering, Biochemistry 27 (1988) 2425–2430. [22] G. Garab, J. Kieleczawa, J.C. Sutherland, C. Bustamante, G. Hind, Organization of pigment-protein complexes into macrodomains in the thylakoid membranes of wild type and chlorophyll b – less mutant of barley as circular dichroism, Photochem. Photobiol. 54 (1991) 273– 281. [23] R. Bassi, P.D. Dainese, A supramolecular light-harvesting complex from chloroplast photosystem – II membranes, Eur. J. Biochem. 204 (1992) 317–326. [24] V. Barzda, L. Mustardy, G. Garab, Size dependency of circular dichroism in macroaggregates of photosynthetic pigment-protein complexes, Biochemistry 33 (1994) 10837–10841. [25] J. Barber, Influence of surface charges on thylakoid structure and function, Annu. Rev. Plant Physiol. 33 (1982) 261–295. [26] G. Garab, Liquid crystal-like domains of the light harvesting chlorophyll a/b protein complex in chloroplast thyalkoid membranes, Mol. Cryst. Liq. Cryst. 152 (1987) 259–266. ´ . Faludi-Da´niel, The light[27] J.G. Kiss, G.I. Garab, Z.M. To´th, A harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field, Photosynth. Res. 10 (1986) 217–222. [28] S.G. Taneva, I. Petkanchin, Surface electric properties of biological systems, Trends Photochem. Photobiol. 6 (1999) 113–139. [29] A.G. Dobrikova, A.G. Ivanov, R. Morgan, I.B. Petkanchin, S.G. Taneva, Contribution of LHC II complex to the electric properties of thylakoid membranes: an electric light scattering study of Chl b-less barley mutant, J. Photochem. Photobiol. B 57 (2000) 33–40. [30] E.E. Gussakovsky, V. Barzda, Y. Shahak, G. Garab, Irreversible disassembly of chiral macrodomains in thylakoids due to photoinhibition, Photosynth. Res. 51 (1997) 119–126. [31] G. Jackowski, P. Olkiewicz, A. Zelisko, The acclimative response of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) to elevated irradiances at the level of trimeric subunits, J. Photochem. Photobiol. B 70 (2003) 163–170.

121

[32] B. Robert, P. Horton, A.A. Pascal, A.V. Ruban, Insights into the molecular dynamics of plant light-harvesting proteins in vivo, Trends Plant Sci. 9 (2004) 385–390. [33] A.G. Dobrikova, R.M. Morgan, A.G. Ivanov, E. Apostolova, I. Petkanchin, N.P.A. Huner, S.G. Taneva, Electric properties of thylakoid membranes from pea mutants with modified carotenoid and chlorophyll-protein complexes composition, Photosynth. Res. 65 (2000) 165–174. [34] A.G. Dobrikova, A.G. Ivanov, E. Apostolova, N. Naydenova, I. Petkanchin, S.G. Taneva, Contribution of LHCII complex to the electric properties of thylakoid membranes, in: N. Go¨zu¨kirmizi (Ed.), Proceedings of the Second Balkan Botanical Congress, Marmara University, Publishing Section, Istanbul, Turkey, 2001, pp. 75–80. [35] M.A. Harrison, A. Melis, Organization and stability of polypeptides associated with the chlorophyll a–b light-harvesting complex of photosystem-II, Plant Cell Physiol. 33 (1992) 627–637. [36] R. Bassi, U. Hinz, R. Barbato, The role of the light harvesting complex and photosystem II in thylakoid stacking in the chlorina-f2 barley mutant, Carlsberg Res. Commun. 50 (1985) 347–367. [37] H.K. Lichtentaler, Chlorophyll and carotenoids: pigments of photosynthetic biomembranes, Methods Enzym. 148 (1987) 350–382. [38] Y. Zeinalov, An equipment for investigations of photosynthetic oxygen production reactions, Bulg. J. Plant Physiol. 28 (3–4) (2002) 57–67. [39] G.H. Krause, E. Weis, Chlorophyll fluorescence and photosynthesis: the basics, Annu. Rev. Plant Physiol. 42 (1991) 313–349. [40] K. Satoh, R.J. Strasser, W.L. Butler, A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts, Biochim. Biophys. Acta 440 (1976) 337–345. [41] R.J. Strasser, W.L. Butler, The yield of energy transfer and the spectral distribution of excitation energy in the photochemical apparatus of flashed bean leaves, Biochim. Biophys. Acta 462 (1977) 295–306. [42] R.J. Strasser, W.L. Butler, Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants, Biochim. Biophys. Acta 462 (1977) 307– 313. [43] R.J. Strasser, M. Tsimilli-Michael, A. Srivastava, Analysis of the chlorophyll a fluorescence transient, in: G. Papageorgiou, Govindjee (Eds.), Chlorophyll Fluorescence: A Signature of Photosynthesis, Kluwer Academic Publishers, The Netherlands, 2004, pp. 321–362. [44] Y. Zeinalov, Mechanisms of photosynthetic oxygen evolution and fundamental hypotheses of photosynthesis, in: M. Pessarakli (Ed.), Handbook of Photosynthesis, second ed., CRC Press, Taylor and Francis Group, Boca Raton, FL, 2005, pp. 3–19. [45] Y. Zeinalov, L. Maslenkova, Mechanisms of photosynthetic oxygen evolution, in: M. Pessarakli (Ed.), Handbook of Photosynthesis, Marcel Dekker, New York, 1996, pp. 129–150. [46] K.D. Allen, L.A. Staehelin, Resolution of 16 to 20 chlorophyllprotein complexes using a low ionic strength native green gel system, Anal. Biochem. 194 (1991) 214–222. [47] B. Bossman, J. Knoetzel, S. Jansson, Screening of chlorina mutants of barley (Hordeum vulgare L.) with antibodies against light-harvesting proteins of PS I and PS II: absence of specific antenna proteins, Photosynth. Res. 52 (1997) 127–136. [48] W.S. Chow, C. Miller, J.M. Anderson, Surface charges, the heterogeneous lateral distribution of the two photosystems, and thylakoid staking, Biochim. Biophys. Acta 1057 (1991) 69–77. [49] A. Telfer, The importance of membrane surface electric changes on the regulation of photosynthetic electron transport by reversible protein phosphorylation, in: J. Biggins (Ed.), Progress in Photosynthesis Research, vol. II, Matinus NijhoffI, Dordrecht, The Netherlands, 1987, pp. 689–696. [50] J. Barber, G.F.W. Searle, Cation induced increase in chlorophyll fluorescence yield and the effect of electrical charge, FEBS Lett. 92 (1978) 5–8.

122

E.L. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 83 (2006) 114–122

[51] A.M. Gilmore, T.L. Hazlett, P.G. Debrunner, Govindjee, Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: photochemical quenching and xanthophyll cycle dependent non-photochemical quenching of fluorescence, Photosynth. Res. 48 (1996) 171–187. [52] J.J. Burke, K.E. Steinback, C.J. Arntzen, Analysis of the lightharvesting pigment-protein complex of wild type and a chlorophyll-bless mutant of barley, Plant Physiol. 63 (1979) 237–243. [53] M.L. Ghirardi, S.W. McCauley, A. Melis, Photochemical apparatus organization in the thylakoid membrane of Hordeum vulgare wild type and chlorophyll b-less chlorina f2 mutant, Biochim. Biophys. Acta 851 (1986) 331–339. [54] N. Shutilova, G. Semenova, V. Klimov, V. Shnyrov, Temperatureinduced functional and structural transformations of the photosystem II oxygen-evolving complex in spinach subchloroplast preparations, Biochem. Mol. Biol. Int. 35 (1995) 1233–1243. [55] Tz. Markova, A. Popova, M. Busheva, M. Velitchkova, N. Naydenova, E. Apostolova, Characteristics of pea chlorophyll mutants: I. Photochemical and physicochemical characteristics of pea mutant with reduced chlorophyll content, Compt. Rend. Acad. Bulg. Sci. 52 (1999) 97–100. [56] Tz. Markova, M. Busheva, A. Popova, M. Velitchkova, N. Naidenova, E. Apostolova, Characteristics of pea chlorophyll mutants: II. Photochemical and physicochemical characteristics of pea mutant with reduced chlorophyll content, Compt. Rend. Acad. Bulg. Sci. 53 (2000) 101–104. [57] I.G. Tremmel, H. Kirchhoff, E. Weis, G.D. Farquhar, Dependence of plastoquinol diffusion on the shape, size and density of integral thylakoid proteins, Bochim. Biophys. Acta 1607 (2003) 97–109.

[58] J. Kurreck, A.G. Seeliger, F. Reifarth, M. Karge, G. Renger, Reconstitution of the endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles, and PS II core complexes from spinach, Biochemistry 34 (1995) 15721–15731. [59] V.P. Shinkarev, Govindjee, Insight into the relationship of chlorophyll a fluorescence yield to the concentration of its natural quenchers in oxygenic photosynthesis, Proc. Natl. Acad. Sci. USA 90 (1993) 7466–7469. [60] S. Styring, A.W. Rutherford, In the oxygen-evolving complex of photosystem II the S0 state is oxidized to the S1 state by D+ (signal IIslow), Biochemistry 26 (1987) 2401–2405. [61] J. Messinger, W.P. Schro¨der, G. Renger, Structure–function relations in photosystem II. Effects of temperature and chaotropic agents on the period four oscillation of flash-induced oxygen evolution, Biochemistry 32 (1993) 7658–7668. [62] J.J. Plijter, A. de Groot, M.A. van Dijk, H.J. van Gorkom, Destabilization by high pH of the S1-state of the oxygen-evolving complex in photosystem II particles, FEBS Lett. 195 (1986) 313–318. [63] C.J. Lockett, C. Demetriou, S.J. Bowden, J.H.A. Nugent, Studies on calcium depletion of PS II by pH 8.3 treatment, Bochim. Biophys. Acta 1016 (1990) 213–218. [64] J. Messinger, G. Renger, Analyses of pH-induced modifications of the period four oscillation of flash-induced oxygen evolution reveal distinct structural changes of the photosystem II donor side at characteristic pH values, Biochemistry 33 (1994) 10896– 10905. [65] N. Tamura, H. Kamachi, N. Hokari, H. Masumoto, H. Inoue, Photoactivation of the water-oxidizing complex of photosystem II core complex depleted of functional Mn, Bochim. Biophys. Acta 1060 (1991) 51–58.