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Keywords:

  • sigma factor;
  • photosystem I;
  • chloroplast;
  • RNA polymerase;
  • transcription;
  • rice

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sigma factors encoded by the nucleus of plants confer promoter specificity on the bacterial-type RNA polymerase in chloroplasts. We previously showed that transcripts of OsSIG1, which encodes one such sigma factor in rice, accumulate relatively late during leaf development. We have now isolated and characterized two allelic mutants of OsSIG1, in which OsSIG1 is disrupted by insertion of the retrotransposon Tos17, in order to characterize the functions of OsSIG1. The OsSIG1−/− plants were found to be fertile but they manifested an approximately one-third reduction in the chlorophyll content of mature leaves. Quantitative RT-PCR and northern blot analyses of chloroplast gene expression revealed that the abundance of transcripts derived from the psaA operon was markedly reduced in OsSIG1−/− plants compared with that in wild-type homozygotes. This effect was accompanied by a reduction in the abundance of the core protein complex (PsaA–PsaB) of photosystem I. Analysis of chlorophyll fluorescence also revealed a substantial reduction in the rate of electron transfer from photosystem II to photosystem I in the OsSIG1 mutants. Our results thus indicate that OsSIG1 plays an important role in the maintenance of photosynthetic activity in mature chloroplasts of rice by regulating expression of chloroplast genes for components of photosystem I.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Gene expression in plastids is mediated by at least two different transcriptional systems based on a plastid-encoded RNA polymerase (PEP) and a nucleus-encoded RNA polymerase (NEP; Shiina et al., 2005; Hajdukiewicz et al., 1997; Hedtke et al., 1997). Plastid-encoded RNA polymerase is a multisubunit eubacterial-type RNA polymerase, with the core subunits being encoded by the plastid genes rpoA, rpoB, rpoC1 and rpoC2. Nucleus-encoded RNA polymerase is a single-subunit bacteriophage-type enzyme that is encoded by a nuclear gene and is similar to a mitochondrial RNA polymerase. Analysis of PEP-deficient plants has suggested that PEP functions predominantly in the expression of photosynthetic genes and that NEP transcribes non-photosynthetic genes in plastids (Liere and Maliga, 1999). Plastid-encoded RNA polymerase requires sigma factors, which are encoded by the nuclear genome, for promoter recognition and initiation of transcription at specific genes. To date, six sigma factors (SIG1, SIG2, SIG3, SIG4, SIG5 and SIG6) have been identified and characterized in the model dicotyledonous plant Arabidopsis thaliana (Fujiwara et al., 2000; Isono et al., 1997; Tanaka et al., 1997). For the model monocotyledonous plant rice (Oryza sativa), six sigma factor genes, OsSIG1 (Os-SigA), OsSIG2A, OsSIG2B, OsSIG3, OsSIG5 and OsSIG6, have also been isolated (Kasai et al., 2004; Kubota et al., 2007; Tozawa et al., 1998) or predicted from the draft sequence of the rice genome and the full-length cDNA sequencing project (Knowledge-Based Oryza Molecular Biological Encyclopedia, http://cdna01.dna.affrc.go.jp/cDNA/CDNA_main_front.html).

Recent studies of gene-disrupted mutants have provided insight into the roles of sigma factors SIG2 to SIG6 of Arabidopsis (Favory et al., 2005; Hanaoka et al., 2003; Ishizaki et al., 2005; Kanamaru et al., 2001; Loschelder et al., 2006; Shirano et al., 2000; Tsunoyama et al., 2004; Yao et al., 2003; Zghidi et al., 2006). SIG2 has thus been shown to regulate the expression of plastid tRNA genes during early development (Hanaoka et al., 2003; Kanamaru et al., 2001). SIG3 and SIG4 contribute specifically to the transcription of psbN (Zghidi et al., 2006) and ndhF (Favory et al., 2005), respectively. SIG5 controls the expression of psbD in response to blue light and various stress conditions such as high light, low temperature, high salt and high osmolarity (Tsunoyama et al., 2004). SIG6 modulates the transcription of many plastid genes such as psaA, psbA, psbD, rbcL, rrn16 and atpB, with the phenotype of SIG6 deficiency being most prominent at the cotyledon stage (Ishizaki et al., 2005; Loschelder et al., 2006).

To clarify the functional roles of SIG1 in chloroplast gene regulation, we screened for and isolated rice SIG1 (OsSIG1) mutants and examined them for chloroplast gene expression. We now describe the phenotypic characterization of OsSIG1mutants and the identification of plastid genes regulated by OsSIG1.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Reduced chlorophyll content in the leaves of rice SIG1-deficient mutants

We screened a rice mutant collection for OsSIG1 mutants by PCR with specific primers (see Experimental procedures) as described (Sato et al., 1999). Of the 27 840 lines screened, 15 mutants with an insertion of the retrotransposon Tos17 at the OsSIG1 locus were identified. Three of these mutants (NE3119, NE8184, NE8287) were found to harbor the insertion in an exon, resulting in disruption of the OsSIG1 open reading frame (ORF). The positions of the Tos17 insertion in two of these latter strains, NE8184 and NE8287, were identical (Figure 1a). The OsSIG1 ORF in NE3119 was disrupted at codon 409, the amino acid encoded by which is located in region 3.1 of OsSIG1 (Helmann and Chamberlin, 1988; Lonetto et al., 1992; Tanaka et al., 1997; Tozawa et al., 1998); the mutant protein would thus be expected to contain 408 residues of OsSIG1 followed by seven amino acids (aa) encoded by the inserted Tos17 sequence. In the case of NE8184 and NE8287, the ORF was disrupted at codon 469, the amino acid encoded by which is located in region 4.1 of OsSIG1 (Tanaka et al., 1997; Tozawa et al., 1998); the mutant protein would thus be expected to contain 468 residues of OsSIG1 followed by the 7 aa encoded by Tos17. In both NE3119 and NE8184 lines, the progeny of heterozygous OsSIG1 mutant (OsSIG1+/−) plants included fertile OsSIG1 mutant homozygotes (OsSIG1−/−), indicating that an intact OsSIG1 protein is dispensable for normal development of rice, at least under our experimental conditions. Northern analysis with an OsSIG1 riboprobe and total RNA isolated from mature leaves revealed that the hybridizing transcript was larger (∼6.2 versus ∼2.1 kb) and greatly reduced in abundance in NE3119 mutant homozygotes compared with that in wild-type (WT) homozygotes (Figure 1b). In contrast, the hybridizing transcript was slightly smaller (∼2.0 kb) and only slightly reduced in abundance in NE8184 mutant homozygotes compared with that in WT homozygotes.

image

Figure 1.  Phenotypic characterization of OsSIG1 mutants. (a) Schematic representation of Tos17 insertion into the OsSIG1 locus of NE3119, NE8184 or NE8287 lines as determined by sequence analysis of DNA fragments amplified by PCR with the primers T17LTR7 F and SigR5. Black boxes represent exons of OsSIG1. (b) Northern analysis of OsSIG1 transcripts. Total RNA (5 μg) isolated from leaves of OsSIG1+/+ or OsSIG1−/− plants of the NE8184 or NE3119 lines was subjected to hybridization with riboprobes specific for transcripts of OsSIG1 or Rac2 (internal control). (c) Leaf color of OsSIG1+/+, OsSIG1+/− or OsSIG1−/− plants of the NE8184 or NE3119 lines. (d) Chlorophyll (b) content in the leaves of OsSIG1+/+ or OsSIG1−/− plants. Progeny seedlings derived from NE3119 (OsSIG1+/−), NE8184 (OsSIG1+/−) or Nipponbare parents and grown at 25°C for 6 weeks under a 16-h light, 8-h dark cycle were analyzed for leaf chlorophyll content. The genotype of each seedling was determined by PCR with the primers used for screening of OsSIG1 mutants. Data are means ± SD of values determined for 40 seedlings of each genotype. (e) Chlorophyll content of OsSIG1 mutants. The chlorophyll a and chlorophyll b contents of leaves from 6-week-old plants were determined for progeny of self-fertilized OsSIG1 mutant heterozygotes of the NE3119 or NE8184 lines. Nipponbare progeny were analyzed as controls.

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The leaf color of OsSIG1−/− progeny of both NE3119 and NE8184 lines was a lighter green than that of their WT (OsSIG1+/+) or heterozygous (OsSIG1+/−) counterparts (Figure 1c). This phenotype of OsSIG1−/− plants was apparent for all expanded leaves, from the seedling stage to that of flag leaf formation. We measured the chlorophyll content of leaf extracts prepared from the different offspring derived from OsSIG1 heterozygotes. The amount of chlorophyll (b) was indeed decreased in the mutant homozygotes to 59% (NE3119) or 65% (NE8184) of the value for WT homozygotes (Figure 1d). The progeny of self-fertilized mutant heterozygotes were examined for the relation between segregation of OsSIG1 and that of chlorophyll content. The 40 progeny each of NE3119 or NE8184 heterozygotes segregated in the Mendelian 1:2:1 ratio in terms of OsSIG1 genotype (data not shown). Furthermore, the segregation of chlorophyll content correlated with that of OsSIG1 genotype, showing that loss of OsSIG1 resulted in a reduction in chlorophyll content (Figure 1e). These results thus indicated that the observed phenotype of the NE3119 and NE8184 lines was attributable to the insertion of Tos17 into the ORF of OsSIG1.

Impaired expression of specific chloroplast genes in OsSIG1 mutants

We previously showed that the maximal level of OsSIG1 expression occurs at a later stage of leaf development than does that of OsSIG2A or OsSIG2B expression (Kasai et al., 2004). To characterize further the expression of OsSIG1, we performed northern analysis with total RNA isolated from three portions of the expanded leaves of 20-day-old Nipponbare (WT) plants. The amount of OsSIG1 mRNA was highest in the middle region of the leaf blades (Figure 2). To examine OsSIG1-dependent gene expression in chloroplasts, we prepared total RNA from the middle region of the leaves of 20-day-old plants and performed quantitative RT-PCR analysis with 81 pairs of gene-specific primers (see Table S1 in Supplementary Material). From initial amplification tests with NE3119 progeny (OsSIG1+/+, OsSIG1−/−), we selected 29 primer sets that yielded reproducible results and performed further analysis of the expression profiles of the corresponding genes in both NE3119 and NE8184 lines. We found that the expression of at least 12 genes was substantially reduced and that of at least 10 genes was substantially increased in OsSIG1−/− plants of both lines (Figure 3; Table S2). The most prominent reduction in expression in OsSIG1−/− plants was observed for the genes psaA, psaB and rps14, which together constitute the psaA operon (Chen et al., 1992). Other genes that showed a reduced level of expression in the OsSIG1 mutant homozygotes were clustered in two distinct operons: psbB (psbB–psbT–psbH–petB–petD) and psbE (psbE–psbF–psbL–psbJ). Compared with WT homozygotes, the reduction in the level of gene expression was ∼64–89% for psaA, ∼41–48% for psbB and ∼15–25% for psbE (Figure 3). The psaA operon of rice encodes PsaA, a subunit of the photosystem I (PSI) reaction-center protein (PSI P-700 apoprotein A1), PsaB (PSI P-700 apoprotein A2) and Rps14 (ribosomal protein S14) (Chen et al., 1992). The genes psbB, psbT, psbH, petB and petD constitute a polycistronic transcription unit not only in rice but also in maize and Arabidopsis (Barkan et al., 1994;Felder et al., 2001; Kanno and Hirai, 1993). These genes of the psbB operon encode subunits of two different protein complexes, photosystem II (PSII: PsbB, PsbT, PsbH) and the cytochrome b6f complex (PetB, PetD). The psbE operon encodes subunits of cytochrome b559 (PsbE, PsbF) and of PSII (PsbL, PsbJ) (Haley and Bogorad, 1990). In contrast to the effect of OsSIG1 deficiency on expression of the genes in the psaA, psbB or psbE operons, the abundance of transcripts of rpl22, rpoA, rpoB, rpoC1, petE, petA, psbG, ORF159, psbZ and psbI was increased in OsSIG1−/− plants (Figure 3; Table S2).

image

Figure 2.  Northern analysis of OsSIG1 transcripts in three regions of the rice leaf blade. (a) Diagram of a rice seedling with a fully emerged third leaf. The third leaves were removed from 20-day-old Nipponbare seedlings and divided into three parts: region I (leaf tip), region II (middle portion) and region III (leaf base). (b) Total RNA (4 μg) isolated from the three leaf portions was subjected to northern analysis with an OsSIG1 riboprobe; the major hybridizing band is indicated by the arrowhead. The region of the ethidium bromide-stained gel containing 25S rRNA is also shown.

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image

Figure 3.  Quantitative RT-PCR analysis of chloroplast gene transcripts in OsSIG1 mutants. Total RNA was isolated from the fully emerged second leaves of 20-day-old NE3119 or NE8184 seedlings grown at 25°C under a 16-h light, 8-h dark cycle, and the amount of transcript for the indicated chloroplast genes was determined by RT and real-time PCR analysis. Data for OsSIG1−/− plants are expressed as a percentage of the corresponding value for OsSIG1+/+ plants and are means ± SD. The original data and n values for each gene are shown in Table S2. Genes in the psaA, psbE or psbB operons are shown in bold.

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Reduced abundance of mRNAs derived from three chloroplast transcription units in OsSIG1 mutants

The abundance of transcripts in plant chloroplasts is determined not only by transcriptional initiation but also by post-transcriptional processing and turnover (Barkan et al., 1994). Indeed, for some genes, post-transcriptional processing of primary transcripts is required for the generation of translation-competent mRNA. We therefore next examined the abundance of mRNAs derived from five chloroplast genes –psaA, psbB, psbE, atpB and rbcL– in OsSIG1-deficient plants by northern analysis. The amount of psaA mRNA (the 5.2 kb RNA is the tricistronic transcript detected by the psaA probe) in OsSIG1−/− plants was only ∼51–54% of that in WT homozygotes (Figure 4a), consistent with the results obtained by quantitative RT-PCR analysis (Figure 3). Multiple bands were detected with the psbB riboprobe in both OsSIG1−/− and OsSIG1+/+ plants (Figure 4b), indicative of post-transcriptional processing of the rice primary psbB transcript (Kanno and Hirai, 1993) similar to that apparent in maize and Arabidopsis (Barkan et al., 1994; Felder et al., 2001). Although the processing of the primary psbB transcript did not appear to be affected in the OsSIG1 mutants, the amount of the major mRNA (∼2 kb) in NE3119 and NE8184 mutant homozygotes was ∼66–77% of that in WT homozygotes. The abundance of psbE mRNA (∼0.9 kb) in OsSIG1−/− plants was ∼72–90% of that in their OsSIG1+/+ counterparts (Figure 4c), with the effect of OsSIG1 deficiency thus being smaller for this mRNA than for psaA and psbB mRNAs, again consistent with the results of RT-PCR analysis. Finally, the amount of atpB mRNA (∼2.6 kb) was increased by ∼25–40% (Figure 4d) whereas that of rbcL mRNA (∼1.9 kb) was largely unchanged (Figure 4e) in OsSIG1−/− plants.

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Figure 4.  Northern analysis of chloroplast gene expression in OsSIG1 mutants. Total RNA (5 μg) isolated as described in Figure 2 was subjected to northern analysis with riboprobes specific for psaA (a), psbB (b), psbE (c), atpB (d) or rbcL (e) mRNAs. The major hybridizing bands are indicated by arrowheads. The region of each ethidium bromide-stained gel containing 25S rRNA is also shown.

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Decrease in the abundance of protein components of PSI in OsSIG1 mutants

We next investigated whether the reduced abundance of transcripts of the psaA operon in the OsSIG1 mutants was accompanied by a decreased number of protein constituents in PSI. We examined the number of PSI components and other thylakoid membrane proteins by non-denaturing green gel electrophoresis (Kashino et al., 1990). The pigment proteins with bound chlorophyll complexes were thus solubilized from thylakoid membranes and separated by polyacrylamide gel electrophoresis under non-denaturing conditions. Whereas seven major pigment proteins were detected in both OsSIG1−/− and OsSIG1+/+ plants, the intensity of a dark green band corresponding to CPI* [PSI–light-harvesting complex I (LHCI) complex] (Jensen et al., 2000) was markedly reduced in the OsSIG1-deficient mutants (Figure 5a). Immunoblot analysis of the same thylakoid membrane protein preparations revealed that the amount of the PSI reaction-center complex (PsaA–PsaB) was substantially reduced (by ∼26%) in OsSIG1−/− plants of both NE3119 and NE8184 lines compared with that in WT homozygotes (Figure 5b). Given that the bacterial homolog of Rps14 is essential for 70S ribosome function (Natori et al., 2007), the reduced expression of rps14, which is included in the psaA operon, in the OsSIG1 mutants might have been expected to be accompanied by an overall reduction in translation activity in chloroplasts. Immunoblot analysis of thylakoid membrane proteins revealed that the abundance of PsbA did not differ between OsSIG1−/− and OsSIG1+/+ plants (Figure 5b). The reduction in the amount of PsaA–PsaB in thylakoid membranes of the OsSIG1-deficient mutants is thus probably due to the reduced transcript level of psaA and psaB, and the reduced transcript level of rps14 does not appear to substantially affect overall translation activity in the mutant chloroplasts.

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Figure 5.  Analysis of thylakoid membrane proteins in OsSIG1 mutants. (a) Green gel electrophoresis. Thylakoid membranes isolated from the leaves of 20-day-old OsSIG1−/− or OsSIG1+/+ plants of the NE3119 or NE8184 lines were solubilized with a solution containing 4% dodecylmartoside, and the solubilized proteins (20 μg) were fractionated by non-denaturing green gel electrophoresis (right). The same gel was stained with Coomassie brilliant blue (left). CPI*, the PSI reaction center complexed with LHCI; CP47/43, the chlorophyll a-containing antenna associated with the PSII reaction center; FP, free pigments. (b) Immunoblot analysis. Proteins (200 ng) solubilized from thylakoid membranes were subjected to immunoblot analysis with antibodies specific for the PsaA–PsaB complex or for PsbA. Arrowheads indicate 66–68 kDa PsaA and PsaB proteins in the upper panel, and 32 kDa PsbA in the lower panel.

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Photosynthetic activities of OsSIG1 mutants

The reduced chlorophyll content, reduced amounts of transcripts of the psaA, psbB and psbE operons, and reduced abundance of PsaA–PsaB in the OsSIG1 mutants suggested that the photosynthetic machinery of these plants might be defective. Indeed, the quantum yield of electron transfer through PSII (ΦII) in OsSIG1−/− plants was found to be only ∼57–64% of that in OsSIG1+/+ plants (Table 1). This decrease in the level of photosynthesis was not due to a decrease in PSII efficiency, given that the maximum (Fv/Fm) or effective (Fv′/Fm′) quantum yields of PSII did not differ between OsSIG1−/− and OsSIG1+/+ plants (Table 1). Photochemical quenching (qP) was also reduced by ∼42–45% in the OsSIG1 mutants, reflecting a reduction in the size of the plastoquinone pool as a result of electrons transferred from PSII, possibly as a result of a defect downstream of PSII.

Table 1.   Photosynthetic activity of OsSIG1 mutants
PlantFv/FmFv′/FmqPΦIIPhoto-oxidizable P-700
  1. Data were obtained with leaves of 20-day-old Nipponbare plants or OsSIG1−/− or OsSIG1+/+ plants of NE3119 or NE8184 lines. Fv/Fm and Fv′/Fm′ indicate maximum and effective quantum yields of PSII, respectively. qP and ΦII indicate photochemical quenching and quantum yield of electron transfer through PSII, respectively. The relative amount of photo-oxidizable P-700 was estimated as signal changes (mV) due to the absorbance increase upon far-red light-induced oxidation of P-700. Data are means ± SD of values from three independent experiments. Numbers in parentheses are percentages of the corresponding value for Nipponbare.

Nipponbare0.788 ± 0.006 (100)0.662 ± 0.013 (100)0.788 ± 0.035 (100)0.522 ± 0.019 (100)2.811 ± 0.428 (100)
NE3119+/+0.786 ± 0.007 (99.7)0.671 ± 0.039 (101.4)0.789 ± 0.009 (100.1)0.530 ± 0.025 (101.5)2.830 ± 0.473 (100.6)
NE3119−/−0.774 ± 0.004 (98.2)0.694 ± 0.042 (104.8)0.441 ± 0.004 (56.0)0.306 ± 0.020 (58.6)0.651 ± 0.037 (23.2)
NE 8184+/+0.783 ± 0.008 (99.4)0.654 ± 0.023 (98.8)0.809 ± 0.025 (102.7)0.529 ± 0.003 (101.3)2.437 ± 0.398 (86.7)
NE 8184−/−0.773 ± 0.023 (98.0)0.727 ± 0.036 (109.8)0.474 ± 0.059 (60.2)0.344 ± 0.030 (65.9)0.912 ± 0.259 (32.4)

Finally, we compared the amount of PSI between OsSIG1−/− and OsSIG1+/+ plants by monitoring changes in absorbance at 830 nm due to the photo-oxidization of P-700, the reaction-center chlorophyll of PSI. The signal due to the oxidization of P-700 was greatly reduced (Table 1), indicating that the amount of functional PSI was lower in the OsSIG1 mutants.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We have isolated and characterized two allelic OsSIG1 mutants of rice, NE3119 and NE8184. Whereas the fertility of the mutant homozygotes did not appear to differ from that of WT homozygotes or heterozygotes, the OsSIG1−/− plants manifest a phenotype characterized by pale green leaves that was shown to result from a reduced chlorophyll content. Furthermore, the leaf chlorophyll content of the two lines was shown to correlate with OsSIG1 genotype.

Analysis of plastid gene expression revealed that the abundance of transcripts derived from three specific operons (psaA, psbB and psbE) was reduced in the middle region of leaf blades of OsSIG1−/− plants. OsSIG1 mRNA accumulated to a greater extent in this region of Nipponbare leaves than in other leaf regions. These results suggest that the promoters of the psaA, psbB and psbE operons are recognized by OsSIG1 in rice chloroplasts, and the greater reduction in the abundance of transcripts of the psaA operon than in that of transcripts of the other two operons in OsSIG1−/− mutants suggests that the specificity of OsSIG1 is highest for the psaA operon. Analysis of an RpoB-deficient tobacco mutant previously showed that transcription of these three operons is dependent on PEP (Hajdukiewicz et al., 1997). The promoter regions of psaA, psbB and psbE operons of land plants contain typical sigma70-type cis (−35 and −10) elements (Figure S1), suggesting that SIG1 family proteins play a common role in regulation of the transcription of these operons in land plants.

The expression levels of some genes, including atpB, rpl22, rpoA, rpoB and rpoC1, were increased in OsSIG1−/− plants. An increase in chloroplast transcript abundance, mostly for NEP-dependent genes, has been described for sig2 or sig6 mutants of Arabidopsis (Ishizaki et al., 2005; Kanamaru et al., 2001), a PEP-deficient mutant of tobacco (Allison et al., 1996; Hajdukiewicz et al., 1997) and a rice virescence mutant (Kusumi et al., 1997). These observations thus suggest the existence of an interaction between PEP-dependent and NEP-dependent transcription in photosynthetically active chloroplasts of land plants.

We found that the abundance of the PsaA–PsaB heterodimer, a component of the PSI reaction center, was also reduced in the OsSIG1 mutants. The amount of PsbA, another thylakoid membrane protein that is a component of the PSII complex, was unaffected by the loss of OsSIG1, suggesting that the reduced abundance of PsaA–PsaB was due to the reduced accumulation of transcripts of the psaA operon. Fluorescence analysis revealed that the activity of PSII (Fv/Fm and Fv′/Fm′) was not affected by the lack of OsSIG1, whereas parameters reflecting qP, ΦII and PSI activity were all reduced in the OsSIG1-deficient mutants compared with those in WT homozygotes. On the other hand, at various intensities of far-red light, the proportion of oxidizable P-700 did not differ between OsSIG1−/− and OsSIG1+/+ plants (data not shown). These results suggest that the OsSIG1 mutants possess a qualitatively normal PSII but are impaired in electron transfer downstream of PSII, probably as a result of a reduction in the abundance of the functional PSI apparatus. The defect in electron transfer might result in a reduction in the size of the plastoquinone pool and induce diffusion of LHCII from PSII.

The reduced level of rps14 mRNA apparent in the OsSIG1−/− plants did not appear to affect translation from chloroplast transcripts. The amount of PsbA (PSII D1 protein) in a thylakoid membrane fraction was thus unaffected in the mutants, indirectly suggesting that the amount of the PSII core complex, which is a scaffold for the D1 protein, is similar in the mutants and their WT counterparts. This finding also suggests that the reduced abundance of psaA operon mRNAs in OsSIG1−/− plants affects only the accumulation of PsaA or PsaB, not that of Rps14. This difference might be due to a difference in protein turnover between the PSI proteins and the ribosomal protein.

In conclusion, we have shown that loss of OsSIG1 results in a reduction in the level of expression of specific chloroplast genes and a reduction in photosynthetic activity as a result of a deficiency of PSI components. The principal role of OsSIG1 in rice may therefore be the maintenance of PSI activity in chloroplasts of mature leaves.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials

Rice plants (O. sativa var. Nipponbare and OsSIG1 mutants) were grown in fields for seed collection or in a growth chamber at 25°C under a 16-h-light, 8-h-dark cycle for RNA preparation and protein analysis. Total RNA for RT-PCR and northern analyses was isolated from leaves of 20-day-old plants.

Screening of Tos17 insertion mutants

For identification of OsSIG1 mutants, we screened a large population of rice mutants generated by Tos17-mediated mutagenesis (Hirochika, 1997). Tos17 insertion in OsSIG1 genomic DNA was surveyed by nested PCR analysis as described previously (Sato et al., 1999). The first PCR was performed with a transposon- specific primer, T17LTR10 F (5′-GCTCTCCACTATGTGCCCTC-3′) or T17LTR8R (5′-CGGTGAAAAGGACAGTGGAG-3′), and the gene-specific primer sigR5 (5′-CAATAATCCTCAAGCAATGC-3′). The second PCR was performed with a transposon-specific primer, T17LTR7 F (5′-CCATCGGATGTCCAGTCCAT-3′) or T17LTR6R (5′-GGACATGGGCCAACTATACAG-3′), and the gene-specific primer sigR8 (5′-CTCCATTGCGATGAGTCCTA-3′). Genomic DNA (∼50 ng) from a pool of 24 plants was used as the template for PCR. The products of the second PCR were subjected to Southern hybridization analysis with a probe prepared from a 792-bp fragment of the OsSIG1 ORF amplified by PCR with the primers sigF4 (5′-GCATTCGCTGAAGATATCAC-3′) and sigR5. Mutant PCR products identified by a positive hybridization signal in the Southern analysis were purified by gel electrophoresis, subcloned into the pCRII vector (Invitrogen, http://www.invitrogen.com/), and sequenced to determine the position of Tos17 insertion in OsSIG1.

Measurement of chlorophyll content

A portion (0.5 cm2) of the middle of leaf blades from 6-week-old plants was soaked overnight in 1 ml of dimethyl formamide. The chlorophyll content of the extract was then determined spectrophotometrically as described previously (Porra et al., 1989).

Quantitative RT-PCR analysis

First-strand cDNA was synthesized from 500 ng of DNase I-treated total RNA with random-hexamer primers and a SYBR Green PCR Master Mix kit (Applied Biosystems, http://www.appliedbiosystems.com/). Real-time PCR was then performed with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) in a reaction mixture (20 μl) containing 20 ng of cDNA, 10 μl of SYBR Green Master mix and 4 μm each of forward and reverse gene-specific primers (Table S1). The amplification protocol comprised an initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Rice chloroplast DNA clones in the BGM vector (Itaya et al., 2005) that cover the entire chloroplast genome of Nipponbare (GenBank accession no. AP006728) were constructed by the inchworm elongation method (Itaya et al., 2005) and used as standard DNA templates to estimate the copy number of gene transcripts (Supplementary Table S3). Data were analyzed with ABI PRISM 7000 SDS Software (Applied Biosystems).

Northern analysis

Isolation of total RNA and northern analysis were performed as previously described (Tozawa et al., 2001). For preparation of riboprobes specific for psaA, psbB, psbE, atpB or rbcL mRNAs, DNA fragments of each gene were amplified by PCR with the primer sets listed in Supplementary Table S4. The PCR products were cloned into the pT7 Blue T-vector (Roche, http://www.roche.com/), and digoxigenin-labeled antisense riboprobes were produced by run-off in vitro transcription with T7 RNA polymerase (Roche). Riboprobes specific for OsSIG1 mRNA or for Rac2 mRNA (internal standard) were also synthesized as described previously (Tozawa et al., 1998). The intensity of hybridizing bands was measured with an image scanner (LAS-1000; Fujifilm, http://www.fujifilm.com/) and IR LAS-1000 Lite V1.31 software (Fujifilm).

Isolation of thylakoid membranes.  Thylakoid membranes were isolated basically as described by Knoetzel and Simpson (1991). Leaves from 20-day-old plants were homogenized with the use of a Physcotron homogenizer (Microtech Nition, http://nition.com/en/) in a solution containing 0.4 m sucrose, 10 mm NaCl, 5 mm MgCl2, 10 mm tricine (pH 7.5) and 10 mm sodium ascorbate. The homogenate was centrifuged at 2000 g for 10 min at 4°C, the chloroplast pellet was resuspended and lysed in 5 mm tricine (pH 7.9), and the resulting lysate was centrifuged at 10 000 g for 10 min at 4°C for isolation of thylakoid membranes. The membrane pellet was resuspended at a chlorophyll concentration of 2 mg ml−1 in ascorbate-free homogenization solution supplemented with 20% glycerol. The membranes were rapidly frozen in liquid N2 and stored at −80°C. They were subsequently solubilized in the dark with an equal volume of TMGD buffer, comprising 0.4 m 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)–maleate (pH 7.0), 68% glycerol, and 4% dodecylmartoside. The extract was centrifuged at 20 400 g for 10 min at 4°C to remove non-solubilized material, and portions of the solubilized membrane protein preparation (20 μg) were subjected to non-denaturing green gel electrophoresis or to immunoblot analysis.

Green gel electrophoresis.  Non-denaturing green gel electrophoresis was performed as described by Knoetzel and Simpson (1991). The separating gel consisted of 11% acrylamide (1.1% T, 2.7% C), 0.24 m TRIS–HCl (pH 8.8), 13% glycerol, 0.05% ammonium persulfate and 0.0625%N,N,N′,N′-tetramethylethylenediamine, and the stacking gel comprised 4% acrylamide (4% T, 1.1% C), 0.25 m TRIS–HCl (pH 8.45), 13% glycerol, 0.08% ammonium persulfate, and 0.08%N,N,N′,N′-tetramethylethylenediamine. Electrophoresis was performed in the dark with a constant current of 10–20 mA for 5–6 h at 4°C and with a solution containing 0.05 m TRIS, 0.49 m glycine and 0.1% SDS.

Immunoblot analysis.  Thylakoid membrane proteins solubilized as described above were mixed with an equal volume of 2× SDS sample buffer and resolved by SDS-PAGE on an 11% gel. The separated proteins were electrophoretically transferred to a polyvinylidenefluoride membrane (Millipore, http://www.millipore.com/) and subjected to immunoblot analysis with antibodies to PsaA–PsaB (Kashino et al., 1990) or to PsbA (Tanaka et al., 2004) as described. The intensity of immunoreactive bands was determined as for northern analysis.

Measurement of photosynthetic activities.  Chlorophyll fluorescence was measured with a pulse-amplitude modulation chlorophyll fluorometer (PAM 101/102/103; Heinz Walz, http://www.walz.com/) as described (Kudoh and Sonoike, 2002). Minimum fluorescence (Fo) was recorded after dark adaptation for 5 min. Maximum fluorescence (Fm) was obtained by application of a 0.8-sec saturating light pulse (5600 μmol photons m−2 sec−1) from a KL 1500 light source (Schott, http://www.galvoptics.fsnet.co.uk). The stable level of fluorescence (Fs) was determined during exposure of a leaf to actinic light with a defined photon flux density (4.06 W m−2) from the same light source. The maximal quantum yield of PSII was calculated as Fv/Fm = (Fm − Fo)/Fm. The maximal quantum yield of PSII during exposure to actinic light was calculated as Fv′/Fm′ = (Fm′ − Fo)/Fm′. Photochemical quenching (qP) and the effective quantum yield of electron transport through PSII (ΦII) were calculated as (Fm′ − Fs)/(Fm′ − Fo′) and (Fm′ − Fs)/Fm′, respectively. The change in absorbance at 830 nm due to P-700 oxidation in vivo was measured with a pulse-modulated system (PAM 101/102, Heinz Walz). P-700 was oxidized by far-red light (13.5 W m−2) from a photodiode (FR-102; Heinz Walz).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank C. Mikami and Y. Ikejiri-Kanno for technical assistance. This study was supported by a grant (Rice Genome Project MP-2115) from the Ministry of Agriculture, Forestry, and Fisheries of Japan (to Y.T.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Comparison of nucleotide sequences of the promoter regions of the chloroplast genes psaA, psbB, and psbE of land plants. The -10 and -35 promoter sequences are boxed. Underlined nucleotide residues are identified transcription start positions (Chen et al., 1992 for psaA of rice; Summer et al., 2000 for psaA of Sinapis alba; Swiatecka-Hagenbruch et al., 2007 for psaA of Arabidopsis and tobacco; Westhoff, 1985 for psbB of spinach; Haley and Bogorad, 1990 for psbE of maize).

References

Chen, S.C.G., Cheng, M.C., Chung, K.R., Yu, N.J. and Chen, M.C. (1992) Expression of the rice chloroplast psaA-psaB-rps14 gene cluster. Plant Sci. 81, 93-102.

Summer, H., Pfannschmidt, T. and Link, G. (2000) Transcripts and sequence elements suggest differential promoter usage within the ycf3-psaAB gene cluster on mustard (Sinapis alba L.) chloroplast DNA. Curr. Genet. 37, 45-52.

Swiatecka-Hagenbruch, M., Liere, K. and Börner, T. (2007) High diversity of plastidial promoters in Arabidopsis thaliana. Mol. Gent. Genomics. DOI 10.1007/s00438-007-0222-4.

Westhoff, P. (1985) Transcription of the gene encoding the 51 kd chlorophyll a-apoprotein of the photosystem II reaction centre from spinach. Mol. Gen. Genet. 201, 115-123.

Haley, J. and Bogorad, L. (1990) Alternative promoters are used for genes within maize chloroplast polycistronic transcription units. Plant Cell2, 323-333.

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