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Annals of Botany, doi:10.1093/aob/mcn234
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© The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

Cristina Pagliano1,{dagger}, Nicoletta La Rocca2,{dagger}, Flora Andreucci1, Zsuzsanna Deák3, Imre Vass3, Nicoletta Rascio2 and Roberto Barbato1,*

1 Dipartimento di Scienze dell'Ambiente e della Vita, Università del Piemonte Orientale, via Bellini 25/G, 15100 Alessandria, Italy
2 Dipartimento di Biologia, Università di Padova, via Bassi 58/B, 35131 Padova, Italy
3 Institute of Plant Biology, Biological Research Center, Temesvári krt. 62, H-6726 Szeged, Hungary

* For correspondence. E-mail roberto.barbato{at}mfn.unipmn.it

Received: 30 July 2008    Returned for revision: 2 September 2008    Accepted: 27 October 2008   


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

Background and Aims: Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca2+ and Cl– as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl– and Ca2+, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides.

Methods: Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used.

Key Results: Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein.

Conclusions: In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.

Key words: Photosystem II, PsbQ, PsbP, halophytes, Salicornia veneta


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Photosystem II (PSII) of oxygenic organisms is a multi-subunit pigment–protein complex that catalyses photo-oxidation of water with concomitant reduction of plastoquinone to plastoquinol (Nelson and Yocum, 2006). The reaction centre complex responsible for these reactions is highly conserved and consists of a minimal structural unit made up of the CP47, CP43, D2, D1 and cytochrome b-559 polypeptides, besides the extrinsic PsbO/OEC1/33-kDa subunit (hereafter referred to as PsbO). This complex occurs in the thylakoid membrane together with many other protein subunits, with roles in light harvesting, photoprotection, structural stability, etc. The oxidizing side of PSII, termed the oxygen-evolving complex (OEC), is a tetra-nuclear manganese cluster (a cubane-like Mn3CaO4 cluster with a mono-µ-oxo bridge to a fourth Mn ion, hereafter referred to as the Ca,Mn cluster) that catalyses the oxidation of water to dioxygen in a multistep process that requires Ca2+ and Cl– for proper function of the catalytic centre. This inorganic cluster is co-ordinated with a number of amino acid residues belonging to the D1 and CP43 polypeptides (Ferreira et al., 2004). Different lines of evidence indicate a role for the PsbO subunit in stabilization of the Ca,Mn cluster (Ghanotakis et al., 1984), although direct interaction of the PsbO subunit with the cluster is not detected (Ferreira et al., 2004). Moreover, when expression of the PsbO protein is prevented by the RNAi technique in higher plants (Yi et al., 2005), PSII centres do not accumulate in the membrane, suggesting other roles for this subunit. In contrast, in cyanobacteria PSII centres are also accumulated when the PsbO protein is completely depleted (Mayes et al., 1991; Philbrick et al., 1991). In higher plants, two additional proteins are associated with the oxidizing side of PSII: the PsbP/OEC2/23-kDa protein (hereafter termed PsbP) and the PsbQ/OEC3/16-kDa protein (hereafter termed PsbQ). Even though these proteins were discovered more than 20 years ago (Akerlund et al., 1982; Miyao and Murata, 1983) and their structure is known at an atomic resolution (Calderone et al., 2003; Ifuku et al., 2004; Balsera et al., 2005), their role in PSII function still remains enigmatic. Recent studies on transgenic plants, with psbP and psbQ genes knocked-out (Yi et al., 2006, 2007) or knocked-down (Ifuku et al., 2005) by the RNAi technique, have not been able to clarify the role of these polypeptides. Mutants lacking the PsbQ protein did not show an altered phenotype (at least when growth light intensity was not very low) and had normal PSII electron transport properties, whereas mutants lacking PsbP showed enhanced photoinhibition and could not survive photoautotrophically. Nevertheless, biochemical and reconstitution studies have unambiguously shown that PsbP and PsbQ proteins modulated the availability of essential Cl– and possibly Ca2+ ions since the in vitro requirement of these co-factors for optimal oxygen-evolving activity showed a ten-fold increase when these polypeptides had been previously removed by NaCl washing (for a discussion see Seidler, 1996; Roose et al., 2007).

Halophytes are a heterogeneous group of plants, with genera widespread in many phylogenetically unrelated families, differing in salt tolerance and the concentration of salt required for optimal growth (O'Leary and Glenn, 1994). Halophytes live in particular environments, such as salt marshes and coastal saline pans, characterized by a total salt concentration up to 1 M (mainly as NaCl, but also Na2SO4, MgSO4, CaSO4, MgCl2, KCl and Na2CO3). Halophytes need ionic and osmotic homeostasis to be salt tolerant. Due to a negative potential inside the plant plasma membrane of between –120 and –200 mV (Niu et al., 1995), passive entry of Na+ into the cytoplasm could make its concentrations even higher than 100 mM, disturbing the normal functioning of the cell (Serrano et al., 1999). In these conditions, the capacity to maintain intracellular K+/Na+ ratio homeostasis is crucial for halophytes. The strategy of maintenance a high K+/Na+ ratio in the cytoplasm includes selective Na+ extrusion and /or the intracellular compartmentalization of Na+ (and chloride), mainly into the vacuole. To balance the osmotic potential of these ions in the vacuole, halophytes accumulate large amounts of osmocompatible organic solutes in the cytoplasm (Flowers et al., 1977; Glenn et al., 1999; Blumwald et al., 2000; Zhu, 2001; Munns, 2002). Little is known about ion distribution in other cellular compartments. By using aqueous fractionation techniques, the chloride concentration in chloroplasts from various species of plants (both glycophytes and halophytes) has been estimated as 40–60 mM in Mesembryanthemum crystallinum (Demmig and Winter, 1986), 60–90 mM in Beta vulgaris (Terry, 1977), 85 mM in Suaeda maritima (Harvey et al., 1981), 80–120 mM in Suaeda australis (Robinson and Downton, 1985), 100 mM in spinach, sugarbeet and pea (Robinson and Downton, 1984), and 100–117 mM in spinach (Robinson et al., 1983). However, when non-aqueous techniques were used chloride concentration in chloroplasts of halophytes was found to be much higher: 383–452 mM in Suaeda maritima (Harvey and Flowers, 1978; Harvey et al., 1981) and 340 mM in Tolypella intricata (Larkum, 1968). Although the estimation of chloroplast chloride concentration is controversial, it seems that even the lowest estimated concentration is sufficient to sustain full oxygen-evolution activity even in the absence of extrinsic PsbP and PsbQ polypeptides. Previous studies with thylakoids isolated from the mangrove Avicennia marina (Andersson et al., 1984; Ball et al., 1984; Ball and Anderson, 1986) suggested that the PsbP protein is necessary at low but not at high chloride concentration, and that thylakoids from this plant are intrinsically much more resistant to salt than thylakoids from glycophytes (Critchley, 1982, 1983). Instead, the PsbQ protein was not necessary at any chloride concentration. Whether PsbP and PsbQ proteins were present in thylakoids from Avinennia marina was not completely clear from this study. In order to clarify the relationships between extrinsic polypeptides and oxygen evolution at high intracellular salt concentrations, the photosynthetic membranes of the extreme halophyte Salicornia veneta were investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Plant material and site description
Fresh samples of the halophyte Salicornia veneta (Pignatti & Lausi, family Chenopodiaceae) were collected in 2005–2008 in the Northern Adriatic region, both in the Natural Reserve of Sacca di Bellocchio and Prato Barenicolo ‘Pietro Zangheri’ (44°36'01'N, 12°17'34'E), during vegetative growth ranging from spring-time to autumn. If not used immediately, samples were frozen in liquid nitrogen and stored at –80 °C.

The elevation of the site, which strongly affects the salinity, ranges from about 1 m at the line of dunes paralleling the coast to a few centimetres below sea level in the depressions. Soil salinity ranges from 6·0 mS cm–1 in the upper marsh to 8·0 mS cm–1 in the lower marsh, whereas water table salinity ranges from 22·7 mS cm–1 for the upper marsh to 33·2 mS cm–1 in the lower one. Average pH of the soil is 7·7 (Andreucci et al., 2000).

Samples of spinach (Spinacia oleracea L., family Chenopodiaceae; hereafter simply referred to as spinach) were collected from local vegetable gardens and used in the studies as a glycophytic control.

Light and electron microscopy
Samples from cortical parenchyma of stems of S. veneta were fixed in glutaraldehyde and processed for light and electron microscopy according to Rascio et al. (1999). For light microscopy, thin sections (1 mm), cut with an ultramicrotome (Ultracut, Jung), were stained with toluidine blue and examined with a light microscope (Ortholux, Leitz). For transmission electron microscopy ultra-thin sections (80 nm), cut with the same ultramicrotome, were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (TEM 300, Hitachi) operating at 75 kV.

Measurements of oxygen evolution, electron transfer and fluorescence
Fluorescence parameters were measured with a portable chlorophyll fluorometer (Hansatech, King's Lynn, UK). Photosynthetic stems were dark adapted for 30 min using leaf clips before measurements, with fluorescence signals recorded up to 1 s at a data acquisition rate of 10 µs for the first 2 ms and every 1 ms thereafter, with a 12-bit resolution, as described by Strasser et al. (1995). Steady-state oxygen evolution with isolated thylakoids was measured in the presence of 0·2 mM 2,6-dichlorobenzoquinone (DCBQ) and 2 mM K3[Fe(CN)6] (FeCN) in 50 mM HEPES-NaOH, pH 7·2, 5 mM MgCl2, 10 mM NaCl, 0·1 M sucrose with a chlorophyll concentration of 10 µg mL–1. Whole-chain electron transport was measured by oxygen uptake using 0·1 mM methyl viologen (MV) in the same buffer containing 0·1 mM sodium azide. When indicated, 5 mM CaCl2 was added. Flash-induced oxygen yield was measured with a home-built bare-platinum electrode system (Vass et al., 1992). Oxygen evolution of isolated thylakoids was induced after 3 min dark adaptation by a series of 20 flashes, given at 1 Hz frequency. The flash patterns were analysed as described by Vass et al. (1992). Light intensities were measured with a digital photoradiometer (Delta OHM).

The flash-induced increase and the subsequent decay of chlorophyll fluorescence yield were measured by a double-modulation fluorometer (Trtilek et al., 1997). For measurements in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), stems were vacuum-infiltrated for 10 min with a solution containing 0·1 mM solution. Data were analysed as described by Vass et al. (1999). Multi-component deconvolution of the measured curves was done by using a fitting function with two exponential components and one hyperbolic component. Very slowly decaying fluorescence is described by a constant A0 amplitude:


Formula

where F(t) is the variable fluorescence yield, F0 is the basic fluorescence before the flash, A0 to A3 are the amplitudes and T1 to T3 are the time constants.

Thylakoid isolation
In order to isolate thylakoid membranes, samples of halophytes stems and spinach leaves were frozen in liquid nitrogen and ground to obtain a fine powder, which was further homogenized in 50 mM HEPES-NaOH, pH 7·2, 5 mM MgCl2, 10 mM NaCl and 0·5 M sucrose. The buffer also contained, in a ratio of 1 : 100 (v/v), protease inhibitors (a mixture of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, bestatin hydrochloride, N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide, leupeptin hemisulfate salt, and pepstatin A, 1,10-phenanthroline). The homogenate was filtered through six layers of cotton cloth, then membranes were pelletted by centrifugation for 10 min at 3000 g at 4 °C. Pellets were resuspended in the previous buffer without sucrose and spun down at 4500 g for 10 min. Finally, thylakoids were resuspended in 50 mM HEPES-NaOH, pH 7·2, 5 mM MgCl2, 10 mM NaCl and 0·1 M sucrose, and the chlorophyll (Chl) concentration measured (Arnon, 1949).

Total protein extracts were prepared by grinding plant leaves or stems in 50 mM HEPES-NaOH, pH 7·2 containing 2 % SDS and protease inhibitors as described above at 4 °C. The homogenates, filtered through six layers of cotton cloth, were centrifuged for 5 min at 3000 g. Supernatants were used for SDS–PAGE.

Treatment with lincomycin and light
D1 turnover was determined by comparing its degradation rate in the presence or absence of 5 mM lincomycin. After overnight incubation, plants were irradiated with a light intensity of 2000 µmol m–2 s–1 for different periods of time (120, 240 and 480 min). Samples were collected at the appropriate irradiation time and thylakoids were immediately isolated as described.

Polyacrylamide gel electrophoresis and immunoblotting
SDS–PAGE was as described by Barbato et al. (1992). After electrophoresis, gels were either stained with Coomassie Blue R-250 or blotted to nitrocellulose membranes (Dunn, 1986). For immunodetection, membranes were saturated with 10 % (w/v) skimmed milk and probed with antibodies to thylakoid proteins (Barbato et al., 1992). Immunodetection was performed by using either chemiluminescence peroxidase substrates or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining.

Immunological detection of extrinsic PsbO, PsbP and PsbQ proteins was carried out with monospecific polyclonal antibodies as described previously (Barbato et al., 1992). Additional antibodies to PsbP and PsbQ were obtained from Dr Inger Carlberg (Stockholm Universitat, Sweden) and Dr Cornelia Spetea (Linkopping Universitat, Sweden), respectively.

Extraction of RNA and RT–PCR analysis of psbO, psbP and psbQ in S. veneta
Total RNA from frozen shoot tissues of S. veneta and spinach was extracted with an RNeasy Plant Mini Kit (Qiagen), according to the manufacturer's instructions. Following DNase treatment, first-strand cDNA synthesis was carried out in triplicate for each sample by reverse transcription (RT) of 2 µg of total RNA, using random hexamers and the ‘ThermoScriptTM RT–PCR System’ Kit (Invitrogen). Samples were then pooled and cDNAs (100 ng) amplified by PCR using primer pairs designed on highly conserved regions of psbO, psbP, psbQ and 18S rRNA gene sequences from spinach (GenBank accessions: X05548 [GenBank] , X05511 [GenBank] , X05512 [GenBank] and L24420 [GenBank] , respectively) and Pisum sativum (GenBank accessions: D13297 [GenBank] , D13296 [GenBank] , AY292531 [GenBank] and U43011 [GenBank] , respectively), plants phylogenetically related to S. veneta. Highly purified salt-free primers for psbO (psbo1f, 5'-CATGACCCGTTTAACCTAC-3'; psbo2r, 5'-AACAAGTTGCTTGATGGTG-3'), psbP (psbp1f, 5'TATGGAGAAGCTGCTAATGT-3'; psbp2r, 5'-TTCCAAAGTAGGCTTGTTT-3'), psbQ (psbq1f, 5'-ATGGCTCAAGCTATGGC-3'; psbq2r, 5'-GCCTCAGCAAGAACAGC-3') and the reference gene 18S rRNA (5'-GCGGATGTTACTTTTAGGAC-3'; 5'-ACCACCCATAGAATCAAGAA-3') were used, generating products ranging in size from 220 to 275 bp. Reaction thermal profiles were as follows: denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C (1 min), 52 °C (30 s), 72 °C (30 s) and a final elongation at 72 °C (10 min). Amplified PCR products were visualized by ethidium bromide staining after electrophoresis in a 1 % agarose gel. Amplicons were then sequenced at the BMR Sequencing Service (University of Padova) on an automated DNA sequencer using the same primers of the amplification.

Sequencing and analysis of psbO, psbP and psbQ of S. veneta
Three OEC genes of S. veneta were sequenced by a combination of RT–PCR and 3'-RACE molecular techniques, followed by automated sequencing of the resulting amplicons. For psbQ, because of absence of mRNA, it was necessary to analyse DNA extracted from fresh shoots of S. veneta with the genomic DNA purification Kit (Fermentas). For all the 3'-RACE reactions, carried out with the FirstChoice RLM-RACE kit (Ambion), several specific couples of primers (beyond those already used for the RT–PCR) were used, chosen every time on a new portion of known gene sequence (for psbO: psbo3f, 5'-GGATGGCATTGACTACG-3'; psbo4f, 5'-TTGAAGGAAAACAACAAGAA-3'; psbo5f, 5'-GAAGAATGGCATTGACTA-3'; psbo6r, 5'-TTGTCATACCCTGTGGAC-3'; for psbP: psbp3f, 5'-GCTGTCATGGTTAGCTCCA-3'; psbp5r, 5'-CTCAAAACTTGACCCGGGTA-3'; for psbQ: psbq3f, 5'-TACCGTCAAAGCCCAACAA-3'; psbq4f, 5'-TTGTTAAGGCTGTTCTTGC-3'). As this sequencing strategy did not allow the sequence of the psbP gene to be completed, a further PCR step was introduced using two new primers (psbp6f, 5'-CTCATTGGTGCTGCTGC-3' and psbp7r, 5'-CTCAAAACTTGACCCGGGTA-3'). Sequencing of all the PCR amplicons used the same primers as amplification. The following GenBank accessions were given to psbO, psbP and psbQ gene sequences of S. veneta, respectively: EF397943 [GenBank] , EF397942 [GenBank] and EF392845 [GenBank] (submitted to NCBI).

Nucleotide sequences of psbO, psbP and psbQ from S. veneta were translated into their corresponding amino acid sequences, using the EMBOSSTranseq software (http://www.ebi.ac.uk/emboss/transeq/), followed by a ‘Swiss Prot’ exhaustive search for PsbO, PsbP and PsbQ amino acid sequences (http://expasy.org/); only sequences belonging to higher plants were further analysed. Precursor polypeptides were cleaved in silico to obtain the mature proteins. Pairwise alignment and identity index were obtained by using the BioEdit suite. Multiple alignments were performed with ClustalX software (Thompson et al., 1997).

Three different matrices were created using all the identity values obtained from each pairwise alignment of all the homologue mature sequences for PsbO, PsbP and PsbQ that were found in database (data not shown).

Quantitative real-time RT–PCR
qRT–PCR reactions used a 7500 Real-Time PCR System (Applied Biosystems), according to the manufacturer's instructions. PCR reactions used gene-specific primers (for psbO: psbo1f, 5'-CATGACCCGTTTAACCTAC-3' and psbo6r, 5'-TTGTCATACCCTGTGGAC-3'; for psbP: psbp1f, 5'-TATGGAGAAGCTGCTAATGT-3' and psbp2r, 5'-TTCCAAAGTAGGCTTGTTT-3'; for psbQ: psbq4f, 5'-TTGTTAAGGCTGTTCTTGC-3' and psbq5r, 5'-AGGTAGAACCTGTCCTTGG-3') and the passive reference dye ROX, a glycine conjugate of 5-carboxy-x-rhodamine with succinimidyl ester (Applied Biosystems), in order to normalize fluorescence across the plate. For each gene, a standard curve was generated using a cDNA serial dilution. Five µL of PCR template, containing 10, 30 and 50 ng of cDNA, were added to 20 µL of mastermix made up of the reaction components of the ‘qPCRTM Core Kit for SYBR® Green I’ (Eurogentec). The following real-time protocol was used: denaturation (95 °C for 10 min), amplification and quantification steps repeated 40 times (95 °C for 1 min, 52 °C for 30 s, 72 °C for 40 s), final extension 72 °C for 10 min, and dissociation (9 5°C for 15 s, 60 °C for 1 min, 95 °C for 15 s).

All reactions were performed in triplicate and a negative water-control was included in each run. Fluorescence was measured at the end of each annealing step. Amplification was followed by a melting-curve analysis with continual fluorescence data acquisition during the 65–95 °C melt. Relative quantification values and standard deviations were calculated using the comparative CT method ({Delta}{Delta} – CT) according to Pfaffl (2001). Values were normalized to the expression of the reference 18S rRNA, as well as the calibrator spinach.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Ultrastructural, photosynthetic characteristics and thylakoid properties of S. veneta
The photosynthetic tissue of S. veneta is in a few cortical layers of the stem cells (Fig. 1A, B), which contain chloroplasts whose organization is similar that of non-halophytic sun plants, with a reduced amount of thylakoid membrane and very large plastoglobules (Fig. 1C). Due to the limited amount of photosynthetic tissue in the stems, the content of photosynthetic pigments was small with a relatively high chlorophyll a/b ratio (3·7; Fig. 1D). Thylakoids were isolated and assayed for functional activity, with spinach thylakoids used for comparison. PSII activity (measured as oxygen evolution with DCBQ and FeCN as the electron acceptor) and whole-chain activity (measured as oxygen uptake with methyl viologen as the electron acceptor) were similar in both plants. For S. veneta and spinach the oxygen-evolution activities were 173 ± 20 and 205 ± 12 µmol O2 mg Chl–1 h–1, respectively, whereas oxygen uptake rates (with methyl viologen as the acceptor) were in the order of 50–60 µmol O2 mg Chl–1 h–1 for both species (49 ± 12 for S. veneta and 57 ± 10 for spinach). When FeCN was used as the electron acceptor, the activity dropped down to 60–70 µmol O2 mg Chl–1 h–1 (68 ± 16 for S. veneta and 67 ± 12 for spinach). Addition of 5 mM CaCl2 (with DCBQ as the acceptor) did not stimulate oxygen-evolution activity in either species (182 ± 8 for S. veneta and 203 ± 12 for spinach). It should also be noted that all electron transport activities were completely blocked by DCMU. The Fv/Fm ratio of isolated thylakoids was also measured and values were 0·76 ± 0·05 for S. veneta and 0·78 ± 0·03 for spinach. Integrity of PSII in isolated tylakoids of S. veneta was further investigated by flash-induced oxygen evolution in isolated thylakoids (Fig. 2). This showed the usual period-four oscillations, with maxima after the 3rd, 7th, 11th ... flashes. While analysing the flash oxygen sequences, a 15 : 85 % distribution of the S0 and S1 states in the dark, as well as 18 % miss and 5 % double-hits were observed (Fig. 2). These values are similar to those obtained in many other photosynthetic systems with a fully intact donor side, and show functional integrity of the Ca,Mn cluster in S. veneta. These data demonstrated that functional thylakoids can be isolated from halophytes.


Figure 1
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  		Fig. 1. The photosynthetic tissue of the Salicornia veneta stem is characterized by a reduced number of cell layers. (A) Light micrograph of a transversal section from the peripheral region of the stem (scale bar = 100 µm); the insert (B) at higher magnification shows the presence of chloroplasts (arrow) in the outer cell layers (scale bar = 50 µm). (C) Transmission electron micrograph of a stem chloroplast showing a reduced amount of regularly organized thylakoid membranes, large plastoglobules and a rather electron-dense stroma; gt, grana thylakoids; st, stroma thylakoids; p, plastoglobule; s, starch. Scale bar = 1 µm. (D) Chorophyll a, chlorophyll b and carotenoid content of stems of S. veneta. Values are the means ±s.d. of seven replicates.

 


Figure 2
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  		Fig. 2. The flash pattern of oxygen evolution in Saliconia veneta thylakoids supports the basic functional integrity of the Ca,Mn cluster. The flash oxygen sequence was measured after a series of single-turnover flashes given at 1 Hz frequency and analysed in terms of the Joliot–Kok model, assuming that in the dark-adapted state PSII centers are either in the S0 or in the S1 state (Vass et al., 1992).

 
PSII in S. veneta lacks the PsbQ polypeptide and contains a reduced amount of the PsbP subunit
The result of an immunoblot made to assess the presence of major polypeptides in thylakoid membranes from S. veneta is given in Fig. 3A (lane 2) and compared with spinach thylakoids (lane 1). Antibodies to the membrane polypeptides PsaC, CP47, CP43, cytochrome f, D2, D1, LHCII, PsbS and {alpha}-subunit of cytochrome b-559 were able to detect their respective antigens, both in thylakoids from S. veneta and spinach and with similar intensities. When antibodies to the extrinsic subunits PsbO, PsbP and PsbQ were used to probe thylakoid membranes (Fig. 3B), PsbO protein was detected both in spinach (lane 1) and S. veneta (lane 2), whereas PsbQ was detected in spinach but not in S. veneta thylakoids. The PsbP protein was detected as a faint band in S. veneta but was abundant in spinach leaves. PsbP was also found in spinach stems together with PsbQ protein (data not shown), indicating that the absence or depletion of these polypeptides in S. veneta were not due to tissue-specific expression. To rule out the possibility that the lack of PsbQ and low amount of PsbP was due to loss of polypeptides during isolation of thylakoids, as reported by Andersson et al. (1984) for Avicennia marina, total stem proteins were loaded onto the gel instead of isolated thylakoids. As shown in Fig. 3C PsbP was again detected as a faint band in S. veneta (lane 2), whereas PsbQ protein was absent. Therefore we conclude that in S. veneta highly modified PSII centres exist, characterized by the lack of the PsbQ subunit. In addition, most PSII centres also lack the PsbP subunit, because its relative abundance is at best 20–30 % of that in spinach. In order to understand whether the reduction or absence of these polypeptides is linked to a failure to accumulate respective transcripts, the presence of mRNAs of psbP and psbQ, together with those of psbO and 18S rRNA, compared to those of spinach, was assessed by RT–PCR (Fig. 3D). The results from this experiment show the specificity of amplification products, as a single band with the expected length was obtained for each gene. As shown in the figure, the amount of mRNA for psbO was similar in the two species (compare lanes 2 and 6); the mRNA for psbP in S. veneta (lane 3) was much less abundant than in spinach (lane 7), and mRNA for psbQ could not be detected in S. veneta (lane 4), whereas it was found in spinach (lane 8). In order to obtain a quantitative estimation of these different mRNAs, the relative abundance of transcripts was measured by using quantitative RT–PCR (qRT–PCR). When normalized to 18S rRNA, the amounts of mRNAs for psbO, psbP and psbQ in S. veneta compared to spinach were found to be 99 %, 33 % and 0·05 %, respectively.


Figure 3
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  		Fig. 3. PSII in Salicornia veneta contains a modified polypeptide composition. (A) Immunoblot of the main thylakoid membrane proteins of leaves of spinach (lane 1) and stems of S. veneta (lane 2). (B) Immunoblot of extrinsic polypeptides from thylakoids of spinach (lane 1) and S. veneta (lane 2). (C) Immunoblot of extrinsic proteins from total protein extracts of leaves of spinach (lane 1) and stems of S. veneta (lane 2). Each lane in (A–C) contains 4 µg of chlorophyll. (D) RT–PCR analysis of psbO, psbP and psbQ expression in S. veneta stems (lanes 2, 3 and 4, respectively) compared to that in spinach (lanes 6, 7 and 8, respectively). RT–PCR was carried out using the amplification of 18S rRNA transcript as a control, for S. (lane 1) and for spinach (lane 5). Mass indicators were loaded on lane 9.

 
Sequences of psbO, psbP and psbQ genes in S. veneta are highly conserved
The amino acid sequences of the PsbP and PsbQ proteins were deduced from the nucleotide sequences of the respective coding region. In Fig. 4, multiple alignments for the mature PsbP (A) and PsbQ (B) proteins are shown, where S. veneta sequences are aligned together with those of other higher plants found in the database. From this figure we conclude that both the PsbP and PsbQ sequences are highly conserved among higher plants (54 % conserved amino acids for PsbP and 45 % for PsbQ). As a measure of sequence similarity, identity indexes for PsbQ were calculated for each of the possible pairwise alignments between S. veneta and any other species, and these give an average value of 0·733 ± 0·043. When pairwise alignments were instead performed using the PsbQ sequence from Chlamydomonas reinhardtii (GenBank accession P12852), this value dropped to 0·253 ± 0·021, indicating that the PsbQ sequence from S. veneta is much more similar to that of any other higher plants than to C. reinhardtii. For the PsbP protein, this bias was even more pronounced, with an average identity index of 0·789 ± 0·034; when the C. reinhardtii sequence was used (GenBank accession P11471 [GenBank] ), the value decreased to 0·353 ± 0·023. A multiple alignment (not shown) was also performed for the sequenced portion of the PsbO protein [GenBank accession P23321 [GenBank] , Arabidopsis thaliana (1-1); Q9S841, Arabidopsis thaliana (1-2); P14226 [GenBank] , Pisum sativum; P23322 [GenBank] , Solanum lycopersicum; P12359 [GenBank] , Spinacia oleracea; Q58H58, Nicotiana benthamiana; Q84QE8, Nicotiana tabacum; P26320 [GenBank] , Solanum tuberosum and P27665 [GenBank] , Triticum aestivum were used]. Identity indexes for PsbO, calculated for each of the possible pairwise alignments between S. veneta and any other species, gave an average value of 0·885 ± 0·049; this value dropped to 0·683 ± 0·020 when pairwise alignments were performed using the PsbO sequence from C. reinhardtii (GenBank accession P12852).


Figure 4
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  		Fig. 4. The sequence of psbP and psbQ genes in Salicornia veneta is highly conserved. (A) Multiple sequence alignment of PsbP proteins from higher plants. Amino acid sequences were downloaded from the ExPASy database (http://expasy.org): Salicornia veneta, ABQ85433 [GenBank] ; Brassica juncea, Q96334 [GenBank] ; Sinapis alba, P11594 [GenBank] ; Arabidopsis thaliana 2-1, Q42029 [GenBank] ; Arabidopsis thaliana 2-2, O49344 [GenBank] ; Solanum lycopersicum, P29795 [GenBank] ; Solanum tuberosum, P93566 [GenBank] ; Nicotiana tabacum 2-1, Q7DM39; Nicotiana tabacum 2-2, P18212 [GenBank] ; Nicotiana benthamiana, Q58H59; Nicotiana tabacum 2-3, Q04127 [GenBank] ; Cucumis sativus, Q9SLQ8; Narcissus pseudonarcissus, Q40407 [GenBank] ; Fritillaria agrestis, O49080 [GenBank] ; Pisum sativum, P16059 [GenBank] ; Spinacia oleracea, P12302 [GenBank] ; Triticum aestivum, Q00434. (B) Multiple sequence alignment of PsbQ proteins from higher plants. Amino acid sequences were downloaded from the ExPASy database: Salicornia veneta, ABQ23675 [GenBank] ; Arabidopsis thaliana 3-1, Q9XFT3; Arabidopsis thaliana 3-2, Q41932 [GenBank] ; Solanum lycopersicum, Q672Q6; Nicotiana tabacum, Q53UI6; Nicotiana benthamiana, Q5EFR5; Pisum sativum, Q7Y1T5; Spinacia oleracea, P12301 [GenBank] ; Zea mays 3-1, Q41048 [GenBank] ; Zea mays 3-2, Q41806. In both (A) and (B) the ‘conserved’ line shows fully conserved residues (*).

 
PSII in S. veneta shows normal electron transport properties
Based on the putative role of PsbQ and PsbP in providing sufficient Cl–, Ca2+ and perhaps Mn ions for PSII functioning, modification of PSII electron transport in S. veneta that lacks all PsbQ and most of PsbP might be expected, similar to that observed after RNAi suppression of PsbQ production in Arabidopsis (Yi et al., 2007). Therefore, we used different biophysical methods to assess electron transport at the donor and acceptor sides of PSII. Illumination with a single-turnover saturating flash induced reduction of QA with an electron extracted from the donor side of PSII, leading to increased fluorescence yield. Subsequent dark reoxidation of QA– resulted in the relaxation of fluorescence yield exhibiting three main decay phases (Vass et al., 1999). The fast phase, A1, contributes to approx. 65 % of total amplitude of decay with a time constant T1 of approx. 0·3 ms, and arises from the reoxidation of QA– by plastoquinone molecules bound to the QB site before the flash. The middle phase, A2, approx. 15 % relative amplitude and T2 of approx. 9 ms, originates from QA– reoxidation by plastoquinone molecules in centres where the QB site was empty at the time of the flash. Finally, the slow phase, A3, approx. 20 % relative amplitude and T3 of approx. 3 s, arises from a back-reaction of the S2 state of the water-oxidizing complex with QA–, which is populated via the equilibrium between QA–QB and QAQB– (Fig. 5A and Table 1). When the QA to QB electron transfer step is blocked by DCMU, the reoxidation of QA– proceeds via charge recombination with donor-side components. The decay was dominated by a 0·6-s component, reflecting the S2QA– recombination (Vass et al., 1999). The fast phase, which is sometimes observed even in the presence of DCMU and arises from the recombination of QA– with Tyr-Z+ in centres with inefficient electron donation from the Ca,Mn cluster to Tyr-Z (Vass et al., 1999), was negligible (below 1 %). These data show that, despite the modified protein composition of the PSII donor side, electron transport from the Ca,Mn cluster to QB is fully functional in S. veneta. Exposure of plants to high light (2000 µmol m–2 s–1 for 2 h) slowed fluorescence relaxation in the absence of DCMU due to the decreased amplitude of the fast phase and the concomitant increase in the amplitude of the middle and slow phases (Fig. 5A). In addition, the time constant of the slow phase was accelerated to the same value as with DCMU (Table 1), showing that in plants treated with high light QB is reduced in most of the centres, and the QA to QB electron transfer is retarded. However, the unchanged fluorescence kinetics in the presence of DCMU showed that high light exposure did not modify the PSII donor side. Previous results on transgenic tobacco plants with severely down-regulated PsbQ and PsbP showed a Ca,Mn cluster that was unstable in the dark (Ifuku et al., 2005). In order to check whether S. veneta shows a similar behaviour, plants were dark-adapted for 2 h. This treatment resulted in the appearance of a very stable fraction of fluorescence relaxation (A0 in Table 1), not decaying in the 100-s time window of the measurement, but eliminated by 5-min exposure of the dark-adapted plants to low light (1–2 µmol m–2 s–1; Fig. 5B).


Figure 5
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  		Fig. 5. Relaxation of flash-induced chlorophyll fluorescence in Salicornia veneta plants shows functionally intact PSII centres. The kinetic traces were measured in intact shoots following excitation by an actinic flash given at 10–3 s. (A) Measurements were performed in plants kept under low-light conditions (circles) or exposed to 2000 mmol m–2 s–1 light for 2 h before the measurements (squares). The shoots were either infiltrated with 100 mM DCMU (open symbols) or not (closed symbols). (B) Shoots kept in low (1–2 mmol m–2 s–1) light intensity (open circles) were dark adapted for 2 h (closed triangles), and were subsequently illuminated with low light for 5 min (closed diamonds). All measurements were performed in the presence of DCMU. The traces are shown after normalization to the same initial amplitude measured 150 ms after the flash.

 


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  		Table 1. QA– reoxidation kinetics in Salicornia veneta in the absence and presence of DCMU: time constants and relative amplitudes of the different phases of chlorophyll-fluorescence relaxation

 
Loss of variable fluorescence is not linked to the loss of oxygen evolution or D1 protein
The results described so far point to the presence in S. veneta of a PSII characterized by a modified donor side. Based on recent suggestions indicating a role for the donor side in light-induced photodamage (Hakala et al., 2005), it may be possible that these plants are affected in their response to high light. This possibility was assessed by analyses of fluorescence, oxygen evolution and immunoblotting with antibodies to D1-protein. After 24 h dark adaptation, a value of 0·81 was observed for the Fv/Fm ratio in S. veneta, similar to values reported for other related halophytes (Redondo-Gomez et al., 2006). Irradiation with a light intensity of about 700 mmol m–2 s–1 for 120 min gave rise to a decrease in the Fv/Fm ratio to 0·64. When plants were irradiated with a light intensity of 2000 mmol m–2 s–1 for 120 min, the Fv/Fm ratio dropped to 0·44. Full relaxation of this strong quenching required many hours of dark incubation. It should also be noted, however, that the quenching of fluorescence was not related to inhibition of oxygen evolution as observed rates were not affected by irradiation, remaining in the order of 10 µmol O2 g–1 f. wt h–1 (with HCO3– as the acceptor). To investigate the role of D1 protein in this phenomenon, the amount of protein was measured by immunoblotting in plants that had been treated with 2000 mmol m–2 s–1 light for different periods of time. As no significant changes in the amount of the D1 protein were detected (Fig. 6, – lincomycin), a lack of correlation between quenching of fluorescence and degradation of D1 exists. The experiment was then repeated with plants pre-incubated with lincomycin, in order to prevent protein resynthesis during irradiation. Again in this case, however, loss of D1 protein from the thylakoid membrane was not evident (Fig. 6, + lincomycin).


Figure 6
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  		Fig. 6. D1 protein is not degraded in high light. Immunoblots with antibodies to D1 of thylakoids isolated from stems of Salicornia veneta exposed to high light intensity (2000 mmol m–2 s–1) for different periods of time: 0 min (lane 1), 120 min (lane 2), 240 min (lane 3) and 480 min (lane 4). + lincomycin refers to plants that had been pre-incubated in the dark with 5 mM lincomycin; – lincomycin refers to plants pre-incubated in water. Each lane contains 4 µg of chlorophyll.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
In this paper we show that thylakoids from the extreme halophyte S. veneta do not contain the PsbQ protein. With spinach taken as a reference, the amount of the PsbP protein in S. veneta is around 30 %, whereas the PsbO polypeptide is present in a normal amount. If the amount of mRNA for extrinsic proteins in spinach is again taken as a reference, then a similar situation is observed: in S. veneta the psbQ gene is not expressed and the amount of the psbP transcript is about 30 % of that observed in spinach, whereas the expression of psbO is unaffected. However, despite the absence of PsbQ and depletion of PsbP, PSII is fully functional. This can be observed for the intact system by single-turnover flash fluorescence decay, and by measuring various functional activities in isolated thylakoids, such as oxygen evolution, S-state turnover, whole-chain electron transfer and variable fluorescence. Therefore it is likely that in S. veneta the PsbQ protein is not necessary and that even sub-stoichiometric amounts of PsbP are sufficient for a normal PSII function. Despite this modified expression pattern, the sequence of extrinsic polypeptides in S. veneta is highly conserved.

As far as we are aware, the absence of PsbQ and depletion of PsbP polypeptides has never previously been reported for plants under natural conditions. Only in plants in which these genes were silenced by the RNAi technique (Ifuku et al., 2005; Yi et al., 2006, 2007) and in the FUD39 mutant of Chlamydomonas reinhardtii (Mayfield et al., 1987) were these polypeptides absent. In their studies, Ifuku et al. (2005) and Yi et al. (2007) observed very high light sensitivity of tobacco plants with very little (less than 5 % of wild type) or no PsbP protein and concluded that this protein is absolutely necessary for photoautotrophic growth. Although S. veneta shows high fluorescence quenching in high light, its oxygen-evolving activity and D1 protein content are unaffected and it can also survive with a small amount of PsbP protein. Yi et al. (2006) also showed that even 5 % of the total amount of PsbP is sufficient for a normal PSII functionality. It could be argued that in S. veneta the very high chloride concentration in the environment, and possibly in the chloroplast (Harvey and Flowers, 1978; Harvey et al., 1981), and the high amount of osmocompatible solutes expected (Glenn et al., 1999) could take on the role of the extrinsic proteins, allowing a normal electron transfer from the Ca,Mn cluster to QB even in the absence of PsbQ and with lower amounts of PsbP.

The occurrence of a non-decaying component of flash-induced fluorescence indicates that in S. veneta the Ca,Mn cluster is replaced by a very stable donor species in a small fraction of PSII centres. This effect may arise from destabilization or release of Mn, or from the reduction of the oxidized form of Tyr-D, which can act as a fast, one-electron donor (Vass et al., 1990). Light-induced reversal of this effect in the presence of DCMU, which only allows a one-step electron transfer, supports the possibility of Tyr-D+ reduction in the dark since photoligation of released Mn would require a multi-step electron transfer through PSII (Bondarava et al., 2005). An increase of the time constant of the main decay phase from 0·62 to 0·74–0·76 s was also observed; however, this change would correspond to less than a 5-meV increase in the energetic stability of the S2QA– charge pair, which is probably negligible at the functional level.

As high salt concentration is the main parameter characterizing the natural environment of S. veneta, it seems reasonable to assume that a high concentration of salt may affect psbP and psbQ gene expression. Accordingly, a recent transcriptomic study (Gong et al., 2005) has shown that in the facultative halophyte Thellungiella halophila (cress) the expression of the psbQ gene is down-regulated upon salt stress. This could be taken as an indication that in high salt conditions the presence of the PsbQ (and at least in part even the PsbP) protein might be dispensable.

The chloride concentration of the chloroplast has been estimated in a wide range of plants. Despite a high degree of variability observed even in the same species [e.g. from 85 mM (Harvey et al., 1981) to 452 mM (Harvey and Flowers, 1978) in Suaeda maritima] depending on the methods used for determination of chloride concentration, values are normally well above the optimal in vitro concentration, which is around 5–10 mM (Seidler, 1996). The observation that the addition of either Ca2+ or Cl– reported here did not stimulate oxygen evolution in isolated thylakoids suggests that in S. veneta the role of these ions is not mediated by PsbQ, and that just 30 % of PsbP is sufficient to fulfil its role. Similar results were reported by Ifuku et al. (2005) with Arabidopsis lacking the PsbQ protein. Therefore, one possibility is that PsbP acts as an assembly factor, a view that would also agree with data from cyanobacteria, where mutants in which homologs of psbP gene have been deleted seem to be affected in the reassembly of PSII during recovery from photoinhibition (Thornton et al., 2006).

Loss of fluorescence in S. veneta plants did not seem to be associated with a massive degradation of reaction-centre D1 protein, as its amount was not affected by lincomycin. A lack of correlation between fluorescence quenching and degradation of D1 protein has also been reported with isolated thylakoids (Santabarbara et al., 2001). The Fv/Fm value observed after light treatment required hours rather than minutes to relax to that of the dark control, suggesting that it is not responsible for the observed phenomenon. It might be expected that, due to the modified polypeptide composition at the donor side, the plant could suffer from donor-side-type photoinhibition. However, as just a slight slowing-down of electron transfer from QA to QB is observed, an acceptor-side-type of photoinhibition could eventually take place (Aro et al., 1993).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The absence of the PsbQ subunit from thylakoid membranes of S. veneta clearly shows that this protein is not necessary for oxygen evolution and that even a relatively low amount of PsbP (when compared with spinach) is sufficient for a normal PSII function. PsbP may be necessary for oxygenic photosynthesis in vivo but not in vitro, when water splitting is still functional because of added Ca2+ and Cl–. One possibility is that a larger concentration of ions (especially Cl–) in the proximity of PSII could modify the requirement for these polypeptides. As the PsbP protein is always found, it is likely that – besides a role in modulation of Ca2+ and Cl– function – it could play additional role(s). Our data are consistent with the possibility that PsbP acts as an assembly factor, as has emerged from recent studies on Arabidopsis (Yi et al., 2007) and cyanobacteria (Thornton et al., 2006).


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Professor Giorgio Forti for carrying out fluorescence and oxygen evolution measurements and for stimulating discussions; Sovrintendente Emanuele Battani and Ispettore Giuseppe Battani (Corpo Forestale dello Stato, Comando Stazione Forestale, Casalborsetti, Ravenna) for plant sampling over the years; and Dr Cornelia Spetea (University of Linkoping) and Dr Inger Carlberg (University of Stockholm) for the gift of antibodies to 33-, 23- and 16-kDa proteins. This work was supported by Fondo Investimento Ricerca di Base (RBAU01E3CX_007 to R.B.), Università del Piemonte Orientale, International Mobility Program 2007 and Associazione Territorio e Formazione, Provincia di Alessandria, and partly by the European Union (grant no. SOLAR-H2, 212508 to I.V. and Z.D.).


   FOOTNOTES
 
{dagger} These authors contributed equally to this work. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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