The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
The chloroplast genome encodes more than 100 genes that are involved in photosynthesis and housekeeping functions in chloroplasts. Marked changes in chloroplast gene expression take place during chloroplast development from the undifferentiated proplastid (for review, see Mullet, 1988). Increase in plastid DNA copy number significantly contributes to this process (Miyamura et al., 1986). Chloroplast development is also accompanied by the shift of transcriptional machinery from the nucleus-encoded phage-type RNA polymerase to plastid-encoded bacterial-type polymerase (Hedtke et al., 1997). While the phage-type RNA polymerase transcribes the housekeeping genes, the bacterial-type RNA polymerase transcribes almost all genes including those functioning in photosynthesis. Once the chloroplast develops, however, chloroplast gene expression is preferentially regulated at post-transcriptional steps (Deng and Gruissem, 1987).
In the chloroplast, a cluster of genes is transcribed from a single promoter as a polycistronic RNA. The precursor RNA undergoes a number of maturation steps: processing in the intergenic region, intron splicing, maturation of 5′ and 3′ ends (often accompanied by the formation of stem and loop structures), RNA editing, and activation of translation. Genetic approaches have identified nuclear mutants affected in these chloroplast RNA maturation steps (for review, see Barkan and Goldschmidt-Clermont, 2000). Thus, the expression of chloroplast genes is regulated by a large number of nucleus-encoded factors.
Chloroplast NDH is a homolog of mitochondrial complex I, NADH dehydrogenase complex (for review, see Peltier and Cournac, 2002; Shikanai and Endo, 2000). Chloroplast transformation using tobacco established the current idea that chloroplastic NDH mediates cyclic electron flow around photosystem I (PSI) in the light and chlororespiratory electron flow in the dark (Burrows et al., 1998; Horváth et al., 2000; Kofer et al., 1998; Shikanai et al., 1998), as does cyanobacterial NDH (Ohkawa et al., 2000). Knockout lines of chloroplast ndh genes are sensitive to supra-saturating light intensity (Endo et al., 1999) and humidity stress (Horváth et al., 2000), suggesting a physiological role for chloroplast NDH in stress tolerance. Unexpectedly, however, even complete disruption of the NDH complex does not affect overall electron transport. We are still not sure of the exact mechanism by which chloroplast NDH is involved in stress tolerance.
Eleven subunits of the NDH complex are encoded by the chloroplast genome. Considering the possible physiological role of NDH in photoprotection, expression of ndh genes may be regulated to cope with changes in environmental conditions. Indeed, oxidative stress stimulates expression of NDH via hydrogen peroxide as a signal molecule in barley (Casano et al., 2001), and the activity is also regulated by phosphorylation of the NdhF subunit (Lascano et al., 2003). However, it is not clear as to what extent the regulation is physiologically significant and information on how environmental cues regulate chloroplast gene expression is still fragmentary.
Cyclic electron flow around PSI is also mediated by ferredoxin-dependent plastoquinone reductase (Arnon et al., 1967). An Arabidopsis mutant proton gradient regulation (pgr5) is defective in this cyclic electron transport activity (Munekage et al., 2002). In contrast to the tobacco lines lacking the NDH complex, proton gradient formation across the thylakoid membranes (ΔpH) is affected in pgr5, leading to a defect in the introduction of thermal dissipation at high light intensity (Munekage et al., 2002). Thus, cyclic electron transport around PSI is mediated by two redundant pathways, the major PGR5-dependent pathway and the minor NDH-dependent pathway. To answer an important physiological question on the redundancy of the electron flow by genetic approach, it is critical to isolate the mutants defective in NDH activity in Arabidopsis. Unfortunately, chloroplast transformation is not routinely feasible, and chloroplast ndh genes cannot be directly disrupted as in tobacco.
Our strategy to answer these fundamental questions is to isolate Arabidopsis nuclear mutants in which the expression of chloroplast ndh genes is specifically affected. This genetic approach enables us to bridge the gap between the molecular biology of chloroplast gene expression and the physiology, thus clarifying the regulatory mechanism of photosynthetic electron transport. To screen mutants affected in NDH activity, we focused on the transient increase in chlorophyll fluorescence after turning off actinic light (AL). This change in post-illumination fluorescence is ascribed to NDH activity, and was clarified by reverse genetic approaches using tobacco (Burrows et al., 1998; Horváth et al., 2000; Kofer et al., 1998; Shikanai et al., 1998). We have improved the chlorophyll fluorescence imaging system (Niyogi et al., 1998; Shikanai et al., 1999) to visualize NDH activity under a charge-coupled device (CCD) camera and thus identify mutants affected in NDH activity. Here, we characterize a mutant, crr2, in which the expression of chloroplast ndhB is specifically affected.
Screening of Arabidopsis mutants defective in NDH activity
To isolate mutants lacking NDH activity, we utilized chlorophyll fluorescence imaging, which allows even subtle changes in photosynthetic electron transport to be detected. At low light intensity, fluorescence is roughly proportional to the degree of reduction of the plastoquinone pool (Krause and Weis, 1991). The plastoquinone pool is also non-photochemically reduced by stromal electrons via NDH activity. This NDH-dependent plastoquinone reduction can be monitored as a fluorescence signal only when photochemical reduction by photosystem II (PSII) is minimized (Shikanai et al., 1998). Thus, fluorescence must be monitored under a measuring light (ML) of low intensity (10 µmol photons m−2 sec−1) so that photochemical reduction of plastoquinone does not mask non-photochemical reduction by NDH activity. It is critical to optimize the light intensity of ML because light intensity less than 1 µmol photons m−2 sec−1 does not produce an increase in fluorescence sufficient to detect non-photochemical reduction by NDH.
Figure 1(a) shows a schematic representation of the screening strategy. Prior to AL illumination, the basal level of chlorophyll fluorescence emitted during exposure to ML is monitored (image 1). Subsequently, AL of 100 µmol photons m−2 sec−1 is turned on for 5 min to build up the stromal pool of reductants (NADPH and reduced ferredoxin). Just after turning on AL, the fluorescence level is maximized by reduction of plastoquinone via the PSII photochemistry, and then subsequently lowered by its oxidation by the downstream electron transport pathway. The fluorescence is near the steady-state level after 5 min. Turning off AL arrests the photochemical reduction of plastoquinone by PSII, and the plastoquinone pool is fully oxidized instantaneously. A subsequent transient increase in fluorescence is because of non-photochemical reduction of plastoquinone by electrons accumulated in the stroma during AL illumination, which depends on the NDH activity. To maximize rather subtle change of chlorophyll fluorescence in Arabidopsis, we used higher light intensity (100 µmol photons m−2 sec−1) than previously used in analysis of tobacco ndhB disruptant (20 µmol photons m−2 sec−1) (Shikanai et al., 1998). It is critical to use AL of this light intensity, as AL of higher intensity transiently lowers the basal fluorescence level after turning off AL and its recovery affects the fluorescence rise caused by NDH activity. Twenty seconds after turning off AL, when the transient reduction level of the plastoquinone pool is maximal, the fluorescence image was captured again (image 2). NDH activity can be estimated by the comparison of two images ((image 2) − (image 1)). Screening of approximately 50 000 M2 seedlings identified 17 crr (chlororespiratory reduction) mutations on at least 11 loci. Here, we characterize two alleles of the mutant, crr2-1 and crr2-2.
NDH activity is specifically affected in crr2
Figure 1(c) shows an image of the difference in fluorescence intensity between images captured before and after AL illumination. In the wild type, the fluorescence level was higher after AL illumination as a result of non-photochemical reduction of plastoquinone by NDH in the dark, resulting in the appearance of the fluorescence image after calculation of ((image 2) − (image 1)). In contrast, for crr2 alleles, there was no image after applying the calculation; the fluorescence level was identical before and after AL illumination presumably because of lack of NDH activity in the mutants.
Genetic analysis established the recessive nature of crr2 mutations (data not shown). F1 plants between crr2-1 and crr2-2 displayed a lack of transient post-illumination increase in chlorophyll fluorescence (data not shown), indicating that mutations occurred in the same gene. Neither allele of crr2 exhibits any visible phenotype, and both grow photo-autotrophically as well as the wild type at 40 µmol photons m−2 sec−1 (Figure 1b).
The crr2 phenotype was further characterized by pulse-amplitude-modulation (PAM) chlorophyll fluorometry. Non-photochemical reduction of the plastoquinone pool by NDH is monitored as a small peak in the fluorescence level, which occurs within a minute after switching off AL in the wild type (Figure 1a). In crr2 alleles, however, this rise in fluorescence level was undetectable, as was in mature leaves of tobacco ndhB disruptant (Shikanai et al., 1998).
To characterize photosynthetic electron transport, light-intensity dependence of two chlorophyll fluorescence parameters was compared between the wild type and crr2 alleles (Figure 2). Electron transport rate (ETR) represents the relative flow of electrons through PSII during steady-state photosynthesis and was not affected in crr2 (Figure 2, upper graph). This result is consistent with the normal growth rate of crr2 in soil (Figure 1b). Lower graph in Figure 2 shows light-intensity dependence of a chlorophyll fluorescence parameter, non-photochemical quenching (NPQ). The main cause of NPQ in higher plants is thermal dissipation, which dissipates excessive light energy as heat from PSII antennae (Krause and Weis, 1991). The process is induced by acidification of the thylakoid lumen to less than pH 6.0 (Munekage et al., 2001), which requires the activity of cyclic electron flow around PSI, as well as linear electron flow (Munekage et al., 2002). Although NDH is involved in cyclic electron flow around PSI, light-intensity dependence of NPQ was not affected in crr2, nor was it in tobacco ndhB disruptants (Joët et al., 2001; Shikanai et al., 1998). We conclude that NDH activity is specifically impaired in crr2, but this impairment scarcely affects total photosynthetic electron flow, at least under the culture conditions used.
Accumulation of the NDH complex is impaired in crr2
To assess the possibility that accumulation of the NDH complex is affected in crr2, Western blots were analyzed using an antibody raised against NdhH (Figure 3). In both alleles of crr2, NdhH was severely reduced. Further diluted loading of wild-type proteins estimated that NdhH level is less than 1/16 in crr2 (data not shown). Consistent with the results of chlorophyll fluorescence analysis (Figure 2), the steady-state protein levels of PsbO (a PSII subunit), Cytf (a subunit of the cytochrome b6f complex), and PsaA/B (reaction center subunits of PSI) were not affected in crr2. Taken together with the chlorophyll fluorescence results (Figures 1a and 2), we conclude that the crr2 phenotype is caused by the specific lack of the NDH complex in chloroplasts.
CRR2 encodes a member of the PCMP family
To identify the gene affected in crr2, crr2-2 (Columbia) was crossed with polymorphic wild type (Landsberg erecta). Analysis of 343 F2 plants identified a 198-kb region spanning three bacterial artificial chromosomes (F18I15, F12A12, and T6H20). As the crr2 phenotype is specific to the chloroplastic NDH level, genes encoding possible chloroplast targeting signals (predicted by Predotar; http://www.inra.fr/Internet/Produits/Predotar/) were sequenced. Finally, mutations were identified in a gene At3g46790 in both alleles.
To verify that the crr2 phenotype is caused by mutations in At3g46790, genomic sequence containing a complete and single gene, At3g46790, was introduced into crr2-2. The transformation fully restored the transient increase in chlorophyll fluorescence after turning off AL (Figure 1a). The NdhH level was also restored to wild-type level in the transformants (data not shown). We conclude that mutations in At3g46790 leads to the drastic reduction of the NDH level.
CRR2 is not interrupted by any intron and encodes a putative protein with 657 amino acids (Figure 4a). The psort program (http://psort.ims.u-tokyo.ac.jp/) predicted the first 40 amino acids to be a transit sequence to chloroplasts. CRR2 is a member of a family called PCMP (plant combinatorial and modular protein) containing more than 200 genes of Arabidopsis (1% of the total genes) (Aubourg et al., 2000). No function for any member of this family has yet been established. CRR2 (AtPCMP H54) belongs to the PCMP-H subfamily, which is characterized by a conserved C-terminal domain containing the motifs D, E, F, G, and H. The motifs were identified by the alignment of 58 AtPCMP members (Aubourg et al., 2000). Motif H in the C-terminal shows especially high sequence conservation and contains a number of conserved cysteine residues. A CxxCH motif is the cytochrome c family heme-binding site signature. This sequence may function in the covalent fixation of heme groups or in S-S bridges, as well as other cysteine and histidine residues conserved in the latter half of the motif H (Aubourg et al., 2000). The N-terminal region (amino acids 41–423) is composed of a reiteration of several motifs, with the pattern of repeats specific to each member. In CRR2, this region is composed of the reiteration of three motifs, A, B and C, in the order 3 × (B-C-A).
Overlapping this reiteration of motifs, a blast search (http://blast.genome.ad.jp/) also found nine pentatrico-peptide repeat (PPR) motifs (Small and Peeters, 2000; Figure 4a,b). Figure 4(c) shows an alignment of all PPR motifs present in the N-terminal region of CRR2. Although the amino acid conservation is rather weak among motifs including the length variation in the first, third and fifth repeats, the conserved amino acids (6th leucine, 14th glycine, 19th alanine, and 26th methionine) are fixed, suggesting that the sequence is related to a PPR motif. Truncation of the four amino acids in third and fifth PPRs is related to the overlapping of the motif C (Figure 4b,c).
In crr2-1, the 167th tryptophan (TGG) was substituted by the stop codon (TGA), truncating the protein and shortening the reading frame to 166 codons (Figure 4a). In contrast, the 309th glycine (GGG) was substituted by arginine (AGG) in crr2-2. Although this glycine site is not included in any reiteration motifs, the NDH level is also severely affected in this allele (Figure 3).
CRR2 is required for the accumulation of specific processed ndhB transcripts
Several proteins containing PPR motifs function in post-transcriptional regulation of organellar gene expression (Bentolila et al., 2002; Coffin et al., 1997; Fisk et al., 1999; Kazama and Toriyama, 2003; Manthey and McEwen, 1995; Meierhoff et al., 2003). It is possible that CRR2 is involved in the post-transcriptional regulation of chloroplast ndh gene(s). To assess this possibility, Northern blots were analyzed by five sets of probes detecting transcripts from 11 chloroplast ndh genes (ndhA–K). Using four of the probes, hybridization patterns were identical between the wild type and crr2 alleles (data not shown). In contrast, using the probe for ndhB, the signal corresponding to the mature form of ndhB was undetectable in crr2 (Figure 5a,b, III). The faint signals detected in crr2 alleles have slightly higher mobility and are a result of cross-hybridization with the abundant rRNA. The signal corresponding to the unspliced form of ndhB was also undetectable in crr2 (Figure 5a,b, II). However, a longer RNA molecule, which was also hybridized by the rps7 probe, accumulated in crr2 alleles as well as in the wild type (Figure 5a,b, I). The RNA was not hybridized by the ndhB intron probe, indicating that this is a tricistronic precursor containing rps12, rps7, and spliced ndhB.
To confirm the conclusion of Northern analysis, RNase protection assay was performed using probes spanning the rps7/ndhB intercistronic region (Figure 5a,c). The protected fragments corresponding to the mature 5′ end of ndhB were missing in crr2 alleles (Figure 5a,c, II + III and IV). We conclude that the intergenic processing between rps7 and ndhB is impaired in crr2.
The rps7 probe detected the same size RNA, probably corresponding to the dicistronic rps7 with rps12, in both the wild type and crr2 alleles (Figure 5a,b, V). Estimating from the RNA size, rps12 is likely to be fully spliced in this molecule. Thus, the expression of rps7 is unlikely to be affected in crr2, which is consistent with the crr2 phenotype specific to the NDH complex (Figures 1–3). The rps7 probe also detected the slightly larger RNA in the wild type, which was undetectable in crr2 (Figure 5a,b, IV). These results indicate that the precursor RNA containing rps7 and ndhB is processed at two sites in the intergenic sequence (Figure 5a). RNA protection assay also confirmed the presence of two 3′-termini of rps7 (Figure 5a,c). Estimating from the size of protected fragments (Figure 5c), the 3′-terminus of short rps7 RNA locates at approximately −180 with respect to the ndhB translation start site (+1), while that of long rps7 RNA locates very closely to the +1 site. We conclude that CRR2 is involved in processing at the site closer to ndhB but not at −180. If this is the case, the longer version of ndhB RNA processed only at −180 should accumulate in crr2 and possibly in the wild type. However, neither Northern analysis nor RNase protection assay detected the signal corresponding to this longer version of ndhB RNA (Figure 5b,c). We consider that processing at the CRR2-dependent site is essential for the stabilization of ndhB RNA, and thus the longer versions of the ndhB transcript are unstable even in the wild type.
To determine the CRR2-dependent processing site at the nucleotide level, the 5′ end of ndhB transcripts was determined using a 5′ Rapid Amplification of cDNA Ends (RACE) system. Consistent with the results of RNase protection assay (Figure 5c), a single fragment was preferentially amplified in the wild type but not in crr2 alleles (Figure 5d). Subsequently, PCR products were cloned and subjected to sequence analysis. Among 41 independent clones, 25 have the identical 5′-termini in the wild type at −12 with respect to the translation initiation codon of ndhB (+1). We consider that the position −12 is a CRR2-dependent cleavage site.
CRR2 is a member of the PCMP family, which is closely related to PPR proteins
CRR2 is a member of the PCMP family whose function has not been clarified. CRR2 belongs to the PCMP-H subfamily, which is characterized by the presence of conserved motifs, D, E, F, G, and H, in the C-terminal region (Aubourg et al., 2000). The N-terminal region consists of the reiteration of three motifs, A, B, and C. The same region was also characterized by the presence of nine PPR motifs. The PPR motif consists of 35 amino acids and is structurally similar to the tetratrico-peptide repeat (TPR) motif consisting of 34 amino acids (Goebl and Yanagida, 1991). While the tandem array of TPR motifs is expected to function in protein–protein interaction (Blatch and Lässle, 1999), PPR motifs and also some TPR motifs (Felder et al., 2001; Vaistij et al., 2000) may function to bind specific RNA sequences, and facilitate RNA maturation steps (Small and Peeters, 2000). The maize PPR protein, CRP1, functions in the processing of petD mRNA in the intergenic region between petB and petD and in petA translation (Barkan et al., 1994; Fisk et al., 1999). HCF152, an Arabidopsis PPR protein, also functions in intergenic RNA processing between psbH and petB and in petB splicing (Meierhoff et al., 2003). Although CRP1 and HCF152 contain rather simple tandem arrays of 13 and 12 PPR motifs, respectively, CRR2 contains three sets of arrays interrupted by sequences that are not related to a PPR motif. Furthermore, alignment of nine PPR motifs revealed rather weak conservation of amino acids among motifs, except for four well-conserved amino acids (Figure 4c). Much higher sequence conservation is observed among PPR motifs in CRP1 and HCF152, and each repeat consists of exactly 35 amino acids. Thus, the PCMP family including CRR2 is rather divergent from the authentic PPR family. As discussed below, however, the function of CRR2 is closely related to that of PPR proteins. We consider that the PCMP family is closely related to PPR proteins functionally, besides showing structural similarity.
Multiple cleavages in the rps7–ndhB intergenic region
The primary transcript including the ndhB sequence is possibly derived from the promoter present in front of 3′rps12 (Hildebrand et al., 1988). We identified at least two processing sites in the intergenic region between rps7 and ndhB (Figure 7). CRR2 is involved in processing at site −12. RNase protection assay also identified the 3′-terminus of long rps7 RNA around site −12 (Figure 5c). The maturation of rps7 also utilizes the additional upstream cleavage site around −180 and does not require CRR2 function. Thus, crr2 defects specifically affect ndhB expression but not rps7 expression.
To explain the fact that the 5′ end of ndhB locates approximately 170 nucleotides downstream of the 3′ end of rps7, there are two possibilities: (i) multiple endonucleotic cleavages generate two distinct termini; or (ii) a single endonucleotic cleavage followed by exonucleotic trimming does. We do not consider that the second possibility is likely for the following reasons: (i) when the CRR2-independent site (−180) is a target of the endonuclease and CRR2 functions to generate the 5′-terminus of ndhB (−12) by protecting the processed transcripts from 5′ to 3′ exoribonuclease activity (Drager et al., 1999), the presence of the longer version of rps7, which is specifically present in the wild type, cannot be explained; (ii) when the CRR2-dependent site (−12) is a target of the endonuclease and 3′ to 5′ exoribonuclease activity generates the 3′ terminus of rps7 at −180, the presence of the mature rps7 RNA in the crr2 mutants cannot be explained. Thus, we conclude that at least two endonucleotic cleavages, CRR2-dependent and CRR2-independent, generate the discontinuous RNA termini between rps7 and ndhB.
Intergenic region between petB and petD is also cleaved at multiple sites in maize (Barkan et al., 1994). The 3′ end of petB RNA locates 30 nucleotides downstream of the 5′ end of petD RNA, indicating that both ends are generated by independent processing. In the mutant, crp1, however, both ends are absent, suggesting that a common processing activity is involved in their formation (Barkan et al., 1994). On the contrary, CRR2 is involved only in the cleavage at −12. The 3′-end maturation at −180 is independent from the CRR2 function and probably requires other factors. Coordinate translation is required in petB and petD, which encode the subunits of the cytochrome b6f complex. However, estimated from the accumulation levels of proteins (Burrows et al., 1998; Klein and Mullet, 1987), efficiency of the rps7 translation is estimated to be roughly 10 times higher than that of ndhB translation. It may be required that the 3′ end of rps7 and the 5′ end of ndhB are processed independently by different factors, facilitating the unbalanced rate of translation.
A possible regulatory mechanism by multiple endonucleotic cleavages in the intergenic region have also been reported in the region between psaC encoding a PSI subunit and ndhD encoding a NDH subunit (del Campo et al., 2002; Hirose and Sugiura, 1997). Based on the stoichiometry of accumulating complexes (Burrows et al., 1998), expression of psaC should be approximately two orders higher than that of ndhD. The alternative cleavage of the intergenic sequence between psaC and ndhD possibly facilitates regulation by activating the translation of one of the two genes (del Campo et al., 2002; Hirose and Sugiura, 1997). In addition to difference in the accumulation level of the gene products between ribosomal proteins and NDH complex subunits, induction of ndh gene expression appears to be mediated by hydrogen peroxide (Casano et al., 2001). This fact suggests that stoichiometry of the rps7 and ndhB expression can vary to cope with changes in environmental conditions. CRR2 may function as a molecular switch to regulate the expression of ndhB without affecting expression of rps7.
Arabidopsis thaliana M2 seeds (Accession Columbia gl1) mutagenized by ethyl methanesulfonate were obtained from Lehle Seeds (Round Rock, TX, USA). Seedlings were cultured in soil in a chamber (40 µmol photons m−2 sec−1, 16-h light/8-h dark cycles at 23°C) for 2 weeks for mutant screening and for 18 days for chlorophyll fluorescence analysis. Prior to analysis, seedlings were dark-adapted for at least 10 min. Mutants were screened using an imaging system for chlorophyll fluorescence (Shikanai et al., 1999), with some modifications as indicated in the text.
Chlorophyll fluorescence analysis
Chlorophyll fluorescence was measured with a MINI-PAM portable chlorophyll fluorometer (Walz, Effeltrich, Germany). The minimum fluorescence at open PSII centers in the dark-adapted state (Fo) was excited by a weak measuring light (650 nm) at a light intensity of 0.05–0.1 µmol photons m−2 sec−1. A saturating pulse of white light (800 msec, 3000 µmol photons m−2 sec−1) was applied to determine the maximum fluorescence at closed PSII centers in the dark-adapted state (Fm) and during actinic light illumination (Fm′). The steady-state fluorescence level (Fs) was recorded during actinic light illumination (15–1000 µmol photons m−2 sec−1). NPQ was calculated as (Fm − Fm′)/Fm′. The quantum yield of PSII (ΦPSII) was calculated as (Fm′ − Fs)/Fm′ (Genty et al., 1989). The relative rate of electron transport through PSII (ETR) was calculated as ΦPSII × photon flux density (PFD) (µmol photons m−2 sec−1). Transient increase in chlorophyll fluorescence after turning off AL was monitored as described by Shikanai et al. (1998).
The crr2 mutation was mapped with molecular markers based on the cleaved amplified polymorphic sequence (Konieczny and Ausubel, 1993). Primer sequences of the markers and the restriction enzymes used are: 5′-GATTAGCATGATCCATCAAGG-3′ and 5′-AGAAGAAAGAGAAGAAGCTCG-3′, AseI for F18L15; and 5′-TGATACAGCAATCAGACTTGG-3′ and 5′-TCTTGGTATGTACTGATCAGG-3′, PvuII for T6H20. Genomic DNA was isolated from F2 plants derived from the cross between crr2-2 and the wild type (Landsberg erecta). Genomic DNA from the wild type and crr2 alleles of At3g46790 was amplified by PCR using ExTaq DNA polymerase (Takara, Kyoto, Japan). The resulting PCR products were directly sequenced using a dye terminator cycle sequencing kit and an ABI PRISM3100 sequencer (Perkin-Elmer, Norwalk, USA).
For complementation of the crr2-2 mutation, the genomic sequence containing wild-type At3g46790 was amplified using primers, 5′-GATCCAAAGGAATGGTTGTA-3′ and 5′-GCAGAAACTGACTACTCTTG-3′, and cloned in pBIN19. The resulting plasmid was introduced into Agrobacterium tumefaciens and then into crr2-2.
Analysis of protein and RNA
Chloroplast proteins were fractionated by 10% SDS–PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and detected using the enhanced chemiluminescence Western blotting kit from Amersham Pharmacia Biotech (Piscataway, USA).
Total RNA was isolated from rosette leaves and subjected to Northern analysis essentially as described by Sambrook et al. (1989). The fragments used as probes were obtained by PCR amplification using oligonucleotides: 5′-ACTCTCCCACTCCAGTCGTTG-3′ and 5′-CCTGAGCAATCGCAATAATCGG-3′ for ndhB 3′ exon; 5′-GGTTCGTTTGAGAAATTCCTACC-3′ and 5′-GAGTCGAAAAGAGGATTCCTCAC-3′ for ndhB intron; and 5′-TCATGTCACGCCCGAGGTACTG-3′ and 5′-GTGCAAAAGCTCTATTTGCCTC-3′ for rps7.
RNase protection assay was performed using an RPA II kit (Ambion, Austin, USA). The rps7/ndhB intergenic sequences were amplified using ndhB reverse primer 5′-TCCATCGAAGAGAAGCAAATG-3′ in combination with two sets of rps7 forward primers 5′-AGGAAAAGCACTTGCCATTCG-3′ (for long probe) and 5′-TTCCGAATTAGTGGATGCTGC-3′ (for short probe), and were subcloned in pGEM-T Easy (Promega, Madison, USA). RNA probes were generated using a SP6/T7 transcription kit (Roche, Mannheim, Germany).
The 5′ end of the monocistronic ndhB RNA was determined using a 5′ RACE system (Invitrogen, Carlsbad, USA). Primers complementary to ndhB RNA are 5′-ACTGGAGTGGGAGATCCTTC-3′, 5′-TTGAACCCAATTCCTACAGTG-3′, and 5′-AACCAGAATAGAAGAGCTTGC-3′. The first primer was designed to bridge two exons to avoid hybridization to the unspliced precursor RNA.
We thank Momoko Miyata for her excellent technical assistance. We appreciate Isao Enami for his gift of antibodies. This work was supported by the Research for the Future Program (JSPS-RFTF00L01604) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (15370024).