Evolutionary Changes in Chlorophyllide a Oxygenase(CAO) Structure Contribute to the Acquisition of a NewLight-harvesting Complex in Micromonas*�
Received for publication, February 19, 2013, and in revised form, May 13, 2013 Published, JBC Papers in Press, May 15, 2013, DOI 10.1074/jbc.M113.462663
Motoshi Kunugi‡, Atsushi Takabayashi‡§1, and Ayumi Tanaka‡§
From the ‡Institute of Low Temperature Science, Hokkaido University, N19 W8 Kita-Ku and the §Japan Core Research forEvolutionary Science and Technology, Sapporo 060-0819, Japan
Background: CAO domain structure has changed during evolution.Results:Micromonas CAO is split into two proteins, both of which are required for chlorophyll b synthesis.Conclusion: Establishment of a new light-harvesting system has been accompanied by changes in the CAO structure duringevolution.Significance: This is the first report showing the relationship between chlorophyll metabolism and structures of the light-harvesting system.
Chlorophyll b is found in photosynthetic prokaryotes and pri-mary and secondary endosymbionts, although their light-har-vesting systems are quite different. Chlorophyll b is synthesizedfrom chlorophyll a by chlorophyllide a oxygenase (CAO), whichis a Rieske-mononuclear iron oxygenase. Comparison of theamino acid sequences of CAOamong photosynthetic organismselucidated changes in thedomain structures ofCAOduring evo-lution. However, the evolutionary relationship between thelight-harvesting system and the domain structure of CAOremains unclear. To elucidate this relationship, we investigatedtheCAOstructure and the pigment composition of chlorophyll-protein complexes in the prasinophyte Micromonas. TheMicromonas CAO is composed of two genes, MpCAO1 andMpCAO2, that possess Rieske and mononuclear iron-bindingmotifs, respectively.Onlywhenboth geneswere introduced intothe chlorophyll b-less Arabidopsis mutant (ch1-1) was chloro-phyll b accumulated, indicating that cooperation between thetwo subunits is required to synthesize chlorophyll b. AlthoughMicromonas has a characteristic light-harvesting system inwhich chlorophyll b is incorporated into the core antennas ofreaction centers, chlorophyll b was also incorporated into thecore antennas of reaction centers of the Arabidopsis transfor-mants that contained the twoMicromonasCAOproteins. Basedon these results, we discuss the evolutionary relationshipbetween the structures of CAO and light-harvesting systems.
Photosynthesis consists of two processes as follows: oneinvolves harvesting light energy and producing ATP andNADPH, and the second involves the fixation of CO2 to sugarusingATP andNADPH. Light energy is harvested by photosyn-
thetic pigments such as chlorophyll, carotenoid, and phycobilin(1, 2). Most oxygenic photosynthetic organisms contain chlo-rophyll a, which is involved in harvesting light energy inantenna systems and in driving electron transfer in the reactioncenters (3), although Prochlorococcus contains 8-vinyl-chloro-phyll a, and Acaryochloris contains chlorophyll d in their reac-tion centers. In addition to chlorophyll a, some photosyntheticorganisms have an additional pigment that exists in the periph-eral antenna complex and plays an important role in absorbinga broad light spectrum for photosynthesis (4). For example,cyanobacteria and red algae contain phycobilins, which form alarge complex called the phycobilisome (5). Fucoxanthin is amajor pigment in brown algae and exists in fucoxanthin-chlo-rophyll-protein complexes (6). The acquisition of these pig-ments has played an essential role in environmental adaptationby establishing new light-harvesting systems.Chlorophyll b is widely distributed in prokaryotes and pri-
mary and secondary endosymbionts (7). However, the light-harvesting systems are quite different among these organisms,although they all contain chlorophyll b as amajor light-harvest-ing pigment. In Prochlorococcus, 8-vinyl-chlorophyll b binds toprochlorophyte chlorophyll-binding protein (Pcb)2 (8) andforms the photosystem (PS)I-Pcb and PSII-Pcb supercom-plexes (9). Pcb belongs to the six-transmembrane helix family,which includes CP43 and CP47 and the iron-stress-inducedprotein A (IsiA) (10). In land plants andmost green algae, chlo-rophyll b binds to Lhc proteins containing three transmem-brane helices that associate with the core antennas of reactioncenters of PSI and PSII (11–13). In the prasinophyte orderMamiellales, the content of chlorophyll b in the light-harvest-ing system is extremely high (14) compared with land plantsand green algae; however, the distribution of chlorophyll b intheir light-harvesting systemhas not beenwell studied. In addi-tion, prasinophyte photosystems contain the prasinophyte-
* This work was supported by Grant-in-aid for Young Scientists 23770035 (toA. Takabayashi) and by Scientific Research 24370017 (to A. Tanaka) fromthe Japan Society for the Promotion of Science.
� This article was selected as a Paper of the Week.1 To whom correspondence should be addressed: Institute of Low Tempera-
specific light-harvesting chlorophyll a/b-protein complex(LHC) type designated LHCP (14), which binds chlorophyll a,chlorophyll b, and chlorophyll c-like pigments and othercarotenoids.Chlorophyll b is synthesized from chlorophyll a by chloro-
phyllide a oxygenase (CAO) (15). CAOcatalyzes two successiveoxygenation reactions, converting a methyl group at the C7position to a formyl group via a hydroxymethyl group to gener-ate chlorophyll b (16). CAO is not structurally related to bciD,which is involved in the C7-methyl oxidation (formylation) inbacteriochlorophyll e synthesis (17). CAO is a unique enzyme,and all the chlorophyll b-containing organisms, without excep-tion, have CAO. However, the structure of CAO is variableamong photosynthetic organisms, in contrast to other chloro-phyll metabolic enzymes whose structures are largely con-served (18). Eukaryotic CAOs, except for that of Mamiellales,consist of three domains as follows: the A, B, and C domains inorder from the N terminus (18). The A domain controls theaccumulation of the Arabidopsis CAO (AtCAO) protein inresponse to the presence of chlorophyll b (19, 20). The Cdomain is the catalytic domain, which contains a Rieske centerand a mononuclear iron-binding motif. The B domain mightserve as a linker between theA andC domains. The prokaryoticProchlorothrix hollandica CAO (PhCAO) has high sequencesimilarity to higher plant CAOs, but it only contains thesequence corresponding to the catalytic domain and has noregulatory domain (21). Prochlorococcus CAOs also lack a reg-ulatory domain and have low sequence homology to otherCAOs, although they contain Rieske and mononuclear iron-binding motifs (22). The low sequence similarity is partlybecause Prochlorococcus uses 8-vinyl-chlorophyll instead ofchlorophyll and therefore had to adapt to fit 8-vinyl-chloro-phyll. Mamiellales has a unique CAO that is quite differentfrom others. The complete sequence of the genomes of theMamiellales Micromonas and Ostreococcus revealed the pres-ence of two genes that have sequence similarity to CAO (23).Interestingly, these two genes conserve either the Rieske centeror mononuclear iron-binding motif, suggesting that these twogene products cooperate in the synthesis of chlorophyll b,although no experimental evidence for this had not been dem-onstrated so far.As mentioned previously, the evolution of the light-harvest-
ing systems of chlorophyll b-containing organisms has beenaccompanied by changes in the CAO structure. In this study,we examined the light-harvesting systems of Micromonas byBN-PAGE and native-green gel followed by pigment analysisand found that Micromonas has a characteristic light-harvest-ing system in which chlorophyll b is incorporated into the coreantennas of reaction centers. When both Micromonas CAOgenes were simultaneously introduced into the Arabidopsischlorophyll b-less (ch1-1) mutant, chlorophyll b was incorpo-rated into the core antennas of reaction centers, indicating thatthe light-harvesting systems of the transgenic plant shared acommon structure with that of Micromonas. Based on theseresults, we discuss the co-evolution of CAO and the light-har-vesting systems.
EXPERIMENTAL PROCEDURES
Plant Materials and Growth Conditions—Arabidopsis thali-ana (Columbia ecotype)was grown in a chamber equippedwithwhite fluorescent lamps under a 16-h photoperiod at a lightintensity of 70�mol photonsm�2 s�1 at 23 °C. TheArabidopsisch1-1 mutant (24) was used as the chlorophyll b-less plant inthis study. Micromonas pusilla (CCMP1545) was inoculatedinto 100 ml of liquid L1 medium (25) in a 300-ml Erlenmeyerflask. All culture experiments with M. pusilla were performedat 23 °C under a 16-h photoperiod of 15�mol photonsm�2 s�1.Artificial genes encoding MpCAO1 (MicpuC2 16022) andMpCAO2 (MicpuC2 60555) were designed to optimize theircodon usage for Arabidopsis (Fig. 3). To construct anMpCAO1-FLAG vector, the full coding region for MpCAO(FeS) was fused with a transit peptide ofAtCAO (1–168 bp) anda C-terminal FLAG tag (GCCTCGTCAGTGATAAAAC-GAGAAGACTACAA) and was subcloned into the pGreenIIvector. To construct an MpCAO2-HA vector, the full codingregion forMpCAO2was fused with a transit peptide of AtCAO(1–168 bp) and a C-terminal HA tag (GCCTCGTCAGT-GATAAAACGAGAAGACTACAA) and was subcloned intothe pGreenII vector. The resulting plasmids were introducedinto ch1-1 plants via Agrobacterium tumefaciens-mediatedtransformation. We named these mutant lines MpCAO1and MpCAO2, respectively. Primary transformants wereselected on agar plates containing 50 mg/liter kanamycin or 20mg/liter hygromycin, respectively. To construct a PhCAO vec-tor, the full coding region for a P. hollandica CAO gene(PhCAO) was fused with a transit peptide ofAtCAO (1–168 bp)and subcloned into the pGreenII vector (21). To construct aBCFLAG vector, the coding region for the B and C domains ofAtCAO (Pro171–Gly535) was fused with a transit peptide ofAtCAO (1–168 bp) and subcloned into the pGreenII vector. Inaddition, we generated double mutants by co-transforma-tion (MpCAO1�MpCAO2–1) and by genetic crossing(MpCAO1�MpCAO2–2). Confirmation of genotypes was per-formed by PCR. Genomic DNA was extracted as described inthe previous report (26). The primers used for amplification ofMpCAO1 andMpCAO2 are as follows:MpCAO1Fw, 5�-ATG-AGATGTGACGCTGAAGGA-3�; MpCAO1Rv, 5�-TTACTT-ATCATCGTCATCTTT-3�; MpCAO2Fw, 5�-ATGGCTCCA-GAAGTATCTTCC-3�; andMpCAO2Rv, 5�-TTATGCGTAA-TCAGGAACATC-3�. The PCR products were separated onethidium bromide-stained 2.0% agarose gels.Expression and Purification of Recombinant AtCAO—The
coding region of AtCAO lacking its transit peptide was ampli-fied by PCR (KOD FX NEO DNA polymerase; Toyobo, Japan)using the following primers: 5�-TAGGCATATGGAGCTCTT-GTTTGATGTGGAGGATCCTA-3� (the underlined region isan engineered SacI site) and 5�-CCTATCTAGACTGCAGT-TAGCCGGAGAAAGGTAGTTTA-3� (the underlined regionis an engineered PstI site) and cloned into the SacI/PstI sites ofthe pCold ProS2 vector (Takara, Japan). The recombinant pro-tein obtained from this plasmid possesses a hexahistidine tag atthe N terminus. The expression plasmid was introduced intoEscherichia coliC41 cells (Avidis SA, France). The transformedcells were grown at 37 °C in 1 liter of Luria-Bertani medium
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containing ampicillin (50mg/liter) and 0.1mM ammonium fer-ric citrate until the absorbance at 600 nm reached 1.5 (27). Theexpression of the recombinant AtCAO gene was induced with0.3 mM isopropyl �-D-thiogalactopyranoside for 18 h at 18 °C.After incubation, the culture was harvested by centrifugation,and the collected cells were resuspended in buffer (20mM imid-azole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.9)) and dis-rupted by sonication. Cells were then centrifuged at 21,600 � gfor 5 min at 4 °C to remove cell debris. The soluble fractioncontaining the recombinant protein was purified with a nickelcolumn (His-Trap HP kit; GE Healthcare), according to themanufacturer’s protocol. The eluent fractions were concen-trated to 0.05ml by a centrifugal filter device (AmiconUltra-15,30,000 MWCO; Millipore) and used for two-dimensionalBN/SDS-PAGE.Isolation of Thylakoid Membranes—Arabidopsis thylakoid
membranes (wild type, MpCAO1� MpCAO2, BCFLAG, andPhCAO) were prepared from the leaves of 5-week-old plants asdescribed previously (28). Micromonas thylakoid membraneswere prepared from 10-day-old cells. Cells were harvested bycentrifugation at 2,500 � g in growth phase and suspended in50 mM Tricine/NaOH (pH 8.0). The cells were broken withglass beads (100 �m in diameter) at 4 °C using a Mini-BeadBeater (Waken B Tech, Japan). After removal of the beads andunbroken cells, the supernatant was centrifuged at 21,600 � gfor 5 min at 4 °C. The green pellets were washed with 5 mM
EDTA-2Na three times and used as Micromonas thylakoidmembranes.Native Green Gel Electrophoresis—Thylakoid membranes
of wild-type and MpCAO1 Arabidopsis and M. pusillaCCMP1545 were solubilized in 1.0% SDS at 4 °C and centri-fuged at 21,600 � g for 5 min at 4 °C. The supernatants wereapplied onto a 8% polyacrylamide disk gel (5 mm in diameter)containing 0.1%SDS. Electrophoresiswas carried out at 4 °C for�1.5 h at 0.5 mA/tube according to the method of Anderson etal. (29), provided that the upper and lower reservoir buffer con-tained 0.1% SDS.One-dimensional SDS-PAGE—The leaf (15 mg flesh weight)
was solubilized by SDS extraction buffer (30) and separatedonto slab gels containing 14% polyacrylamide using the Laem-mli buffer system (31). For the immunoblot analysis of PsaA/PsaB (CP1), the leaves were separated on 14% polyacrylamidegels that contained 4 M urea.Two-dimensional SDS/SDS-PAGE—The total leaf proteins
were extracted from the leaf (100 mg flesh weight) of 25-day-old plants (wild type,MpCAO1�MpCAO2) using LDS samplebuffer (63 mM Tris-HCl (pH 6.8), 10% glycerol, and 0.5% LDS).After centrifugation at 18,800 � g for 5 min at 4 °C, the super-natants were loaded onto an SDS-polyacrylamide gel (14%acrylamide gel) at 4 °C for 4 h at 6mA using the Laemmli buffersystem (31). For the second dimension using SDS-PAGE, theSDS gel strips were soaked for 1 h at room temperature in asolution that contained 1% (w/v) SDS and 1% (v/v) 2-mercap-toethanol and run on a second dimension SDS-polyacrylamidegel (14% polyacrylamide gels that contained 4 M urea) using theLaemmli buffer system (31). After the second dimension SDS-PAGE, gels were stainedwith the Pierce Silver Stain kit formass
spectrometry (Thermo Fisher Scientific, Japan) according toinstructions.Two-dimensional BN/SDS-PAGE—BN-PAGE was per-
formed essentially according to themethod described by Taka-bayashi et al. (28). Briefly, the thylakoid membrane proteins(which corresponded to 5 �g of Chl) of Arabidopsis wild-type,MpCAO1�MpCAO2,BCFLAG, andPhCAOplants or the puri-fied recombinant AtCAO proteins (which corresponded to 4�g of protein) were suspended in ice-cold resuspension bufferA that contained 50 mM imidazole-HCl (pH 7.0), 20% glycerol,5 mM 6-aminocaproic acid, and 1 mM EDTA-2Na and werethen solubilized with 1% (w/v) �-dodecyl maltoside on ice for 5min. For the second dimension using SDS-PAGE, the BN gelstrips were soaked for 1 h at 37 °C in a solution that contained1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol and run on asecond dimension SDS-polyacrylamide gel (14% polyacryl-amide) using the Laemmli buffer system (31).Immunoblot Analysis—Immunoblot analysis was performed
essentially according to the method described by Takabayashiet al. (28). The transferred proteins were detected with an anti-AtCAO rabbit primary antibody (19), an anti-PsaA/PsaB (CP1)antibody (32), an anti-CP47 antibody (Agrisera, Sweden), ananti-Lhcb2 antibody (Agrisera, Sweden), an anti-FLAG anti-body (Sigma), an anti-HA antibody (Funakoshi, Japan), andPenta-His HRP conjugate kit (Qiagen, Japan).Co-immunoprecipitation—The thylakoid membrane pro-
teins (which corresponded to 5 �g of Chl) of Arabidopsis wild-type and MpCAO1�MpCAO2 plants were suspended in ice-cold resuspension buffer A and solubilized with 1% (w/v)�-dodecyl maltoside on ice for 5 min. Extracts were incubatedwith anti-DYKDDDDK beads (Wako, Japan) and anti-HAbeads (Wako, Japan) for 8 h at 4 °C. Supernatants were dis-carded, and beads were washed with buffer A three times.Bound complexeswere elutedwith SDS extraction buffer for 10min at 100 °C. After centrifugation at 21,600 � g for 5 min at4 °C, the supernatants were separated on slab gels containing14% polyacrylamide.Pigment Determination—Leaves were weighed and pulver-
ized in acetone using the ShakeMaster grinding apparatus (Bio-Medical Science, Japan), and the extracts were centrifuged at21,600� g for 5min at 4 °C. The supernatant was loaded onto aSymmetry C8 column (4.6 � 150 mm; Waters, Japan) asdescribed previously (33). The elution profiles were monitoredby measuring the absorbance at 650 nm using a diode arraydetector (SPD-M20A, Shimadzu, Japan), and the pigmentswere identified by their retention times and spectral patterns.Pigment quantification was performed using the areas of thepeaks, described previously (34). All reported Chl quantitieswere the means of three biologically independent samples.Multiple Alignment of Amino Acid Sequences—The amino
acid sequences listed below were retrieved from GenBankTM(www.ncbi.nlm.nih.gov) and Joint Genome Initiative (jgi.doe.gov). The amino acid sequences were aligned with the ClustalWprogram (35) via the BioEdit program (36).Accession Numbers—The amino acid sequences used in
this work are as follows. GenBankTM identification numbers:A. thaliana (At), AtCAO,15219408; Chlamydomonas rein-hardtii (Cr), CrCAO,159463890; P. hollandica (Ph), PhCAO,
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38488612; M. pusilla (Mp) protein IDs are from the JointGenome Initiative: MpCAO (Fe-S), 16022 and MpCAO (Fe),60555.
RESULTS
Diversity of CAODomain Structure inChlorophyll b-contain-ing Organisms—CAOs of vascular plants andmost of the greenalgae have the same domain structure, which consists of theregulatory domain (A domain), catalytic domain (C domain),and the linker (B domain) (Fig. 1C). The amino acid sequence ofthe B domain is quite different, likely because this domain has
no function besides linking theA andCdomains (18). Sequencevariations are observed in the A domain between vascularplants and the green algae Chlamydomonas (Fig. 2), likely dueto different regulatory mechanisms of chlorophyll b synthesis.Prochlorophyte and Mamiellales CAOs have no sequence cor-responding to the regulatory and linker domains (Fig. 1C). Theamino acid sequences of the catalytic domains of CAOs arehighly conserved except for the Prochlorococcus (22) andMamiellales CAOs (Fig. 2). InMicromonas, CAO is composedof two subunits, one (MpCAO1) that contains only a Rieskecenter motif and another (MpCAO2) that contains only amononuclear iron-binding motif (Fig. 1, A and B).Cooperation betweenMpCAO1 andMpCAO2 in Chlorophyll
b Synthesis—CAO, which contains both Rieske and mononu-clear iron-binding motifs, is not found in the Micromonas orOstreococcus genomes, and MpCAO1 and MpCAO2 are theonly genes that have sequence similarity toCAO in these organ-isms. This suggests that MpCAO1 andMpCAO2 are responsi-ble for chlorophyll b synthesis in these organisms, although thestructure of these proteins is largely different fromotherCAOs.Introduction of a gene to chlorophyll b-less organisms ormutants is a convenientmethod to determinewhether the genein question is responsible for chlorophyll b synthesis or not (21,37). However, the GC content of theMicromonas genes is high(25), and the gene is not thought to be efficiently translated inArabidopsis. Therefore, the codons ofMpCAO1 andMpCAO2were optimized for expression inArabidopsis (Fig. 3) and fusedwith FLAG and HA tags, respectively.The synthetic DNAs encoding these proteins were expressed
under the control of the cauliflower mosaic virus 35S promoterin the Arabidopsis ch1-1 mutant, which lacks chlorophyll b.PCR experiments showed that the MpCAO1 and MpCAO2genes were successfully introduced into the Arabidopsis ch1-1mutant (Fig. 4B). Fig. 4A shows the plants 25 days after germi-nation. The ch1-1mutant plants were small compared with theWT and exhibited a pale green color due to the lack of chloro-phyll b. The transgenic plants with either MpCAO1 orMpCAO2 exhibited the same phenotype as the ch1-1 mutant.When both MpCAO1 and MpCAO2 genes were introducedinto the ch1-1 mutant, the size and the color of the transgenicplants became similar to that of theWT.Next, we examined theaccumulation of the transgene products by immunoblottingusing antibodies against the FLAG (MpCAO1) or HA(MpCAO2) tags (Fig. 4C). As shown in Fig. 4, theMpCAO1 andMpCAO2 proteins accumulated in the transgenic plants har-boring MpCAO1 or MpCAO2 genes, respectively. NeitherMpCAO1 norMpCAO2was detected in theWT and the ch1-1mutant plants, and two lines of MpCAO1�MpCAO2 plantsaccumulated both proteins.Next, we determined the chlorophyll content of the trans-
genic plants byHPLC (Table 1). The chlorophyll a/b ratio of theWT was 3.03, whereas chlorophyll b was not detected in thech1-1 mutant as reported previously. The chlorophyll contentof ch1-1was lower thanWTdue to the deficiency in LHCII (28),which is amajor chlorophyll-binding complex. The chlorophyllcontent of the transgenic plants that have either MpCAO1 orMpCAO2 was similar to that of the ch1-1 mutant, indicatingthat neitherMpCAO1norMpCAO2alone can synthesize chlo-
FIGURE 1. Comparison of the domain structure of the CAO protein amongphotosynthetic organisms. A, comparison of amino acid sequences of theRieske center in CAO proteins. B, comparison of amino acid sequences of themononuclear iron-binding motif in CAO proteins. In both panels, identicalresidues are shown in white type on a black background. C, domain structure ofCAO proteins. Rieske center (Fe-S) and mononuclear iron (Fe)-binding motifare in the C domain. AtCAO, A. thaliana CAO; CrCAO, C. reinhardtii CAO;MpCAO1, M. pusilla CAO (Fe-S); MpCAO2, M. pusilla CAO (Fe); PhCAO, P. hol-landica CAO. D, schematic drawing of core/peripheral antenna complexes ofArabidopsis, Chlamydomonas, Micromonas, and Prochlorothrix.
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rophyll b. In contrast, a large amount of chlorophyll b accumu-latedwhenMpCAO1 andMpCAO2were co-introduced. Theseresults clearly indicate that cooperation of MpCAO1 andMpCAO2 is indispensable for the synthesis of chlorophyll b. Itshould be noted that the chlorophyll a/b ratio of theMpCAO1�MpCAO2plantswas significantly lower than that oftheWT. Itwas reported that inArabidopsis, the chlorophylla/bratio does not significantly change even when full-lengthAtCAO is overexpressed because the A domain strictly regu-lates chlorophyll b synthesis by a negative feedbackmechanism.Therefore, the expression of Micromonas MpCAO1 andMpCAO2 might not be properly regulated in Arabidopsis.Subunit Structures of CAO—As part of the catalytic activity
of CAO, an electronmust be transferred to amononuclear ironfrom aRieske center. For efficient electron transfer, it is reason-able to assume that the two subunits, MpCAO1 andMpCAO2,form a complex. To examine this possibility, we carried out aco-immunoprecipitation experiment using anti-FLAG oranti-HA antibodies. MpCAO1-FLAG and MpCAO2-HA wereco-expressed in the ch1-1 mutant, and the thylakoid mem-
branes of the transgenic plants were isolated. Solubilized thyla-koid membranes were subjected to co-immunoprecipitationwith anti-FLAG antibodies. Co-precipitated proteins wereeluted and subjected to immunoblot analysis. As shown in Fig.5A, MpCAO2-HA co-precipitated with the anti-FLAG anti-body, indicating that MpCAO2 interacted withMpCAO1. Thesame result was obtained when MpCAO2 was precipitated bythe anti-HA antibody (Fig. 5B). These experiments showed thatMpCAO1 and MpCAO2 form a complex.Next, we examined whether MpCAO1 and MpCAO2
form a heterodimer by BN-PAGE followed by immunoblotanalysis. Thylakoid membranes were isolated fromMpCAO1�MpCAO2 plants and subjected to BN-PAGE fol-lowed by immunoblot analysis using anti FLAG or anti-HA tagantibodies. Both MpCAO1 andMpCAO2 bands were found at�40 kDa, corresponding to monomeric MpCAO1 andMpCAO2 (Fig. 6A). In addition to these monomeric bands, an80-kDa band was observed with both anti-FLAG and -HA anti-bodies (Fig. 6A), suggesting that an MpCAO1 and MpCAO2heterodimer was formed. Interestingly, higher molecular
FIGURE 3. Sequences of synthetic genes of MpCAO1 and MpCAO2.
FIGURE 2. Multiple amino acid sequence alignment of CAO proteins. Identical residues are shown in white type on a black background. The AtCAO sequencescorresponding to the A domain, B domain, and C domain are shown. Asterisks and closed squares show binding sites of Rieske center and mononucleariron-binding motif, respectively.
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weight bands than the heterodimer were found, and the immu-noblot profile with anti-FLAG tag antibodies was almost thesame as that with anti-HA tag (Fig. 6A).We then examined the oligomeric state of Arabidopsis CAO
in chloroplasts. Because full-length AtCAO (ABC) is under thedetectable level of immunoblotting as we reported previously(19), we expressed the AtCAO protein without the A domainfused with a FLAG tag (BCFLAG) in the Arabidopsis ch1-1mutant. The thylakoid membranes were solubilized and ana-lyzed by two-dimensional BN/SDS-PAGE followed by immu-noblotting. Almost the same profiles were observed with theanti-CAO and -FLAG antibodies (Fig. 6B). The smallest bandwas �50 kDa, which likely represents monomeric BCFLAGprotein (42 kDa) (Fig. 6B). In addition to the monomeric form,higher molecular weight bands were observed on the gel, esti-mated as �160, 240–300, 500, and 750 kDa (Fig. 6B). CAO has
sequence homology to dicamba monooxygenase and 2-oxo-quinoline 8-monooxygenase (Fig. 7), which contain a Rieske-mononuclear iron oxygenase motif also present in CAO. Thecrystal structure of the oxygenase component of dicambamonooxygenaseand2-oxoquinoline8-monooxygenase revealedahomotrimer and showeda ring-shaped, symmetric arrangement(39). Considering this report, it is reasonable to assume that the160-kDa band corresponds to a trimeric form of AtCAO(BCFLAG). If this assumption is true, then the 240–300-, 500-,and 750-kDa bands probably correspond to a 2 � 3-mer, 3 �3-mer, and 5 � 3-mer, respectively.The experimental results shown in Fig. 6B do not necessarily
exclude the possibility that full-length CAO does not form thesame oligomeric structures with the A domain-deleted AtCAOor that AtCAO assembled with other proteins to form a largecomplex. To examine this possibility, we expressed full-length AtCAO fused with FLAG (AtCAO-FLAG) in E. coli.The soluble fraction of the E. coli lysate was subjected toBN-PAGE followed by immunoblotting (Fig. 6C). A largecomplex and trimeric forms were observed as in the experi-ments with transgenic plants (Fig. 6B). These results clearlyshowthat full-lengthCAOiscapableof formingahomotrimerandlarger complexes.P. hollandica is a cyanobacterium that contains chlorophyll
b. Prochlorothrix CAO (PhCAO) has no regulatory or linkerdomains and contains only the catalytic domain. To examinewhether PhCAO forms trimeric and multimeric complexes asAtCAO does, we expressed PhCAO in the Arabidopsis ch1-1mutant and examined the oligomeric state of PhCAO (Fig. 6D).A large amount of the PhCAO protein was detected between170 and 1,200 kDa in addition to the trimeric and monomericPhCAO (Fig. 6D).Pigment Composition of Chlorophyll Protein Complexes of
Micromonas and the Arabidopsis Transformants HarboringMicromonas CAO—In green plants, it is reported that chloro-phyll b exists in peripheral antennas such as LHCI and LHCII.However, when the A domain deleted-CAO (B and C domainsonly) is expressed inArabidopsis, chlorophyll bwas distributednot only in the peripheral antenna but also in the inner antennaof both photosystems (40), indicating the close relationshipbetween the CAO structure and the distribution of chlorophyllb among various light-harvesting complexes. To examinewhether a different structure of the light-harvesting system is
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FIGURE 4. Generation of MpCAO overexpression of Arabidopsis lines. A,phenotypes of wild-type and transgenic lines. 1, wild type; 2, MpCAO1�MpCAO2–1; 3, MpCAO1�MpCAO2–2; 4, ch1-1; 5, MpCAO1; and 6, MpCAO2. B,confirmation of transgenic lines by PCR. C, confirmation of transgenic lines byimmunoblot analysis. The MpCAO1-FLAG and MpCAO2-HA proteins weredetected using anti-FLAG or anti-HA antibodies, respectively. Lane 1, wildtype; lane 2, ch1-1; lane 3, MpCAO1; lane 4, MpCAO2; lane 5, MpCAO1�MpCAO2–1; lane 6, MpCAO1�MpCAO2–2.
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FIGURE 5. Co-immunoprecipitation of MpCAO1-FLAG and MpCAO2-HAproteins. The solubilized thylakoid membrane proteins from wild type orMpCAO1�MpCAO2 plants were co-immunoprecipitated with the anti-FLAGantibody (FLAG) or anti-HA antibody (HA) and subjected to SDS-PAGE. Detec-tion of MpCAO1-FLAG and MpCAO2-HA proteins was performed by immu-noblot analysis with anti-FLAG antibody (A) or anti-HA antibody (B), respec-tively. Lane 1, wild type; lane 2, MpCAO1�MpCAO2.
TABLE 1Chlorophyll content of wild-type and transgenic Arabidopsis plantsThe value given is the mean � S.D. of three experiments. ND stands for not detected;FW is flesh weight.
constructed in Micromonas due to the characteristics of itsCAO, Micromonas chlorophyll-protein complexes were sepa-rated by native-green gel (Fig. 8A). Six green bands wereresolved. Bands 1 and 4 are the core complexes of photosystemI and II, respectively. Bands 3 and 5 are the trimeric and mono-meric LHCII, respectively. The level of monomeric LHCII wasvery low, indicating that the LHCII trimer is more stable com-pared with that of other green plants. The separation profile ofthese bands was very close to that of Arabidopsis and othergreen plants except for the LHCII band between the PSI coreantennas and LHC trimer. Band 6 is a free pigment band. Next,pigments were extracted from these bands and analyzed byHPLC. The chlorophyll a/b ratios of the trimeric and mono-meric LHCII bands were 0.98 and 1.38 (Table 2), respectively,which is consistent with that of higher plants. 8-Vinyl-proto-chlorophyllide and prasinoxanthin are the characteristic pig-ments of Micromonas. These pigments were found only inLHCII and not in the core antennas of reaction centers. In con-trast, �-carotene is predominantly found in the core antennasof both photosystems. The chlorophyll a/b ratios of the coreantennas of PSI and PSII were 3.22 and 5.73, respectively, whichare low values compared with those of other photosyntheticorganisms, indicating that chlorophyll b is significantly incor-porated into core antennas of reaction centers inMicromonas.One of the reasons for the distribution of chlorophyll b in thecore antennas of reaction centers might be the different struc-ture of CAO. To examine this possibility, we introducedMicromonas MpCAO1 and MpCAO2 into the Arabidopsisch1-1 mutant and analyzed the pigment composition of thegreen bands (Fig. 8B). The chlorophyll a/b ratios of the coreantennas of PSI and PSII of wild type Arabidopsis plantswere 21.35 and 13.09 (Table 3), respectively, indicating thatthese core antennas of reaction centers consist primarily ofchlorophyll a, and the level of chlorophyll b is very low. In con-trast, the ratios of the transgenic plants with MpCAO1 andMpCAO2 were 3.43 and 3.50, respectively. This indicates thatchlorophyll b is incorporated into core antennas of reactioncenters when AtCAO is substituted with Micromonas CAO,suggesting a strong evolutionary relationship between theCAOstructure and light-harvesting systems.Although the green band of CP1 (heterodimer of PsaA/PsaB)
was reported to contain only the core antennas of PSI (41),co-migration of LHC on this region cannot be completelyexcluded because the CP1-LHCI band was close to CP1 on thegel (Fig. 8B). To purify the core antennas of PSI without LHC,thylakoid membranes were solubilized in 0.5% SDS and sepa-rated by Tris/glycine SDS-PAGE for 4 h. The CP1 band wasclearly separated from trimeric LHC with this electrophoresis,and the CP1-LHCI bands disappeared (Fig. 8, C and D). Sec-ond-dimensional SDS-PAGE visualized with silver stainingclearly shows that CP1 bands did not contain LHC in both wildtype and MpCAO1�MpCAO2 plants, indicating that PsaA/PsaB was purified by this method. Chlorophyll a/b ratio of WTCP1 band was 19.30, which is consistent with the previousreport (37). Chlorophyll a/b ratio of the CP1 band of theMpCAO1�MpCAO2 plant was 4.46, indicating that chloro-phyll b was incorporated into the core antennas of PSI. Theincorporation of chlorophyll b into the core antennas of reac-
FIGURE 6. Analysis of the oligomeric states of the CAO proteins. A, two-dimensional BN/SDS-PAGE followed by immunoblotting indicated thatMpCAO1 and MpCAO2 proteins accumulated in monomeric, heterodimeric(86 kDa), and higher molecular weight forms in MpCAO1�MpCAO2 plant. B,two-dimensional BN/SDS-PAGE followed by immunoblotting indicated thatBCFLAG proteins accumulated in monomeric (43 kDa), trimeric (126 kDa), andhigher molecular weight forms in the BCFLAG overexpression plants. TheBCFLAG proteins were detected using an anti-FLAG antibody or anti-CAOantibody. C, two-dimensional BN/SDS-PAGE followed by immunoblottingindicated that recombinant AtCAO protein formed higher molecular weightprotein complexes in E. coli. The AtCAO proteins were detected using an anti-His antibody. D, two-dimensional BN/SDS-PAGE followed by immunoblottingindicated that PhCAO formed monomeric (41 kDa), trimeric (123 kDa, andhigher molecular weight complexes. The PhCAO proteins were detectedusing an anti-CAO antibody.
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tion centers was also observed in the BC-Arabidopsis plants inwhich A domain of CAO is deleted (40). It should be noted thatLHCII amounts were slightly high and PsaA/B amounts wereslightly low in the MpCAO1�MpCAO2 plant compared withwild type (Fig. 8E). This is consistent with the idea that the levelof LHCII is regulated by the amount of chlorophyll b.
DISCUSSION
Oligomeric Structure of CAO—Dicambamonooxygenase and2-oxoquinoline monooxygenase are Rieske-mononuclear ironoxygenases (39). Their crystal structures show a symmetrictrimer and ring-like structure. In the trimeric form, an electronis transferred from theRieske center to themononuclear iron inthe neighboring subunit, where the catalytic reaction takesplace. It is reasonable to speculate that CAO has the same olig-omeric structure and electron transfer chain because CAO is aRieske-mononuclear iron oxygenase and has sequence similar-ity to dicamba monooxygenase and 2-oxoquinoline monooxy-genase. BN-PAGE also indicated that bothAtCAOandPhCAOform a trimer and larger complexes (Figs. 6 and 7). Interest-ingly, CAO in Arabidopsis formed complexes larger than thetrimer. Recombinant CAO protein expressed in E. coli alsoshowed the same electrophoretic profile on the BN-gel, indicat-ing that AtCAO and PhCAO proteins form trimers and multi-mers. It should be noted that neither AtCAO nor PhCAO asso-ciated with LHCII or PSII to form large complexes becausethese complexes did not co-migrate with the CAO protein onthe BN gels, suggesting that the trimeric CAO, a unit structureof CAO, assembles into a large complex by itself. Although it isstill unclear whether phytylation or formylation is the last step
of chlorophyll b biosynthesis (44), CAO is one of the enzymesthat catalyze the last part of chlorophyll synthesis. This suggeststhe involvement of CAO in the formation of LHC.One hypoth-esis is that a large complex contributes to supplying sufficientchlorophyll b for the formation of LHC, which requires �7chlorophyll b molecules. The other hypothesis is that a largecomplex is an inactive form.At present, the biochemical impor-tance of this large complex is not clear. Further study is neces-sary to clarify the importance of the large complex.Subunit Structure of Micromonas CAO—Micromonas CAO
is composed of two proteins, which contain either Rieske-(MpCAO1) or mononuclear iron (MpCAO2)-binding motifs.Neither of these proteins can synthesize chlorophyll b by itself.Onlywhen the twoproteinswere simultaneously expressedwaschlorophyll b accumulated. Co-immunoprecipitation and BN-PAGE followed by immunoblotting showed that the MpCAO1andMpCAO2 proteins form a heterodimer. As far as we know,theRieske-mononuclear iron oxygenase forms a homotrimer ofc3 symmetry (45–47). The Micromonas CAO may be the firstexample of a Rieske-mononuclear iron oxygenase that forms aheterodimer. Based on the present results and previous reportof the Rieske-mononuclear iron oxygenase (39), the electrontransfer route between the CAO subunits is proposed in Fig. 9.In the trimeric formofCAO,Rieske is reduced from ferredoxin,and then the electron is transferred to a mononuclear iron inthe neighboring subunit, converting chlorophyll a into chloro-phyll b. In theMicromonasCAO, ferredoxin reduces the Rieskecenter in MpCAO1, which transfers electron to the mononu-clear iron in MpCAO2 where chlorophyll b is synthesized.
FIGURE 7. Multiple sequence alignment of AtCAO, dicamba monooxygenases (ddmC), and 2-oxoquinoline 8-monooxygenase (oxoO) proteins. Iden-tical residues are shown in white type on a black background. Red asterisks and red circles show binding sites of Rieske center and mononuclear iron-bindingmotif, respectively.
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Evolution of CAOs and Light-harvesting Systems—It has beenreported that chlorophyll metabolism is closely related to theformation and degradation of chlorophyll-protein complexes.Direct interaction between light-harvesting complexes and achlorophyll metabolic enzyme was reported for chlorophyll breductase, which converts chlorophyll b to 7-hydroxymethyl
chlorophylla in the chlorophyll cycle (48). Chlorophyll b reduc-tase directly catalyzes the reduction of chlorophyll b in theLHC, which is the first step of LHCII degradation (27). For thisreason, LHC is never degraded in the cbr mutant (38). WhenCAO was overexpressed in Arabidopsis, the LHC levelincreased (42). Interestingly, when the A domain-deleted CAOwas introduced into the ch1-1 mutant, an unusual light-har-vesting system was constructed, and chlorophyll b was incor-porated not only into the LHCbut also into the core antennas ofboth photosystems (21, 43). These results lead us to speculatethat the evolution of CAO is closely related to that of the light-harvesting systems.
MpCAO1+MpCAO2
WT
CP1*CP1
LHCII3
CPa
LHCII1
FC
A
123
Micromonas
45
6
B
MpCAO1+MpCAO2WT
PsaA/B
CP47
Lhcb2
E
CP1 LHCII3 LHCII1 CP1 LHCII3 LHCII1
LHCIILHCII
C D
FIGURE 8. Separation of chlorophyll-protein complexes. Thylakoid mem-branes of M. pusilla (A), A. thaliana (WT) (B, C, and E), and MpCAO1�MpCAO2plants (B, D, and E) were prepared. Chlorophyll-protein complexes of thoseplants were separated by native green gel electrophoresis (A and B). Theidentity of the chlorophyll-protein complexes corresponding to the bandsare presented on the left side of the gel. CP1*, CP1 (PsaA/B)-LHCI complexes;CPa, core complexes of PSII (CP43/CP47); LHCII1 and LHCII3, monomeric andtrimeric LHCII, respectively; FC, free Chl. In addition, chlorophyll-protein com-plexes of WT and MpCAO1�MpCAO2 plants were also analyzed by two-di-mensional SDS/SDS-PAGE, followed by silver staining (C and D). The amountsof PsaA/B, CP47, and Lhcb2 proteins were estimated by SDS-PAGE, followedby immunoblot analysis with their specific antibodies (E).
OryzaArabidopsis
Micromonas
Chlamydomonas
Prochlorothrix
Prochlorococcus
A B
FeS
Fe
FeS Fe
Trimeric structures of CAO(AtCAO, PhCAO)
Dimeric structures of CAO(MpCAO)
FIGURE 9. Evolution of CAO structure. A, prochlorophyte CAOs have onlythe C domain (catalytic domain) and form a trimer. CAOs in green algae andland plants have A, B, and C domains and form a trimer. Mamiellales Micromo-nas CAOs are composed of two proteins with either a Rieske (Fe-S)- or mono-nuclear iron (Fe)-binding motifs. Neither protein has an A or B domain. Theyinteract with each other to form a dimer. B, in each case, electrons must betransferred to a mononuclear iron (Fe) from a Rieske center (Fe-S) across theprotein-protein interface.
TABLE 2Chlorophyll a/b ratios and other pigments of chlorophyll-protein com-plexes in MicromonasChlorophyll-protein complexes were separated by native green gel electrophoresisand were extracted from the gel. Their chlorophyll a/b ratios and other pigmentswere determined by HPLC analysis. The band numbers correspond to Fig. 8A. Chl,Chlorophyll; �-car, �-carotene; DVP*, 8-vinyl-protochlorophyllide; Pra*, prasinox-anthin; ND, not detected.
Band no. Chl a/b �-car*/Chl a DVP*/Chl a Pra*/Chl a
TABLE 3Chlorophyll a/b ratios of chlorophyll-protein complexes in WT andMpCAO1�MpCAO2 plantsChlorophyll-protein complexes were separated by native green gel electrophoresisand were extracted from the gel. CP1 is a heterodimer of PsaA/PsaB. Their chloro-phyll a/b ratios were determined by HPLC analysis. Chl, chlorophyll.
The evolution of CAOs and light-harvesting systems is sum-marized in Fig. 9. Prochlorophyte CAOs consist of only cata-lytic domains, and they have no sequence corresponding to theA and B domains. In these organisms, chlorophyll b does notexist in the LHCbut is found in Pcb (10), which has no sequencesimilarity to LHC but is similar to core antennas (CP43/CP47)of photosystem II. The Prochlorothrix CAO has high sequencesimilarity to the C domain of higher plants, but ProchlorococcusCAO has less similarity (22). This may be at least partly due tothe different chlorophyll species used by each enzyme. Prochlo-rococcus use 8-vinyl-chlorophyll, whereas other oxygenicorganisms use chlorophyll. In eukaryotes, photosyntheticorganisms acquired an LHC with a three-membrane helix, andmost chlorophyll b is incorporated into the LHC. At this stageof evolution, CAO acquired the A domain, which might beimportant for preferential incorporation of chlorophyll b intothe LHC as well as the regulation of chlorophyll synthesis. Thishypothesis is supported by the finding that when full-lengthCAO (with A, B, and C domains) was substituted by the Adomain-deleted CAO, chlorophyll b accumulates in excess andis incorporated into the core antennas of reaction centers (21).The A domain is conserved in land plants and green algae inwhich LHCII is a major light-harvesting complex, and chloro-phyll b does not exist in the core antennas of reaction centers.However, in prasinophytes, the A domain was lost, and CAOseparated intoMpCAO1 andMpCAO2 over the course of evo-lution. Acquisition of the two separate genes encoding CAOmight not be an evolutionarily difficult process because an elec-tron is transferred from Rieske and mononuclear iron betweentwo neighboring CAO, which was already the case in the trim-eric CAO.Interestingly, the light-harvesting system of Micromonas
also drastically changed, although chlorophyll b is amajor light-harvesting pigment as in other green algae and land plants. Thechlorophyll a/b ratio ofMicromonas is low, and chlorophyll b isincorporated into core antennas of reaction centers. This char-acteristic organization of the light-harvesting system ofMicromonas might be at least partly related to the changes inthe CAO structure. This hypothesis is supported by experi-ments showing that when the Micromonas CAO was intro-duced into Arabidopsis, the chlorophyll a/b ratio became low,and chlorophyll b was incorporated into the core antennas ofreaction centers, which is similar to light-harvesting systems ofMicromonas.
Acknowledgments—We thank Dr. Hisashi Ito (Hokkaido University)for technical assistance, and we also thank Dr. Ryouichi Tanaka(Hokkaido University) for helpful discussions.
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