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First published online July 8, 2009; 10.1104/pp.109.139683 Plant Physiology 151:114-128 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Phosphofructokinase B-Type Carbohydrate Kinase Family Protein, NARA5, for Massive Expressions of Plastid-Encoded Photosynthetic Genes in Arabidopsis1,[W],[OA]Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630–0101, Japan (T.O., K.N., T.A., H.A., A.Y.); and Research Institute of Innovative Technology for the Earth, Kizugawa, Kyoto 619–0292, Japan (H.T., K.-I.T.)
To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Here, we describe a screening method based on the toxic accumulation of ammonia in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase, during photorespiration initiated by the oxygenase reaction of Rubisco in Arabidopsis (Arabidopsis thaliana). Five recessive mutants with decreased amounts of Rubisco were identified and designated as nara mutants, as they contained a mutation in genes necessary for the achievement of Rubisco accumulation. The nara5-1 mutant showed markedly lower levels of plastid-encoded photosynthetic proteins, including Rubisco. Map-based cloning revealed that NARA5 encoded a chloroplast phosphofructokinase B-type carbohydrate kinase family protein of unknown function. The NARA5 protein fused to green fluorescent protein localized in chloroplasts. We conducted expression analyses of photosynthetic genes during light-induced greening of etiolated seedlings of nara5-1 and the T-DNA insertion mutant, nara5-2. Our results strongly suggest that NARA5 is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome, particularly rbcL.
Plastids are DNA-containing organelles that show several types of morphology and functions depending on environmental stimuli and tissue specificity (López-Juez and Pyke, 2005  ). Chloroplasts are one type of plastid that accumulate massive amounts of proteins that are essential for photosynthesis. The enzyme responsible for photosynthetic CO2 fixation is Rubisco. This enzyme accounts for 20% to 35% of total leaf nitrogen in higher plants and is the most abundant protein on earth (Ellis, 1979; Evans, 1989). The complex transcriptional, translational, and folding-assembly machineries in chloroplasts, therefore, have evolved to support this massive accumulation of photosynthetic proteins (Mullet, 1993; Mayfield et al., 1995; López-Juez and Pyke, 2005).
Cyanobacteria have approximately 3,000 to 7,000 genes in their genomes and produce sufficient proteins for cells to adapt to their environment. The chloroplast genome contains only about 100 genes, and the genes for the remaining 3,000 chloroplast proteins are located in the chromosomal DNA (Leister, 2003
The concentration of the Rubisco protein in the stroma of chloroplasts is approximately 200 mg mL–1 (Yokota and Shigeoka, 2008 To understand the coordinated synthesis of Rubisco and other photosynthetic proteins in chloroplasts, we can use new screening techniques to isolate mutant plants. To our knowledge, screening for mutants that are defective in the large-scale accumulation of Rubisco has never been reported. Here, we describe a method to screen for Arabidopsis (Arabidopsis thaliana) mutants that cannot accumulate high concentrations of Rubisco due to a mutation in a gene necessary for the achievement of Rubisco accumulation (nara). The method relies on the toxic functions of NH3 that accumulate in the presence of Met sulfoximine (MSX), a specific inhibitor of Gln synthetase (GS), during photorespiration initiated by the oxygenase reaction of Rubisco. Of the five mutants with decreased amounts of Rubisco, we selected the mutant with the lowest amount of Rubisco in chloroplasts for detailed analyses. This mutant was defective in the function of a phosphofructokinase B (pfkB)-type carbohydrate kinase family protein that is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome.
Evaluation of the Screening Method
It has been estimated that Rubisco catalyzes the oxygenase reaction in photosynthesizing Arabidopsis leaves at 13 µmol g–1 fresh weight h–1 (Sweetlove et al., 2006
The wild type and the rca mutant of Arabidopsis were grown in air supplemented with 5% CO2 at 50 µmol photons m–2 s–1 for 2 weeks. The medium contained 30 mM Gln with or without 50 µM MSX (Fig. 2A ). The plants were then transferred to ambient air and grown at 130 to 190 µmol photons m–2 s–1. The rca mutant was used as the positive control in our screening experiments. This mutant lacks Rubisco activase, which promotes carbamylation of the Rubisco active sites (Somerville et al., 1982 ; Orozco et al., 1993). There was no visible effect of MSX on the wild type and rca in the presence of 5% CO2 at both the low and high light intensities (Fig. 2A, left; Supplemental Fig. S1A). After wild-type plants were transferred to ambient air conditions with high light intensity, plants grown on MSX-containing medium died within 100 h (Fig. 2A, right). At low light intensity, however, the wild-type plants grown in ambient air conditions grew, although slowly (Supplemental Fig. S1B). The inhibitory effects of MSX on wild-type plants indicate that the Rubisco oxygenase reaction has a role in the observed growth inhibition. Consistent with this, the growth of the rca mutant, which has little Rubisco activity in air (Salvucci et al., 1986), was not visibly affected by MSX. These results suggest that MSX-resistant Arabidopsis mutants screened under photorespiratory conditions have lower activities of the oxygenase reaction of Rubisco.
To examine the inhibitory effect of MSX on GS activity under these screening conditions, we measured the amount of NH4+ in plants grown in the presence of 50 µM MSX under photorespiratory conditions (Fig. 2B). In wild-type plants, the level of NH4+ was constantly low at approximately 4 µmol g–1 fresh weight in the presence of 5% CO2 at 50 µmol photons m–2 s–1. In wild-type plants, this level increased to approximately 210 µmol g–1 fresh weight at illumination levels of 130 to 190 µmol photons m–2 s–1 in ambient air, but in rca mutants grown under the same conditions, the level increased to only 30 µmol g–1 fresh weight. The rate of NH3 release from wild-type plants was 4 to 5 µmol g–1 fresh weight h–1. The absence of MSX did not cause any increase in NH3 in these experiments.
Sterilized M2 seeds (approximately 20,000) of ethylmethanesulfonate (EMS)-treated Arabidopsis were germinated on agar plates to screen for mutants with lower levels of Rubisco. Seedlings were grown for 14 d under nonphotorespiratory conditions, then were grown under screening conditions for 100 to 150 h to select for mutants that survived the photorespiratory conditions (Figs. 1B and 3 ). The frequency of MSX-resistant plants was two to three per 1,000 seeds, but many failed to grow or were sterile after transplanting into soil. We collected M3 seeds of 12 MSX-resistant mutants and assessed repeatability of resistance (Fig. 3). Five of the selected mutants showed the recessive phenotype. We measured fresh weight of aerial parts, Rubisco activity, and amount of Rubisco protein in 14-d-old mutant lines. Compared with the wild type, the mutants were smaller and had lower fresh weights (Fig. 4A ). Lines 4 and 5 contained 30% and 20% of the amount of Rubisco in wild-type plants, respectively (Fig. 4B). Accumulation of Rubisco was moderately suppressed in lines 3, 7, and 10. Although rca had a lower activation state of Rubisco, this was not the case in the mutant lines selected in this study. The observed lower initial and total activities of Rubisco were due to decreased amounts of the Rubisco protein in these mutants (Fig. 4B). These mutants were designated as nara mutants.
To examine genetic dominance and genetic segregation of the mutated genes, these mutants were crossed with the wild-type Landsberg erecta. Plants of all F1 progeny showed no detectable growth phenotype, but the mutant phenotype appeared in one-third of plants of nara3 to nara5, nara7, and nara10 in F2 progeny (Table I ). This result indicates that phenotypes in these mutants are due to recessive mutations at single genetic loci.
Map-Based Cloning of Genetic Loci in the nara Mutants
We identified genetic loci by map-based cloning of the mutations in the nara3, -5, -7, and -10 mutants. We found a T-to-C substitution at +2,047 of the At5g16180 coding sequence in nara3 (Table II
; Supplemental Fig. S2). At5g16180 encodes Chloroplast RNA Splicing1 (AtCRS1), which participates in splicing of the intron in atpF and protein translation in chloroplasts (Till et al., 2001
The nara5 mutant showed a severely impaired growth phenotype with a very low level of Rubisco. Map-based cloning showed that the genetic locus of NARA5 was in a 120-kb-long genomic sequence located between two polymorphic markers on the two bacterial artificial chromosome clones, M4I22 and T27E11, on chromosome IV. Sequencing of genes with annotations and plastid-leading sequences in this region revealed a G-to-A substitution at +626 of the At4g27600 coding sequence (Fig. 5A ; Supplemental Fig. S2). This substitution changes Gly at the 209th residue to Glu (Fig. 5, A and B). We introduced the full-length cDNA of At4g27600 under the control of the cauliflower mosaic virus 35S promoter into the nara5 mutant. This resulted in complete recoveries in appearance and Rubisco content to those of the wild type (Fig. 6, A and C ). Thus, we concluded that the phenotype observed in the nara5 mutant was due to the point mutation in At4g27600. At4g27600 encodes a 471-amino acid pfkB-type carbohydrate kinase family protein with unknown function, as described later. This newly found protein was named NARA5.
Map-based cloning of the nara7 mutant identified a G-to-A substitution at +1,156 of the At2g13360 coding sequence (Table II). At2g13360 encodes alanine:glyoxylate aminotransferase1 (AGT1), which catalyzes the aminotransferase reaction between Ser and glyoxylate in the photorespiratory pathway (Fig. 1A). Growth inhibition of the nara7 mutant was completely removed by 0.3% CO2, as observed previously in an AGT1-deficient mutant (Somerville and Somerville, 1980 ; Liepman and Olsen, 2001). The mutation in At2g13360 caused AtAGT1 to have Thr in place of a highly conserved Ala at the 386th residue. The similar severe phenotypes of the nara7 mutant and AGT1-deficient mutants (Supplemental Fig. S3) suggest that Ala-386 has a pervasive influence on AGT1 activity.
The nara10 mutant had a C-to-T point mutation at +929 in At5g64050, which encodes glutamyl-tRNA synthetase (GluRS; Table II). This mutation substituted Phe for Ser at the 310th residue, which is highly conserved among GluRS enzymes of other organisms. The GluRS is essential for protein synthesis in both chloroplasts and mitochondria and for chlorophyll synthesis in chloroplasts (Duchene et al., 2005 Of all the nara mutants, nara5 showed the greatest decrease in Rubisco content; therefore, it was selected for further detailed analyses and characterization.
To predict the function of NARA5, homologous genes were surveyed using the deduced whole amino acid sequence as the query in the National Center for Biotechnology Information genome database (http://www.ncbi.nlm.nih.gov/). Multiple alignments revealed that NARA5 showed 67%, 61%, and 51% amino acid sequence identity to Vitis vinifera hypothetical protein (GenBank: CAN82032), Oryza sativa hypothetical protein (GenBank: NP_001065468), and Physcomitrella patens predicted protein (GenBank: XP_001764628), respectively (Fig. 5B). Gly-209 was completely conserved in these sequences. The sequences most homologous to NARA5 in the Arabidopsis genome were a pfkB-type carbohydrate kinase family protein (At1g19600) and adenosine kinase 2 (Moffatt et al., 2000
NARA5 had two conserved motifs, the NXXE motif (Maj et al., 2002
We analyzed another nara5 mutant to understand the physiological significance of NARA5. The GABI-KAT line 718C05 was tagged by T-DNA in the first intron of the NARA5 gene (Fig. 5A). The tagged mutant was a null mutant of NARA5 (Fig. 6, A and B). An allelism test by outcrossing the nara5 homozygote and the heterozygote of the tagged mutant indicated that line 718C05 was a mutant allele of nara5 (Supplemental Fig. S4). The nara5 EMS mutant was renamed nara5-1, and the T-DNA insertion mutant was renamed nara5-2. Compared with nara5-1, nara5-2 was yellower and smaller when grown heterotrophically on Suc and could not grow autotrophically on soil. The phenotype of nara5-2 was recovered by introducing the full-length NARA5 fused to a His tag and to synthetic GFP (sGFP) at the C terminus (Fig. 6A). Thus, NARA5 encodes a gene that is essential for autotrophic photosynthetic growth of Arabidopsis, and nara5-1 was a leaky mutant with a normal transcription level of nara5 (Fig. 6B).
In 8-d-old wild-type plants, the NARA5 mRNA content was 65 times higher in illuminated shoots than in roots. In shoots, the mRNA levels were highest in rosette leaves, with 20% lower levels in the cauline leaves and inflorescence tissues (Supplemental Fig. S5). NARA5 mRNA levels were lowest in the stems. This expression pattern is comparable to that of the Rubisco gene transcripts in higher plants (Manzara and Gruissem, 1988 The long N-terminal nonconserved sequence (+1 to +117) of NARA5 and its function proposed in this study suggest that NARA5 is localized in chloroplasts (Fig. 5B). Indeed, TargetP predicted that the N-terminal sequence was a chloroplast transit sequence. To analyze the localization of NARA5, we introduced a binary vector containing the sGFP gene fused to the C terminus of the full-length NARA5 sequence under the control of the cauliflower mosaic virus 35S promoter into plants with the nara5 alleles. The NARA5-sGFP fusion protein completely complemented the phenotype and the Rubisco content in the mutants (Fig. 6A). NARA5-sGFP signals were visualized as green spots clearly superimposed on the red fluorescence from protochlorophyllide and chlorophyll (Fig. 7 ). These results suggest that NARA5 is localized in chloroplasts.
Phenotype Analysis of nara5 Mutants
We compared chlorophyll fluorescence parameters between the wild type and nara5-1 (Table III
). Compared with the wild type, the maximum quantum yield (Fv/Fm) and the quantum efficiency of PSII (
Western blotting of chloroplast proteins encoded by the chloroplast and chromosomal genomes in both alleles of nara5 is shown in Figure 6. Compared with the wild type, the levels of D1, D2, cytochrome (Cyt) b6, and PsaA, as well as LSU of Rubisco, all encoded by the plastid genome, were reduced by approximately 80% in nara5-1 and by up to 90% in nara5-2. Such severe reductions in levels of photosynthetic proteins other than Rubisco were not observed in the rbcL-deficient mutant of tobacco (Allahverdiyeva et al., 2005 ). In addition to these plastid-encoded proteins, the nucleus-encoded  -subunit of ATP synthase showed similarly reduced levels. In protein complexes such as ATP synthase, which include subunits encoded by both nuclear and plastid genomes, the expression of the -subunit gene in nuclei may have been affected by expression of the partner gene in chloroplasts. On the other hand, the expression of the nucleus-encoded chloroplast proteins Fru-bisP aldolase (FBA) and ferredoxin-NADP+ oxidoreductase (FNR) was unaffected in these mutants. These results suggest that NARA5 is involved in the synthesis and/or maintenance of proteins coded by the plastid genome.
During greening of leaves, large amounts of photosynthetic proteins including Rubisco are synthesized in a relatively short time (Sasaki et al., 1985
To determine the mechanism by which expression of plastid-encoded photosynthetic genes is repressed in nara5 mutants, we measured transcript levels of these genes in dark-grown and greening nara5 mutants using real-time PCR. Since these photosynthetic genes are transcribed by PEP, we also measured transcripts of clpP, a plastid-encoded gene transcribed by PEP and nucleus-encoded RNA polymerase (NEP), and rpoB transcribed only by NEP (Fig. 8B). Expression data are relative values and are compared with the level in dark-grown wild-type plants at time 0. The expression of genes transcribed by PEP was more severely suppressed in nara5 mutants than those transcribed by NEP/PEP and NEP. Among the genes examined, the transcript level of rbcL was most severely suppressed in nara5 alleles. The transcript level of the nucleus-encoded RbcS in nara mutants was lowered after 10 h of illumination compared with the wild type. This may have been caused by the suppression of plastid gene transcription in the mutant, as has been reported (Rapp and Mullet, 1991 ). The severe suppression of rbcL transcription may have been a major cause of the decreased Rubisco accumulation in nara5 mutants. Next, we examined the effect of greening on expression of the NARA5 gene (Fig. 8C). A basal level of NARA5 transcripts was found in etiolated cotyledons, but this level increased 4.5-fold during greening under illumination for 60 h. Together, these results indicate that NARA5 has a role in expression of PEP-transcribed photosynthetic genes, particularly rbcL.
Here, we describe a new method to screen for Arabidopsis mutants with decreased Rubisco activity (Fig. 1). We identified mutants with low Rubisco activity by selecting plants that were resistant to the inhibitory effects of MSX on photorespiratory metabolism. Wild-type Arabidopsis plants are killed by MSX as a result of excessive NH3 accumulation from photorespiration, which relies on the Rubisco oxygenase activity. The rate of accumulation of NH3 by the wild type in the presence of MSX (Fig. 2B) was similar to that reported for glycolate metabolism in Arabidopsis (Sweetlove et al., 2006 ). Both NH3 and its ionized form NH4+ are harmful to plant growth (van der Eerden, 1982; Britto and Kronzucker 2002). The rca mutant survived in the presence of MSX (Fig. 2), because this mutant has lower Rubisco activity in vivo (Fig. 4), as reported previously (Salvucci et al., 1986; Eckardt et al., 1997). On the other hand, wild-type plants were resistant to MSX when grown with higher concentrations of CO2 (i.e. when the Rubisco oxygenase reaction is blocked by competitive inhibition of CO2; Fig. 2A). The five mutants screened in this study had lower levels of Rubisco protein compared with wild-type Arabidopsis (Fig. 4). These results indicate that our positive screening method can identify mutants defective in various factors associated with activity or biosynthesis of Rubisco.
The reduced Rubisco levels in the nara3, -4, -5, -7, and -10 mutants examined were accompanied by impaired growth phenotypes and yellow/pale green foliage (Fig. 3). Mutants with similar phenotypes can occur without influence on Rubisco content. For example, Rubisco levels in the chaperonin 60β-subunit and Hsp100-deficient mutants were relatively unchanged (Ishikawa et al., 2003
Among the nara mutants, nara5 showed the most severely defective phenotype for accumulation of plastid-encoded photosynthetic proteins, including Rubisco (Figs. 4, 6, and 8). Many mutants of higher plants that show severely decreased amounts of photosynthetic proteins are defective in factors involved directly in the biosynthetic process. Mutants with defects in genes encoding BSD2, Toc159, and APG3, which participate in either translational or posttranslational processes in chloroplasts, show albino or yellowish phenotypes and accumulate very low levels of Rubisco (Brutnell et al., 1999
The pfkB family of proteins contains many carbohydrate kinases, such as RK, ADK, GSK, fructokinase, tagatose-6-P kinase, and others (Bork et al., 1993
Although the catalytic function of NARA5 is unclear, our data suggest two possible roles for this protein in influencing expression of chloroplast genes. First, NARA5 may be involved in the PEP-dependent transcription of plastid-encoded photosynthetic genes. In support of this, two pfkB-type carbohydrate kinase family proteins coded by At1g69200 and At3g54090 were identified together with PEP subunits in a transcriptionally active chromosome complex from Arabidopsis and mustard (Sinapis alba) chloroplasts (Pfalz et al., 2006
The second possibility is that NARA5 may stabilize transcripts of photosynthetic genes, mainly rbcL. Stabilization of mRNA is an important factor in defining transcript levels in chloroplasts. It has been known that trans-acting factors bind to cis-sequences in the target mRNAs and protect them from degradation by endoribonucleases and exoribonucleases (Herrin and Nickelsen, 2004 In summary, NARA5 is a pfkB-type carbohydrate kinase family protein that is indispensable for massive accumulation of plastid-encoded photosynthetic proteins. Our findings suggest that it participates in the transcription and/or posttranscriptional steps of photosynthetic gene expression such as rbcL in chloroplasts. Future studies will also continue to analyze these proteins and screen for additional Rubisco-deficient nara mutants in a bid to identify key proteins that regulate its synthesis (and that of other proteins) in chloroplasts to expand our understanding of this complex process.
Plant Materials
We used Arabidopsis (Arabidopsis thaliana ecotype Columbia [Col-0]) as the wild type, the mutant lines rca (Somerville et al., 1982
We screened nara mutants by uniformly spreading 40 to 50 seeds of EMS-mutagenized M2 plants in the Col-0 genetic background (Lehle Seeds) on selective plates. The selection medium was NH4NO3- and KNO3-free Murashige and Skoog medium supplemented with 50 µM MSX, 30 mM Gln, 3% (w/v) Suc, 19 mM KCl, 0.5 g L–1 MES (pH 5.7), 0.8 µM nicotinic acid, 0.4 µM pyridoxine hydrochloride, 2.4 µM thiamine hydrochloride, 550 µM myoinositol, and 0.8% (w/v) agar. Plants were initially grown for 2 to 3 weeks in the presence of 5% CO2 under a 20-h photoperiod (50 µmol photons m–2 s–1)/4 h of dark at 23°C. Plates were then transferred to continuous irradiation at 130 to 190 µmol photons m–2 s–1 in ambient air. After 5 to 6 d, MSX-resistant mutants with green or pale green leaves were selected. Plants were then grown on selection medium without MSX for 2 to 3 weeks and then on soil (Metro-Mix 350; Scot-Sierra Horticultural Products) until self-fertilization, both in 5% CO2.
Homozygous nara mutants were crossed with Landsberg erecta ecotypes. The F1 plants were self-pollinated, and the resulting F2 plants were used for segregation analyses and mapping of nara mutations. Genomic DNA was isolated according to Edwards et al. (1991)
For expression of NARA5, NARA5-sGFP, and NARA5-His in plants with nara5 alleles, a series of Gateway binary vectors (pGWBs) were used as described by Nakagawa et al. (2007)
Shoots were homogenized in liquid nitrogen with a mortar and pestle and then dissolved in extraction buffer (50 mM HEPES-KOH [pH 8.0], 1 mM EDTA, 1 mM dithiothreitol [DTT], 1 mM MgCl2, and 2 mM phenylmethylsulfonyl fluoride). Rubisco activity was spectrophotometrically assayed by measuring the rate of NADH oxidation in the presence of phosphoglycerate kinase and glyceraldehyde 3-P dehydrogenase (Sharkey et al., 1991 To determine Rubisco content, shoots were homogenized on ice with a mortar and pestle and dissolved in extraction buffer (50 mM HEPES-KOH [pH 8.0], 1 mM EDTA, 1 mM DTT, and 1 mM MgCl2) supplemented with complete protease inhibitor cocktail tablets (Roche). The homogenate was then centrifuged at 18,800g for 20 min at 4°C. Total proteins in the supernatant were separated by native-PAGE on a 3% to 10% gradient gel (PAGEL NPG-310L; ATTO) at 4°C. Gels were stained with Coomassie Brilliant Blue R-250. The prominent band intensity corresponding to the Rubisco holoenzyme was measured by a scanning densitometer and quantified using NIH Image software (National Institutes of Health). To compare the amount of Rubisco among the mutants, we constructed a calibration curve from the staining intensities of Rubisco protein bands of wild-type plants. For each mutant, we calculated relative amounts of Rubisco per 2 µg of total soluble protein and then expressed the amount as a percentage of that in wild-type plants.
Extraction of proteins from shoots was modified slightly depending on the localization of individual proteins in plant cells. Plant shoots were homogenized and centrifuged as described above. Soluble proteins in the supernatant were quantified as described by Bradford (1976)
Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen). We treated RNA with FPLC-pure DNase I (Amersham Pharmacia Biotech) for 30 min at 37°C, and then RNA was purified by phenol/chloroform extraction and ethanol precipitation. Single-strand cDNA was synthesized from 0.5 µg of total RNA using an oligo(dT) primer and ReverTraAce (Toyobo), except that cDNA of plastid-encoded genes was synthesized using a random primer (Toyobo) in place of the oligo(dT) primer. Semiquantitative reverse transcription (RT)-PCR was performed using the following specific primer pairs: for NARA5, 5'-TGAATTCATACAAGTCCATGCTAAT-3'/5'-TCATCCACAACTCCAGAGAAATC-3'; for Act8, 5'-GACATCGTTTCCATGACGGGATCA-3'/5'-CGCTGTAACCGGAAAGTTTCTCAC-3'.
Quantitative RT-PCR was carried out using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Each 25-µL PCR mixture contained gene-specific primer sets (0.2 µM each), 12.5 µL of Power SYBR Green PCR Master Mix (Applied Biosystems), and 1 µL of a 64-fold dilution of the cDNA. Thermal cycling consisted of an initial step at 95°C for 10 min, followed by 40 cycles of 5 s at 95°C and 20 s at 60°C. The following specific primers were used: for rbcL, 5'-TGACCGAGATCTTTGGAGATGA-3'/5'-CAAGATCACGTCCCTCATTACG-3'; for RbcS, 5'-CCGCTCAAGTGTTGAAGGAAG-3'/5'-GGCTTGTAGGCAATGAAACTGA-3'; for petB, 5'-AGTGCTAGTGTTGGACAATCCAC-3'/5'-AGGGACCAGAAATACCTTGCTTAC-3'. Amplicon size and specificity for each primer pair were confirmed by gel electrophoresis. Quantitative RT-PCR of psbA was conducted with primers described by Loschelder et al. (2006)
Chlorophyll fluorescence parameters were measured with a MINI-PAM portable chlorophyll fluorometer (Walz) as described elsewhere (Munekage et al., 2002
NARA5-sGFP fluorescence was observed with an LSM510 confocal laser scanning microscope (Carl Zeiss Microimaging). An argon laser was used as the excitation light source (488 nm), and sGFP signal was detected at an emission band of 500 to 530 nm. Chlorophyll autofluorescence was observed using an LP 650 optical filter.
The following materials are available in the online version of this article.
We are grateful to Professor Archie R. Portis Jr. (University of Illinois) for providing us with rca seeds, Professor Toru Hisabori (Tokyo Institute of Technology) for the antibodies against the -subunit of ATP synthase, Professor Toshiharu Hase (Osaka University) for FNR, Dr. Tsuyoshi Nakagawa (Shimane University) for the pGWB series plasmids, and Dr. Miyo Terao Morita (Nara Institute of Science and Technology) for help with fine-mapping. We also gratefully acknowledge the generous supply of the nara5-2 line by the GABI-Kat team at the Max-Planck-Institute fuer Zuechtungsforschung. We thank Mayumi Aihara (our laboratory) for technical assistance. Received April 8, 2009; accepted July 2, 2009; published July 8, 2009.
1 This work was supported by the Japan Society for the Promotion of Science (grant nos. 17208031 and 18688021 for Scientific Research), by the Asahi Glass Foundation (grant no. FY2004–2006 for General Science and Technology), and by the Nissan Science Foundation (grant no. FY2005–2007). 
2 Present address: Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1–1 Nanjyo, Sogabe, Kameoka, Kyoto 621–8555, Japan.
3 Present address: Plant High Technology Institute, Ltd., Takayama Science Plaza, 8916–12 Takayama, Ikoma, Nara 630–0101, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Akiho Yokota (yokota{at}bs.naist.jp).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.139683 * Corresponding author; e-mail yokota{at}bs.naist.jp.
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ABSTRACT
RESULTS
PSII) were severely reduced in the nara5-1 mutant, and the reduction level of the primary electron-accepting plastoquinone of PSII (1 – qP), was increased. These results suggest that, in addition to Rubisco accumulation, photochemical activities are also impaired in nara5-1. 