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Journal of Plant Physiology 169 (2012) 111– 116

Contents lists available at SciVerse ScienceDirect

Journal of Plant Physiology

jou rn al h o mepage: www.elsev ier .de / jp lph

hloroplast ultrastructure and thylakoid polypeptide composition are affected byifferent salt concentrations in the halophytic plant Arthrocnemumacrostachyum

ndrea Trottaa,1 , Susana Redondo-Gómezb , Cristina Paglianoc , Manuel Enrique Figueroa Clementeb ,icoletta Rasciod, Nicoletta La Roccad, Alessia Antonaccia, Flora Andreuccia, Roberto Barbatoa,∗

Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale„ viale Teresa Michel 11, 15121 Alessandria, ItalyDepartamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, SpainDipartimento di Scienza dei Materiali e Ingegneria Chimica, Politecnico di Torino, ItalyDipartimento di Biologia, Università di Padova, Padova, Italy

r t i c l e i n f o

rticle history:eceived 25 July 2011eceived in revised form 2 November 2011ccepted 3 November 2011

a b s t r a c t

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosyntheticcomplexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied.Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found thatthe PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in

eywords:rthrocnemum macrostachyumhloroplast structurealophyteshotosystem IIhylakoid composition

a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunitswith a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, aswell as the organization of thylakoid membranes were affected by NaCl concentration, with many granacontaining few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mMNaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.

© 2011 Elsevier GmbH. All rights reserved.

ntroduction

Photosystem II (PSII) is the water splitting enzyme, able toxtract electrons from water and use them to reduce a plasto-uinone pool, with the production of molecular dioxygen (Barber,009). The reaction centre of PSII is composed of the D1 (PsbA), D2PsbD), CP47 (PsbB), CP43 (PsbC) and � and � subunits (PsbE andsbF) of cytochrome b559 intrinsic polypeptides, to which a num-er of extrinsic subunits, i.e. PsbO, PsbP, PsbQ and likely the PsbRrotein are associated at the luminal side (Suorsa and Aro, 2007).ater oxidation is carried out by an inorganic cluster composed of

Mn and 1 Ca ions (CaMn4), which are co-ordinated to a number

f amino acid residues located in the luminal exposed regions ofhe intrinsic D1 and CP43 polypeptides. However, it has also longeen known that in order to optimize the water splitting reaction of

Abbreviations: Chl, chlorophyll; PAM, pulse amplitude modulated fluorimeter;S, photosystem; RC, reaction centre; SDS-PAGE, sodium dodecyl sulphate polyacry-amide gel electrophoresis.∗ Corresponding author. Tel.: +39 0131 360294; fax: +39 0131 360390.

E-mail address: roberto.barbato@mfn.unipmn.it (R. Barbato).1 Present address: Molecular Plant Biology, Department of Biochemistry and Foodhemistry, University of Turku, Finland.

176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.jplph.2011.11.001

PSII, the presence of Cl− and Ca2+ is required (see Homann, 2002).The PsbO protein stabilizes the CaMn4 cluster, whereas, based onin vitro studies, PsbP and PsbQ are thought to modulate the func-tional roles of Ca2+ (PsbP) and Cl− (PsbP and PsbQ) (see Seidler,1996). Recently, using the RNAi technique, it was shown that PsbQis completely dispensable in intact plant (Ifuku et al., 2005; Yi et al.,2006), as long as the light intensity for growth was not too low (i.e.5 �mol m−2 s−1) (Yi et al., 2006), whereas PsbP is necessary at leastto some extent (Ifuku et al., 2005; Ishihara et al., 2005; Yi et al.,2007). Moreover, PsbQ/PsbQ interaction (De Las Rivas et al., 2007;Anderson et al., 2008) or PsbP-PsbX (where X is any other unspec-ified PSII protein) interaction (Yi et al., 2009) are supposed to playan important role in grana stacking. These findings suggest com-plex roles for these subunits, in addition to those in Ca2+ and Cl−

homeostasis.Halophytes are a phylogenetically heterogeneous group of

plants adapted to salty environments. They may tolerate, or evenrequire, high salt concentrations during growth (O’Leary and Glenn,1994). The mechanism by which they survive in saline environ-ments is based on the selective absorption of K+ over Na+ and

compartmentalization and/or secretion of most of the absorbedsalts (Flowers et al., 1977; Glenn et al., 1999; Flowers and Colmer,2008). Usually, salts are accumulated in the vacuole and a highlynegative water potential, necessary to prevent dehydration, is

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12 A. Trotta et al. / Journal of Pla

eveloped by the synthesis of osmocompatible solutes. The wayn which other cellular compartments, chloroplasts in particular,dapt to these osmotic conditions, as well as their actual concen-ration of ions, remain to be established.

Recently, it has been shown that in the extreme halophytealicornia veneta living in its natural salty environment the PsbQrotein is totally absent and only 30% of the PsbP polypeptidewhen compared with the spinach glycophyte) could be detectedPagliano et al., 2009). The conclusion was that in such growingonditions PsbQ was largely dispensable, whereas even a limitedmount of PsbP was sufficient for normal growth, consistently withesults derived from studies with knocked-down or knocked-outransgenic plants (Ifuku et al., 2005; Yi et al., 2006, 2007).

With the above findings in mind we undertook a study on theffect of growing another halophyte Arthrocnemum macrostachyumwhich is phylogenetically closely related to S. veneta) in differentmounts of NaCl approximately ranging from 6 mM to 1030 mMo understand the possible effects of varying external salts on thehloroplast ultrastructure and the composition and functionality ofhylakoids in this extreme halophyte.

aterials and methods

lant material and growth conditions

Seeds of Arthrocnemum macrostachyum were collected inctober 2006 from Odiel Marshes (37◦15′N, 6◦58′W, SW Penin-

ula Iberica). Seeds were placed in a germinator (ASL Aparatosientificos M-92004, Spain), and subjected to a 10 h light35 �mol m−2 s−1)/14 h dark regime for 30 days. Temperature was0 ◦C during the light period and 5 ◦C during the dark phase. Thesehotoperiodic and temperature conditions were chosen to mimiche autumn conditions in Odiel marsh, and brought about the ger-

ination of a high percentage of seeds (Redondo-Gómez et al.,007). Seedlings were then planted in individual plastic pots filledith perlite and 20% Hoagland’s nutrient solution was added. Potsere placed in a glasshouse, where temperature was 21–25 ◦C, rel-

tive humidity 40–60%; irradiation was from natural daylight andeached a maximum photon flux of 1000 �mol m−2 s−1. At desiredime, pots were subjected to five different NaCl treatments, i.e. 0,0, 170, 510 and 1030 mM, using shallow trays (three pots per tray,ne tray per saline treatment), lasting for 30 days. Salinity treat-ents were given by combining 20% Hoagland solution with the

ppropriate amount of NaCl, that means that no addition of NaClorresponds to the salinity of 20% Hoagland solution, i.e. about

mM total salt. At the beginning of the experiment 3 L of the appro-riate solution was placed in each tray. During the experiment, the

evel of solution was maintained constant by adding 20% Hoaglandolution with the appropriate NaCl amounts. The saline solutionas renewed every 2 weeks. Other halophyte samples (i.e. Sal-

cornia veneta, Salicornia emerici, Halocnemum strobilaceum) wereollected at Bellocchio marsh (Italy), whose ecological characteris-ics have previously been described (Pagliano et al., 2009). Spinachlants, used as a control, were purchased at a local market.

ransmission electron microscopy

Samples from stem cortical parenchyma of A. macrostachyumlants grown on different salt concentrations were fixed in glu-araldehyde and processed according to Rascio and Cuccato (1999).n order to make sure that chloroplast shape was not affected dur-

ng fixation by osmolarity, different concentrations of cacodylateuffer (0.1–0.6 M) was used.

Ultrathin sections (800 nm), cut with an ultramicrotomeUltracut-Jung), were stained with uranyl acetate and lead citrate

siology 169 (2012) 111– 116

and examined with a transmission electron microscope (TEM 300,Hitachi) operating at 75 kV.

Fluorescence analysis

Pulse-modulated fluorescence parameters were measuredusing a Dual-PAM-100 chlorophyll (Chl) fluorimeter (Walz). Theminimum Chl fluorescence at an open PSII centre (Fo and Fo′) wasdetermined using light at an intensity of 1 �mol m−2 s−1. A satu-ration pulse of white light (3000 �mol m−2 s−1 for 1.0 s) was thenapplied to determine the maximum Chl fluorescence at closed PSIIcentres in the dark (Fm) and during actinic light illumination (Fm′).The steady state of the Chl fluorescence level (Fs) was recorded dur-ing actinic light illumination (3–800 �mol m−2 s−1). The Fv/Fm and˚PSII were calculated as (Fm − Fo)/Fm and (Fm′ − Ft)/Fm′, respec-tively (Maxwell and Jonhson, 2000).

Thylakoids isolation, chlorophyll and electron transfermeasurements

Thylakoids were isolated by grinding photosynthetic stemsof A. macrostachyum from different salt conditions in a buffermade of 50 mM Hepes pH 7.2, 5 mM MgCl2, 10 mM NaCl, 0.5 Msucrose. To prevent protein degradation the buffer also con-tained, with a ratio 1/100, a cocktail of protease inhibitors (amixture of [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlo-ride], bestatin hydrochloride, N-(trans-epoxysuccinyl)-l-leucine4-guanidinobutylamide, leupeptin hemisulfate salt, pepstatinA, 1,10-phenanthroline) (Sigma). The homogenate was filteredthrough 6 layers of cotton cheese cloth and membranes pellet-ted by centrifugation for 10 min at 3000 × g at 4 ◦C. Pellets weresuspended in the same buffer, without sucrose, and spun down at4500 × g for 10 min. Finally, thylakoids were resuspended in 50 mMHepes–NaOH, pH 7.2, 5 mM MgCl2, 10 mM NaCl, 0.1 M sucroseand the Chl concentration measured according to Arnon (1949).The same procedure was used to isolated thylakoids from otherhalophytes here considered. For spinach, thylakoids were isolatedas described in Barbato et al. (1992). Steady state oxygen evolu-tion with isolated thylakoids was determined in the presence of0.2 mM 2,6-dichlorobenzoquinone in 50 mM Hepes–NaOH, pH 7.2,5 mM MgCl2, 10 mM NaCl, 0.1 M sucrose with a Chl concentrationof 10 �g ml−1.

SDS-PAGE and immunoblotting

SDS-PAGE was carried out as described by Barbato et al. (1992).After electrophoresis, gels were either stained with Coomassie BlueR-250 or blotted to nitrocellulose membranes according to Dunn(1986). For immunodetection, membranes were saturated with10% skimmed milk and probed with antibodies to thylakoid pro-teins as described previously. Immunodetection was performedby the alkaline phosphatase conjugate method, with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as chromogenicsubstrates. Properties of polyclonal antibodies raised in rabbits todifferent antigens (CP47, CP43, D2, D1, PsbS, PsbO, PsbP, PsbQ,cytochrome f) were previously reported (Barbato et al., 1992). Prop-erties of antibodies to a PSI-200 preparation, obtained by sucrosegradient ultracentrifugation of Triton X-100 solubilized unstackedthylakoids (Mullet et al., 1980), were described in Paolo et al. (1990).Properties of PsbR antiserum were described by Suorsa et al. (2006).Estimation of relative amounts of different proteins (on a Chl basis)was performed by digitalization of several blots for each anti-

body. Intensities of immunoreactions were compared with thoseobserved in spinach, assumed to represent 100% of the protein.See legend of Fig. 5 for further details. The Quantity One (BioRad)software was used.

nt Physiology 169 (2012) 111– 116 113

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Fig. 1. Transmission electron micrographs of chloroplasts from Arthrocnemummacrostachyum plants grown on different NaCl concentrations. (A) Broadenedchloroplasts with thick grana (g) and numerous swollen thylakoids (arrows) from aplant grown on 0 mM NaCl. (B) The chloroplast of a plant grown on 80 mM NaCl stillshowed some swollen thylakoids. (C) The inner membrane system of the chloroplastfrom a plant grown on 510 mM NaCl was well organized, with flattened thylakoidsand numerous although small grana. (D) The chloroplast from a plant grown on1030 mM NaCl exhibits lengthened shape and thylakoids with very numerous but

A. Trotta et al. / Journal of Pla

esults

Chloroplast ultrastructure and functional properties of the pho-osynthetic apparatus of A. macrostachyum are affected by growthn different salt conditions.

In the green stem of A. macrostachyum the photosynthetic activ-ty is provided by the peripheral region of the cortical cylinder,

hose cells contain several chloroplasts (Castroviejo et al., 1990).nalysis by electron microscopy showed that chloroplasts of thehotosynthetic cells (Fig. 1) were affected by external salt con-entration. At 510 mM NaCl, the optimal concentration for growth,hloroplasts (Fig. 1C) displayed a normal lenticular shape and aypical thylakoidal system, characterized by the presence of manyrana and stroma lamellae. At variance, when salt concentrationas higher (Fig. 1D) or lower (Fig. 1A and B) than the optimal one,

hloroplast ultrastructure was affected. At low salt concentrationshe chloroplasts exhibited an inner membrane system with largeranal stacks and swollen thylakoids. Thylakoids from grana endsere more affected than internal ones, and this effect was moreronounced at 0 mM (Fig. 1A) than 80 mM NaCl (Fig. 1B). At 170 mMnot shown), chloroplast structure was similar to that observed at10 mM NaCl. At 1030 mM NaCl, chloroplasts became longer andhinner than in other samples and the inner membrane systemas composed by a high number of grana, each containing a few

hylakoids (Fig. 1D). Interestingly, large and faintly electron-denselastoglobules were usually present in stroma of all chloroplasts,hich resembled the same kind of inclusions already noticed in

he chloroplasts from the related halophyte S. veneta (Paglianot al., 2009). Different osmolarity during fixation did not affecthe shape of chloroplasts, excluding that the observed changesere due to artefact during fixation itself. In Table 1, some gen-

ral photosynthetic characteristics of A. macrostachyum (obtainedoth from in vivo and in vitro measurements) are reported. Plantsrown in 0 mM NaCl showed photosynthetic properties clearlyifferent from those of salt treated samples: a lower thylakoidshl a/b ratio (i.e. 2.5 vs. 3.0–3.2), a halved electron transfer ratemeasured with isolated thylakoids as oxygen evolution with 2,6-ichlorobenzoquinone as electron acceptor), and lower Fv/Fmatios of dark-adapted samples and lower ˚PSII, indicating that, ateast in this condition, PSII is strongly affected.

hotosystem II, but not Photosystem I or cytochrome b6/f, isffected by different salt conditions

Polypeptide composition of thylakoid membranes isolated fromlants grown under different salt conditions is reported in Figs. 2–4.

n Fig. 2, a Coomassie stained SDS/PAGE of A. macrostachyum thy-akoids from plants grown at different concentrations of NaCllanes 1–5) is shown, together with spinach thylakoids (lane 6)s comparison, whereas in Figs. 3 and 4, immunoblots with anti-odies to different thylakoid polypeptides are reported. On theasis of apparent molecular weights and reactivity to antibodies,he presence/absence and the identity of a number of thylakoidsolypeptides could be established. These included: the CP1 andHCI subunits of PSI (Figs. 2 and 3A), � and � subunits of ATPaseFig. 2), cytochrome f (Fig. 3B), and some PSII proteins, such as PsbBCP47), PsbC (CP43), PsbD (D2), PsbA (D1), LHCII, PsbS, PsbR, PsbO,nd PsbP (Fig. 4). PSI and cytochrome f proteins were present in

similar amount among different NaCl treated samples. At vari-nce, the relative amount of a number of PSII proteins was foundo be influenced in a complex way by salt concentration. Thus themount of PsbO, PsbS and PsbR, seemed to be independent of the

alt treatment, as their level remained rather constant, whereasariation of the amount of LHCII proteins was rather small, in therder of 10% or less. All PSII reaction centre proteins were foundo decrease in the 0 mM NaCl treated plants. The slightly higher

reduced granal stacks. In all the organelles large and faintly electron-dense globularinclusions (gi) can be seen (Bars = 1 �m).

abundance of LHCII in the 0 mM NaCl treated sample paralleled bythe decreased amount of PSII centres, could account for the lowerChl a/b ratio observed in thylakoids from this sample (Table 1). The

amount of PsbP protein showed a different behaviour: the highestamount was observed at 80–510 mM NaCl, whereas a decrease atboth higher and lower concentrations of external NaCl occurred.Due to this complex behaviour, a semi-quantitative approach was

114 A. Trotta et al. / Journal of Plant Physiology 169 (2012) 111– 116

Table 1Properties of Arthrocnemum macrostachyum plants and isolated thylakoids grown at different concentrations of NaCl.

NaCl (mM) 0 80 170 510 1030

Fv/Fm 0.74 ± 0.03 0.80 ± 0.01 0.81 ± 0.01 0.77 ± 0.02 0.79 ± 0.02˚PSII

a 0.31 ± 0.02 0.48 ± 0.02 0.50 ± 0.02 0.52 ± 0.02 0.54 ± 0.01Chl a/b 2.53 ± 0.03 3.15 ± 0.04 2.94 ± 0.04 3.19 ± 0.03 3.05 ± 0.07O2 evolutionb 86 ± 4 199 ± 20 198 ± 17 196 ± 9 178 ± 15

a ˚PSII was measured during irradiation with a light intensity of 100 �mol m−2 s−1.b Oxygen evolution was measured in the presence of 2,6-dicholorobenzoquinone and

4–6 independent measurements.

Fig. 2. Coomassie stained SDS-PAGE containing thylakoids from Arthrocnemummp4

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nificant changes were observed in Arthrocnemum plants grown inmedia supplemented with 80, 170, 510 and 1030 mM NaCl indicat-ing that this halophyte is able to adapt to salt, allowing the assemblyof a normal PSII.

acrostachyum; the position of PsbQ is marked on lane 6; lanes 1–5 thylakoids fromlants treated with 0, 80, 170, 510 and 1030 mM NaCl, lane 6 spinach thylakoids;

�g Chl per lane were loaded.

aken to investigate the ratio between extrinsic and intrinsic pro-eins in Arthrocnemum, using spinach as a reference. Triplets 1–3n Fig. 5 shows that when different amounts of chlorophyll wereoaded onto the gel, a proportional increase in immunostaining wasbserved for antibodies to CP47, PsbO and PsbP, indicating that ouretection system was in the linear range of response. Therefore, the

mmunostaining intensity observed with 1.5 �g Chl from Arthroc-emum sample grown at different concentrations of salt was usedo estimate (at least approximately) the level of PSII proteins. Themount of PsbP was in between 20% (in the 0 mM NaCl treated sam-le) and about 60% (in the 80–510 mM NaCl treated samples) wheneferred to spinach. The level of the PsbO protein was around 60%ith respect to spinach, irrespectively of salt concentration in the

rowth medium, whereas variation of other proteins (PsbR, PsbS)as not so pronounced, as stated above. The level of CP47, taken

s an indication of the amount of RCII, was in between 28% (in the mM NaCl sample) and 78% (in the 80 mM NaCl sample) wheneferred to spinach. However, the most striking difference with thelycophyte, was the absence of PsbQ from any of thylakoid extractsf the salt treated A. macrostachyum samples (Fig. 4).

he PsbQ protein is absent from thylakoids of other halophytes

In Fig. 6 an immunoblot with antibodies to PsbQ and PsbO pro-eins of thylakoids from a number of other halophytes collectedn their natural environment is shown, with spinach as reference.learly, the PsbO protein is detected in all species whereas the PsbQ

is expressed as �mol O2 mg Chl−1 h−1. Values are average ± standard deviation of

protein is depleted in thylakoids of all halophytes and is detectedonly in spinach (lane 5).

Discussion

In this paper it is shown that not PSI or cytochrome b6/f, butboth PSII and chloroplast ultrastructure were significantly affectedin A. macrostachyum when grown in different salt conditions, asjudged by polypeptide composition, electron transfer, fluorescenceand electron microscopy. In particular, 0 mM NaCl external concen-tration, when compared with the other tested ones, was the mostsevere condition, as a marked loss of PSII reaction centres and adecrease of the quantum yield of remaining centres was observed inplants grown without addition of external salt. In this condition, anuneven degradation of reaction centre polypeptides was observed,which brought about a marked loss of PSII centres (>50%) as judgedby immunoblots, which could explain the decrease in oxygen evo-lution displayed by this sample. As highlighted by Redondo-Gómezet al. (2010), low salt content in the growing medium broughtabout an increased stomata resistance which, in turn, could lead tothe over-reduction of electron acceptors, increasing the probabilityof photoinhibition (Powles, 1984; Aro et al., 1993; Takahashi andBadger, 2011) even under moderate light intensities, which couldalso explain the observed decrease in ˚PSII. At variance, no sig-

Fig. 3. (A) Immunoblotting with antibodies to PSI and (B) cytochrome f; lanes 1–5thylakoids from plants treated with 0, 80, 170, 510 and 1030 mM NaCl, lane 6 spinachthylakoids; 1.5 �g Chl was loaded per gel lane.

A. Trotta et al. / Journal of Plant Physiology 169 (2012) 111– 116 115

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Fig. 5. Densitometric analysis of immunological reaction for some selected anti-bodies. In order to evaluate the linearity of our detection method, three differentamounts of spinach thylakoids (corresponding to 2.0, 1.5 and 1.0 �g chlorophyllin triplets 1–3 respectively) were loaded onto the gel; immunodetection with anti-CP47 (closed columns), anti-PsbO (grey columns) and anti-PsbP (open columns) wasthen performed and blots digitalized. From triplets 1 to 3, an increase of immunos-taining proportional to the increase of chlorophyll loaded onto the gel is observed.In triplets 4–8 the immunostaining intensity of the same antibodies is reportedfor Arthrocnemum samples from 1030 (triplet 4), 510 (triplet 5), 170 (triplet 6),80 (triplet 7) and 0 mM NaC (triplet) is reported. 1.5 �g Chl was loaded for these

particular, PsbQ–PsbQ interactions in the light would enhancegrana stability (De Las Rivas et al., 2007; Anderson et al., 2008).Anyway, Yi et al. (2009) have shown that in Arabidopsis thaliana

ig. 4. Immunoblotting with antibodies to PSII proteins; lanes 1–5 thylakoids fromlants treated with 0, 80, 170, 510 and 1030 mM NaCl, lane 6 spinach thylakoids.5 �g Chl was loaded per gel lane.

Surprisingly, but consistently with earlier findings in the halo-hyte S. veneta (Pagliano et al., 2009), also in A. macrostachyum thesbQ protein was never present, even when NaCl was not added tohe growth medium. Therefore it seems likely that, as previouslybserved for S. veneta, the lack of protein could be paralleled by aack of its specific mRNA and that the concentration of NaCl in therowing medium is not a main determinant in the regulation ofsbQ gene expression.

The other two extrinsic polypeptides PsbO and PsbP showed different behaviour. As the amount of PsbO protein was almostndependent of the salt concentration, and the amount of PsbP was

odulated by salt in a way similar to reaction centre proteins, aifferent ratio between the two extrinsic subunits and the reac-ion centre (RC) polypeptides would be expected. Roughly, ouralculation from data in Fig. 6 indicates a PsbO/PsbP/RCII ratio of/0.5/1 for the 0 mM NaCl treated sample and a ratio of 1/0.5/1or all other samples, suggesting for the presence of an extra PsbOopy in the 0 mM NaCl treated sample. Whether this extra copy ofrotein is either assembled with PSII or free in the lumen, cannote said currently (see Ettinger and Theg, 1991; Hashimoto et al.,996, 1997); however, evidence exists both for 1 copy of PsbO perC (Murata et al., 1984; Enami et al., 1991) and 2 copies of PsbOer RC (Seidler, 1994; Leuschner and Bricker, 1996; Betts et al.,997; Popelkova et al., 2008), suggesting for the possibility of atoichiometry between extrinsic an intrinsic subunits modulatedy environmental conditions which could contribute to PSII het-rogeneity (Giardi et al., 1992; Baroli and Melis, 1996; Mathur et al.,

011).

Plants, both halophytes and glycophytes, growing under differ-nt salt concentrations are known to accumulate different amountsf salt in their leaves (Glenn et al., 1999). Therefore, the possibility

samples. Columns represented the average value (expressed as relative units) cal-culated from three independent blots, error bar represented the SD from thesemeasurements.

that the level of sodium and chloride ions is constant in the pho-tosynthetic tissue is not likely, as shown by the different shape ofchloroplasts in differently salt treated samples. Indeed, measure-ment of chloride concentration in chloroplasts led to a number ofvery different values, ranging from a few mM in glycophytes to upto 400 mM in halophytes, with most of measurements falling intothe 30–100 mM range (Harvey et al., 1981; Demmig and Gimmler,1983; Robinson et al., 1983; Robinson and Downton, 1984, 1985).Several in vitro biochemical studies have clearly shown that thepresence of PsbQ and PsbP is not necessary when the chloride con-centration in the medium is around these values (30–100 mM),which are high enough to provide PSII with essential chloride,even in the absence of high affinity sites generated by the pres-ence of PsbP and/or PsbQ subunits (Seidler, 1996). One possibleexplanation for the absence of PsbQ in Arthrocnemum is that at anyof the ionic concentrations tested, the chloride concentration inthe lumen of the chloroplast could be high enough to make PsbQdispensable and, partially, even PsbP.

Recently, it has been hypothesized that normal thylakoid archi-tecture requires the presence of PsbP and PsbQ proteins. In

Fig. 6. Immunoblotting with antibodies to PsbO and PsbQ of thylakoids isolatedfrom Salicornia veneta (lane 1), Arthrocnemum macrostachyum (lane 2), Halocnemumstrobilaceum (lane 3), Salicornia emerici (lane 4) and spinach (lane 5). 1.5 �g Chl oneach lane of the gel were loaded.

1 nt Phy

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16 A. Trotta et al. / Journal of Pla

utant depleted of the PsbQ protein (growing under standardight conditions), thylakoid structure was not affected, whereas in

utants with the PsbP protein knocked-down (13% of wild type), aarked effect on thylakoid structure was observed. Accordingly, in

. macrostachyum a normal thylakoid structure is observed in sam-les depleted of PsbQ but containing significant amount of PsbP (80,70, 510 and 1030 mM NaCl treated samples). Thylakoids architec-ure was modified in the 0 mM NaCl treated sample, where the levelf PsbP dropped down to about 10% as compared with its counter-art in spinach. Finally, the absence of PsbQ from thylakoids of all

nvestigated halophytes, suggested that this could be a commoneature of these plants. Whether the absence of this protein coulde an advantage for halophytes growing in strong salty, remains toe established.

cknowledgements

This work was supported by funding to R.B. by the DiSAV, Uni-ersità del Piemonte Orientale (Ricerca Locale) and by ATF programf the Provincia of Alessandria. The program ‘International Mobil-ty’ of the Università del Piemonte Orientale allowed the start of theollaboration between Alessandria and Sevilla labs, and is greatlycknowledged. Prof. James Barber (Imperial College, London, UK) ishanked for critical reading of the manuscript. Prof. Eva-Mari AroUniversity of Turku, Finland) and Prof. Roberto Bassi (Universityf Verona, Italy) are thanked for making us available antibodies tosbR and PSI respectively.

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