Báo cáo khóa học: Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ pdf

In the absence of psbL, no PSII core dimers or PSII–light harvesting complex LHCII supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile.. In

Protein assembly of photosystem II and accumulation of subcomplexes

in the absence of low molecular mass subunits PsbL and PsbJ

Marjaana Suorsa1, Ralph E Regel2, Virpi Paakkarinen1, Natalia Battchikova1, Reinhold G Herrmann2 and Eva-Mari Aro1

1

Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Finland;2Botanisches Institute der Ludwig-Maximilians Universita¨t, Mu¨nchen, Germany

The protein assembly and stability of photosystem II (PSII)

(sub)complexes were studied in mature leaves of four plastid

mutants of tobacco (Nicotiana tabacum L), each havingone

of the psbEFLJ operon genes inactivated In the absence of

psbL, no PSII core dimers or PSII–light harvesting complex

(LHCII) supercomplexes were formed, and the assembly of

CP43 into PSII core monomers was extremely labile The

assembly of CP43 into PSII core monomers was found to be

necessary for the assembly of PsbO on the lumenal side of

PSII The two other oxygen-evolving complex (OEC)

pro-teins, PsbP and PsbQ, were completely lackingin DpsbL In

the absence of psbJ, both intact PSII core monomers and

PSII core dimers harboringthe PsbO protein were formed,

whereas the LHCII antenna remained detached from the

PSII dimers, as demonstrated by 77 K fluorescence

meas-urements and by the lack of PSII–LHCII supercomplexes

The DpsbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins In con-trast, very young, vigorously expanding leaves of all psb-EFLJoperon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were pre-sent and correctly assembled in all psbEFLJ operon mutants Keywords: oxygen-evolving complex; photosystem II assembly; photosystem II small subunits; psbEFLJ operon; tobacco

Photosystem II (PSII) is a multisubunit pigment–protein

complex that catalyses electron transfer from water to the

plastoquinone pool with concomitant evolution of oxygen

The PSII reaction center core consists of the D1 and

D2 proteins, cytochrome b559 (Cyt b559), the

chloro-phyll a-bindingantenna proteins CP43 and CP47, and a

number of low molecular mass (LMM) proteins, the

functions and locations of which in PSII are still largely

unknown They include both chloroplast-encoded (PsbH, I,

J, K, L, M, N, T and Z) and nucleus-encoded (PsbR, W and

X) proteins with generally only one membrane-spanning

helix [1] Duringthe past few years, enormous progress has

been made in determiningthe structure of PSII [2–4] The

functional form of PSII is apparently a dimer [5] The

oxygen-evolving complex (OEC) situated on the lumenal

side of PSII is composed of the PsbO (33 kDa), PsbP

(23 kDa) and PsbQ (17 kDa) proteins in higher plants PSII

dimers further associate with the light-harvesting complex II (LHCII) to form PSII–LHCII supercomplexes, the minor antenna proteins CP24, CP26 and CP29 probably servingas linker proteins [2,5,6] It has been suggested that several LMM proteins participate in PSII dimerization [7,8] However, despite the available structure of PSII at 3.8 and 3.7 A˚ resolution [3,4], the exact locations and roles of most of the LMM proteins in the assembly and stability of PSII remain to be determined

Today it is a challenge to resolve the assembly steps of PSII Various approaches have been fruitful in analysingthe primary assembly steps of PSII [9] The best-characterized LMM proteins of PSII, the a and b subunits of Cyt b559, probably function as an assembly core, which is required for the synthesis of the D2 protein [10] Indeed, it has been shown that Cyt b559and the D2 protein exist as a complex

in etiolated barley leaves [11] The full-length D1 protein, however, is synthesized only in the light and is cotransla-tionally associated with the D2–Cyt b559 complex [12] Radiolabelingexperiments have demonstrated that the subsequent assembly steps include association of CP47 followed by that of CP43 [13] Labelingexperiments, however, are unable to reveal all the different steps in the sequential and hierarchical assembly of multisubunit PSII

In particular, the assembly of the LMM subunits, except the Cyt b559 subunits, has been difficult to address This is because separation of the various subcomplexes after assembly of each of the LMM subunits is not possible

Correspondence to E.-M Aro, Department of Biology, Plant

Physio-logy and Molecular BioPhysio-logy, FIN-20014 University of Turku,

Finland Fax: + 358 2333 5549, Tel.: + 358 2333 5931,

E-mail: evaaro@utu.fi

Abbreviations: BN, Blue-native; Cyt, cytochrome; LHCII,

light-harvestingcomplex II; LMM, low molecular mass; OEC,

oxygen-evolvingcomplex; PSI, photosystem I; PSII, photosystem II.

(Received 5 September 2003, revised 28 October 2003,

accepted 4 November 2003)

because of resolution problems and, furthermore, only some

of the LMM subunits of PSII incorporate [35S]methionine

Another approach to understandingthe assembly of

LMM subunits into PSII is to use specific PSII protein

deletion mutants and to analyse the ability of such mutants to

form various PSII subassemblies This approach has only

seldom been taken because of technical problems, and, when

applied, the fractionation of PSII subcomplexes has been

based in sucrose-density centrifugation with limited

resolu-tion capacity [14] Moreover, none of the numerous studies

with Synechocystis 6803 mutants of the LMM subunits of

PSII has addressed the PSII assembly process as such, but

instead the focus has been on functional properties of PSII

and the overall synthesis or composition of thylakoid

polypeptides Furthermore, despite remarkable similarities

between cyanobacterial and chloroplast PSIIs [15], many of

the PSII LMM subunits, which are completely dispensable

for the assembly of PSII in Synechocystis, are necessary for

the formation of functional PSII in the respective LMM

mutants of Chlamydomonas reinhardtii Representative

examples of differential effects on the formation of functional

PSII in Synechocystis and Chlamydomonas are the deletion

mutants of psbH [16,17], psbI [18,19] and psbK [20,21]

However, it is not known at which assembly step these

proteins are crucial for the formation of functional PSII in

Chlamydomonas So far only a few studies have seriously

searched for PSII assembly intermediates in the absence of

any particular LMM subunit, in either Synechocystis or

chloroplasts of Chlamydomonas and higher plants

The psbEFLJ operon of plant chloroplasts encodes four

distinct LMM subunits of PSII, the a and b subunits of

Cyt b559 (encoded by the psbE and psbF genes) and two

other small subunits, PsbL and PsbJ Deletion of the psbE

gene in Chlamydomonas [22] or the psbF gene in

Synecho-cystis[23] resulted in loss of PSII activity Similarly, the psbL

deletion mutant of Synechocystis was not capable of PSII

oxygen evolution [24] The crucial role of PsbL has been

suggested to be related to the function of the acceptor side of

PSII at the level of QA[25,26] On the other hand, the psbJ

deletion mutants of cyanobacteria were capable of slow

photoautotrophic growth [27,28], whereas the growth of

DpsbJ tobacco mutants was completely dependent on an

external energy source [28,29], possibly because of an

incorrectly assembled OEC [29]

Recently, very youngleaves of tobacco psbEFLJ operon

mutants were characterized in terms of functional, structural

and biogenetic aspects [14] To differentiate the mechanisms

related to the rapid growth and division of chloroplasts in

youngleaves from mechanisms of the PSII assembly process

as such, we mainly focused on mature, but not old, leaves of

tobacco psbEFLJ operon mutants, where partial

disassem-bly and assemdisassem-bly of PSII is constantly occurringbecause of

turnover of the reaction center D1 protein [30] In

particular, the role of PsbL and PsbJ in the assembly and

stability of PSII was addressed To maximize separation of

PSII subcomplexes, we applied 2D Blue-native (BN) gel

electrophoresis followed by protein identification with

immunoblottingand MS In addition, comparative analysis

of both very youngand mature leaves was performed to

examine the developmental aspects of PSII core and OEC

protein accumulation in the psbEFLJ operon mutants with

impaired PSII assembly

Materials and methods

Transformation of tobacco chloroplasts Tobacco (Nicotiana tabacum cv Petit Havanna) psbEFLJ operon mutants were constructed by replacingportions of the four individual genes of the operon with a terminator-less aadA gene cassette A similar cassette with a terminator was also inserted into an EcoRV site, located in the 3¢ UTR

of the operon, to generate the RV control plants The plasmid construct and the transformation, selection and culture of the transformants is described in detail elsewhere [14,28,31] Mutants and controls (wild-type and RV plants) were aseptically grown in MS medium [32] supplemented with 3% (w/v) sucrose under low light conditions ( 10 lmol photonsÆm)2Æs)1) at 25C Mature, fully expanded green leaves, but not senescing ones (hereafter referred to as mature leaves), were used for all experiments except the one in Fig 1B where, for comparison, rapidly expandingsmall and very youngleaves (hereafter referred to

as youngleaves) were used

Isolation of thylakoid membranes Leaves were briefly homogenized in 50 mM Hepes/KOH,

pH 7.5, containing330 mMsorbitol, 2 mM EDTA, 1 mM MgCl2, 5 mM ascorbate, 0.05% BSA and 10 mM NaF, filtered through Miracloth and centrifuged at 2500 g for

4 min at 4C The pellet was resuspended in 50 mMHepes/ KOH, pH 7.5, containing5 mMsorbitol and 10 mMNaF and centrifuged at 2500 g for 4 min at 4C The thylakoid pellet was resuspended in 50 mM Hepes/KOH, pH 7.5, containing100 mM sorbitol, 10 mM MgCl2 and 10 mM NaF, centrifuged at 2500 g for 3 min at 4C, and finally resuspended in the same buffer Chlorophyll was extracted

in 80% (v/v) buffered acetone (2.5 mM Hepes/NaOH,

pH 7.5) and quantitated as described [33]

BN-PAGE, SDS/PAGE and protein identification Blue-native PAGE (BN-PAGE) was performed as des-cribed previously [34] with slight modifications Thylakoid membrane suspensions containing20 lgchlorophyll were used as startingmaterial Thylakoids were washed with

50 mM BisTris/HCl, pH 7.0, containing330 mM sorbitol and 0.25 lgÆlL)1Pefabloc (Roche), sedimented at 3500 g for 2 min at 4C, and resuspended in 25 mMBisTris/HCl,

pH 7.0, containing20% (w/v) glycerol and 0.25 lgÆlL)1 Pefabloc Thylakoids were then solubilized with 1% (w/v) n-dodecyl b-D-maltoside (0.5 mgchlorophyllÆmL)1) and incubated on ice for 2 min After centrifugation at 18000 g for 15 min at 4C, the supernatant was supplemented with 0.1 vol sample buffer (100 mMBisTris/HCl, pH 7.0, 0.5M e-amino-n-caproic acid, 30% (w/v) sucrose, 50 mgÆmL)1 Serva blue G) and subjected to BN-PAGE with a gradient

of 5–12% acrylamide in the separation gel The electro-phoresis was performed at 2C, 95 V overnight, followed

by a progressive increase in voltage to 200 V for 4–5 h After the run, a lane of BN-PAGE was cut out, solubilized with 5% (v/v) 2-mercaptoethanol in the sample buffer [35] for 40 min and run in the second dimension in SDS/PAGE with 15% acrylamide and 6 urea After electrophoresis,

gels were silver-stained or electroblotted on to a

poly(viny-lidene difluoride) membrane Western blottingwith

chemi-luminescence detection was performed with standard

techniques usingprotein-specific antibodies (D1, D2, PsbE,

CP43, CP47, PsbO, PsbP, PsbQ, Cyt f, Lhcb1,2, CP26,

CP29) or an antibody raised against the PSI complex The

AIS Analytical Imaging Station (version 3.0 rev 1.7;

Imaging Research Inc., Brock University, St Catharines, Ontario, Canada) was used for quantitation of the Western blots For each quantitation, a minimum of three inde-pendent Western blots was used

Several protein components of PSII, OEC and LHCII complexes as well as Cyt b6fand PSI were also identified by

MS MALDI-TOF analysis Protein in-gel digestion with modified trypsin (Promega) and sample preparation for MS analysis were performed manually [36] Samples were loaded on to the target plate by the dried droplet method using a-cyano-4-hydroxycinnamic acid as a matrix MALDI-TOF analysis was performed in reflector mode

on a Voyager-DE PRO mass spectrometer (Applied Bio-systems, Foster City, CA, USA) Internal mass calibration

of spectra was based on trypsin autodigestion products (842.5094 and 2211.1046 m/z) Proteins were identified as the highest rankingresult by searchingin the NCBI database usingMascot (http://www.matrixscience.com) The search parameters allowed for carbamidomethylation

of cysteine, one miscleavage of trypsin, and 50 p.p.m mass accuracy For positive identification, the score of the result [)10 · log(P)] where P is the probability that the observed match is a random event had to be over the significance threshold level (P < 0.05)

Fluorescence measurement Fluorescence emission spectra at 77 K were measured on a diode array spectrophotometer (S200; Ocean Optics, Dun-edin, FL, USA) equipped with a reflectance probe [37] Fluorescence was excited with visible light below 500 nm, which was defined by usingLS500S and LS700S filters (Corion Corp., Holliston, MA, USA) in front of the slide projector The emission between 600 and 780 nm was recorded Thylakoid samples (100 lL) contained 10 lg chlorophyll per mL in 50 mM Hepes/KOH, pH 7.5, containing100 mM sorbitol, 10 mM MgCl2, and 10 mM NaF Three independent measurements were made from each tobacco line

Results

Polypeptide composition of thylakoid membranes The protein composition of thylakoids from mature leaves

of psbEFLJ operon mutants and the controls, wild-type and the RV plants (see Materials and methods), was first determined using1D SDS/PAGE and immunoblottingwith protein-specific antibodies DpsbE and DpsbF thylakoids were practically devoid of all PSII core proteins tested (includingD1, D2, CP43, CP47, PsbE and PsbZ, Fig 1A) Similarly, all three OEC proteins, PsbO, PsbP and PsbQ, were completely missingfrom thylakoids of these two mutants (Fig 1A) PsbW, on the other hand, represented a PSII LMM protein that was present at reduced amounts in the thylakoids of both DpsbE and DpsbF (33 ± 11 of that in the control thylakoids) To investigate the apparent devel-opmental control of the accumulation of PSII proteins, we also isolated thylakoids from very young, rapidly expanding leaves of DpsbE and DpsbF and analysed their protein composition (Fig 1B) In contrast with mature leaves, the youngleaves of both DpsbE and DpsbF accumulated all

Fig 1 Immunoblots of thylakoid membrane proteins of the four tobacco

psbEFLJ operon mutants and the controls (wild-type and RV)

Thyla-koids were isolated from mature green leaves (A) and rapidly

expandingyoungleaves (B) Proteins were separated by SDS/PAGE,

electroblotted on to a poly(vinylidene difluoride) membrane and

pro-bed with antisera against different thylakoid membrane proteins.

Chlorophyll (1 lg) was loaded in each well, except for PsbW (0.3 lg)

and PsbO and PsbP (0.5 lg).

OEC proteins and also traces of D2 and the CP43 protein

(Fig 1B) Interestingly, traces of PsbE protein (the a

subunit of Cyt b559) could also be distinguished in young

leaves of DpsbF (Fig 1B) Other PSII core proteins (D1,

CP47) were, however, similarly missingfrom both the

youngand mature leaves of the DpsbE and DpsbF mutants

As to the DpsbJ and DpsbL mutants, the thylakoids from

mature leaves contained all major PSII core proteins

(Fig 1A), but in lower amounts than in the controls The

mean content of the major PSII core proteins (D1, D2,

PsbE, CP43 and CP47) in DpsbL and DpsbJ was 14 ± 5%

and 57 ± 18% of that in the control thylakoids,

respect-ively Interestingly, the recently identified small PSII protein

PsbZ [38–40] was present in both DpsbL and DpsbJ, in

quantities related to the amount of the D1 protein present

in the thylakoid membrane (18% and 88% of that in the

control thylakoids in DpsbL and DpsbJ, respectively) Also

PsbW was present in both DpsbL and DpsbJ, amountingto

69 ± 4% of that in the control thylakoids As to the OEC

proteins, the thylakoids from mature leaves of the DpsbL

and DpsbJ mutants clearly differed from both each other

and the controls Only scarce amounts of PsbO were found

in thylakoids isolated from DpsbL (Fig 1A; 11% of that in

the control), while other OEC proteins were missing

Thylakoids of DpsbJ, on the other hand, contained

consid-erable amounts of PsbO and also some PsbQ (up to 100%

and 6%, respectively, compared with the control

thyla-koids), whereas the PsbP protein was completely missing, in

accordance with earlier observations [29] It is noteworthy

that, when the immunoblots were heavily overexposed

showingtraces of PsbP even in DpsbE, DpsbF and DpsbL,

the PsbP protein could not be detected in DpsbJ thylakoids

(data not shown) Youngleaves of both DpsbL and DpsbJ,

on the other hand, accumulated all OEC proteins in

considerable amounts However, the DpsbJ mutant was

again the exception, accumulating only traces of PsbP

compared with the other mutants (Fig 1B) Otherwise the

pattern of PSII proteins in youngleaves of DpsbL and DpsbJ

resembled that of the mature leaves (Fig 1B)

As well as the PSII core and OEC proteins, we

investigated the amounts of the LHCII, CP26, Cyt f, LHCI

and PsaA/B proteins in mature leaves of the psbEFLJ

operon mutants All mutants were capable of accumulating

these proteins and no clear differences were recorded

compared with thylakoids isolated from control plants

(Fig 1A)

Assembly of thylakoid membrane protein complexes

inpsbEFLJ operon mutants

Simple detection of thylakoid proteins by immunoblotting

does not reveal whether the proteins are assembled into

complexes or whether they exist as free proteins in the

membrane or lumen The general assumption that good

quality control in chloroplasts results in rapid degradation

of unassembled proteins [41] does not always hold true In

rapidly expandingyoungleaves in particular, some of the

PSII core proteins and all of the OEC proteins can

accumulate in thylakoids in the absence of any assembly

of PSII, as was evident for the DpsbE and DpsbF tobacco

mutants (Fig 1B) Thus, to understand the role of various

LMM subunits in the stable assembly of PSII, it is necessary

to isolate various PSII assembly intermediates For these experiments we used only mature leaves to avoid accumu-lation of PSII proteins that do not become assembled One-dimensional separation of thylakoid protein com-plexes in BN gels had already revealed major differences in the capacity for PSII assembly in the psbEFLJ operon mutants Clear separation of intact PSII core monomers, PSII core dimers and PSII–LHCII supercomplexes was typical only for the control thylakoids (Fig 2), whereas DpsbJ and DpsbL, and particularly DpsbE and DpsbF, showed clear deficiencies in their PSII assemblies The PSII monomer was missingfrom DpsbE and DpsbF and was present only in minor amounts in DpsbL In contrast, the two other thylakoid electron-transfer complexes, the PSI and Cyt b6fcomplexes, were present in similar amounts in all the mutants and control plants (Figs 2 and 3)

More detailed information about various PSII (sub)assemblies and their polypeptide composition was obtained from 2D gel analysis (BN-PAGE followed by SDS/PAGE) combined with immunochemical detection (D1, D2, CP43, CP47, PsbE) and MS analysis (MALDI-TOF) of various PSII core and OEC proteins (Figs 3 and 4)

In wild-type plants, the intact PSII core monomers, the PSII core dimers and PSII–LHCII supercomplexes (confirmed

by immunoblottingto contain the D1, D2, PsbE, CP47 and CP43 proteins) were detected, and only a very minor amount of CP43-less PSII monomers was present (Fig 3) The absence of free PSII core proteins after 2D electro-phoresis (see the immunoblots below the silver-stained gels) was an indication of the general stability of PSII core complexes on dodecyl maltoside solubilization and subse-quent electrophoretic separation of thylakoid protein com-plexes Of the OEC proteins, the PsbO subunit was always detected in association with the PSII–LHCII supercom-plexes (Fig 4A) The Cyt b6fcomplex was present in wild-type thylakoids mainly as a dimer, and the PSI complex

Fig 2 BN-PAGE of thylakoid protein complexes from mature leaves of the four tobacco psbEFLJ operon mutants and the wild-type and RV controls Thylakoids (20 lgchlorophyll per well) were solubilized with 1% n-dodecyl maltoside before BN-PAGE For identification of the complexes, see Fig 3.

Fig 3 Two-dimensional gel analysis of the thylakoid protein complexes from mature leaves of wild-type and DpsbF, DpsbL and DpsbJ mutants of tobacco Thylakoids were solubilized and subjected to BN-PAGE separation of the protein complexes as described in Fig 2 After the run, a lane of BN-PAGE was cut out, solubilized with 5% (v/v) 2-mercaptoethanol, and placed horizontally on the top of the SDS/polyacrylamide gel After electrophoresis, the gel was silver-stained Similar gels were also electroblotted on to poly(vinylidene difluoride) membranes and probed with antisera against D1, D2, CP43, CP47 and PsbE (Cyt b 559 a subunit) Strips of such immunoblots are presented below the correspondingsilver-stained gels Some of the immunoblots are overexposed and thus cannot be compared quantitatively The D1, D2, CP43 and CP47 proteins from the PSII complexes (PSII core monomers, CP43-less core monomers, PSII core dimers and PSII–LHCII supercomplexes) are circled Positions of PSI, Cyt b 6 f dimers and various LHCII subassemblies are circled in the silver-stained gel of the DpsbF mutant lackingall PSII complexes and were identified by MALDI-TOF MS and immunoblotting(data not shown).

Fig 4 Presence of the 33-kDa PsbO protein of OEC in different PSII assemblies of the control and DpsbJ and DpsbL mutant thylakoids isolated from mature leaves (A) Protein components of PSII (sub)complexes from wild-type and DpsbJ and DpsbL mutants of tobacco The gels for the wild-type and DpsbJ mutant are enlargements from Fig 3 (the 27–50-kDa region) The corresponding region from the DpsbL mutant was obtained after only partial solubilization of thylakoid complexes with n-dodecyl b- D -maltoside and separation of the complexes with a mini-gel system, which allowed disclosure of the PSII core monomer complex with attached PsbO Arrows indicate the location of PsbO protein in PSII–LHCII supercomplexes of the wild-type control thylakoids and in the PSII core dimer or in a distinct PSII core monomer complex of the DpsbJ and DpsbL mutants, respectively Cyt f of the Cyt b 6 f dimer complex (identified by both immunoblottingand MS; not shown) is indicated in the silver-stained gels with

an asterisk (B) Representative mass spectrum and the peptide masses of the PsbO protein (straight arrows with closed square) and the overlapping D2 protein (tilted arrows with open circles) from the PSII–LHCII supercomplex of control thylakoids Tilted arrows with a cross show the trypsin self-digest products used for MS calibration.

migrated in a BN gel in close proximity to the PSII dimer

(Fig 3) LHCII proteins, despite forming the PSII–LHCII

supercomplexes, were present in various subcomplexes

detached from PSII

In the absence of either PsbF (Fig 3) or PsbE (not

shown) the 2D BN-PAGE profiles of the main thylakoid

protein complexes were very similar No PSII core proteins

were found assembled into any kind of complexes, neither

did they accumulate as free proteins Other thylakoid

protein complexes, such as Cyt b6fdimer, PSI and various

LHCII subassemblies, were present in DpsbE and DpsbF in

comparable amounts to that in the wild-type Complete

(DpsbE and DpsbF) or partial (DpsbL and DpsbJ; Fig 4)

depletion of the PSII complexes thus had no effect on the

assembly and accumulation of other multiprotein

photo-synthetic complexes in the thylakoid membrane This differs

from a recent study in which the amounts of some PSI

proteins were reduced in tobacco DpsbJ mutant [29]

Analysis of DpsbJ by 2D BN-PAGE revealed that both

PSII core monomers and dimers were correctly assembled

(Fig 3) Considerable amounts of PSII monomers lacking

CP43 were, however, also present, although the relative

amount of free CP43 was much less than in DpsbL (see

below) It is noteworthy that not even traces of PSII–LHCII

supercomplexes were present in DpsbJ thylakoids In the

absence of PSII–LHCII supercomplexes, the PsbO protein

of the OEC was found to be associated with the PSII core

dimers (Fig 4A) in the thylakoid membranes of DpsbJ

The DpsbL mutant was capable of partial assembly of the

PSII core monomers, whereas PSII core dimers and

supercomplexes were completely missing(Fig 3) Small

amounts of both types of PSII core monomers, an intact

PSII monomer and a CP43-less monomer, were observed

(Fig 3) It is noteworthy that, in DpsbL, the portion of free

CP43 compared with that assembled into the PSII core

monomer was extremely high (91 ± 5%) In wild-type

thylakoids, only a minor amount (2 ± 1%) of CP43 was

found free and unassembled into the PSII complexes under

similar experimental conditions This indicates that, in the

absence of PsbL, the assembly of CP43 and thus the

formation of stable intact PSII core monomers is severely

impaired None of the other PSII proteins were found free

after 2D BN-PAGE of DpsbL thylakoids (except for a tiny

amount of PsbE; Fig 3), indicating no general disassembly

of PSII core complexes duringelectrophoretic separation

Further, the presence of a small amount of PsbO detected

by immunoblottingof DpsbL thylakoid proteins (Fig 1A)

prompted us to search for a PSII subcomplex with attached

PsbO protein Only after usinga mini-gel system and partial

solubilization of the thylakoid complexes for fast and gentle

separation of the PSII subcomplexes did we succeed in

isolatinga novel PSII core monomer with attached PsbO

(Fig 4) This complex migrated slightly more slowly in the

BN-gel than the normal intact PSII core monomer

The gentle separation system did not reveal the presence

of this novel PSII core monomer–PsbO protein complex in

the control or DpsbJ thylakoids (data not shown) It did

confirm the association of PsbO with PSII–LHCII

super-complexes in the wild-type and with the PSII core dimers in

DpsbJ, as well as the absence of PSII–LHCII

supercom-plexes from DpsbJ, and both the supercomsupercom-plexes and PSII

core dimers from DpsbL (Fig 4A) However, although

useful in detectingthe PSII core monomer–PsbO protein complex in DpsbL, the gentle mini gel system could not be used for PSII assembly studies in general because of a background smear and tailing of protein bands

77 K fluorescence emission spectra All mutant thylakoids harbored considerable amounts of LHCII complexes, which, however, could not be isolated in supercomplexes with PSII cores To investigate whether there was energy transfer from LHCII to the PSII core, the fluorescence emission spectra at 77 K were recorded from thylakoids of the four psbEFLJ operon mutants after excitation with visible light below 500 nm The wild-type and RV thylakoids showed well-defined PSII emission peaks at 685 nm (CP43) and 695 nm (CP47) as well as the PSI emission peak at 735 nm (Fig 5) [42] DpsbE, DpsbF and DpsbL lacked the emission peaks at 685 and 695 nm and instead had a prominent peak at 680 nm, characteristic

of free LHCII The 730-nm PSI peak was shifted to a lower wavelength Interestingly, in DpsbJ, the 680-nm (LHCII), 685-nm (CP43) and 695-nm (CP47) 77 K fluorescence emission peaks were all present, in addition to the promi-nent PSI emission peak

Fig 5 77 K fluorescence emission spectra of thylakoid membranes of tobacco psbEFLJ operon mutants and controls (wild-type and RV) Thylakoids were excited with visible light below 500 nm.

PSII contains several chloroplast-encoded and

nuclear-encoded LMM subunits, the role of which in the assembly

and stability of the complex has remained poorly

under-stood We have used a reverse genetics approach to

elucidate the role of proteins encoded by the psbEFLJ

operon, with special attention to PsbL and PsbJ, in the

stable assembly process of the PSII core subunits, the

LHCII antenna polypeptides, and the proteins of the OEC

PsbJ is essential for correct association of LHCII

Although stable PSII core dimers were assembled in DpsbJ,

the PSII–LHCII supercomplexes were completely missing

This indicates the importance of PsbJ in the steady-state

higher organization of the PSII complexes This conclusion,

deduced from the 2D gel analysis (Fig 3), was further

supported by the 77 K fluorescence emission spectrum of

DpsbJ revealinga distinct emission peak directly from

LHCII at 680 nm, in addition to the two emission peaks

from the PSII core (685 nm and 695 nm referringto CP43

and CP47, respectively; Fig 5) This strongly suggests that

the light energy absorbed by LHCII is not properly

transferred to the PSII reaction center In variance with a

recent study with tobacco DpsbJ mutant [29], we did not

find any reduction in the contents of CP26 (Fig 1A),

the minor LHCII antenna protein thought to mediate the

transfer of excitation energy from LHCII antennae to the

PSII reaction center [6] PsbJ is therefore probably essential

in providingthe PSII core dimer with a conformation that

allows correct association with the LHCII complex and

thereby efficient capture of excitation energy for PSII

Whether PsbJ exerts its effect on LHCII association directly

or via its effects on the assembly of OEC remains to be

resolved

PsbL is required for stable assembly of CP43

Comparison of the assembly of PSII in DpsbL and DpsbJ

clearly demonstrates that PsbL is essential at earlier

assembly steps than PsbJ, and therefore probably also

represents a more intrinsic core protein than PsbJ in the

structural hierarchy of PSII Stable PSII core dimers were

formed despite the absence of PsbJ, whereas in the absence

of PsbL the PSII core proteins accumulated in minor

amounts and successfully assembled only into PSII core

monomers with unstable association of CP43 (Fig 4) As

shown with wild-type thylakoids, the correctly assembled

PSII core monomers preserve their intactness during

electrophoretic separation, whereas there are large amounts

of free CP43 with the DpsbL thylakoids It is thus

conceivable that PsbL is an essential protein component

of PSII for ensuringthe stable assembly of CP43, and

therefore, in DpsbL, the CP43 protein readily becomes

detached from the PSII core monomer duringthe

elec-trophoretic run On the basis of the crystal structure of PSII

[3], it was suggested that a transmembrane a-helix in the

vicinity of CP47 possibly represents PsbL We are inclined,

however, to suggest that, rather than being located in the

vicinity of CP47, PsbL is one of the unassigned

transmem-brane a-helices in the vicinity of CP43 and D1 [3] This

suggestion is also supported by the fact that CP47 stably assembles with PSII core monomers even in the absence of PsbL (Fig 3)

Recently there has been a growing consensus in favour of PSII dimers beingthe functional forms of PSII [2,5,6] Whether PsbL has a direct role in PSII dimerization, as was suggested by Barber and coworkers [7], is difficult to assess

It is probable that problems in stable assembly of CP43 exert secondary effects on PSII dimerization, and thus the role of PsbL in the dimerization process itself may be indirect The exact mechanism of PSII dimerization is not known but it is conceivable that several small PSII subunits collectively control the successful dimerization of PSII [7,8]

The presence of CP43 in PSII is a prerequisite for association of PsbO whereas PsbL and PsbJ are needed for correct association of PsbP and PsbQ

Three-dimensional OEC structures from spinach [2], Chlamydomonas and Synechococcus elongatus [43] were recently published In all of these evolutionarily divergent species, the PsbO protein was suggested to be located towards the CP47/D2 side of the PSII reaction center core whereas the PsbQ and PsbP proteins (in cyanobacteria PsbV and PsbU, respectively) were located towards the N-terminal lumenal loop of the D1 protein Such structures are in accordance with our results on the assembly of PsbO with the PSII core monomer in the mature leaves of DpsbL mutant A lack of PsbL still allows a stable assembly and orientation of the CP47 side of the PSII core, which probably is required for stable association of PsbO It should be noted, however, that the novel PSII core monomer–PsbO complex could be demonstrated only when CP43 was also present in the complex (Fig 4A) Indeed, PsbO was found to be absent (as assessed by MALDI-TOF analysis and silver staining) from the CP43-less PSII core monomer It is thus conceivable that the extended lumenal loops of CP43 are also involved in stabilization of the attachment of PsbO to the PSII core In fact, the close proximity of PsbO and CP43 has been predicted previously from various in vitro studies with PSII membranes [44–47]

On the other hand, the lability and possibly incorrect conformation of the D1/CP43 side seems to prevent the assembly of PsbP and PsbQ with the PSII core monomer, despite the presence of PsbO, as evidenced by the complete absence of these OEC proteins from DpsbL The mature leaves of DpsbJ showed a more stable association of CP43 than DpsbL, and indeed traces of PsbQ were also present, in addition to PsbO, whereas PsbP was completely missing There seems to be no tight mutual control in the assembly

of the three OEC proteins Although the binding of PsbO apparently occurs first [48] and may be a prerequisite for the assembly of the other OEC proteins, it does not seem to provide any direct bindingsite for either PsbP or PsbQ, which does not support the previous suggestion [49] It is evident that the presence of both PsbL and PsbJ is critical in providingproper dockingsites, either directly or indirectly,

by modifyingthe conformation of PSII on the lumenal side, makingefficient bindingof PsbQ and PsbP of the OEC possible

A fundamental difference in the DpsbJ mutants between cyanobacteria and the chloroplasts of higher plants was

recently described: only the cyanobacterial mutant is

capable of slow photoautotrophic growth [28] This is

reflected in the capacity of the mutants to oxidize QA– It is,

however, likely that the water splittingand donation of

electrons to P680+also play a role in the better performance

of the cyanobacterial than the tobacco DpsbJ mutant

Requirements for OEC proteins in cyanobacteria seem to be

less stringent than in eukaryotes In cyanobacteria, the

presence of either PsbO or Cyt c550(PsbV) confers

photo-autotrophic growth [50,51], whereas three distinct proteins

form the OEC in eukaryotes [51] Both PsbO and to a lesser

extent PsbQ were present in mature leaves of tobacco DpsbJ

mutant (Figs 1A and 4A), but, owing to the lack of PsbP,

the oxygen-evolving capacity of this mutant is severely

hampered [29] In line with this notion, a Chlamydomonas

mutant lackingPsbP was defective in oxygen evolution,

which, however, could be restored by the addition of

chloride ions [52]

The absence ofpsbEFLJ operon encoded proteins

affects the accumulation of PSII core and OEC proteins

in a development-dependent manner

Studies with Chlamydomonas have demonstrated a

com-plete lack of PSII assembly in the absence of Cyt b559[22]

An absolute requirement for PSII assembly of both the a

and the b subunit of Cyt b559, encoded by the plastome psbE

and psbF genes, was corroborated by this study using

mature tobacco leaves In fact, no PSII core or OEC

proteins accumulated in thylakoids of mature leaves of the

psbE and psbF inactivation mutants (Fig 1A) This is

completely opposite to the situation in youngleaves, which,

despite the lack of PSII assembly, kept accumulatingall

three proteins of the OEC and traces of the D2 and CP43

core proteins as well (Fig 1B) The presence of minor

amounts of D2 in both DpsbE and DpsbF supports the

suggestion that the D2 protein is a component of the

primary receptor for the synthesis and cotranslational

assembly of D1 [53,54] In addition, Cyt b559has been found

in barley etioplasts as a complex with D2 [11], emphasizing

the role of these two subunits as primary assembly partners

for construction of the PSII complexes Indeed, the PsbE

protein was also present in tiny amounts in the thylakoid

membranes of young, developing leaves of DpsbF Of the

internal antenna proteins of PSII, the assembly of CP47

possibly also occurs cotranslationally because no free

protein was found in the membrane, whereas the assembly

process of CP43 seems to be less stringent [9,54,55] and

some free CP43 was found in the thylakoid membrane of

youngdevelopingleaves (Fig 1B)

Apparently a change in the developmental program upon

leaf maturation and cessation of chloroplast division leads

to down-regulation of both the chloroplast-encoded and

nucleus-encoded PSII proteins (Fig 1A), avoiding the

wasteful synthesis of proteins when their assembly into

functional complexes is prohibited The possible signaling

mechanisms leadingto complete down-regulation of PSII

core and OEC proteins in the absence of PSII assembly,

manifested in DpsbE and DpsbF upon leaf maturation

(Fig 1A,B), are not known However, the notion of the

strict regulation of OEC protein synthesis in mature leaves

also is supported by the identification of PSII subcomplexes

that bind the PsbO protein in DpsbL and DpsbJ thylakoids (Fig 4) Demonstration of the association of PsbO with PSII subcomplexes implies that free OEC proteins do not accumulate in the thylakoid lumen of mature leaves, in contrast with rapidly expandingyoungleaves (Fig 1B)

Fig 6 Scheme demonstrating the ability of mature leaves of the DpsbE, DpsbF, DpsbL and DpsbJ mutants to form PSII–LHCII assemblies In wild-type thylakoid membranes, PSII core dimers together with associated LHCII and OEC proteins form PSII–LHCII supercom-plexes In the absence of PsbJ, the PSII core dimers can harbor the oxygen-evolving PsbO protein and also some PsbQ, but the LHCII complexes remain completely detached Lack of PsbL results in more severe problems for the assembly of PSII: only PSII core monomers can be assembled with labile association of CP43, and, of the oxygen-evolvingproteins, only PsbO is attached to the core monomer, pro-vided that CP43 is also present Mature leaves of the DpsbE and DpsbF mutants do not accumulate any PSII core or OEC proteins but the LHCII complexes remain free in the thylakoid membrane Oxygen-evolvingproteins are shown as O (PsbO), P (PsbP) and Q (PsbQ) For clarity, only the major subunits are included.

When assembly partners are not available, the

chloro-plast-encoded major PSII core proteins (D1, D2, CP47) are

typically down-regulated at the level of translation (for

reviews, see [9,10,54]) The regulation of the synthesis of

nuclear-encoded OEC proteins is still not understood, but

may occur at the level of transcription Accordingto our

results, it is likely that the regulation mechanisms for

chloroplast-encoded PSII core and nuclear-encoded OEC

proteins are different and independent of each other, as

suggested previously [54] Another explanation for the

observed differences between youngand mature leaves may

be the enhanced proteolytic activity in mature leaves

sufferingfrom photo-oxidative stress because they either

lacked PSII (DpsbE and DpsbF) or had a defectively

assembled PSII (DpsbL and DpsbJ), as was discussed in

the recent report on the DpsbJ tobacco mutant with

dramatically reduced photosynthetic performance [29]

Lessons frompsbEFLJ operon mutants on the role

of PsbW and PsbZ subunits

PSII assembly studies on psbEFLJ operon mutants also

provided some information on the two other small PSII

proteins, PsbW and PsbZ Nuclear-encoded PsbW has been

found to accumulate in the thylakoid membranes of both

mature (Fig 1A) and young [14] leaves of psbEFLJ operon

mutants, even in the complete absence of PSII complexes

(DpsbE and DpsbF) Similarly, PsbW was present, but at

reduced amounts, in tobacco DpsbA mutant with no PSII

assembly and activity [56] Less stringent mutual regulation

of the accumulation of PsbW and the other PSII core

proteins was also evident in psbW antisense mutants of

Arabidopsis[8] All this suggests that PsbW is not under the

same strict regulation and/or quality control as the other

PSII core proteins and the OEC proteins in mature leaves

Recently characterized chloroplast-encoded PsbZ [38–

40], on the other hand, accumulated in mature leaves of

DpsbL and DpsbJ, in comparable amounts to assembled

PSII complexes, while beingabsent from DpsbE and DpsbF

(Fig 1A) The presence of PsbZ even in DpsbL may suggest

the location of PsbZ in a very central core of PSII Such a

central location in PSII, however, seems to contradict the

fact that PsbZ is not required for correct assembly of the

oxygen-evolving PSII complexes and photoautotrophic

growth of mutant plants [38–40]

Concluding remarks

The capacity of mature leaves of the psbEFLJ operon

mutants to assemble PSII (sub)complexes is schematically

presented in Fig 6 When either PsbE or PsbF is missing,

the synthesis and accumulation of other PSII core proteins

and the OEC proteins are strictly prevented Thus, in

contrast with youngleaves [14], the absence of PSII

assembly partners in mature leaves either evokes a signal

to prevent the synthesis of other PSII proteins, of either

chloroplast or nuclear origin, or enhances the proteolytic

activity Such control is, however, not exerted on the

synthesis and assembly of the nuclear-encoded LHCII

polypeptides or PSII LMM protein PsbW Association of

PsbL with PSII subcomplexes, in particular, promotes the

stable and correct assembly of CP43 and thereby probably

also facilitates the dimerization of PSII Assembly of PsbJ is

a subsequent step to the association of PsbL, and probably occurs only after the assembly of the PSII core monomer, or even the dimer, is accomplished Of the OEC proteins, the bindingof PsbO is clearly dependent on the presence of CP43 in the PSII core complex, whereas the correct association of PsbP and PsbQ additionally requires the presence of both the PsbL and PsbJ subunits It remains to

be investigated whether the PsbL and/or PsbJ proteins offer

a direct dockingsite for PsbP and PsbQ or whether PsbL and PsbJ only modulate the structure and mutual orienta-tion of the PSII proteins on the lumenal side, makingthe association of PsbP and PsbQ feasible Finally, PsbJ is clearly required for stable formation of PSII–LHCII supercomplexes, thereby allowing greater organization of PSII complexes in the thylakoid membrane

Acknowledgements Elena Baena-Gonzalez and Mika Kera¨nen are thanked for help with the 77 K fluorescence measurements, and Drs Roberto Barbato, Toril Hundal, Stefan Jansson, Wolfgang Schro¨der and Francis-Andre Wollman for the gifts of antibodies This work was supported by the Academy of Finland, the Finnish Ministry of Agriculture and Forestry (NKJ project), the German Research Foundation (SFB-TR1) and Fonds der Chemischen Industrie.

References

1 Hankamer, B., Morris, E., Nield, J., Carne, A & Barber, J (2001) Subunit positioningand transmembrane helix organisation in the core dimer of photosystem II FEBS Lett 504, 142–152.

2 Nield, J., Orlova, E.V., Morris, E.P., Gowen, B., van Heel, M.

& Barber, J (2000) 3D map of the plant photosystem II super-comples obtained by cryoelectron microscopy and single particle analysis Nat Struct Biol 1, 44–47.

3 Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W & Orth, P (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A˚ resolution Nature 409, 739–743.

4 Kamiya, N & Shen, J.-R (2003) Crystal structure of oxygen-evolvingphotosystem II from Thermosynechococcus vulcanus at 3.7-A˚ resolution Proc Natl Acad Sci USA 100, 98–103.

5 Hankamer, B., Nield, J., Zheleva, D., Boekema, E., Jansson, S & Barber, J (1997) Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo Eur J Biochem 243, 422–429.

6 Boekema, E.J., Roon, H & Dekker, J.P (1998) Specific associa-tion of photosystem II and light-harvesting complex II in partially solubilized photosystem II membranes FEBS Lett 424, 95–99.

7 Zheleva, D., Sharma, J., Panico, M., Morris, H.R & Barber, J (1998) Isolation and characterization of monomeric and dimeric CP47-reaction center photosystem II complexes J Biol Chem.

273, 16122–16127.

8 Shi, L.-X., Lorkovic, J.L., Oelmuller, R & Schro¨der, W.P (2000) The low molecular mass PsbW protein is involved in the stabili-zation of the dimeric photosystem II complex in Arabidopsis thaliana J Biol Chem 275, 37945–37950.

9 Baena-Gonzalez, E & Aro, E.-M (2002) Biogenesis, assembly and turnover of photosystem II units Philos Trans R Soc Lond.

B 357, 1451–1461.

10 Zerges, W (2002) Does complexity constrain organelle evolution? Trends Plant Sci 7, 175–182.