the plastid genome-encoded ycf4 protein functions as a ...photosystem biogenesis in the thylakoid...

The Plastid Genome-Encoded Ycf4 Protein Functions as aNonessential Assembly Factor for Photosystem I inHigher Plants1[W]

Katharina Krech, Stephanie Ruf, Fifi F. Masduki, Wolfram Thiele, Dominika Bednarczyk2,Christin A. Albus, Nadine Tiller, Claudia Hasse, Mark A. Schöttler, and Ralph Bock*

Max-Planck-Institut für Molekulare Pflanzenphysiologie, D–14476 Potsdam-Golm, Germany

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly ofnucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such aschromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and theidentity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterizedthe chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode aprotein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stabletransformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotianatabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable ofphotoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential forphotosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unalteredexpression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein inthe PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSIassembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis isrestricted to young leaves.

The light reactions of photosynthesis are performedby a highly complex macromolecular machinery thatresides in the thylakoid membrane. In photosyntheticeukaryotes, the thylakoids are encapsulated in a ded-icated organelle, the chloroplast. Over the past years,remarkable progress has been made with resolving thecomposition and three-dimensional structure of themajor thylakoidal protein complexes involved inphotosynthetic electron transfer and ATP synthesis(for review, see Nelson and Ben-Shem, 2004; Nelsonand Yocum, 2006). In contrast, we still know very littleabout the biogenesis of these big multiprotein com-plexes. Their assembly requires the coordinated syn-thesis of many protein subunits, some of which areencoded in the chloroplast genome and others in thenuclear genome. These proteins need to be insertedinto the thylakoid membrane in a sequential order

(Ossenbühl et al., 2004; Rokka et al., 2005). Moreover,hundreds of cofactors, such as chlorophylls, carote-noids, quinones, and iron-sulfur clusters, need to findtheir correct place in the complexes. How the cellaccomplishes the daunting task of assembling thephotosynthetic complexes in the thylakoid membraneis still largely a mystery.

PSI, the plastocyanin-ferredoxin oxidoreductase ofthe photosynthetic electron transport chain, is one ofthe largest multiprotein complexes known to reside inbiological membranes. In photosynthetic eukaryotes,PSI is composed of 15 protein subunits (PsaA–PsaLand PsaN–PsaP). Five of these subunits (PsaA–PsaC,PsaI, and PsaJ) are encoded in the chloroplast genomeof higher plants; the others are encoded by nucleargenes and posttranslationally imported into thechloroplast compartment. Four subunits (PsaG, PsaH,PsaN, and PsaO) represent evolutionarily new acqui-sitions in photosynthetic eukaryotes, whereas onesubunit (PsaM) found in cyanobacterial PSI was lostand is not present in eukaryotic PSI complexes(Amunts et al., 2007, 2010; Busch and Hippler, 2011;Schöttler et al., 2011). In addition to the 15 subunitsconstituting the catalytically active PSI core complex,at least four stably bound light-harvesting complexproteins forming the PSI antenna (LhcA1–LhcA4) areassociated with each monomeric PSI unit. PSI alsoharbors a huge number of cofactors, including at least173 chlorophylls, two phylloquinones, three iron-sulfurclusters, and 15 carotenoids (Amunts et al., 2010).

1 This work was supported by the Max Planck Society and by theDeutsche Forschungsgemeinschaft (grant no. SFB 429 A12).

2 Present address: Department of Plant Sciences, Weizmann Insti-tute of Science, Rehovot 76100, Israel.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ralph Bock ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.196642

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PSI complex assembly is only poorly understood.One reason for this is that assembly intermediatescannot be readily identified, presumably because theassembly process occurs very fast (Ozawa et al., 2010).Also, assembly intermediates cannot be easily resolvedby molecular mass-based separation techniques (suchas gradient centrifugation or native electrophoresis),because PSI biogenesis begins with the formation ofthe large PsaA/PsaB reaction center heterodimer, whichaccounts for almost half the total molecular mass of PSI.Afterward, beginning with the three extrinsic subunitsof the so-called stromal ridge involved in ferredoxinbinding (PsaC, PsaD, and PsaE), only low-molecular-mass subunits are added to the reaction center, so that aresolution of the different assembly intermediates ismuch more challenging than in the case of other pho-tosynthetic complexes (Schöttler et al., 2011).

In recent years, forward and reverse geneticsapproaches have provided a promising entry pointinto the study of PSI assembly. The analysis of mutantsdeficient in PSI accumulation has led to the discoveryof several proteins that are required for efficient PSIbiogenesis without being part of the active PSI com-plex (Boudreau et al., 1997; Ruf et al., 1997; Göhreet al., 2006; Stöckel et al., 2006; Albus et al., 2010). Themolecular defects in these mutants fall into at leastthree different categories: (1) defects in cofactor syn-thesis; (2) defects in cofactor insertion; and (3) defectsin protein subunit incorporation. As phylloquinone isa cofactor required only in PSI, mutants in phylloqui-none biosynthesis show a specific deficiency in PSIaccumulation (Lohmann et al., 2006). An example for agene involved in cofactor insertion is Hcf101, whichencodes a scaffold protein for [4Fe-4S] cluster assembly(Stöckel and Oelmüller, 2004; Schwenkert et al., 2010).A few proteins have been implicated in the assemblyof the PSI protein subunits. ALBINO3 (ALB3) mediatesthe membrane insertion of the two large reactioncenter proteins, PsaA and PsaB (Göhre et al., 2006). It isnot just involved in PSI biogenesis but presumablyalso in the assembly of all other protein complexes ofthe photosynthetic electron transport chain (Ossenbühlet al., 2004; Pasch et al., 2005; Schöttler et al., 2011). Incontrast, PALE YELLOW GREEN7 (PYG7; Stöckelet al., 2006) acts in a PSI-specific manner. It containsthree tetratricopeptide repeat domains, structural mo-tifs known to mediate protein-protein interactions.Like ALB3, PYG7 is encoded in the nuclear genome.

Surprisingly, two other factors that have been im-plicated in PSI biogenesis are encoded in the chloro-plast (plastid) genome. Knockout of the open readingframe ycf3 (for hypothetical chloroplast reading frame)in the higher plant tobacco (Nicotiana tabacum; Rufet al., 1997) and the unicellular green alga Chlamydo-monas reinhardtii (Boudreau et al., 1997) led to a com-plete and specific loss of PSI. Ycf3 was shown to act inPSI biogenesis at the posttranslational level (Boudreauet al., 1997; Ruf et al., 1997), and, like PYG7, it is atetratricopeptide repeat protein. In Chlamydomonas,Ycf3 interacts with the PSI subunits PsaA and PsaD

(Naver et al., 2001) and thus may chaperone the correctassociation of these proteins with each other and/orother subunits in the stromal ridge of the complex.Interestingly, transplastomic tobacco plants with re-duced Ycf3 expression displayed a nearly proportionalreduction in PSI accumulation (Petersen et al., 2011),possibly indicating a rate-limiting role of Ycf3 in PSIbiogenesis. A search for protein interaction partners ofYcf3 in tobacco led to the discovery of a novel (nucleus-encoded) assembly factor, designated Y3IP1 (for Ycf3-interacting protein 1; Albus et al., 2010).

Reverse genetics in Chlamydomonas has providedevidence for a second plastid genome-encoded factorinvolved in PSI biogenesis: Ycf4 (Boudreau et al.,1997). Loss of ycf4 gene function resulted in the com-plete loss of PSI activity and, hence, loss of autotrophicgrowth. Consistent with this, earlier work in the cya-nobacterium Synechocystis sp. PCC 6803 had shownthat inactivation of the ycf4 homolog leads to an in-creased PSII-to-PSI ratio (Wilde et al., 1995). In Chla-mydomonas, the thylakoid membrane-intrinsic Ycf4protein was detected in complexes with the PSI sub-units PsaA to PsaF and the opsin-related eyespotprotein COP2 (Ozawa et al., 2009). The COP2 proteinmay be unlikely to function in PSI biogenesis, becausesilencing of the Cop2 gene by RNA interference did notaffect PSI accumulation in the alga (Ozawa et al.,2009). Interestingly, Ycf4 was also identified as a pro-tein component of the eyespot in Chlamydomonaschloroplasts (Schmidt et al., 2006), possibly suggestinga second function of Ycf4 (in association with COP2) inthe eyespot. Because embryophytes do not have eye-spots, the data raise questions about the validity of thefindings in Chlamydomonas for higher plants.

Here, we have performed a functional analysis ofthe open reading frame ycf4 in the plastid genome oftobacco plants. Using chloroplast transformation, wehave generated stable knockout mutants for ycf4. Weshow that the Ycf4 protein is specifically involved inPSI assembly and is present in protein complexes thatare associated with the thylakoid membrane. How-ever, unlike in Chlamydomonas, Ycf4 is not essential forphotosynthesis in tobacco, and the ycf4 knockoutmutants are capable of assembling sufficient amountsof PSI to allow for slow autotrophic growth.

RESULTS

Targeted Inactivation of the ycf4 Gene in the TobaccoPlastid Genome

The ycf4 gene is part of a gene cluster in the largesingle-copy region of the tobacco plastid genome,which comprises the psaI gene (encoding a small non-essential subunit of PSI; Schöttler et al., 2011) upstreamof ycf4 and the genes ycf10 (encoding a nonessentialmembrane protein possibly involved in inorganic car-bon uptake into the chloroplast; Rolland et al., 1997)and petA (encoding the cytochrome f subunit of thecytochrome b6f complex [Cyt bf]) downstream of ycf4

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(Fig. 1A). To construct a knockout allele for ycf4, thecorresponding region was cloned from the tobaccoplastid DNA, and most of the ycf4 reading frame wasdeleted from the cloned fragment and replaced with achimeric aadA cassette (Fig. 1A). The aadA gene servesas selectable marker for the isolation of chloroplast-transformed (transplastomic) cells (Goldschmidt-Clermont, 1991; Svab and Maliga, 1993) and encodes

the enzyme aminoglycoside 399-adenylyltransferase,which confers resistance to the aminoglycoside-typeantibiotics spectinomycin and streptomycin. TheaadA cassette was inserted in the same transcrip-tional orientation as ycf4 (Fig. 1A) to avoid the gen-eration of antisense RNA that potentially couldinterfere with the expression of the neighboringgenes.

Figure 1. Targeted inactivation of the plastid genome-encoded open reading frame ycf4 by stable transformation of thechloroplast genome. A, Physical maps of the ycf4-containing region of the tobacco plastid genome and of the transformedplastid genome in Nt-Dycf4 transplastomic lines. All genes shown are transcribed from left to right. Restriction sites relevant forcloning and/or RFLP analysis of transplastomic plants are indicated. Hybridization probes used for Southern-blot and northern-blot analyses are denoted by horizontal bars. The expected sizes of hybridizing fragments in RFLP analysis of wild-type andtransplastomic plants are indicated in kb. In Nt-Dycf4 transplastomic plants, most of the ycf4 coding region is deleted andreplaced with the selectable marker gene aadA. The expression of aadA is driven by a chimeric rRNA operon promoter (Prrn)and the 39 untranslated region from the psbA gene (Svab and Maliga, 1993). B, RFLPanalysis of transplastomic lines carrying theycf4 knockout allele. Total cellular DNA was digested with XhoI and HindIII and hybridized to a radioactively labeled probedetecting the accD region of the plastid genome, which flanks the transgene insertion site. Fragment sizes are given in kb. Thesize difference of 0.7 kb corresponds to the difference between the wild-type ycf4 gene and the mutant allele disrupted withthe aadA selectable marker cassette (compare with A). C, Analysis of ycf4 mRNA accumulation (left panel) and analysis of theexpression of petA, an essential gene for Cyt bf function located downstream of ycf4 (right panel), by northern blotting. As acontrol, RNA from a previously generated knockout line for ycf3, a plastid gene encoding an essential factor for PSI assembly(Ruf et al., 1997), was also loaded. Sizes of the RNA marker bands are given in kb. The absence of ycf4 transcripts from the Nt-Dycf4 transplastomic lines confirms the loss of ycf4 function from the plastid genome. As expected, expression of the aadAselectable marker gene cassette from the strong constitutive Prrn promoter causes increased accumulation of petA transcripts,due to read-through transcription (Zoubenko et al., 1994; Wurbs et al., 2007). To control for equal loading, the ethidiumbromide (EtBr)-stained agarose gels photographed prior to blotting are also shown. WT, Wild type. D, Absence of detectableYcf4 protein from Nt-Dycf4 transplastomic plants. To reveal the detection limit of the anti-Ycf4 antibody, a dilution series ofthylakoid proteins from wild-type plants was loaded (20 mg chlorophyll = 100%), and the amount of loaded extract from theknockout plant was increased up to 1,000%. To control for loading, a membrane stained with Ponceau red (P.) is shown belowthe blot.

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The ycf4 knockout vector was used to biolisticallybombard tobacco leaves followed by selection forspectinomycin-resistant cell lines on a plant regener-ation medium. Several independent spectinomycin-resistant clones were obtained and passed throughthree additional rounds of regeneration under strin-gent antibiotic selection to eliminate residual wild-typecopies of the plastid genome and purify homoplasmictransplastomic clones (Bock, 2001; Maliga and Bock,2011). Successful transformation of the plastid genomeand faithful integration of the transforming DNA byhomologous recombination was confirmed by DNAgel-blot analysis (Fig. 1B). As often seen in RFLPanalyses of plastid transformants, faint hybridizationsignals corresponding in size to the wild-type DNAwere detected in addition to strong signals for thetransgenic plastid genome (Fig. 1B). These signals arenot normally indicative of heteroplasmy (i.e. low-levelpresence of residual copies of the wild-type plastidgenome) but rather correspond to so-called promis-cuous DNA: chloroplast DNA pieces that have inte-grated into the nuclear genome (Ayliffe et al., 1998;Hager et al., 1999; Ruf et al., 2000; Bock and Timmis,2008). To confirm homoplasmy of the ycf4 knockoutplants, two independently generated transplastomiclines were selected (subsequently referred to as Dycf4-1 and Dycf4-2) and analyzed by northern blotting (Fig.1C). Hybridization of RNA gel blots to a ycf4-specificprobe revealed the complete loss of ycf4 expression(Fig. 1C), as expected. To confirm that the knockout ofycf4 does not impair the expression of adjacent genes(Fig. 1A), petA mRNA accumulation was analyzed.petA was chosen because, unlike psaI and ycf10 (whoseknockout does not result in a mutant phenotype;Rolland et al., 1997; Schöttler et al., 2011; S. Ruf, K.Krech, D. Bednarczyk, M.A. Schöttler, and R. Bock,unpublished data), it encodes an essential protein forphotosynthesis. Northern-blot analysis using a petA-specific probe revealed similar petA transcript patternsin the wild type and the Dycf4 mutants. The majortranscript species accumulating in the wild type (morethan 4 kb) corresponds to the tetracistronic mRNAcovering all genes of the operon from psaI to petA (Fig.1, A and C). The same tetracistronic transcript is alsodetected by the ycf4-specific probe in the wild type(Fig. 1C). The Dycf4 mutants accumulated even morepetA transcripts than the wild type (Fig. 1C), which ismost probably due to read-through transcription fromthe upstream aadA gene (which is driven by the strongribosomal operon promoter Prrn; Svab and Maliga,1993). Similar accumulation of read-through tran-scripts has been observed previously in many trans-plastomic studies (Zoubenko et al., 1994; Ruf et al.,1997).

To confirm the homoplasmy of the transplastomicDycf4 mutants, seeds were harvested and germinationassays were performed on spectinomycin-containingsynthetic medium. The progeny of Dycf4mutant plantsturned out to be uniformly resistant to spectinomy-cin (Supplemental Fig. S1). This lack of phenotypic

segregation in the next generation provides stronggenetic evidence of homoplasmy (Bock, 2001; Maliga,2004; Ruf et al., 2007).

To be able to analyze the expression of ycf4 at theprotein level, we generated specific antibodies againstYcf4-derived specific peptide sequences. With theseantibodies, the Ycf4 protein could be readily detectedin thylakoids of wild-type tobacco plants, demon-strating that the protein is membrane associated (Fig.1D). Complete absence of a hybridization signal fromwestern blots with thylakoid proteins of the Dycf4 linesconfirmed the specificity of the antibody and alsoprovided additional evidence of homoplasmy of ourtransplastomic ycf4 knockout lines (Fig. 1D).

To ultimately prove homoplasmy and the lack ofexpression of the promiscuous ycf4-like sequences pre-sumably present in the nucleus (Fig. 1B), we conductedquantitative real-time (qRT) PCR experiments. Whereasin the wild type ycf4 transcripts were detected at highsensitivity (even in a 1:100 cDNA dilution), no specificamplification was detectable with cDNA from thetransplastomic Dycf4 mutants (Supplemental Fig. S2),thus confirming the absence of ycf4 gene product fromour knockout lines.

Phenotype of Transplastomic ycf4 Knockout Plants

The successful generation of stable ycf4 knockoutplants enabled us to analyze the phenotypic conse-quence of the loss of ycf4 gene function. To this end,mutant and wild-type plants were grown side-by-sidein a controlled-environment chamber. The Dycf4 plantsproved to be extremely sensitive to light and wereunable to grow at light intensities higher than 80 mEm22 s21. However, under low-light conditions (40–50mE m22 s21), Dycf4 mutant plants grew photoautotro-phically. Although their growth and developmentwere severely retarded (Fig. 2A), the plants eventuallyreached the reproductive stage and set a small numberof flowers (Fig. 2B).

Our finding that ycf4 knockout plants can growautotrophically is in stark contrast to the situation inthe unicellular green alga Chlamydomonas, where ycf4mutant strains have been reported to be incapable ofphotoautotrophic growth (Boudreau et al., 1997). Aswork in Chlamydomonas had revealed an involvementof the Ycf4 gene product in PSI biogenesis, we alsowanted to compare the phenotype of our Dycf4 plantswith a mutant that is deficient in PSI accumulation. Tothis end, we selected a tobacco mutant that, underautotrophic growth conditions, has only 10% of the PSIlevels of the wild type (on a leaf area basis) due toreduced translation initiation efficiency of the psaAmRNA encoding one of the two reaction centerproteins of PSI (D. Bednarczyk, R. Bock, and M.A.Schöttler, unpublished data). Interestingly, the phe-notype of this mutant (referred to as KD-psaA forknockdown of psaA) was very similar to that ofthe Dycf4 plants (Fig. 2A). Both mutants showed


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comparably slow growth and severe pigment defi-ciency, suggesting that their photosynthetic perfor-mance is similarly strongly affected.

Photosynthesis in ycf4 Knockout Plants

To characterize the mutant phenotype of the Dycf4plants in more detail, several parameters related to theefficiency of the light reactions of photosynthesis weremeasured (Fig. 3). To minimize secondary effects fromcarbon starvation and/or photooxidative damage, allparameters were measured not only in plants grownautotrophically in soil but also in plants raised on Suc-containing synthetic medium in sterile containers(mixotrophic growth conditions; Fig. 3). As expectedfrom the pale-green phenotype of the Dycf4 mutantplants, their chlorophyll content was significantly lowerthan in wild-type plants. Also, the chlorophyll a/b ratiowas reduced in the mutants, possibly indicating a de-ficiency in the (mainly chlorophyll a-containing) pho-tosystem core(s) relative to the antennae (Fig. 3). Themaximum quantum efficiency of PSII was also sig-nificantly reduced in the Dycf4 plants, supporting adefect in the photosynthetic apparatus in the thyla-koid membrane. To test if the defect can be attributedto a specific complex, we next determined the contentsof the components of the photosynthetic electrontransport chain (PSII, Cyt bf, plastocyanin, and PSI;Fig. 3) by spectroscopic methods (Schöttler et al.,2007a, 2007b). These analyses revealed that the Dycf4mutants displayed a specific reduction in PSI contents(Fig. 3). PSI levels in plants raised under mixotrophicconditions reached only approximately 15% to 20% ofwild-type levels. Plants grown in soil were even morestrongly affected and had less than 10% of the wild-type levels of PSI. Under mixotrophic conditions,none of the other components of the electron transportchain were significantly affected by the loss of ycf4function, whereas under autotrophic conditions, theCyt bf was strongly decreased (Fig. 3). Importantly,

this reduction was also seen in the KD-psaA controlplants, clearly indicating that the lowered Cyt bfcontent is an indirect consequence of a severe PSIdeficiency. Whether this loss of Cyt bf is triggered by acarbon starvation signal (released in the mutants uponautotrophic growth but suppressed by the sugar inthe medium upon mixotrophic growth) or ratherby increased oxidative stress under our autotrophicgrowth conditions remains to be investigated.

To further analyze the photosynthesis-deficientphenotype of the Dycf4 plants, chlorophyll a fluores-cence emission measurements at 77 K were conducted.At this low temperature, the characteristic long-wavelength emission from PSI-LHCI can be readilyanalyzed and compared with the shorter wavelengthfluorescence emission originating from PSII (Krauseand Weis, 1991). Recording of 77 K chlorophyll afluorescence emission spectra of wild-type plants andthe Dycf4 mutants revealed that the maximum fluo-rescence emission from PSI (peaking at 733 nm in thewild type) was significantly shifted to shorter wave-lengths in the Dycf4 mutant plants, and a very similarshift was also seen in the PSI-deficient KD-psaA mu-tant (Fig. 4). This suggests an altered functional orga-nization of PSI (Jensen et al., 2004; Schöttler et al.,2007b) and indicates that a substantial proportion ofthe PSI antenna is not connected to PSI reaction centers(Stöckel et al., 2006). Taken together, the results fromour spectroscopic analyses (Figs. 3 and 4) support aspecific function of the Ycf4 protein in the biogenesis ofPSI cores.

Accumulation of Thylakoid Protein Complexes inDycf4 Plants

To confirm the PSI deficiency in Dycf4 mutant plantsat the molecular level, thylakoids were isolated fromsoil-grown plants and analyzed immunobiochemicallyusing a set of specific antibodies raised against diag-nostic subunits of all four multiprotein complexes in

Figure 2. Phenotypes of Dycf4 transplastomic to-bacco lines. A, Wild-type (WT) and Dycf4 trans-plastomic knockout plants are shown after 8 weeks ofgrowth in soil under low-light conditions (40–50 mEm22 s21). For comparison, a PSI-deficient mutantgenerated by knockdown of the gene encoding thereaction center subunit PsaA (KD-psaA; for details,see text) is also shown. B, After extended growthunder low-light conditions (for 16 weeks), Dycf4transplastomic lines reached the reproductive stagebut developed only very few flowers.



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the thylakoid membrane (PSII, Cyt bf, PSI, and ATPsynthase). Five subunits of PSI were investigated: thereaction center protein PsaB, the plastocyanin-dockingprotein PsaF, and the three small subunits PsaG, PsaK,and PsaL. At least the reaction center protein PsaB isdiagnostic of PSI complex accumulation in that no PSIcan assemble in thylakoid membranes of psaB knock-out mutants (Schaffner et al., 1995). This holds truealso for the tested subunits of the other thylakoidalprotein complexes: PsbD and PsbO (diagnostic ofPSII), PetA and PetC (diagnostic of Cyt bf), and AtpB(diagnostic of the ATP synthase).

Immunobiochemical analysis of thylakoid proteinsfrom two Dycf4 lines and the KD-psaA control mutantrevealed a strong reduction in PSI amounts comparedwith wild-type tobacco plants (Fig. 5). The reductionwas slightly stronger in the Dycf4 plants than in theKD-psaA plants, supporting the quantitative data ofour spectroscopic measurements (Fig. 3). As the thy-lakoids were isolated from soil-grown plants, thewestern blots also showed the reduction in the Cyt bfcomplex secondarily occurring under autotrophicconditions (Fig. 3). Importantly, this reduction wasalso seen in the KD-psaA control plants, indicating thatit represents a secondary consequence of strongly de-creased PSI levels. In the Dycf4 lines and the KD-psaAmutant, the diagnostic subunits of PSII and the ATPsynthase accumulated to similar or slightly elevatedlevels compared with the wild type (Fig. 5), confirmingthat Ycf4 is specifically required for PSI biogenesis andis unlikely to be directly involved in the biogenesis ofthe other complexes of the photosynthetic electrontransport chain.

To confirm the preliminary conclusion from ourspectroscopic data (Fig. 4) that the Ycf4 protein specif-ically functions in the biogenesis of PSI cores, we alsoinvestigated the accumulation of the PSI antenna pro-teins LhcA1, LhcA2, and LhcA4 using specific anti-bodies. LhcA proteins have been shown to accumulate

Figure 4. The 77 K chlorophyll a fluorescence emission measure-ments. The fluorescence emission signals were normalized to the PSIIemission maximum at 685-nm wavelength. The PSI emission signalpeaks at 733 nm in the wild type (WT). The blue-shifted wavelength ofthe PSI emission maximum in the Dycf4 knockout mutants and the KD-psaA control plants indicates that PSI antenna organization is alteredand suggests that a significant proportion of PSI antenna proteins arenot attached to PSI core complexes.

Figure 3. Analysis of chlorophyll contents and various photosyntheticparameters in transplastomic Dycf4 knockout mutants and wild-type (WT)as well as KD-psaA control plants. Data sets are shown for plants grownunder 40 to 50 mE m22 s21 in either synthetic Suc-containing medium(mixotrophic conditions) or soil (autotrophic conditions) for 6 weeks. Foreach plant line, three or more different plants were measured, and datawere subjected to one-way ANOVA using a pairwise multiple comparisonprocedure (Holm-Sidak method) in SigmaPlot. Highly significant differ-ences are indicated by asterisks (P , 0.05). Error bars represent SD. Pho-tosynthetic complexes were quantified from difference absorbancemeasurements of cytochrome b559 (PSII), Cyt bf, plastocyanin, and P700(PSI) in isolated thylakoids.


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independently of the PSI reaction center (Stöckel andOelmüller, 2004; Stöckel et al., 2006). In contrast to allPSI core subunits tested, accumulation of the threeproteins of the PSI antenna (LHCI) was unaffected inthe transplastomic Dycf4 lines (and, as expected, also inthe KD-psaA control mutant; Fig. 5), suggesting thatthe function of Ycf4 in PSI biogenesis is restricted tothe assembly of the core complex.Interestingly, accumulation of the Ycf4 protein was

not reduced in the psaA knockdown mutants (KD-psaA; Fig. 5). This shows that Ycf4 accumulates in thethylakoid membrane independently of PSI, a findingthat is well compatible with an auxiliary role of Ycf4 inPSI biogenesis.

Faithful Expression of Plastid PSI Genes in ycf4Knockout Plants

We next wanted to test whether Ycf4 controls PSIaccumulation at the posttranslational level (i.e. at thelevel of complex assembly or stability). To this end, wesought to exclude the alternative possibilities that the

Ycf4 protein is involved in the transcription of PSIgenes, the accumulation of stable PSI mRNAs, and/orthe translation of PSI subunits. As Ycf4 is encoded inthe plastid genome and the protein therefore cannot bepresent in the nucleocytosolic compartment, only thePSI genes in the plastid genome are relevant to anypossible function of Ycf4 in transcription, RNA stabil-ity, or translation. The tobacco plastid genome encodesthree essential subunits of PSI: PsaA, PsaB (the tworeaction center proteins of PSI encoded by the psaA/Boperon and translated from a large polycistronictranscript; Meng et al., 1988), and PsaC (an essentialiron-sulfur cluster-containing PSI protein; Takahashiet al., 1991). The other two small plastid-encoded PSIsubunits, PsaJ and PsaI, are nonessential for PSI accu-mulation, and their loss has only very minor effects onPSI accumulation, which cannot account for the strongphotosynthetic phenotype observed here (Schöttleret al., 2007b, 2011). We additionally included the plastidycf3 gene in our analyses, because the Ycf3 protein haspreviously been shown to be an essential chloroplastgenome-encoded assembly factor for PSI (Ruf et al.,1997; Albus et al., 2010).

We compared transcript patterns and accumulationlevels for these four essential plastid PSI-related genesin Dycf4 lines and wild-type plants. To test for a pos-sible involvement of Ycf4 in the translation of plastidPSI mRNAs, the ribosome association of the psaA/B,psaC, and ycf3 mRNAs in Dycf4 plants and wild-typeplants was also comparatively assessed by polysome-loading experiments. When the transcripts were in-vestigated by northern blotting, none of them showedany reduction in RNA abundance or alteration in RNAprocessing patterns (Fig. 6), indicating that Ycf4 actsneither at the transcriptional level nor at the level ofmRNA maturation or stability. Interestingly, the psaA/B mRNA level was found to be significantly higher inthe Dycf4 mutant than in the wild type. This couldindicate that the plant responds to the PSI deficiencyby transcriptionally up-regulating the operon com-prising the genes for the two PSI reaction center sub-units. This transcriptional up-regulation was alsoclearly seen when ribosome-associated mRNAs wereanalyzed (Fig. 6). Polysomes are complexes of mRNAscovered with translating ribosomes, and their migra-tion into continuous Suc gradients upon ultracentri-fugation correlates with the number of ribosomesbound to the mRNA molecule (in that intenselytranslated mRNAs are loaded with many ribosomesand migrate deeper into the gradient than weaklytranslated mRNAs). For all the PSI-related plastidmRNAs analyzed, the polysome profiles (i.e. themRNA distribution across the Suc gradient) in wild-type and Dycf4 mutant plants were very similar (Fig.6), indicating that PSI mRNA translation proceeds atcomparable efficiency. These data suggest that Ycf4also does not act as a translation factor for plastidgenome-encoded PSI mRNAs.

Taken together, our analyses of PSI gene transcrip-tion and translation strongly suggest that the PSI

Figure 5. Immunoblot analysis of diagnostic components of the multi-protein complexes in the thylakoid membrane in transplastomic Dycf4knockout mutants, wild-type plants (WT), and KD-psaA control plantsgrown under autotrophic conditions. Isolated thylakoid proteins wereseparated by SDS-PAGE, blotted, and probed with antibodies against theproteins indicated on the left side of each panel. Equal amounts ofchlorophyll were loaded. For quantitative assessment of protein accu-mulation in the mutants, a dilution series of the wild-type sample (100%,50%, and 25%) was loaded. A Ycf4-specific antibody generated in thisstudy was used to assess the accumulation of the ycf4 gene product.



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deficiency in Dycf4 mutants is not caused by a defect inplastid gene expression. Instead, the data are consis-tent with a function of Ycf4 at the posttranslationallevel, either in PSI assembly or PSI stability.

Association of the Ycf4 Protein with High-Mr Complexes

The successful generation of a sensitive Ycf4-spe-cific antibody enabled us to assess the possible asso-ciation of the Ycf4 protein with higher Mr complexesin the thylakoid membrane. To this end, we separatedthylakoidal protein complexes by blue-native (BN)PAGE, blotted the gels, and hybridized the mem-branes to our anti-Ycf4 antibodies. In these experi-ments, the bulk of Ycf4 protein migrated with thenon-complex-associated fraction (“free” proteins; Fig.7A). A smaller fraction was associated with at leasttwo different high-Mr complexes, one of which cor-related in migration with PSI. This raises the possi-bility that a subfraction of Ycf4 is associated with PSIparticles, either because it is involved in late steps ofPSI assembly or because it helps in stabilizing theassembled complex.

To confirm this result and supply additional evi-dence for the association of Ycf4 with PSI particles, weperformed two-dimensional gel electrophoresis ex-periments followed by western blotting and immu-nological detection of the Ycf4 protein and the PSIreaction center protein PsaA as a diagnostic subunit ofPSI complexes. These analyses confirmed the comi-gration of a subfraction of the Ycf4 protein with PSI(Fig. 7B), thus providing further evidence for a phys-ical interaction of Ycf4 with PSI complexes. Moreover,

identical electrophoretic migration of the PSI com-plexes from wild-type and mutant plants and stableassociation of the small G-subunit with the complexalso in the transplastomic Dycf4 knockout confirmedthat the composition of the PSI complexes in the mu-tant is unaltered compared with the wild type (Fig.7C).

Differential Accumulation of PSI and Its Assembly Factorsduring Plant Development

The sensitive detection of the Ycf4 protein by ourspecific antibody enabled us to investigate the rela-tionship between Ycf4 protein accumulation and PSIaccumulation during leaf ontogenesis. To conduct amore systematic analysis of PSI assembly during de-velopment, we also included two previously identifiedPSI assembly factors: the chloroplast genome-encodedYcf3 (Ruf et al., 1997) and its nucleus-encoded inter-action partner Y3IP1 (Albus et al., 2010). As PSI ac-cumulation is strictly dependent on Ycf3 and Y3IP1function, comparison of Ycf3 and Y3IP1 protein accu-mulation with PSI subunit accumulation may alsoprovide information about the turnover rate of the PSIcomplex. For Ycf3, we had previously constructed anepitope-tagged transplastomic line expressing a Ycf3protein with a C-terminal FLAG epitope (Albus et al.,2010). The use of this line enabled us to follow the fateof Ycf3 during development. Because for Y3IP1 nospecific antibodies or tagged lines were available, wegenerated specific antibodies in rabbits. Testing of theantibodies in the wild type and Y3IP1 knockdownlines (Albus et al., 2010) confirmed their specificity and

Figure 6. Unaltered gene expression of all essential plastid-encoded PSI genes in Dycf4 knockout mutants. Translational ef-ficiency for the essential plastid genome-encoded PSI-related genes psaA/B, psaC, and ycf3 was analyzed by polysome-loadingassays. Collected fractions from the Suc density gradients are numbered from top to bottom. Equal aliquots of extracted RNAsfrom all fractions were separated by denaturing agarose gel electrophoresis, blotted, and hybridized to gene-specific radiola-beled probes. The arrows at the top indicate the gradient in Suc density (from low to high). As a control, a sample was treatedwith the antibiotic puromycin to cause the dissociation of ribosomes from the mRNAs. Ribosome distribution in the gradients isrevealed by ethidium bromide (EtBr) staining of the agarose gels prior to blotting. PSI transcript patterns and mRNA accu-mulation levels were also determined (middle panel) for the three essential plastid genome-encoded PSI subunits (psaA/B andpsaC) and the ycf3 gene encoding an essential PSI assembly factor (Ruf et al., 1997). To confirm equal loading, an ethidiumbromide-stained agarose gels is also shown. WT, Wild type.


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revealed that they detect the Y3IP1 protein at highsensitivity (Fig. 8).

In order to analyze Ycf3, Y3IP1, and Ycf4 expressionin relation to PSI accumulation during plant develop-ment, we compared the accumulation of all threeproteins with that of the PSI reaction center subunitPsaA in a developmental series of leaves harvestedfrom 12-week-old tobacco plants. In these plants, leaf1 represents the oldest leaf and leaves 10 and 11 (whichhad to be combined due to their small size; Fig. 8)represent the youngest leaves at the top of the plant.Analysis of PsaA accumulation revealed comparablyhigh amounts of PSI over the entire developmentalseries analyzed, well in line with previous observa-tions (Schöttler et al., 2007b). A similar constitutiveaccumulation was seen for Ycf3. Interestingly, Y3IP1,the interaction partner of Ycf3, exhibited a very dif-ferent pattern. Its accumulation was highest in veryyoung leaves, continuously declined with leaf age, andwas undetectable in older leaves (Fig. 8). As Y3IP1 isessential for PSI accumulation (Albus et al., 2010), thissuggests that PSI biogenesis ceases in old leaves. AsPSI levels remain unaltered, this in turn indicates avery high stability of the PSI complex. The Ycf4 proteinshowed a similar age-dependent decline in proteinaccumulation as Y3IP1 (Fig. 8). This indicates that Ycf4is not required for PSI stability and suggests that Ycf4acts as an assembly factor for PSI.

DISCUSSION

In this work, we have investigated the function ofthe open reading frame ycf4 in the plastid genome ofthe higher plant tobacco. Previous work in the uni-cellular alga Chlamydomonas had revealed that the Ycf4gene product is essential for PSI biogenesis and pho-tosynthetic activity (Boudreau et al., 1997). Our datademonstrate that, in the seed plant tobacco, the Ycf4protein is neither essential for PSI biogenesis nor for

Figure 7. Detection of Ycf4-containing high-Mr complexes by BN gelelectrophoresis. A, One-dimensional BN gel electrophoretic separationof thylakoidal protein complexes. Transplastomic Dycf4 knockout mu-tants, wild-type plants (WT), and KD-psaA control plants were analyzed.Arrows denote Ycf4-containing fractions. The asterisk indicates a non-specific immunological cross-reaction (as evidenced by the presence ofthe signal in the Dycf4 knockout mutants). D, Dimer; M, monomer; SC,supercomplex; T, trimer. B, Two-dimensional electrophoretic analysis ofPSI complexes and Ycf4-containing complexes in wild-type and Dycf4transplastomic knockout plants. The first dimension was BN gel elec-trophoresis, and the second dimension was SDS-PAGE (see “Materialsand Methods”). Nonspecific cross-reactions are indicated by the asterisk,and the Ycf4 protein is indicated by an arrow. C, Two-dimensionalelectrophoretic analysis of residual PSI complexes accumulating in theDycf4 knockout mutant. The gels were blotted, and the blots wereprobed with antibodies against the PSI reaction center protein PsaA andthe small PSI subunit PsaG. The identical electrophoretic migration ofthe PSI complexes from wild-type and mutant plants and the stable as-sociation of the small G-subunit with the complex also in the Dycf4knockout suggest that the composition of the PSI complexes in themutant is unaltered compared with the wild type.

Figure 8. Developmental regulation of PSI assembly factors. Trans-plastomic Ycf3-FLAG lines (Albus et al., 2010) were used to follow thefate of three assembly factors for PSI: the plastid genome-encodedproteins Ycf3 and Ycf4 and the nuclear genome-encoded interactionpartner of Ycf3, Y3IP1 (Schottler et al., 2011). A developmental seriesof leaves from a tobacco plant prior to flowering is shown. Leaves arenumbered from bottom to top. The two youngest leaves (nos. 10 and11) had to be combined due to their small size. The assembly factorswere detected with specific antibodies (for details, see text). Forcomparison, PSI accumulation was followed with an antibody againstthe essential reaction center subunit PsaA.



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photoautotrophic growth. Although the Dycf4 trans-plastomic mutants generated in this work had reducedlevels of PSI and their growth was severely retarded,they assembled sufficient amounts of functional PSIcomplexes to enable autotrophic growth in soil. Thispoints to mechanistic differences in PSI biogenesisbetween algae and higher plants and may indicate thathigher plants have evolved additional auxiliary func-tions (proteins?) that can partially replace Ycf4 in PSIassembly. The identity of these additional accessoryfactors and their possible interplay with Ycf4 in one ofthe Ycf4-containing high-Mr complexes (Fig. 7) re-mains to be investigated. It is noteworthy in this re-spect that the ycf4 gene, although otherwise wellconserved in the green lineage, has been lost from thechloroplast genome in the legume species Lathyrusodoratus and separately in three other groups of le-gumes (Magee et al., 2010). A nuclear copy of ycf4could not be identified in Lathyrus (Magee et al., 2010),making it unlikely that a functional gene copy wastransferred to the nuclear genome. Although thisconclusion still awaits ultimate confirmation fromwhole-genome sequencing, it lends support to the ideathat Ycf4 function can be replaced by other factor(s)acting in PSI assembly.

The study of algal strains expressing tagged andpoint-mutated versions of the Ycf4 protein has pro-vided some evidence for a function of Ycf4 in the PSIassembly process (Onishi and Takahashi, 2009; Ozawaet al., 2009) but did not directly exclude a function inthe translation of plastid genome-encoded PSImRNAs. Performing polysome-loading analyses, wehave shown here that, at least in higher plants, Ycf4 isunlikely to be involved in the synthesis of PSI subunits(Fig. 6). This suggests a posttranslational cause of thePSI deficiency in Dycf4 mutants, well compatible witha function of Ycf4 in PSI assembly and/or stabilization.At the moment, we can only speculate which specificsteps of PSI biogenesis are supported by Ycf4. InChlamydomonas, Ycf4 was found to be part of a PSIassembly intermediate complex comprising the threestromal ridge subunits and PsaF (Ozawa et al., 2009).Therefore, a role in either the formation of the stromalridge or the insertion of PsaF seems conceivable(Ozawa et al., 2010). However, in our two-dimensionalgel electrophoresis experiments, whereas most of theYcf4 protein was found to be present as free protein, asignificant subfraction of Ycf4 comigrated with themature PSI complex (Fig. 7B). This may suggest a rolerather late in PSI biogenesis, for example, in the at-tachment of the small peripheral subunits and/or theantenna proteins. A somewhat different function ofYcf4 in higher plants from the proposed function inChlamydomonas would also be compatible with thedifferent phenotypic effects of ycf4 inactivation inChlamydomonas and tobacco.

Our analysis of Ycf4 expression during developmentsupports a function of Ycf4 in PSI assembly rather thanstability. Whereas PSI is still present in high amountsin mature and old leaves, the level of Ycf4 (and also

that of Y3IP1; Fig. 8) declines continuously. The highrequirement for PSI synthesis in young developingleaves is expected to coincide with a high demand forthe PSI assembly factors Ycf3, Ycf4, and Y3IP1, thuspotentially explaining their high abundance in veryyoung leaf tissue (Fig. 8). The strong decline in Ycf4and Y3IP1 accumulation during leaf development in-dicates that PSI biogenesis is restricted to youngleaves, suggesting that PSI is highly stable. The evo-lutionary optimization of both the efficiency of theelectron transfer reactions in PSI and the stability of thePSI core complex may have allowed plants to keep PSIturnover at a minimum and avoid the costly resyn-thesis of this gigantic pigment-protein complex. Theaccumulation of high amounts of PSI in the virtualabsence of Y3IP1 and Ycf4 in old leaves furthermoresuggests that the function of these factors is confined tothe PSI assembly process and seems to exclude animportant function in PSI stability.

The lack of coregulation of Ycf3 with its interactionpartner Y3IP1 and with Ycf4 is somewhat surprising.Whether there is a continued demand for Ycf3 functionin old leaves and in the absence of the other PSI as-sembly factors remains to be determined. One possi-bility could be that some components of the PSIcomplexes in mature and old leaves suffer photooxi-dative damage and need to be exchanged by newlysynthesized molecules through the action of Ycf3 in aY3IP1- and Ycf4-independent fashion. Indications forphotoinhibition of the PSI acceptor side (Kudoh andSonoike, 2002; Scheller and Haldrup, 2005; Sonoike,2011) and a repair cycle replacing the stromal ridgesubunits, which are most sensitive to oxidative dam-age, have recently been reported (Oh et al., 2009). In-terestingly, Ycf3 has been specifically implicated in theassembly of PsaD into PSI (Naver et al., 2001) andtherefore might also play an important role in the re-pair of the stromal ridge.

In sum, our work establishes the chloroplast-encodedYcf4 protein as an important, but nonessential, factorfor PSI assembly of higher plants. Together with pre-viously identified assembly factors (Ruf et al., 1997;Stöckel et al., 2006; Albus et al., 2010; Schöttler et al.,2011), we now know at least a small set of proteins thatare specifically involved in PSI biogenesis. The bigchallenge for the future will be to determine the rela-tionship between these factors and reveal the mecha-nistic details of the assembly process.

MATERIALS AND METHODS

Plant Material

Tobacco (Nicotiana tabacum ‘Petit Havana’) plants were aseptically grownby germinating surface-sterilized seeds on agar-solidified Murashige andSkoog (MS) medium (Murashige and Skoog, 1962) with 30 g L21 Suc.Homoplasmic transplastomic lines were rooted and propagated on the samemedium. Rooted homoplasmic plants were transferred to soil and grownunder low-light conditions (40–50 mE m–2 s–1) in the greenhouse (day tem-perature, 20°C; night temperature, 18°C). For photosynthetic measurementswith plants grown on Suc-containing synthetic medium, the sterile containers


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were kept in a controlled-environment chamber, and similar growth temper-atures and light intensity as for autotrophic growth were chosen, except thatthe day temperature was 25°C and the night temperature was 20°C. Day-length was set to 16 h of light.

Cloning Procedures

To construct the chloroplast transformation plasmid pDycf4, the cloningvector pBluescript II SK+ was digested with EcoRV and HincII and religated toeliminate the restriction site for ClaI. Subsequently, the ycf4-containing regionof the tobacco plastid genome was cloned into the ClaI(2) pBluescript II SK+vector as a 3,251-bp BamHI fragment (nucleotide positions 60,864–64,115 inthe tobacco ptDNA; Yukawa et al., 2005; Fig. 1A). The resulting plasmid wasdigested with ClaI and treated with Klenow DNA polymerase to fill in theoverhanging ends. An aadA cassette (Svab and Maliga, 1993) was ligated intothe blunted ClaI site as an Ecl136II/DraI fragment generating the final plastidtransformation vector pDycf4.

Transformation of Tobacco Chloroplasts

Plastid transformation was carried out using the biolistic protocol (Svab andMaliga, 1993). Briefly, young leaves from tobacco plants grown under asepticconditions were bombarded with plasmid DNA-coated 0.6-mm gold particlesusing a biolistic gun (PDS1000He; Bio-Rad). Primary spectinomycin-resistantlines were selected on regeneration medium containing spectinomycin (500mg L21; Svab and Maliga, 1993). Plastid transformation was preliminarilyconfirmed by double resistance tests on regeneration medium with bothspectinomycin and streptomycin (500 mg L21 each; Bock, 2001). Several in-dependent transplastomic lines were subjected to three additional rounds ofregeneration on spectinomycin-containing MS medium to eliminate residualwild-type plastome copies and obtain homoplasmic tissue. Homoplasmy wasconfirmed by inheritance assays (Bock, 2001), in which seeds were germinatedon MS medium containing spectinomycin (500 mg L21).

Isolation of Nucleic Acids and Gel-Blot Analyses

Total DNA from tobacco leaf samples was isolated by a cetyl-trimethylammoniumbromide-based method (Doyle and Doyle, 1990). Totalcellular RNA was extracted using the peqGOLD TriFast reagent (Peqlab). ForSouthern-blot analysis, samples of 5 mg of total DNA were digested with therestriction enzymes XhoI and HindIII (Fig. 1), separated by agarose gel elec-trophoresis on 1% agarose gels, and transferred onto Hybond nylon mem-branes (GE Healthcare) by capillary blotting. Total cellular RNA samples wereelectrophoresed on formaldehyde-containing 1% agarose gels and blottedonto Hybond nylon membranes. Hybridizations were performed at 65°C us-ing standard protocols. Hybridization probes were purified by agarose gelelectrophoresis after extraction of the DNA fragments of interest from excisedgel slices using the GFX PCR (DNA and Gel Band Purification) kit (GEHealthcare). A 943-bp XhoI/BglII restriction fragment containing the 39 part ofthe accD coding region was used as an RFLP probe to verify plastid trans-formation and assess homoplasmy. A ClaI restriction fragment (Fig. 1A) wasused as a hybridization probe for the detection of ycf4 transcripts in northern-blot analyses, and a petA-specific probe was prepared by PCR amplificationusing total DNA as a template and the gene-specific primers PpetA5 (59-GCGACTGGGCGTATTGTATGTGC-39) and PpetA3 (59-CGCCCTCGGAAA-CAAGAAGTTCTG-39). [a-32P]dCTP-labeled probes were generated byrandom priming (Multiprime DNA labeling system; GE Healthcare). Hy-bridization signals were analyzed using a Typhoon Trio+ variable mode im-ager (GE Healthcare).

cDNA Synthesis and qRT-PCR

For cDNA synthesis, total plant RNA was isolated using the NucleoSpinRNA Plant kit (Macherey-Nagel) following the instructions of the supplier.To fully remove contaminating DNA, a second DNase digest was done usingrDNase (Macherey-Nagel). cDNA synthesis was performed with randomhexamer primer (3 mg per reaction) and the SuperScript III reverse tran-scriptase enzyme (Life Technologies) following the instructions of themanufacturer. qRT-PCR assays were performed according to standardprotocols using SYBR Green to monitor the amplification process. ycf4 wasamplified with primers Pycf4-5 (59-GGCGATCAGAACATATATGGATAG-

39) and Pycf4-3 (59-CCAACTAATAAGAAGCCTAATGAACC-39), atpA withprimers PatpA5 (59-TTCTACCGTGAGAGGAGCTGATTGG-39) and PatpA3(59-GCCTTTGCACAATTTGCTTCTGATC-39), and EF1-a with primersPef1-5 (59-TGAGATGCACCACGAAGCTC-39) and Pef1-3 (59-CCAA-CATTGTCACCAGGAAGTG-39).

Polysome-Loading Assays

Isolation of polysomes and RNA extraction from polysomal gradientfractions were performed as described previously (Kahlau and Bock, 2008;Rogalski et al., 2008). Gradient fractionation was carried out using the AutoDensi-Flow (Labconco) and the Pharmacia LKB RediFrac fraction collector (GEHealthcare). RNA pellets were dissolved in 30 mL of sterile water, and 5-mLaliquots were used for northern-blot analyses. As a control, 4 mg of total RNAfrom wild-type and mutant plants was analyzed.

Isolation of Thylakoids and Immunoblotting

Thylakoid proteins from wild-type and transplastomic plants were isolatedfrom total leaf material using published procedures (Machold et al., 1979;Schöttler et al., 2004). For western blotting, samples were normalized to chlo-rophyll, electrophoretically separated on SDS-polyacrylamide gels (Laemmli,1970), and transferred to Hybond-P polyvinylidene difluoride membranes (GEHealthcare) using standard protocols. Immunoblot detection was performedwith specific antibodies using the ECL PLUS system (GE Healthcare). Poly-clonal antibodies against PsaL, PsaG, PsaA, PsaB, PsaF, PsaK, PsbD, PsbO,PetC, and AtpB (produced in rabbits) were purchased from Agrisera. Goatanti-rabbit IgG (H + L)-horseradish peroxidase conjugate (Bio-Rad) was usedas a secondary antibody. The Ycf3-FLAG protein (Albus et al., 2010) wasdetected with an affinity-purified anti-FLAG M2 monoclonal antibody (Agi-lent Technologies) and anti-mouse IgG peroxidase conjugated (Sigma). Thechemiluminescence signal was visualized by exposure to Kodak Biomax XARfilm (Sigma-Aldrich).

Generation and Affinity Purification of Antibodies againstYcf4 and Y3IP1

Polyclonal antibodies against Ycf4 were produced in rabbits using thesynthetic peptide sequences CVGSGYDRFDRKEGI-amide and CTDENLT-PREIEQKA-amide and LPH as the carrier protein. Specific antibodies wereenriched by purification of serum samples with antigen-coupled HiTrap NHS-activatedHP columns following the instructions of the supplier (GEHealthcare;Antibody Purification Handbook).

For the generation of Y3IP1 antibodies, a 172-amino acid sequence repre-senting the hydrophilic part of the mature tobacco Y3IP1 protein was chosen asan epitope. The respective coding sequence was amplified from tobacco cDNAwith the primers 59-TTCCATGGGGAAAGAAGAAGACAGTGCAACC-39and 59-TTCTCGAGACCCAAAGCTGGAGGAACATC-39, introducing the re-striction sites NcoI and XhoI, respectively. The resulting PCR product wascloned in frame into the vector pET-28a(+) (Novagen) via the NcoI and XhoIrestriction sites. The resulting plasmid encoding the C-terminally His6-taggedY3IP1 fragment was introduced into Escherichia coli BL21 cells. After iso-propylthio-b-galactoside induction, the overexpressed protein was isolated ina two-step purification by nickel-nitrilotriacetic acid agarose (Qiagen) andsubsequent separation of the eluate by SDS-PAGE and recovery of the purifiedprotein from the gel. To this end, the excised gel slice containing the Y3IP1protein fragment was macerated in 50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM

EDTA, pH 7.5, and incubated overnight at 30°C. The protein-containing bufferwas then filtered (Whatman filter; 0.45-mm cutoff) to remove the gel matrix,concentrated by ultrafiltration (Microcon YM-3; Millipore), tested for identityand purity by tandem mass spectrometry, and subsequently used for theimmunization of rabbits (BioGenes).

BN Gel Electrophoresis

One- and two-dimensional BN gels were prepared according to publishedprocedures (Dietzel et al., 2011) with the sole exception that the gels were runin the PROTEAN II j Cell gel system (Bio-Rad). For immunoblot analysis ofthylakoid membrane proteins, one-dimensional BN gels were transferredonto polyvinylidene difluoride membranes (GE Healthcare) and destainedaccording to published protocols (Wittig et al., 2006). For two-dimensional



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BN-PAGE, the second dimension was performed as in standard SDS-PAGE(Laemmli, 1970).

Pigment Analysis and Photosynthesis Physiology

Chlorophyll contents were determined in 80% (v/v) acetone (Porra et al.,1989). The 77 K chlorophyll a fluorescence emission spectra of isolated thylakoids(equivalent to 10 mg chlorophyll mL21) were determined using a Jasco F-6500fluorimeter. Chlorophyll a fluorescence was excited at 430-nm wavelength (10-nm spectral bandwidth), and fluorescence emission was determined with aspectral bandwidth of 1 nm in a wavelength range from 660 to 800 nm.

Chlorophyll fluorescence was recorded with a pulse amplitude-modulatedfluorimeter (Dual-PAM-100; Heinz Walz) on intact wild-type and mutantplants at room temperature. Plants were dark adapted for 1 h prior to mea-surement of maximum quantum efficiency of PSII. The contents of PSII, Cyt bf,plastocyanin, and PSI were determined in thylakoids prepared as describedpreviously (Schöttler et al., 2004). PSI was quantified from P700 differenceabsorption signals at 830 to 870 nm in solubilized thylakoids using the Dual-PAM instrument (Schöttler et al., 2007a, 2007b). Contents of PSII and Cyt bfwere determined from difference absorption measurements of cytochrome b559and Cyt bf, respectively. Measurement procedures and data deconvolutionmethods have been described in detail previously (Kirchhoff et al., 2002;Schöttler et al., 2007b).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Confirmation of homoplasmy and uniparentalmaternal inheritance of the ycf4 knockout allele.

Supplemental Figure S2. qRT-PCR assays to confirm the absence of de-tectable ycf4 transcripts in Dycf4 knockout plants.

ACKNOWLEDGMENTS

We are grateful to Steffi Seeger (Max-Planck-Institut für Molekulare Pflan-zenphysiologie) for help with tissue culture and plant transformation.

Received March 2, 2012; accepted April 17, 2012; published April 19, 2012.

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