Supplemental Data. Yamamoto et al. (2011). Plant Cell /tpc NADH. e - NAD + Nqo5. Nqo4 Nqo6. Nqo11. Nqo10 Nqo7.

Transkript

1 A NADH Cytoplasm e - Nqo FMN Nqo2 Nqo3 Nqo5 Nqo9 Nqo5 Nqo4 Nqo6 NAD + Membrane Nqo8 Nqo Nqo Nqo7 Nqo4 Nqo3 Nqo2 Periplasm B e - Stroma 3-kD complex? Thylakoid Lumen Fd CRR3 CRRJ CRRL M,N,O H,I,J,K A,B,C,D L E,F,G Membrane subcomplex Subcomplex A Subcomplex B Lumen subcomplex PPL2,PsbQ-F,F2, Tlp2,FKBP6-2 NDF,2,4 NDH8,NDF6 Lhca5,6 LHCI Fd P7* PSI P7 LHCI Supplemental Figure. Schematic Models of Bacterial Complex I and Chloroplast NDH-PSI (A) Structural model of complex I from Thermus thermophilus based on Efremov et al. (2). Subunits involved in NADH oxidation are shown in red. (B) Structural model of chloroplast NDH-PSI based on Peng et al. (29). The localizations of CRR3, CRRJ and CRRL are based on the present study. Electrons derived from PSI are transferred to NDH via Fd. Homologous subunits between two complexes are surrounded by dashed boxes.

2 A 2 D2 ETR (relative) crr3- crr3-2 ETR (relative) crrj- crrl- B Light intensity (μmol photons m -2 s - ) E Light intensity (μmol photons m -2 s - ) NPQ.5.5 crr3- crr3-2 NPQ.5.5 crrj- crrl- C Light intensity (μmol photons m -2 s - ) F Light intensity (μmol photons m -2 s - ).8.8 -ql crr3- crr3-2 -ql crrj- crrl- G Light intensity (μmol photons m -2 s - ) Plants F v/f m Light intensity (μmol photons m -2 s - ).788 ±.8 crr ±.3 crr ±.7 crrj-.79 ±.5 crrl-.789 ±.3 Supplemental Figure 2. Chlorophyll Fluorescence Parameters of, crr3-, crr3-2, crrj- and crrl- Leaves. (A and D) Measurement of relative electron transport rate (ETR). ETR is presented as values relative to the maximum ETR in (%). (B and E) Measurement of non-photochemical quenching (NPQ). (C and F) Measurement of a fraction of closed center of PSII (-ql). Leaves of, crr3- and crr3-2 were used in A-C, whereas those of, crrj- and crrl- were used in D-F. -ql was calculated as described (Miyake et al., 29). (G) Maximum quantum yield of photosystem II (F v /F m ). Values are means ± SD (n=4). 2

3 I II 3+ c rr 3- CRR3 + CR R3 Δ c rr 3c rr 23/ 4-3 c rr c rr ndh l E5 7-L 68 PSII supercomplexes PSI + PSII dimer PSII monomer LHCII trimer LHCII monomer Supplemental Figure 3. Analysis of thylakoid protein complexes in crr3-. Chloroplasts were isolated from various genotypes as indicated across the top. Thylakoid protein extract corresponding to µg chlorophyll was loaded onto each lane. The positions of band I and II and other protein complexes are indicated based on Peng et al. (29). 3

4 At CRR3 Vv CRR3 Pt CRR3_ Pt CRR3_2 Os CRR3 Sb CRR3 Zm CRR3 Plastid targeting signal Repeat : ---MATSSITIPTIRTPIHRSKFLGQTHQFSTVNRSVFPPPKQQSKLYQVKAMGKFNLWEVMGGRGLCNGEKGIEKELQRNIED : 8 : ---MAYSFTVPSLQRPLPHKSHFLGQGHFPNNIQKASLSRTRTP---LPVKASAKFDLFGIMGGRGLCNGEEGLQQELKRNIEP : 78 : MASSITLQSTLLRSSFLGQNNFPNHPHKPYSLIPKEHR--LKIKTCAKFDPFEILGGRGLCNGEKGVQQELQRNIEE : 75 : MASSITLQSTLLRSNFLGQNNCFNHPHKPYSLIPKDHR--LKLKTCAKLDLFEILGGRGLCNGEKGVQQELKRNIEE : 75 : MAPTTPASFLRPPPLPHHHHPRIVRLPPPSATFRVADLLGGRGLCNGEVGIRKELASDSPA : 6 : MAPPPTSSSFLRPPPLPHHPHPRLHFRPP-SASFRVSEILGGRGLCNGEVGVRKELTSGS-- : 59 : MAPPPTSPSFLRPPPLPHHPHPRLLLRPP-SASFRVAEILGGRGLCNGEVGVRKELASG--- : 58 : MAAMTGICRAPLSLALQRESSFWGSNVGVHQQQKSASSNSGGSTGGVRVGIRAEGFDFWQVLGGRGLKGGEDGLKQEKTARVLQ : 84 Pp CRR3 T.BP- : : - N.72 : : - S.683 : : - Repeat 2 At CRR3 : EQETSKAENNETERESDDSNLSFKVPEDGFEKEMMGLTGGFPGGEKGLKTFIEKNPPPPPP---PPPAKQGSDASAVATDKKPK : 62 Vv CRR3 : ----APSPDSVKDEEKPALAAVDDVPEDGFDKELLGLTGGFPGGEKGLKQFLEKNPPPE KTSGNIIENARLRKPK : 49 Pt CRR3_ : E-----APPAAGEEEYSGNLEISSVPEDGFEKELMGLTGGFPGGEKGLEKFIEENPPPK KQPAAKLTITNKPK : 43 Pt CRR3_2 : Q-----ASSTAGREENSGSLEKSSVPDDAFEKELMGLTGGFPGGEKGLKRFIEENPSPK KQSVPKLTITSRPK : 43 Os CRR3 : APPSTTTSSDEPAESPPPPPAASGVDPDAFDKEMMGLTGGFPGGEVGLKDFVAKNPPPPKP---AHRKG----LAAAATVERPR : 38 Sb CRR3 : -SASTTTSSPAPSPSPSTESPPPAVDLDAFDKEMMGLTGGFPGGEVGLKDFVAKNPPPPRS---KKSNSQLVAPQATTLSAPPR : 39 Zm CRR3 : ----STTSSPAPSPPPSTDSPPPAVDPDAFEKEMMGLTGGFPGGEVGLKDFVAKNPPPPKK---SESQP------QATLSAPPR : 29 Pp CRR3 : EAKKNLVVEKKKKGSVEGNVEAAEGLPGTFNKELGGGGFPGGEKGLRQFVQSNPPPAKASQMSNEIRKLQDSISRPLKPRAP : 68 T.BP- : MIRPIADTYPLLPLSKAQMGQRQEIINSHKRLWDKTMA : 38 N.72 : : - S.683 : : - SH3-domain like * * * * * * * At CRR3 : APKLPLLMPGMIAIVKNQNSPYHMYCGIVQRITDGKAGVLFEGGNWDRLITFRLEELERREKGPPGKNPKSCILEPLIEQMQKE : 246 Vv CRR3 : PPELPLLMPGMIAIVKNPNNPFYMYCGIVQRITDGKAGVLFEGGNWDRLITFRLEELQRRDKGPPMKNPKSAILETLLEQEA-- : 23 Pt CRR3_ : PPELPLLLPGMIAIVKNPNNPFYMYTGIVQRITDGKAGVIFEGGNWDRLVTFRLEELERREKGPPGKNPRSAIIEEFYGTES-- : 225 Pt CRR3_2 : PPELPLLLPGMIAIVKNPNNPFYMYTGIVQRITDGKAGVIFEGGNWDKLVTFRLEELERREKGPPGKNPRSAIIEAFYEAESQS : 227 Os CRR3 : APELPLFLPGMVVLVKNPNNAYHMYCGIVQRVTDGKVGVLFEGGIWDRLITFDLDELEGREKGPPMVNPKSVLLESLAAEMEDD : 222 Sb CRR3 : TPELPLFLPGMVVLVKNPNNAYYMYCGIVQRVTDGKVAVLFEGGIWDRLITFNLDELEGREKGPPMVNPKSVVLEDIVAQLEDD : 223 Zm CRR3 : PPELPLFLPGMVVLVKNPNNAYHMYCGIVQRVSDGKVAVLFEGGVWDRLITFNLDELEGREKGPPMANPKSVVLEDLVAELEDD : 23 Pp CRR3 : S--PPLLMPGMTVKVISPSNPYFEFIGIVQRVTDGKVGVIFEGGNWDKLVSFKLQDLERTSQGPPMSNPKSAILERMIVPEGTS : 25 T.BP- : TDLIMTILPGMTVKVTNPNDTYYQFQGIVQRITDGKVAVLFEGGNWDKLVTFQASELEPVVVTPKEKAKAKK : N.72 : -----MILPGATVRVKNPNDTYYRYEGLVQRVTDGKVAVIFEGGNWDKIITFRLSELELVEITAGKKKGK : 65 S.683 : -----MIFPGATVRVTNVDDTYYRFEGLVQRVSDGKAAVLFENGNWDKLVTFRLSELEAVKPI : 58 At CRR3 : EAAP : 25 Vv CRR3 : : - Pt CRR3_ : : - Pt CRR3_2 : SS : 229 Os CRR3 : VAKEEEGEEAKKKKEEEGTAAAA : 245 Sb CRR3 : DDDKED-EAAKKEKEPEGAAAAA : 245 Zm CRR3 : DDDKED-EAAKKKEPEGAAAAAT : 235 Pp CRR3 : SPEGASS : 257 T.BP- : : - N.72 : : - S.683 : : - Supplemental Figure 4. A Multiple Alignment of Amino Acid Sequences of CRR3 Homologs from Plants and Cyanobacteria. The sequences of At CRR3 (NP_942., AT4G2389, A. thaliana), Vv CRR3 (XP_ , Vitis vinifera), Pt CRR3_ (XP_ , Populus trichocarpa), Pt CRR3_2 (XP_23456., P. trichocarpa), Os CRR3 (NP_599., Os7g962, Oryza sativa), Sb CRR3 (XP_ , Sorghum bicolor), Zm CRR3 (NP_43622., Zea mays), Pp CRR3 (XP_77269., Physcomitrella patens subsp. patens), T. BP- (NP_68425., TLR636, Thermosynechococcus elongatus BP-), N. 72 (NP_ , ASR654, Nostoc sp. PCC 72) and S. 683 (NP_ , SSL352, Synechocystis sp. PCC 683) were aligned using ClustalW2. Black boxes indicate strictly conserved amino acids, whereas grey boxes indicate closely related amino acids. The predicted plastid-targeting signal and cleavage site of At CRR3 are indicated by a bar and an arrowhead in green above the sequence, respectively. Tandem repeat sequences, repeat and repeat 2 (red dashed boxes), were predicted by Radar ( The highly conserved C-terminal regions predicted to have a SH3 domain-like fold are indicated by a brown dashed box. Blue asterisks indicate the highly conserved positively charged residues in the C- terminal regions of CRR3 homologs of land plants. 4

5 A At CRRJ : : - Vv CRRJ : : - Pt CRRJ : : - Os CRRJ : : - Sb CRRJ : : - Zm CRRJ : : - Pp CRRJ : MSSSYENGNENGTRTKIKIVNERKEKGLGMEDEDKGWMKSIMKYQRESPTWNATSTWSRSGVVASAPHVSHGTDSLQICAIVKQVDR : 87 Plastid targeting signal At CRRJ : MAYATSTYARTSCIILPKIQNGAHFTDDTKAFRRITAR RVTRIYASQG-PTKPSKP : 55 Vv CRRJ : MASTAAPPPSFSLLLKTQNTTNQLRSVTPSRRRRRS--SSSQGGFWILAAAQDSKNPRQRA : 59 Pt CRRJ : MASTTATQAPFSLFLRTQNGSGHRRKKHLSIRKAPSGRTGNRGRAFGVYAEQGSPEKPQRA : 6 Os CRRJ : MAASTASAPFTPLLHRRRASVHGRRGSGRAFVAVVVAAAAGGAPETEPSPATAAGAAA-QGK : 6 Sb CRRJ : MAASTASPTFTPFLHRHQQRRT NAVVVVAAASNPGAEPTPATAADGAGPAGK : 52 Zm CRRJ : MLPAQTLTHTFPMAASTASRIFTPFLHRHQRR TKAVVVAAASPPDAEPTPATADGAG--PGK : 6 Pp CRRJ : FCEGSKWNLGHSDLGSRIYGFWRGELYSGKRRPVLVLDWSLRNAGIQWCDRMERWRAVDFENVQLHSVDPTSTSSAGSTSGDGDGGS : 74 J domain ### At CRRJ : SPGVDTRIHWESPDEGWIGGR--SDPAKSVDEDKTNLLSDEKFAELIKDSFDSHYQFLGVSTDADLEEIKSAYRRLSKEYHPDTTSL : 4 Vv CRRJ : PPGVDTRIHWDNPDEGWIGGS--SSKPKLNGEEEEKNLLGEEFSDLLDDSSDSHYQFLGISAQADLEEIKAAYRRLSKEYHPDTTSL : 44 Pt CRRJ : PPGVDTRIHWDNEDEGWVGGSSTSSEQTKQAEDEQKDMLGKRFADLLNDSSDSHYQFLGVSADADMEEIKTAYRRLSKEYHPDTTSL : 48 Os CRRJ : KKTVDTRIHWSDPDEGWVGGN-AKKDGGGRKK----EPLGGRFADLINNPSESHYQFLGVEPKADIEEIKAAYRRLSKEYHPDTTSL : 43 Sb CRRJ : KKTVDTRIHWSNPDEGWVGGN-AKKEGGGSGGGSKNEPLGGRFADLINNASESHYQFLGIAPEADIEEIKAAYRRLSKEYHPDTTTL : 38 Zm CRRJ : KKTVDTRIHWSNPDEGWVGGK-AKKEGDGRGS-SKNEPLGGRFADLINNAAESHYQFLGIAPEADIEEIKTAYRRLSKEYHPDTTTL : 45 Pp CRRJ : KKHVDTRIHWGDSAEGWIGID---CDDETSSQFRSWGRRGRSNSSLLDQASDSHYRYMGLSSDAELEEIKSAYRRLSKLYHPDTTQL : 258 At CRRJ : PLKTASEKFMKLREVYNVLSDEETRRFYDLAQEVASRQAEKMRMKLEDPKEQDFRGYESIPDMVDR--LGGRNMELSDQAMTALT : 225 Vv CRRJ : PLKAASDKFLKLREVYDVLCDEERRRFYDLAQEAASRKAEKMKMKLEDPYQKDVQNWESIPDMVDR--LGGRNMELSDQAVTALT : 229 Pt CRRJ : PLKSASEKFMRLREVYDILSNDEKRKFYDLAQEAASRQAEQMRMRLEDPYEQEINRYESVPDMVDR--LGGRNMGLSDQAMSALT : 233 Os CRRJ : PLREASERFIRLREAYNVLSREESRRFYDLAQEAESRRLQQLRSRLEDPYQQDLDSYQSVPDMVDR--LGGRNMDLSDQAMTALT : 228 Sb CRRJ : PLKVASQKFIRLREVYNVLSKEETRRFYDLAQEAESRRLQQLRSRLEDPYEQDIINYKSVPDMVDR--LGGKNMELSDQAMTALT : 223 Zm CRRJ : PLKVASQKFIRLREVYNVLSKEETRRFYDLAQEAESRRLQQLRSRLEDPYEQDIINYKPVPDMVDR--LGGKNMKLSDQAMTALT : 23 Pp CRRJ : PLEIAAQKFMRLKDAYDTLSNEELRNLYDSRLENKIVMNNEGSLYGYPTSSQQGYNSPTQKRPERVDVDTLGGDNMPLSDQALTALG : 345 Transmembrane At CRRJ : FDILIVLFAVCCIAFVIVFKDPSY : 249 Vv CRRJ : FDILIIIFAICCIIYVLVFKEPNY : 253 Pt CRRJ : FDIFIILFSIGCLIFVLFFKEPY- : 256 Os CRRJ : IDIGIIIFCVFCLIYAVFFKEQY- : 25 Sb CRRJ : FDIVIIIISICSIIYAVYFKEQY- : 246 Zm CRRJ : IDIVIIIISICCIIYAVFFKEQY- : 253 Pp CRRJ : FDLLVFVICVLVIIYVAYFKHAA- : 368 Supplemental Figure 5. Multiple Alignments of Amino Acid Sequences of CRRJ and CRRL Homologs from Plants. (A) The sequence alignment of CRRJ homologs, At CRRJ (NP_92673., AT4G935, A. thaliana), Vv CRRJ (XP_ , V. vinifera), Pt CRRJ (XP_ , P. trichocarpa), Os CRRJ (NP_6758., O. sativa), Sb CRRJ (XP_ , S. bicolor), Zm CRRJ (NP_48857., Z. mays) and Pp CRRJ (XP_ , P. patens subsp. patens). (B) The sequences of CRRL homologs, At CRRL (NP_85862., AT5G243, A. thaliana), Vv CRRL (XP_ , V. vinifera), Pt CRRL (XP_23947., P. trichocarpa), Rc CRRL (XP_25625., Ricinus communis), Os CRRL (NP_6682.2, O. sativa), Sb CRRL (XP_24432., S. bicolor), Zm CRRL (NP_48857., Z. mays), Pp CRRL_ (XP_ , P. patens subsp. patens) and Pp CRRL_2 (XP_ , P. patens subsp. patens). In (A) and (B), sequences were aligned using ClustalW2. Black boxes indicate strictly conserved amino acids, whereas grey boxes indicate closely related amino acids. The predicted plastid-targeting signals and their cleavage sites in At CRRJ and At CRRL are indicated by bars and arrowheads in green above the sequences, respectively. Putative transmembrane domains and J- or J-like domains are indicated by pink bars and dashed blue boxes, respectively. A HPD motif in CRRJ homologs is marked with a red #. 5

6 B Plastid targeting signal At CRRL : MASLSTITQPSLVHIPGESVLHHVPSTCSFPWKPTINTKRIICSPARNSSEVSAEAETE GG : 6 Vv CRRL : MAVTSTSATASLLRPNFRTSPPRHGGQFSNSIAIAFKPRRFRIR------SSGDAPLETTD ES : 57 Pt CRRL : RSSSDGAAETAATE VE : 6 Rc CRRL : MAVSSTSATIYILRQNFPISTTKNTSVLLNFAPRKTCRHVIRSSSSDGTAESTAATKVQEE EE : 63 Os CRRL : MAAVGVVSPPAPPAVAAASTSSPRRRVRLPLGRVSSSAASFRARCAAAAEDGGATAAPEDAAAAAAVAEAVEGDP----EAGTD : 8 Sb CRRL : MAAVGITSPPAPAPHALAPAASPSASSYSTLGLRRLRLSPRLAAGGASFRAR-RCAAAADAGAAASEGASAVADP----DAGTD : 79 Zm CRRL : MAAVGIPSP--PAPHVFAPAASPSSSTYSTLAPRRLRLSTRLGLAAAAASCRTRCAAAADGGAAASEGASAVAELEGDPDAGTD : 82 Pp CRRL_ : MVQCLPAQCQLVSPVSTQIGPRTRRLTCNAARISHSRRGLGLAVRAESKSASTSTVEPAGPAP T : 64 Pp CRRL_2 : MMQCLPAQCQLLSPVSTNVTSRTQRPSCSITRISQSRRASGLVVRAESTSAPTSTVETESPTP T : 64 J-like domain At CRRL : SSTAVDEAPKESPSLISALNVERALRGLPITDVDHYGRLGIFRNCSYDQVTIGYKERVKELKEQGLDEEQLKTKMDLIKESYTI : 45 Vv CRRL : DSNSPVETPKGPPSLISALNVEKALRGIAITDADHYGRLGLQRECSYDQVTVAYKRKVEELKSQELDEEEANNKLGLLKESYSI : 4 Pt CRRL : DSENYIEVPGGPPSLISALNVERALRGIPITDVDHYGRLGIQRRSSYEEVTVAYKNKVDELLNQGLDEAEVREKMELLKESYTI : Rc CRRL : ESETLMEVPEGP-SLISALNVERALRGIPITDVDYYGRLGVKRGCSNEEVTVAYKNKVEELLKQGLDEEDVRKEMELLKESYAI : 46 Os CRRL : VAGGAATSTRPPYSLISADNVQKAMRGLAITECDHYGRLGITRSASTDEVKAAYEKKCEELNSKGLEEEEINKEHDLLKESFTI : 64 Sb CRRL : VAGGAATSTRPPYSIISADNVQKAMRGLAITDADHYGRLGVTRLASTDEVKAAYEKKCEELNSKELEEEELHKEYDLIKESFTI : 63 Zm CRRL : VAGGAATSTRPPYSLISADNVQKAMRGLAITDADHYGRLGVTRLASTDEVKAAYEKMCEELNSKELEEEELNKEHELLKESFTI : 66 Pp CRRL_ : EEVTLTPTAPPSKSLINEENILSAISGRNIIVEDHYGRLRVNPEASSSEIVKAYQERCAEVRQQNLGESEAQEILNKLKDSVDL : 48 Pp CRRL_2 : EAVALTPTAPASKSLINEENILSAITGQNIIVEDHYGRLGVNPEASSSEIVKTYQERCAEVRQKNLSESETEEILNKLKDSVDL : 48 Transmembrane At CRRL : LSTVEERRMYDWSLA-RSEKAERYVWPFEVDIMEPSREE-PPPQEPEDVGPTRILGYFIGAWLVLGVALSVAFNR : 28 Vv CRRL : LSSPKERRLYDWSLA-RSEKRDRYAWPFEVEKAKTPTEE-PPAQEPEDVRPTILVGYFLFAWLILSFTLSIALNR : 24 Pt CRRL : LSSRDERRMYDWSLV-RSEKPERYMWPYEVDFTPTAP---PPPQEPEDEGPTRLVGYFALAWLVLAVTLSVTLNR : 7 Rc CRRL : LSSEDERRMYDWSLA-RSENPDRYAWPYETDKVKPSPD--TPPPVQENPGGRNQKTWKRQDWWDTFYWVG : 23 Os CRRL : LEEERRLYDWSLA-RSGKPERYVWPFEVDPMELAPD---PPKEPEDEFPTKLVGYFFLAWFILSVALSVTLNR : 235 Sb CRRL : LSTEEERRLYDWSLS-RSGQPERYVWPFQVDPLEMAPD---PPQEPEDELPTKLVGYFLLAWFILSVALSVTLNK : 234 Zm CRRL : LSTEEERRLYDWSLS-RSGQPERYVWPFEVDPLELAPD---PPKEPEDEFPTKLVGYFLLAWFILSVGLSVALNK : 237 Pp CRRL_ : LTSEEERRLYDWCLLRKATHTVDYAWPYEADLTQRLQDPTTLVVPPEDDGVNDQVGLFFAGWFVLSCVLSIVIGHGWDKCL : 229 Pp CRRL_2 : LTSEEERRLYDWCLLRKATHTVDYAWPYEADITQRLQDPTSLVVPPEDDGVNDKVGLFFAGWFVLSCILSIVIGHGWEKSM : 229 Supplemental Figure 5. Multiple Alignments of Amino Acid Sequences of CRRJ and CRRL Homologs from Plants. (A) The sequence alignment of CRRJ homologs, At CRRJ (NP_92673., AT4G935, A. thaliana), Vv CRRJ (XP_ , V. vinifera), Pt CRRJ (XP_ , P. trichocarpa), Os CRRJ (NP_6758., O. sativa), Sb CRRJ (XP_ , S. bicolor), Zm CRRJ (NP_48857., Z. mays) and Pp CRRJ (XP_ , P. patens subsp. patens). (B) The sequences of CRRL homologs, At CRRL (NP_85862., AT5G243, A. thaliana), Vv CRRL (XP_ , V. vinifera), Pt CRRL (XP_23947., P. trichocarpa), Rc CRRL (XP_25625., Ricinus communis), Os CRRL (NP_6682.2, O. sativa), Sb CRRL (XP_24432., S. bicolor), Zm CRRL (NP_48857., Z. mays), Pp CRRL_ (XP_ , P. patens subsp. patens) and Pp CRRL_2 (XP_ , P. patens subsp. patens). In (A) and (B), sequences were aligned using ClustalW2. Black boxes indicate strictly conserved amino acids, whereas grey boxes indicate closely related amino acids. The predicted plastid-targeting signals and their cleavage sites in At CRRJ and At CRRL are indicated by bars and arrowheads in green above the sequences, respectively. Putative transmembrane domains and J- or J-like domains are indicated by pink bars and dashed blue boxes, respectively. A HPD motif in CRRJ homologs is marked with a red #. 6

7 A /8 /4 /2 crrj- crrj- + CRRJ crrj- + CRRJ H34Q # crrj- + CRRJ H34Q #2 crrl- crrl- + CRRL CRRJ CRRL CRR3 NdhL NdhH Cytf B Chlorophyll fluorescence (a.u.) crrj- crrj- + CRRJ H34Q # crrj- + CRRJ H34Q #2 min Time C I II I II crr23/ndhl crr4-3 crrj- crrj- + CRRJ H34Q # crrj- + CRRJ H34Q #2 * * D crrj- crrj- + CRRJ crrj- + CRRJ H34Q # crrj- + CRRJ H34Q #2 CRRJ ACT8 Supplemental Figure 6. The HPD Motif Is Essential for CRRJ Function. (A) Immunodetection of CRR3, CRRJ, CRRL and NDH subunits. Chloroplasts were isolated from various genotypes as indicated on the top. Protein levels were analyzed using antibodies as indicated on the right. Membrane protein corresponding to 2 µg chlorophyll was loaded on each lane, as well as a dilution series of protein. Cytf was detected as a loading control. (B) Monitoring of in vivo NDH activity by chlorophyll fluorescence. a.u., arbitary units. Fluorescence levels were normalized by the F m levels., crrj- and two-independent transgenic lines (crrj- + CRRJ H34Q # and #2) were analyzed. The HPD motif in CRRJ is essential for NDH activity. (C) Magnification of the top part of the BN-PAGE gel. Chloroplasts were isolated from various genotypes as indicated across the top. Thylakoid protein extract corresponding to µg chlorophyll was loaded on each lane. The BN-PAGE gel (upper panel) was stained with CBB (bottom panel). The positions of band I and II are indicated. Asterisks indicate bands probably corresponding to aggregated proteins. (D) RT-PCR analysis of CRRJ mrna in, crrj- and crrj- transformed with CRRJ or CRRJ H34Q. ACT8 was amplified as a control. 7

8 2.5 A NPQ pgr5 crr4-2 pgr5 crr3- pgr5 crrj- pgr5 crrl- pgr5 crr3- crrj- crrl- -ql Light intensity (μmol photons m -2 s - ) B Light intensity (μmol photons m -2 s - ) C Plants F v/f m.79 ±.7 pgr5.782 ±. crr4-2 pgr5.734 ±.27 crr3- pgr5.778 ±.3 crrj- pgr5.74 ±. crrl- pgr5.764 ±.8 crr ±.5 crrj-.78 ±.7 crrl-.778 ±.9 Supplemental Figure 7. Chlorophyll Fluorescence Parameters of, crr3- pgr5, crrj- pgr5 and crrl- pgr5 Leaves. (A) Light-intensity dependence of non-photochemical quenching of chlorophyll fluorescence (NPQ) in and mutant leaves. (B) Measurement of a fraction of closed PSII centers (-ql) in and mutant leaves. (C) Maximum quantum yield of PSII (F v /F m ). Values are means ± SD (n=3). 8

9 Supplemental Table. Primers List and Sequence The following primers used for RT-PCR analysis: CRR3 RT-Fw 5 GGCGACTTCTTCGATCACTATCCCG3 CRR3 RT-Rv 5 CACTTATGGTGCTGCCTCTTCC3 CRRJ RT-Fw 5 GCCCGGACGAAGGCTGGATCGGAGG3 CRRJ RT-Rv 5 CGATCAAAATATCGAACGTGAGAGC3 CRRL RT-Fw 5 ACGATAACTCAACCATCTTTAGTCC3 CRRL RT-Rv 5 TCCATGATGTCAACCTCAAATGGCC3 ACT8 RT-Fw 5 GAGAGATTCAGGTGCCCAG3 ACT8 RT-Rv 5 AGAGCGAGAGCGGGTTTTCA3 The following primers used for complementation vector construction: attb-crr3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAGCAGGAAG AAGATGTTGGGTTTGGG3 attb2-crr3 5 GGGGACCACTTTGTACAAGAAAGCTGGGTCATACAGAGA AGAGAAGATCACTTATGGTGC3 attb-crrj 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATAAGGAT TGGAAAAAATATGGC-3 attb2-crrj 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTTACTTCCCAAA CTAGTGAATTCCGC3 attb-crrl 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTGACTCTGTAG ATATCGAAGCATGTGC3 attb2-crrl 5 GGGGACCACTTTGTACAAGAAAGCTGGGTGGGTAAATAA ACATCGGATTTGAAGTGAGG3 The following primers used for mutagenesis of CRR3 and CRRJL: CRR3del-FW 5 GGCCATGGGTAAATTCAATCTATGGCTCATGCCGGGGATGA TTGCTATAGTCAAG3 CRR3del-Rev 5 CTTGACTATAGCAATCATCCCCGGCATGAGCCATAGATTGA ATTTACCCATGGCC3 CRRJH34QFw 5 CCGGAGATTATCTAAAGAGTATCAACCAGATACAACTTCAC TACC3 CRRJH34QRv 9

10 5 GGTAGTGAAGTTGTATCTGGTTGATACTCTTTAGATAATCTC CGG3 The following primers used for protein expression vector construction: CRR3F(NdeI) 5 AACATATGGCCATGGGTAAATTCAATCTATGGG3 CRR3R-(Hind) 5 TTAAGCTTTGGTGCTGCCTCTTCCTTTTGCATC3 CRRJ-F(NdeI) 5 AACATATGGCGTCACAAGGCCCGACTAAAC3 CRRJ-R(Hind) 5 TTAAGCTTCATTGCTTGATCACTAAGCTCC3 CRRL-F(NdeI) 5 AACCATGGAACATATGGCGGAGGCAGAAACCGAAGG TGG3 CRRL-R(Bam) 5 TTGAATTCGGATCCCTACCTCGTTGGTCCTACATCTTCC GG3 Restriction enzyme site, attb or attb2 sequences used to insert the cloned cdna into the respective vectors are underlined.