OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function

Authors


Authors for correspondence:
Clive Lo
Tel: + 852 2299 0337
Email: clivelo@hkucc.hku.hk

Yuezhi Tao
Tel: + 86 571 56404203
Email: taoyz@zaas.org

Summary

  • The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant.
  • Rice (Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant.
  • Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected.
  • Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants.

Introduction

Chloroplasts are plant- and algal-specific organelles which are responsible for essential metabolic processes such as photosynthesis and the biosynthesis of amino acids and fatty acids. The chloroplast genomes of higher plants encode approx. 100 proteins as well as the tRNAs and rRNAs required for protein synthesis. However, chloroplasts are clearly not autonomous as the vast majority of the over 2000 proteins that function in chloroplasts are encoded by nuclear genes, translated in the cytosol and subsequently imported into the organelle (Abdallah et al., 2000). The chloroplast translation process itself requires the import of initiation, elongation and termination factors, additional ribosomal proteins, and a number of RNA/protein modifying and processing enzymes derived from the nuclear genome (Subramanian, 1993). In addition, there is growing biochemical and genetic evidence that supports the role of nuclear-encoded proteins as regulators of chloroplast protein synthesis, often by affecting the translation of a single mRNA species through interaction with the 5′-untranslated regions (Marín-Navarro et al., 2007).

Consistent with the proposed endosymbiotic origin of chloroplasts from ancestral free-living cyanobacteria, chloroplast translation shares many of the features typical of prokaryotic protein synthesis, for example, the 70S-type ribosomes, the uncapped and polycistronic mRNAs, and the Shine–Dalgarno interaction with the 16S rRNA for start codon selection in some genes (Marín-Navarro et al., 2007). The molecular machineries for translation are not interchangeable between the cytosol and the chloroplasts. Recently, an Arabidopsis homolog of the bacterial YqeH protein, AtRIF1, was demonstrated to be required for proper ribosome functions in chloroplasts (Flores-Pérez et al., 2008). YqeH is a circularly permuted GTPase (cGTPase) belonging to the YlqF/YawG family (Leipe et al., 2002). In cGTPases, the normal G1-G2-G3-G4-G5 orientation of the GTP-binding domain has been rearranged to G4-G5-G1-G2-G3. In Bacillus subtilis, YqeH is essential for proper 70S ribosome formation and 30S subunit assembly or stability (Uicker et al., 2007). In Arabidopsis, AtRIF1 is a nuclear-encoded protein targeted to the chloroplast stroma. Loss of AtRIF1 functions resulted in decreased levels of a few chloroplast-encoded proteins, including RBCL, AtpB and PsbA (Flores-Pérez et al., 2008). AtRIF1 was identified after analysis of the Arabidopsis rif1-1 mutant, which was obtained following screening for resistance to fosmidomycin (FSM). In addition, elevated accumulation of two chloroplast-targeted methylerythritol phosphate (MEP) pathway enzymes, which were believed to be responsible for the FSM resistance, was detected in the mutant. Expression of a chloroplast-targeted YqeH–green fluorescent protein (GFP) fusion in rif1 was able to complement the mutant phenotypes and restore the FSM sensitivity, demonstrating that AtRIF1 and YqeH share similar biochemical functions.

Interestingly, AtRIF1 was initially reported as AtNOS1 which was described as an arginine-dependent nitric oxide synthase (NOS) targeted to mitochondria in Arabidopsis (Guo et al., 2003; Guo & Crawford, 2005). Reduced NOS activities and nitric oxide (NO) levels in the nos1 mutant have been demonstrated by independent laboratories (Guo et al., 2003; He et al., 2004; Zeidler et al., 2004; Zhao et al., 2007). A variety of noticeable phenotypes, ranging from inhibition of seed germination, reduced shoot and root growth, and leaf yellowing to impaired fertility, were first observed in the mutant and they could be rescued by exogenous application of sodium nitroprusside (SNP) which is an NO donor (Guo et al., 2003). The role of NOS1 was later expanded to providing the major source of NO needed for some defense responses (Zeidler et al., 2004). However, conflicting results were obtained for the difference between wild-type and nos1 mutant plants in their NO accumulation response to different hormones or oxidative stress (Gas et al., 2009). More critically, the NOS activities of the recombinant AtNOS1 protein could not be reproducibly demonstrated, leading to the subsequent renaming of the protein as AtNOA1 (NITRIC OXIDE ASSOCIATED PROTEIN1) (Crawford et al., 2006; Zemojtel et al., 2006). Furthermore, recent molecular and transgenic analyses provided conclusive results that AtNOA1/RIF1 has GTPase activity and is targeted to chloroplasts – both features are necessary for proper expression of the protein functions in Arabidopsis (Flores-Pérez et al., 2008; Sudhamsu et al., 2008; Moreau et al., 2008). The biological role of AtNOA1/RIF1 is currently believed to be primarily associated with chloroplast ribosome functions (Flores-Pérez et al., 2008; Gas et al., 2009). However, defective NO production in the mutant is likely to be an indirect consequence of altered chloroplast metabolism owing to the loss of AtNOA1/RIF1 function (Zemojtel et al., 2006; Gas et al., 2009).

During the time when the emerging evidence was placing AtNOS1 into a pivotal role in NO-mediated physiological responses in Arabidopsis, investigations on a potential functional homolog in rice, the monocot model, were underway in our laboratories. In the present study, we report that a rice NOA1/RIF1 homolog (OsNOA1/RIF1), which is encoded by the nuclear gene Os02g01440 (TIGR Gene Identifier; http://rice.plantbiology.msu.edu), is a chloroplast-localized protein capable of complementing most of the defects in an Arabidopsis rif1 mutant. The gene is constitutively expressed in different rice tissues and is light-inducible in etiolated seedlings following illumination. Targeted gene suppression mediated by RNA interference (RNAi) was used to investigate the biological functions of OsNOA1/RIF1 in the rice plant. The RNAi lines were chlorotic, with lower levels of photosynthetic pigments, lower photosystem II (PSII) efficiencies, and reduced abundance of the chloroplast 16S rRNA. Quantitative proteomics analysis revealed proteome changes that support the functional role of OsNOA1/RIF1 in chloroplast ribosome biogenesis and/or translation. The relevance of several classes of proteins that showed significant fold-changes following OsNOA1/RIF1 suppression will be discussed.

Materials and Methods

Plant materials and growth conditions

Ozyza sativa L. cv Nipponbare was used for all the rice experiments. Wild-type and transgenic rice plants were grown in a glasshouse at 30°C during daytime under natural sunlight and 25°C at night. Arabidopsis plants (Col-0) were grown under a 16 h : 8 h (light : dark) cycle at 22°C in a growth chamber. The Arabidopsis rif1 mutant used was an insertion allele in the Salk collection (SALK_047882; Arabidopsis Biological Resource Center, Columbus, OH, USA) and was named rif1-2 (Flores-Pérez et al., 2008). To observe the root morphology, Arabidopsis seeds were surface-sterilized in 70% ethanol for 5 min and then in 5% bleach solution for 15 min. After rinsing in sterile water, the seeds were then plated on germination medium as described previously (Guo et al., 2003). Plates were kept at 4°C for 2 d and then germinated vertically at 24°C under continuous light.

Vector construction

For complementation of the Arabidopsis rif1-2 mutant, a full-length cDNA fragment encoding OsNOA1/RIF1 was amplified from rice cDNA using the primers 5′-CCT CCT CCT GCT CCT AGT A-3′ and 5′-TCA GTA ATG CCA TTT AGG TCT C-3′. The PCR product was cloned between the CaMV 35S promoter and the NOS-3′ terminator in the binary vector pCAMBIA1301–35S-NOS. For the OsNOA1/RIF1-EYFP fusion construct, the OsNOA1/RIF1 coding region was cloned in-frame with the EYFP gene in the pCAMBIA1301-35S-NOS vector. The OsNOA1/RIF1 coding region was amplified with the primers 5′-GAG CTC ATG GCG GCG CCT CCT CTG CTG-3′ and 5′-GGT ACC GTA ATG CCA TTT AGG TCT CA-3′ using rice cDNA as the template. For the OsNOA1/RIF1 RNAi gene suppression vector, a 251-bp cDNA fragment between the 597th and 847th positions in the OsNOA1/RIF1 coding region was amplified using the primers 5′-GCT GGT TGA CAT AGT TGA CTT-3′ and 5′-TCT TTT CCT GCT GAA TCT C-3′. The second intron of the maize gene NIR1 was inserted between the sense and antisense cDNA fragments. The cassette was then cloned into the pCAMBIA1301–35S-NOS construct for rice transformation.

Plant transformation

Transgenic Arabidopsis plants were generated by Agrobacterium-mediated transformation using the floral dip method (Clough & Bent, 1998). Harvested seeds were surface-sterilized and germinated on Murashige and Skooge (MS; Sigma) agar containing 3% (w : v) sucrose and 25 μg ml−1 hygromycin (Sigma). Resistant seedlings were transplanted and placed in a growth chamber.

To generate gene suppression lines, 2–3-wk-old calli derived from scutellum of rice seeds (O. sativa spp japonica cv Nipponbare) were inoculated with Agrobacterium cultures carrying the RNAi binary vector as described earlier. Rice tissue culture, selection, and regeneration of transgenic plants were performed essentially as described by Lee et al. (1999). Stable transformants were selected for hygromycin resistance (these primary transformants were designated as the T0 generation). Transgenic plants were grown to maturity in growth chambers or a glasshouse (28°C, 12 h-light : dark) for two generations to collect the T1 and T2 seeds.

Measurement of photosynthetic pigments and chlorophyll a fluorescence parameters

Pigments were extracted from second leaf of 2- to 4-wk-old rice seedlings with 96% ethanol. Absorbance of the extract was measured spectrophotometrically at 470, 649 and 665 nm. Pigment contents (mg g−1 FW) were determined according to the following formulae:

image

Chlorophyll fluorescence measurements were performed using a PAM-101 fluorometer (Walz, Effeltrich, Germany). The maximum photochemical yield of PSII (Fv/Fm = (Fm − F0)/Fm) was measured as variable chlorophyll fluorescence of dark-adapted (30 min) leaves at room temperature. The minimum fluorescence (F0) was obtained upon excitation of leaves with a weak beam (650 nm, 0.8 μmol photons m−2 s−1). The maximum fluorescence (Fm) was measured with 100-kHz modulation frequency and the leaves were elicited by saturating flashes of 0.8-s duration from a built-in miniature halogen lamp (> 4000 μmol photons m−2 s−1). A 15-min illumination with actinic light at 200 μmol photons m−2 s−1 was used to drive the electron transport between the two photosystems for measuring the effective quantum yield of PSII (ΦPSII) which is defined as F/F ′m = (F ′mFs)/F ′m. The fluorescence parameters F ′m and Fs are the maximum and the steady state fluorescence levels under light, respectively.

RNA experiments

Total RNA was isolated from rice tissues using the RNAgents total RNA isolation system (Takara, Dalian, China). DNAase I-treated RNA samples were reverse transcribed by M-MLV reverse transcriptase (Promega). The following gene-specific primers were used for cDNA amplification: Actin, 5′-GCA TCT CTC AGC ACA TTC CA-3′ and 5′-CTG GTA CCC TCA TCA GGC AT-3′; OsNOA1/RIF1, 5′- AGC GGC TAC TCT TCC TCT CC-3′and 5′-CGA CTT TGT GGT GCA AGA GA-3′. Northern blot analysis was carried out using digoxigenin (DIG)-labeled DNA probes as described previously (Huang et al., 2000). The following primers were used to prepare PCR-amplified probes for the northern hybridization experiments: 16S rRNA, 5′-CAA GCT GGA GTA CGG TAG GG-3′ and 5′-GCT CGT TGC GAG ACT TAA CC-3′; rbcL, 5′-GCT GCC GAA TCT TCT ACT GG-3′ and 5′-TAC CTG CAG TCG CAT TCA AG-3′; atpB, 5′-CGT GCT AGC TTC ATC GAT GT-3′ and 5′-AAG CAG GAT CGG AGG TAT CT-3′; psaA, 5′-CAT GTG GAT TGG CGG ATT TC-3′ and 5′-CAA CTT TGC CGC CTA CTG CTA C-3′; psbA, 5′-ATT CGT GAG CCT GTT TCT GG-3′ and 5′-CCC TAC TAC AGG CCA AGC AG-3′.

Protein and immunoblot analysis

Two-week old wild-type and RNAi mutant rice seedlings were collected for protein extraction. Crude protein extracts were obtained by grinding tissues in liquid nitrogen followed by resuspension in ice-cold sodium phosphate buffer (100 mM, pH 7.5) containing 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 10% sucrose, 100 μg ml−1 phenylmethylsulfonyl fluoride, 0.2% Triton X-100 and 1× protease inhibitor cocktail (Roche). Protein concentrations were determined by the Bio-Rad protein assay. Protein samples were separated by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by Coomassie blue staining. For immunoblot analysis, proteins on the gel were transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences) using a Mini Trans-Blot-Electrophoretic Transfer Cell (Bio-Rad). The protein blot was then incubated with the DXS antibody (P. Leon, Instituto de Biotechnologia-UNAM, Cubernavaca, Mexico). Hybridization signals were detected using the enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer’s instructions.

Quantitative proteomics analysis of rice seedlings

Protein samples (100 μg) prepared as outlined earlier were reduced, alkylated, and trypsin-digested according to the manufacturer’s instructions in the iTRAQ reagents 8-plex kit (Applied Biosystems, Foster City, CA, USA). The digested peptides were then labeled with four iTRAQ tags (wild type 115, mutant 116; wild type 119; mutant 121) and pooled. The combined iTRAQ samples were fractionated by strong cation exchange (SCX) chromatography. Eleven SCX fractions were subject to LC-MS/MS analysis. Separation was performed on reverse-phase capillary columns (150 μm internal diameter 200 mm) connected to an Agilent 1100 capillary LC system. The eluents were coupled online to a QSTAR XL quadrupole-time-of-flight hybrid mass spectrometer (Applied Biosystems) through an electrospray interface. The acquired MS/MS data were analysed and quantified using the Paragon algorithm in proteinpilot 2.0.1 software (Applied Biosystems). Detailed descriptions of the quantitative proteomics experiments are provided in the Supporting Information Methods S1.

Results

Sequence, gene expression, and localization analyses of OsNOA1/RIF1

A blast search against the TIGR rice genome annotation database using the AtNOA1/RIF1 sequence (561 aa) retrieved the accession Os02g01440 encoding a protein (547 aa) with 62% sequence identity. The rice gene, which we name OsNOA1/RIF1, is a single-copy gene containing 13 exons. In addition, a number of highly conserved sequences from plant species of diverse taxonomic groups were retrieved from the NCBI nonredundant protein database. The closest homolog of OsNOA1/RIF1 is a sequence from sorghum (97.2% identity) which belongs to the same family (Poaceae). Other homologs with high sequence identity include those from maize (84.4%), Nicotiana benthamiana (66.1%), grapevine (64.3%), castor bean (63.1%), and Sitka spruce (70.3%). All the newly identified sequences were predicted to be targeted to chloroplasts by the TargetP (http://www.cbs.dtu.dk/services/TargetP/) and WoLF PSORT (http://wolfpsort.org/) algorithms (data not shown). Multiple alignments of the different NOA1/RIF1 homologs revealed high levels of homology throughout their entire sequences (Fig. 1a). All of them contained the central GTP-binding region (CPG domain) with the G4-G5-G1-G2-G3 arrangement characteristic of cGTPases which belong to the YlqF/YawG family (Leipe et al., 2002). In addition, the N-terminal zinc binding region (ZBD) containing four conserved cysteine residues and the C-terminal domain (CTD) were identified. All the above features are conserved in the B. subtilis and Geobacillus stearothermophilus YqeH proteins (Moreau et al., 2008; Sudhamsu et al., 2008). Phylogenetic analysis showed that the dicot sequences clustered together while OsNOA1/RIF1 and the other two cereal sequences formed a separate clade (Fig. 1b). By contrast, the Sitka spruce homolog is more distantly related to the other plant sequences identified.

Figure 1.

 (a) Multiple alignment of OsNOA1/RIF1 with different plant homologs by the Clustalw method (http://www.ebi.ac.uk/clustalw). The proteins can be divided into three conserved domains: the zinc-binding domain (ZBD, CxxCx(26–34)CxxC), the circularly permuted G-domain (CPG) with GTP-binding regions (green), and the C-terminal domain (CTD). The four conserved cysteine residues in ZBD are indicated by the open arrowheads. Plant sequences were retrieved from the NCBI nonredundant protein database (grape, XP_002268388; castor bean, XP_002510962; Nicotiana benthamiana, BAF93184; maize, ACN26917; sorghum, XP_002451348; Sitka spruce, ANR16951). (b) An unrooted phylogenetic tree of the above sequences. The tree was constructed by MEGA3.1 based on the neighbor-joining method (Kumar et al., 2004). Numbers at the nodes represent bootstrap values from 1000 replication. Scale bar represents 0.05 substitutions per site.

OsNOA1/RIF1 was expressed constitutively in all rice tissues examined but higher levels of transcript accumulation were generally detected in the leaves of different plant ages (Fig. 2a). Etiolated rice seedlings gradually turn green following light exposure and gene expression analysis was performed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) using RNA samples prepared from tissues harvested at different time points. As shown in Fig. 2(b), there was only low level of OsNOA1/RIF1 expression when the plants were kept in darkness (0 h). Light-inducible expression was detected as early as 4 h after illumination. OsNOA1/RIF1 transcript levels reached a maximum level at 12 h and then remained relatively constant during the rest of the 24-h period.

Figure 2.

 Expression of OsNOA1/RIF1 in rice (Oryza sativa cv Nipponbare). (a) Tissues at the following plant stages were collected for reverse-transcription polymerase chain reaction (RT-PCR) expression analysis: R2, root (2 wk); S2, stem (2 wk); L2, leaf (2 wk); R4, root (4 wk); S4, stem (4 wk); L4, leaf (4 wk); S10, stem (10 wk); L10, leaf (10 wk); P10, panicle (10 wk). (b) Rice seeds were germinated in the dark for 10 d and the etiolated seedlings were then placed under continuous light. Semiquantitative RT-PCR (28 cycles) analysis revealed the light-inducible OsNOA1/RIF1 expression at the indicated time-points (h) after illumination. Actin expression was used as the internal control.

To determine the subcellular location of OsNOA1/RIF1, a translational fusion with EYFP at the C-terminus driven by the CaMV 35S promoter was constructed and analysed in transgenic Arabidopsis plants. T1 seedlings (1–2 wk old) were examined by confocal microscopy for fluorescence expression. As shown in Fig. 3, the OsNOA1/RIF1-EYFP fluorescent signals were detected in large organelles of mesophyll cells consistent with the sizes and chlorophyll autofluorescence of chloroplasts. Chloro-plast localization of OsNOA1/RIF1-EYFP in transgenic Arabidopsis was further confirmed by immunogold electron microscopy (see Fig. S1).

Figure 3.

 Localization analysis of OsNOA1/RIF1-EYFP fusion protein in transgenic Arabidopsis. (a) OsNOA1/RIF1-EYFP fluorescent signals observed by confocal microscopy through a green fluorescent protein (GFP) filter (i) appeared to overlap with chlorophyll autofluorescence from chloroplasts (ii) in mesophyll cells. Merged images of (i) and (ii) are shown in (iii). (b) An untransformed plant as a negative control. Bars, 10 μm.

Complementation of Arabidopsis rif1-2 mutant

To investigate whether OsNOA1/RIF1 may have conserved functions with AtNOA1/RIF1, the rice gene was overexpressed in the Arabidopsis rif1-2 mutant (=Atnoa1 in Guo et al., 2003) for complementation analysis. As shown in Fig. 4(a), several growth-related mutant pheno-types were complemented by the rice gene, including the yellow first true leaves and the reduced growth of shoot, root and inflorescence. A number of NO-associated phenotypes were initially described in the Arabidopsis rif1-2 mutant (Guo et al., 2003). Using the NO-sensitive fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2-DA), NO production in root tips of young Arabidopsis rif1-2 mutant seedlings was found to be restored by OsNOA1/RIF1 overexpression (Fig. 4b). Recently, the Arabidopsis rif1-1 mutant was demonstrated to have differential changes in chloroplast protein profiles (Flores-Pérez et al., 2008). In the complemented lines, the RBCL levels were restored, as detected by Coomassie blue staining after SDS-PAGE of the protein samples (Fig. 4c). Conversely, the upregulated 1-deoxyxylulose 5-phosphate synthase (DXS) levels were reduced, as revealed by immunoblot analysis using a specific antibody against this MEP pathway enzyme (Fig. 4c). The DXS levels were lower in the OsNOA1/RIF1 over-expression rif1-2 lines than the wild-type plants, possibly as a consequence of enhanced translation of chloroplast-encoded proteases that target this MEP enzyme.

Figure 4.

 Complementation analysis of OsNOA1/RIF1 in the Arabidopsis rif1-2 mutant. (a) Restoration of normal growth phenotypes (roots, shoot, and inflorescence) in the transgenic rif1-2 mutant plants overexpressing OsNOA/RIF1. (b) Complementation of nitric oxide (NO) production in mutant roots. Endogenous NO production was examined in roots by the NO-sensitive dye DAF-2 DA.The production of NO (shown as green fluorescence from the dye) was diminished in the mutant but was restored in the complemented lines. (c) Protein analysis of RBCL (Coomassie blue staining) and DXS (immunoblot hybridization) in the complemented lines. WT, wild type; M, rif1-2; C1 and C2, complemented rif1-2 lines overexpressing OsNOA1/RIF1.

Generation and characterization of OsNOA1/RIF1 RNAi gene suppression lines

To analyse the OsNOA1/RIF1 gene functions in rice, an RNAi binary vector was constructed for the generation of gene suppression mutants by Agrobacterium-mediated transformation. Approx. 20 independent lines were regenerated from hygromycin-resistant calli and they were confirmed to be true transgenic lines by glucuronidase (GUS) staining and genomic PCR. All the primary transformants showed different degrees of chlorotic phenotypes. Most of them did not survive to maturity and only two lines produced enough seeds for our subsequent experiments. The T1 seedlings resembled the highly chlorotic phenotype of the primary transformants (Fig. 5a). A few green seedlings were also present among the population (Fig. 5a) but they were found to be nontransgenic (not shown). Pigment analysis of the chlorotic RNAi seedlings revealed substantial decreases in Chla, Chlb and total carotenoid concen-trations (Fig. 5b), suggesting a reduction in thylakoid antenna protein levels. Chlorophyll fluorescence induction experiments were then performed to determine the photosynthetic capacity of the RNAi mutant. The maximum efficiency of PSII (Fv/Fm) and operating efficiency of PSII (ФPSII) represent the capacity of photon energy absorbed by PSII for photochemistry under dark- and light-adapted conditions, respectively. As shown in Fig. 5(b), the Fv/Fm and ФPSII values for the mutant seedlings were approx. 50% of those obtained for the wild type, suggesting some deficiencies in the energy transfer within PSII in the mutant seedlings as a result of OsNOA1/RIF1 suppression.

Figure 5.

 Phenotypic analysis of OsNOA1/RIF1 RNAi mutants. (a) A population of 2-wk-old RNAi T1 seedlings with chlorotic phenotype is shown in the left panel. Several green seedlings were also present but they were nontransgenic. A representative RNAi mutant seedling is shown in the right panel. (b) Chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoid (car) levels, Fv/Fm ratios, and ФPSII (operating efficiency of photosystem II) values in mutant (R1, dark grey bars, and R2, light grey bars) and wild-type (mid grey bars) seedlings. Data are expressed as mean SD. FW, fresh weight.

Molecular analysis of OsNOA1/RIF1 suppression mutants

As revealed by RT-PCR analysis, OsNOA1/RIF1 expression was completely suppressed in the chlorotic RNAi seedlings (Fig. 6a). Protein extracts were also prepared from wild-type and mutant seedlings and separated by SDS-PAGE. As shown in Fig. 6(b), RBCL levels were considerably reduced in the mutant seedlings. By contrast, immunoblot analysis demonstrated that the nuclear-encoded MEP enzyme DXS was produced in levels exceeding those in the wild type (Fig. 6b), consistent with results obtained for the Arabidopsis rif1-1 mutant (Flores-Pérez et al., 2008). However, unlike the Arabidopsis mutant, green pigmentation could not be restored in the rice mutant seedlings following treatment with 100 μM of the NO donor SNP for > 3 wk since germination (data not shown). Thus, the chlorotic phenotype was not likely to be a consequence of NO deficiency.

Figure 6.

 Molecular analysis of OsNOA1/RIF1 RNAi mutant seedlings. T1 seedlings (2 wk old) of two RNAi lines (R1 and R2) were collected for RNA and protein extraction. (a) OsNOA1/RIF1 expression was suppressed in the RNAi lines as detected by reverse-transcription polymerase chain reaction (RT-PCR). Actin expression was used as an internal control. (b) Analysis of RBCL and DXS protein accumulation. The RBCL levels (Coomassie blue staining) were reduced while DXS levels (immunoblot analysis) were elevated in the RNAi lines. (c) Abundances of chloroplast rRNA species (16S and 23S*) appear to be reduced in the RNAi seedlings as detected by ethidium bromide staining. 23S* is a fragment of the 23S rRNA. (d) Northern blot analysis of 16S rRNA levels and expression levels of selected chloroplast genes.

Interestingly, the denaturing RNA gel photograph shows that the smaller-sized rRNA signals were weakly or barely detected in the mutant samples (Fig. 6c). They were designated as chloroplast rRNA species, the 16S rRNA and a fragment of the 23S rRNA (Williams & Barkan, 2003). We confirmed by northern analysis that the 16S rRNA abundances were reduced substantially in the mutant samples (Fig. 6d). The transcripts of several chloroplast-encoded genes involved in photosynthesis were also detected by hybridization experiments. As shown in Fig. 6(d), rbcL, atpB, psaA and psbA were all found to have high expression levels in the mutant seedlings.

Quantitative protein expression analysis in the OsNOA1/RIF1 RNAi mutant

To understand the effects of OsNOA1/RIF1 gene suppression on proteome changes in rice seedlings, LC-MS/MS analysis was performed following labeling of wild-type and mutant trypsin-digested proteins with an isobaric labeling reagent (iTRAQ; Applied Biosystems). The samples were labeled with four iTRAQ tags (wild type 115, mutant 116; wild type 119; mutant 121) and pooled for fractionation by SCX chromatography. A total of 11 selected fractions were determined to contain sufficient amounts of peptides for LC-MS/MS analysis, which was performed twice in two separate experiments. Thus, the mutant to wild type protein expression (M : W) ratios were measured up to four times. The acquired MS/MS spectra were searched against the TIGR Rice Annotation Release 6.1 Pseudomolecules (67 393 entries) (http://rice.plantbiology.msu.edu). A total of 1805 rice accessions were identified from the quantitative proteomics analysis, among which 893 (49%) are common to both LC-MS/MS experiments. Among them, 1405 had in two or more instances at least three peptides contributing to their quantitative information. A total of 211 protein sequences were demonstrated to have statistically significant (P < 0.05) M : W ratios of ≥ 1.3 or ≤ 0.7 in at least two out of the four measurements (Tables 1, 2, S1). Representative MS/MS spectra for a fructose 1, 6-bisphosphatase and an NADPH-dependent FMN reductase domain-containing protein showing their identification and relative abundances in wild-type and mutant plant samples are shown in Fig. S2.

Table 1.   Chloroplast proteins with significant fold changes in the OsNOA1/RIF1 RNAi mutant seedlings (P < 0.05; M : W ≥ 1.3 or ≤ 0.7)
Protein nameTIGR accessionCt-encoded (accession)Arabidopsis homologa Functional classificationbM : W ratioc
  1. Ct, Chloroplast; PS, photosystem; N/A, not available.

  2. aBest Arabidopsis homologs as judged by blastE-value.

  3. bFunctional classification based on mapman BIN system.

  4. cMutant to wild-type expression ratio (average values of at least two measurements).

  5. dThe TIGR accession for the nuclear insertion of the corresponding chloroplast-encoded gene.

  6. eAn alternative splice form is also detected with similar fold changes and only one form is reported here.

  7. *mapman annotation for AT5G26742, as retreived from the Plant Protoeme Database (Mapman: 27.5* RNA.DEAD/DEAH BOX helicase, http://ppdb.tc.cornell.edu/dbsearch/gene.aspx?id=23459).

Upregulated proteins (9)
Enoyl-acyl-carrier-protein reductase NADH, chloroplast precursor, expressedLOC_Os08g23810 AT2G0599011.1.6Lipid metabolism.FA synthesis and FA elongation.enoyl ACP reductase1.331
2-Isopropylmalate synthase B, putative, expressedLOC_Os12g04440 AT1G7404013.1.4.4.1Amino acid metabolism.synthesis.branched chain group.leucine specific.2-isopropylmalate synthase1.369
D-3-Phosphoglycerate dehydrogenase, chloroplast precursor, putative, expressedLOC_Os04g55720 AT4G3420013.1.5.1.1Amino acid metabolism.synthesis.serine–glycine–cysteine group.serine.phosphoglycerate dehydrogenase1.478
ABC transporter, ATP-binding protein, putative, expressedLOC_Os03g21490 AT3G1067014S-assimilation1.454
DEAD-box ATP-dependent RNA helicase 3, putative, expressedLOC_Os03g61220 AT5G2674227.5*RNA. DEAD/DEAH BOX helicase1.366
Peptide chain release factor protein, putative, expressedLOC_Os11g43600 AT3G6291029.2.5Protein.synthesis.release1.388
Chaperonin, putative, expressedLOC_Os02g54060 AT5G2072029.6Protein.folding1.472
Chaperonin, putative, expressedLOC_Os10g41710 AT3G6021029.6Protein.folding1.344
Dihydroxy-acid dehydratase, putative, expressedLOC_Os08g44530 AT3G2394035.1Not assigned.no ontology1.313
Downregulated proteins (119)
Chlorophyll ab binding protein, putative, expressed (LHCB5)LOC_Os11g13890 AT4G103401.1.1.1PS.light reaction.PSII.LHC-II0.488
Chlorophyll ab binding protein, putative, expressed (LHCB2.1)LOC_Os03g39610 AT2G051001.1.1.1PS.light reaction.PSII.LHC-II0.451
Chlorophyll ab binding protein, putative, expressed (LHCB6)LOC_Os04g38410 AT1G158201.1.1.1PS.light reaction.PSII.LHC-II0.403
Chlorophyll ab binding protein, putative, expressed (LHCB1)LOC_Os09g17740 AT2G344301.1.1.1PS.light reaction.PSII.LHC-II0.413
Chlorophyll ab binding protein, putative, expressed (LHCB4)LOC_Os07g37240 AT5G015301.1.1.1PS.light reaction.PSII.LHC-II0.309
Chlorophyll ab binding protein, putative, expressed (NPQ4/PSBS)LOC_Os01g64960 AT1G445751.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.340
Oxygen-evolving enhancer protein 1, chloroplast precursor, putative, expressed (PSBO)LOC_Os01g31690 AT3G508201.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.483
Oxygen-evolving enhancer protein 3 domain containing protein, expressed (PSBQ)LOC_Os07g36080 AT4G212801.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.336
PsbP, putative, expressedLOC_Os07g04840 AT1G066801.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.376
Photosystem II 11 kDa protein, putative, expressedLOC_Os03g21560 AT1G036001.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.534
Photosystem II 10 kDa polypeptide, chloroplast precursor, putative, expressed (PSBR)LOC_Os07g05360 AT1G790401.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.258
Photosystem II 5 kDa protein, chloroplast precursor, putative, expressed (psbTn-2)LOC_Os02g37060 AT1G514001.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.222
Thylakoid lumenal 20 kDa protein, putative, expressedLOC_Os01g59090 AT3G566501.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.536
Photosystem Q, putative (PSBA/D1)LOC_Os10g21192dLOC_Osp1g00110ATCG000201.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.343
Photosystem II D2 protein, putative (PSBD/D2)LOC_Os02g24634dLOC_Osp1g00170ATCG002701.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.239
Photosystem II P680 chlorophyll a apoprotein, putative, expressed (psbB)LOC_Os10g21310dLOC_Osp1g00600ATCG006801.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.338
Photosystem II reaction center protein H, putative, expressed (PSBH)LOC_Os08g15296dLOC_Osp1g00620ATCG007101.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.186
Photosystem II 44 kDa reaction center protein, putative (PSBC)LOC_Os10g21212dLOC_Osp1g00180ATCG002801.1.1.2PS.light reaction.PSII.PSII polypeptide subunits0.241
Chlorophyll ab binding protein, putative, expressed (LHCA3)LOC_Os02g10390 AT1G615201.1.2.1PS.light reaction.photosystem I.LHC-I0.379
Photosystem I reaction center subunit II, chloroplast precursor, putative, expressed (PSAD)LOC_Os08g44680 AT1G031301.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.437
Photosystem I reaction center subunit III, chloroplast precursor, putative, expressed (PSAF)LOC_Os03g56670 AT1G313301.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.264
Photosystem I reaction center subunit, chloroplast precursor, putative, expressed (PSAG)LOC_Os09g30340 AT1G556701.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.303
Photosystem I reaction center subunit N, chloroplast precursor, putative, expressed (PSAN)LOC_Os12g08770 AT5G640401.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.214
Photosystem I P700 chlorophyll a apoprotein A1, putative (PSAA)LOC_Os10g38229dLOC_Osp1g00340ATCG003501.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.184
Photosystem I iron–sulfur center, putative (PSAC)LOC_Os10g21406dLOC_Osp1g00920ATCG010601.1.2.2PS.light reaction.photosystem I.PSI polypeptide subunits0.177
Cytochrome b6f complex iron–sulfur subunit, chloroplast precursor, putative (PETC)LOC_Os07g37030 AT4G032801.1.3PS.light reaction.cytochrome b6f0.253
Apocytochrome f precursor, putative (PETA)LOC_Os06g39738dLOC_Osp1g00480ATCG005401.1.3PS.light reaction.cytochrome b6f0.301
Apocytochrome f precursor, putative (PETA)LOC_Os10g21290dLOC_Osp1g00480ATCG005401.1.3PS.light reaction.cytochrome b6f0.218
ATP synthase F1, delta subunit family protein, putative, expressed (ATPD)LOC_Os02g51470 AT4G096501.1.4PS.light reaction.ATP synthase0.414
ATP synthase B chain, chloroplast precursor, putative, expressed (ATPG)LOC_Os03g17070 AT4G322601.1.4PS.light reaction.ATP synthase0.245
ATP synthase gamma chain, putative, expressed (ATPC)LOC_Os07g32880 AT4G046401.1.4PS.light reaction.ATP synthase0.258
ATP synthase subunit alpha, putative, expressed (ATPA)LOC_Os04g16740dLOC_Osp1g00310ATCG001201.1.4PS.light reaction.ATP synthase0.208
ATP synthase subunit beta, putative (ATPB)LOC_Os06g39740dLOC_Osp1g00410ATCG004801.1.4PS.light reaction.ATP synthase0.160
ATP synthase subunit beta, putative (ATPB)LOC_Os10g21266dLOC_Osp1g00410ATCG004801.1.4PS.light reaction.ATP synthase0.233
PGR5, putative, expressedLOC_Os08g45190 AT2G056201.1.40PS.light reaction.cyclic electron flow-chlororespiration0.324
Expressed protein (PGR5-LIKE A)LOC_Os03g64020 AT4G228901.1.40PS.light reaction.cyclic electron flow-chlororespiration0.310
Plastocyanin, chloroplast precursor, putative, expressed (PC-2)LOC_Os06g01210 AT1G761001.1.5.1PS.light reaction.other electron carrier (ox/red).plastocyanin0.350
2Fe–2S Iron–sulfur cluster binding domain containing protein, expressed (FED A)LOC_Os08g01380 AT1G609501.1.5.2PS.light reaction.other electron carrier (ox/red).ferredoxin0.473
Ferredoxin–NADP reductase, chloroplast precursor, putative, expressedLOC_Os02g01340 AT1G200201.1.7PS.light reaction.ferredoxin reductase0.382
Ferredoxin–NADP reductase, chloroplast precursor, putative, expressedLOC_Os06g01850 AT1G200201.1.7PS.light reaction.ferredoxin reductase0.517
4-Nitrophenylphosphatase, putative, expressedLOC_Os04g41340 AT5G367001.2.1PS.photorespiration.phosphoglycolate phosphatase0.442
Calvin cycle protein CP12, putative, expressedLOC_Os03g19380 AT3G624101.3PS.Calvin cycle0.345
Ribulose bisphosphate carboxylase large chain precursor, putative, expressedLOC_Os10g21268dLOC_Osp1g00420ATCG004901.3.1PS.Calvin cycle.rubisco large subunit0.103
Ribulose bisphosphate carboxylase large chain precursor, putativeLOC_Os12g10580dLOC_Osp1g00420ATCG004901.3.1PS.Calvin cycle.rubisco large subunit0.176
Ribulose bisphosphate carboxylase small chain, chloroplast precursor, putative, expressedLOC_Os12g17600 AT1G670901.3.2PS.Calvin cycle.rubisco small subunit0.070
Ribulose bisphosphate carboxylase small chain, chloroplast precursor, putative, expressedLOC_Os12g19470e AT1G670901.3.2PS.Calvincycle.rubisco small subunit0.147
Glyceraldehyde-3-phosphate dehydrogenase, putative, expressedLOC_Os03g03720 AT1G429701.3.4PS.Calvin cycle.GAP0.504
Glyceraldehyde-3-phosphate dehydrogenase, putative, expressedLOC_Os04g38600 AT3G266501.3.4PS.Calvincycle.GAP0.341
Triosephosphate isomerase, chloroplast precursor, putative, expressedLOC_Os09g36450 AT2G211701.3.5PS.Calvincycle.TPI0.560
Fructose-bisphospate aldolase isozyme, putative, expressedLOC_Os11g07020 AT4G389701.3.6PS.Calvincycle.aldolase0.402
Fructose-1,6-bisphosphatase, putative, expressedLOC_Os03g16050 AT3G540501.3.7PS.calvin cycle.FBPase0.447
Transketolase, chloroplast precursor, putative, expressedLOC_Os06g04270 AT2G452901.3.8PS.Calvin cycle.transketolase0.523
Fructose-1,6-bisphosphatase, putative, expressedLOC_Os04g16680 AT3G558001.3.9PS.Calvincycle.seduheptulose bisphosphatase0.372
Ribose-5-phosphate isomerase A, putative, expressedLOC_Os07g08030 AT3G047901.3.10PS.Calvincycle.Rib5P Isomerase0.411
Ribulose-phosphate 3-epimerase, chloroplast precursor, putative, expressedLOC_Os03g07300 AT5G614101.3.11PS.calvin cyle.RPE0.422
Phosphoribulokinase/Uridine kinase family protein, expressedLOC_Os02g47020 AT1G320601.3.12PS.Calvin cyle.PRK0.331
Uncharacterized kinase mug58, putative, expressedLOC_Os01g48990 AT1G803801.3.12PS.Calvincycle.PRK0.589
AAA-type ATPase family protein, putative, expressedLOC_Os11g47970 AT2G397301.3.13PS.Calvin cycle.rubisco interacting0.368
Kinase, pfkB family, putative, expressedLOC_Os06g12600 AT1G664302.2.1.1Major CHO metabolism.degradation.sucrose.fructokinase0.655
Uridylyltransferase-related, putative, expressedLOC_Os08g14440 AT1G168803.8.3Minor CHO metabolism.galactose.galactose-1-phosphate uridyl transferases0.584
Carbonic anhydrase, chloroplast precursor, putative, expressedLOC_Os01g45274 AT5G147408.3TCA/org. transformation.carbonic anhydrases0.221
Ferredoxin-dependent glutamate synthase, chloroplast precursor, putative, expressedLOC_Os07g46460 AT5G0414012.2.1N-metabolism.ammonia metabolism.glutamate synthase0.582
Glutamine synthetase, catalytic domain containing protein, expressedLOC_Os04g56400 AT5G3563012.2.2N-metabolism.ammonia metabolism.glutamine synthase0.362
Cysteine synthase, mitochondrial precursor, putative, expressedLOC_Os01g74650 AT3G5976013.1.5.3.1Amino acid metabolism.synthesis.serine–glycine–cysteine group.cysteine.OASTL0.623
Glyoxalase family protein, putative, expressedLOC_Os02g17920 AT1G6728013.2.3.2Amino acid metabolism.degradation.aspartate family.threonine0.449
Expressed proteinLOC_Os05g27100 AT5G1491015.2Metal handling.binding, chelation and storage0.312
Magnesium-protoporphyrin O-methyltransferase, putative, expressedLOC_Os06g04150 AT4G2508019.11Tetrapyrrole synthesis.magnesium protoporphyrin IX methyltransferase0.685
Thioredoxin, putative, expressedLOC_Os02g42700 AT4G0352021.1Redox.thioredoxin0.641
Thioredoxin, putative, expressedLOC_Os12g08730 AT3G1536021.1Redox.thioredoxin0.469
Thioredoxin, putative, expressedLOC_Os07g29410 AT1G7608021.1Redox.thioredoxin0.521
Thioredoxin, putative, expressedLOC_Os01g68480 AT5G1640021.1Redox.thioredoxin0.626
Ferredoxin-thioredoxin reductase, variable chain, putative, expressedLOC_Os02g42570 AT5G2344021.1Redox.thioredoxin0.614
Peroxidase precursor, putative, expressedLOC_Os04g51300 AT4G0901021.2.1Redox.ascorbate and glutathione.ascorbate0.534
OsAPx8 – Thylakoid-bound Ascorbate Peroxidase encoding gene 5,8, expressedLOC_Os02g34810 AT1G7749021.2.1Redox.ascorbate and glutathione.ascorbate0.594
Peroxiredoxin, putative, expressedLOC_Os06g09610 AT3G2606021.5Redox.periredoxins0.392
Peroxiredoxin, putative, expressedLOC_Os02g09940 AT3G5296021.5Redox.periredoxins0.441
Dehydrogenase, putative, expressedLOC_Os08g29170 AT1G2374026.7Misc.oxidases – copper, flavone, etc.0.391
3-Beta hydroxysteroid dehydrogenase/isomerase family protein, putative, expressedLOC_Os12g23180 AT1G0934027RNA0.422
NAD-dependent epimerase/dehydratase family protein, putative, expressedLOC_Os07g11110 AT3G6314027RNA0.348
Endoribonuclease, putative, expressedLOC_Os07g33240 AT3G2039027.1; 27.1.19RNA.processing; RNA.processing.ribonucleases0.616
RNA recognition motif containing protein, putative, expressedLOC_Os04g50110 AT2G3541027.4RNA.RNA binding0.542
RNA recognition motif containing protein, putative, expressedLOC_Os09g39180 AT4G2477027.4RNA.RNA binding0.472
Hypothetical proteinLOC_Os07g43860 AT2G3722027.4RNA.RNA binding0.229
Chloroplast 30S ribosomal protein S4, putativeLOC_Os10g38214dLOC_Osp1g00360ATCG0038029.2.1.1.1.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit0.256
Chloroplast 30S ribosomal protein S18, putative, expressedLOC_Os08g15308dLOC_Osp1g00570ATCG0065029.2.1.1.1.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit0.342
Chloroplast 30S ribosomal protein S19, putative, expressedLOC_Os10g21352dLOC_Osp1g01130ATCG0082029.2.1.1.1.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit0.456
Chloroplast 30S ribosomal protein S7, putativeLOC_Os10g21372dLOC_Osp1g00820ATCG0090029.2.1.1.1.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit0.322
RNA recognition motif containing protein, putative, expressedLOC_Os09g10760 AT3G5215029.2.1.1.1.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit0.477
S10/S20 Domain containing ribosomal protein, putative, expressedLOC_Os03g10060 AT3G1312029.2.1.1.1.1.10Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit.S100.403
30S ribosomal protein S31, chloroplast precursor, putative, expressedLOC_Os05g09400 AT2G3814029.2.1.1.1.1.31Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit.S310.225
Ribosomal protein, putative, expressedLOC_Os03g34040e AT2G3380029.2.1.1.1.1.5Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit.S50.303
Ribosomal protein S6, putative, expressedLOC_Os03g62630 AT1G6451029.2.1.1.1.1.6Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30S subunit.S60.612
Chloroplast 50S ribosomal protein L14, putativeLOC_Os10g21342dLOC_Osp1g00710ATCG0078029.2.1.1.1.2Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit0.262
L1P family of ribosomal proteins domain containing protein, expressedLOC_Os05g32220 AT3G6349029.2.1.1.1.2.1Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L10.623
Ribosomal protein L10, putative, expressedLOC_Os03g17580 AT5G1351029.2.1.1.1.2.10Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L100.393
Ribosomal protein L7/L12 C-terminal domain containing protein, expressedLOC_Os01g47330 AT3G2785029.2.1.1.1.2.12Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L120.446
Ribosomal protein L13, putative, expressedLOC_Os01g54540 AT1G7863029.2.1.1.1.2.13Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L130.385
50S Ribosomal protein L15, chloroplast precursor, putative, expressedLOC_Os03g12020 AT3G2592029.2.1.1.1.2.15Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L150.481
50S Ribosomal protein L17, putative, expressedLOC_Os03g60100 AT3G5421029.2.1.1.1.2.17Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L170.384
Ribosomal L18p/L5e family protein, putative, expressedLOC_Os03g61260 AT1G4835029.2.1.1.1.2.18Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L180.468
50S Ribosomal protein L19, chloroplast precursor, putative, expressedLOC_Os02g43600 AT4G1756029.2.1.1.1.2.19Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L190.352
Ribosomal protein L29, putative, expressedLOC_Os02g51790 AT5G6522029.2.1.1.1.2.24Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L240.523
Ribosomal protein L24, putative, expressedLOC_Os06g46930 AT5G5460029.2.1.1.1.2.24Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L240.44
Ribosomal protein L27, putative, expressedLOC_Os01g69950 AT5G4095029.2.1.1.1.2.27Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L270.282
Ribosomal protein L3, putative, expressedLOC_Os02g04460 AT2G4303029.2.1.1.1.2.3Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L30.492
Ribosomal protein L4, putative, expressedLOC_Os03g15870 AT1G0732029.2.1.1.1.2.4Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L40.445
Ribosomal protein L5, putative, expressedLOC_Os03g03360 AT4G0131029.2.1.1.1.2.5Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L50.501
Ribosomal protein L6, putative, expressedLOC_Os03g24020 AT1G0519029.2.1.1.1.2.6Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L60.458
Ribosomal L9, putative, expressedLOC_Os02g57670 AT3G4489029.2.1.1.1.2.9Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L90.49
S1 RNA binding domain containing protein, expressedLOC_Os03g20100 AT5G3051029.2.1.1.3.1.1Protein.synthesis.ribosomal protein.prokaryotic.unknown organellar.30S subunit.S10.244
Ribosome, putative, expressedLOC_Os02g09590 AT3G2716029.2.1.1.3.1.21Protein.synthesis.ribosomal protein.prokaryotic.unknown organellar.30S subunit.S210.245
Methionyl-tRNA synthetase, putative, expressedLOC_Os04g23820 AT3G5998029.2.4Protein.synthesis.initiation0.407
Phosphoglycerate kinase protein, putative, expressedLOC_Os05g41640 AT3G1249029.5.3Protein.degradation.cysteine protease0.447
Peptidyl-prolyl cis-trans isomerase, FKBP-type, putative, expressedLOC_Os08g42850 AT2G4356029.6Protein.folding0.691
Arsenate reductase, putative, expressedLOC_Os02g49680 AT5G2306030.3Signalling.calcium0.569
Calvin cycle protein CP12, putative, expressedLOC_Os01g19740 AT2G4740035.1Not assigned.no ontology0.347
CbbY, putative, expressedLOC_Os03g36750 AT3G4842035.1Not assigned.no ontology0.374
Enzyme of the cupin superfamily protein, putative, expressedLOC_Os04g36760 AT4G1030035.2Not assigned.unknown0.448
Expressed proteinLOC_Os05g27100 N/A35.2Not assigned.unknown0.312
Table 2.   Nonchloroplast proteins with significant fold changes in the OsNOA1/RIF1 RNAi mutant seedlings (P < 0.05; M : W ≥ 1.3 or ≤ 0.7)
Protein nameTIGR accessionArabidopsis homologa Functional classificationbM : W ratioc
  1. N/A, not available.

  2. aBest Arabidopsis homologs as judged by blastE-value.

  3. bFunctional classification based on MapMan BIN system.

  4. cMutant to wild type expression ratio (average values of at least two measurements).

  5. dAn alternative splice form is also detected with similar fold changes and only one form is reported here.

Upregulated proteins (54)
Kinase, pfkB family, putative, expressedLOC_Os08g02120AT4G102602.2.1.1Major CHO metabolism.degradation.sucrose.fructokinase1.328
Sucrose synthase, putative, expressedLOC_Os03g28330AT3G431902.2.1.5Major CHO metabolism.degradation.sucrose.sucrosesynthase1.379
Enolase, putative, expressedLOC_Os06g04510AT2G365304.12Glycolysis.enolase1.977
Pyruvate kinase, putative, expressedLOC_Os11g05110AT3G529904.13Glycolysis.PK1.337
Fructose-bisphospate aldolase isozyme, putative, expressedLOC_Os05g33380AT2G364604.7Glycolysis.aldolase1.557
Fructose-bisphospate aldolase isozyme, putative, expressedLOC_Os01g02880AT2G011404.7Glycolysis.aldolase1.516
Glyceraldehyde-3-phosphate dehydrogenase, putative, expressedLOC_Os08g03290AT3G041204.9Glycolysis.glyceraldehyde 3-phosphate dehydrogenase1.378
6-Phosphogluconate dehydrogenase, decarboxylating, putative, expressedLOC_Os06g02144AT3G023607.1.3OPP.oxidative PP.6-phosphogluconate dehydrogenase1.331
Cytochrome c, putative, expressedLOC_Os05g34770AT4G100409.6Mitochondrial electron transport/ATP synthesis.cytochrome c1.599
OsTIL-1 Temperature-induced lipocalin-1, expressedLOC_Os02g39930AT5G5807011Lipid metabolism1.426
Acyl CoA-binding protein, putative, expressedLOC_Os08g06550AT1G3181211.1.13Lipid metabolism.FA synthesis and FA elongation.acyl-CoA binding protein1.383
NADPH-dependent FMN reductase domain containing protein, expressedLOC_Os01g57570AT5G5450011.8Lipid metabolism.exotics (steroids, squalene etc.)2.254
Glutamine synthetase, catalytic domain containing protein, expressedLOC_Os02g50240AT5G3760012.2.2N-metabolism.ammonia metabolism.glutamine synthase1.510
Phenylalanine ammonia-lyase, putative, expressedLOC_Os02g41630AT2G3704016.2.1.1Secondary metabolism.phenylpropanoids.lignin biosynthesis.PAL1.305
Caffeoyl-CoA O-methyltransferase, putative, expressedLOC_Os08g38900AT4G3405016.2.1.6Secondary metabolism.phenylpropanoids.lignin biosynthesis.CCoAOMT1.339
Chalcone–flavanone isomerase, putative, expressedLOC_Os12g02370AT5G0527016.8.2Secondary metabolism.flavonoids.chalcones1.547
Heat shock protein, putativeLOC_Os09g30439AT5G5603020.2.1Stress.abiotic.heat1.538
Heat shock 22 kDa protein, mitochondrial precursor, putative, expressedLOC_Os02g52150AT5G5144020.2.1Stress.abiotic.heat1.717
Pathogenesis-related Bet v I family protein, putative, expressedLOC_Os04g39150AT1G2402020.2.99Stress.abiotic.unspecified1.851
Thioredoxin, putative, expressedLOC_Os07g08840AT3G5103021.1Redox.thioredoxin1.589
OsAPx1 – Cytosolic Ascorbate Peroxidase encoding gene 1-8, expressedLOC_Os03g17690AT1G0789021.2.1Redox.ascorbate and glutathione.ascorbate2.003
Glutathione S-transferase, N-terminal domain containing protein, expressedLOC_Os05g02530AT1G7527021.2.1Redox.ascorbate and glutathione.ascorbate1.326
Catalase isozyme A, putative, expressedLOC_Os02g02400AT4G3509021.6Redox.dismutases and catalases1.313
Glyceraldehyde-3-phosphate dehydrogenase, putative, expressedLOC_Os04g40950AT1G1340027.3.99RNA.regulation of transcription.unclassified1.457
tRNA synthetase class II core domain containing protein, expressedLOC_Os05g05840AT3G0276029.1.21Protein.amino acid activation.histidine-tRNA ligase1.372
40S Ribosomal protein S13, putative, expressedLOC_Os08g02410AT4G0010029.2.1.2.1.13Protein.synthesis.ribosomal protein.eukaryotic.40S subunit.S131.474
40S Ribosomal protein S17, putative, expressedLOC_Os10g27190AT3G1061029.2.1.2.1.17Protein.synthesis.ribosomal protein.eukaryotic.40S subunit.S171.342
40S Ribosomal protein S21, putative, expressedLOC_Os03g46490AT5G2770029.2.1.2.1.21Protein.synthesis.ribosomal protein.eukaryotic.40S subunit.S211.421
40S Ribosomal protein S28, putative, expressedLOC_Os10g27174AT3G1009029.2.1.2.1.28Protein.synthesis.ribosomal protein.eukaryotic.40S subunit.S281.340
60S Acidic ribosomal protein, putative, expressedLOC_Os01g09510AT2G2771029.2.1.2.2.82Protein.synthesis.ribosomal protein.eukaryotic.60S subunit.P21.599
60S Ribosomal protein L27-3, putative, expressedLOC_Os02g18380AT4G1500029.2.1.2.2.27Protein.synthesis.ribosomal protein.eukaryotic.60S subunit.L271.441
Ribosomal protein L14, putative, expressedLOC_Os04g43540AT2G2045029.2.1.2.2.14Protein.synthesis.ribosomal protein.eukaryotic.60S subunit.L141.542
Ribosomal protein L37, putative, expressedLOC_Os08g03450AT1G1525029.2.1.2.2.37Protein.synthesis.ribosomal protein.eukaryotic.60S subunit.L371.343
DEAD-box ATP-dependent RNA helicase, putative, expressedLOC_Os06g48750AT3G1392029.2.3Protein.synthesis.initiation1.376
Elongation factor protein, putative, expressedLOC_Os07g46750AT5G1211029.2.4Protein.synthesis.elongation1.328
26S Protease regulatory subunit 4, putative, expressedLOC_Os03g18690AT4G2904029.5.11.20Protein.degradation.ubiquitin.proteasome1.541
Peptidase, T1 family, putative, expressedLOC_Os06g06030AT1G1306029.5.11.20Protein.degradation.ubiquitin.proteasome1.439
Chaperonin, putative, expressedLOC_Os07g44740AT1G1498029.6Protein.folding1.732
Chaperonin, putative, expressedLOC_Os03g25050AT1G1498029.6Protein.folding1.514
DnaK family protein, putative, expressedLOC_Os02g53420AT5G0959029.6Protein.folding1.377
T-complex protein, putative, expressedLOC_Os03g59020AT3G0396029.6Protein.folding1.345
14-3-3 Protein, putative, expressedLOC_Os08g33370AT3G0252030.7Signalling.14-3-3 proteins1.312
Actin, putative, expressedLOC_Os12g06660AT5G0981031.1Cell.organisation1.483
Actin, putative, expressedLOC_Os03g50885AT3G1211031.1Cell.organisation1.378
Cell division control protein 48 homolog E, putative, expressedLOC_Os03g05730AT5G0334031.2Cell.division1.747
Mitochondrial glycoprotein, putative, expressedLOC_Os01g05060AT2G3979535.1Not assigned.no ontology1.544
PDI, putative, expressedLOC_Os03g29240AT1G6042035.1Not assigned.no ontology2.987
PDI, putative, expressedLOC_Os03g29190AT1G6042035.1Not assigned.no ontology1.358
Salt stress root protein RS1, putative, expressedLOC_Os02g18410AT4G2026035.1Not assigned.no ontology1.311
Transferase family protein, putative, expressedLOC_Os08g44840AT5G2394035.1Not assigned.no ontology2.198
WD40-like Beta Propeller Repeat family protein, expressedLOC_Os03g19990AT1G2168035.1Not assigned.no ontology1.679
Gibberellin receptor GID1L2, putative, expressedLOC_Os07g06840AT5G0657035.1Not assigned.no ontology1.301
Sex determination protein tasselseed-2, putative, expressedLOC_Os07g46852dAT4G0341035.1Not assigned.no ontology1.426
Retrotransposon protein, putative, unclassifiedLOC_Os11g14960N/A Others1.418
Downregulated protein (29)
Hydroxyacid oxidase 1, putative, expressedLOC_Os03g57220AT3G144201.2.2PS.photorespiration.glycolate oxidase0.542
Hydroxyacid oxidase 1, putative, expressedLOC_Os07g05820AT3G144201.2.2PS.photorespiration.glycolate oxidase0.402
Aminotransferase, classes I and II, domain containing protein, expressedLOC_Os07g01760AT1G233101.2.3PS.aminotransferases peroxisomal0.444
Aminotransferase, putative, expressedLOC_Os08g39300AT2G133601.2.3PS.aminotransferases peroxisomal0.430
Glycine cleavage system H protein, putative, expressedLOC_Os10g37180AT1G324701.2.4PS.photorespiration.glycine cleavage0.433
Serine hydroxymethyltransferase, mitochondrial precursor, putative, expressedLOC_Os03g52840AT4G379301.2.5PS.photorespiration.serine hydroxymethyltransferase0.612
Erythronate-4-phosphate dehydrogenase domain containing protein, expressedLOC_Os02g01150dAT1G680101.2.6PS.photorespiration.hydroxypyruvate reductase0.483
Fructose-1,6-bisphosphatase, putative, expressedLOC_Os01g64660AT1G436702.1.1.3Major CHO metabolism.synthesis.sucrose.FBPase0.301
Fructose-bisphospate aldolase isozyme, putative, expressedLOC_Os06g40640AT4G265304.7Glycolysis.aldolase0.371
Triosephosphate isomerase, cytosolic, putative, expressedLOC_Os01g05490AT3G554404.8Glycolysis.TPI0.571
Lactate/malate dehydrogenase, putative, expressedLOC_Os03g56280AT5G096606.3Gluconeogenesis.Malate DH0.453
Periplasmic beta-glucosidase precursor, putative, expressedLOC_Os03g53860AT5G2095010.6.1Cell wall.degradation.cellulases and beta-1,4-glucanases0.680
Aminomethyltransferase, putative, expressedLOC_Os04g53230AT1G1186013.2.5.2Amino acid metabolism.degradation.serine–glycine–cysteine group.glycine0.436
Osmotin, putative, expressedLOC_Os12g38150AT2G2879020.2Stress.abiotic0.324
Peroxidase precursor, putative, expressedLOC_Os06g20150AT1G0526020.2.2Stress.abiotic.cold0.360
Cupin domain containing protein, expressedLOC_Os08g35760AT1G7261020.2.99Stress.abiotic.unspecified0.588
Catalase isozyme B, putative, expressedLOC_Os06g51150AT4G3509021.6Redox.dismutases and catalases0.621
Catalase domain containing protein, expressedLOC_Os03g03910AT4G3509021.6Redox.dismutases and catalases0.392
LTPL52 – Protease inhibitor/seed storage/LTP family protein precursor, expressedLOC_Os03g26820AT5G6408026.21Misc.protease inhibitor/seed storage/lipid transfer protein (LTP) family protein0.433
60S Acidic ribosomal protein, putative, expressedLOC_Os06g48780AT4G2589029.2.2Protein.synthesis.misc ribososomal protein0.613
Transportin-2, putative, expressedLOC_Os04g59494AT2G1695029.3.1Protein.targeting.nucleus0.398
Expressed proteinLOC_Os01g66980AT2G3069529.6Protein folding0.625
Light-induced protein 1-like, putative, expressedLOC_Os01g01340AT3G2674030.11Signalling.light0.478
Gamma-tubulin complex component 6, putative, expressedLOC_Os04g47906AT3G4361031.1Cell.organization0.682
Kinesin motor domain containing protein, expressedLOC_Os04g28260AT3G2367031.1Cell.organization0.174
Expressed proteinLOC_Os05g48630AT1G7983035.1Not assigned.no ontology0.348
Expressed proteinLOC_Os12g37900AT1G0779535.2Not assigned.unknown0.261
BBTI4 – Bowman–Birk type bran trypsin inhibitor precursor, expressedLOC_Os01g03340N/A Others0.320
Retrotransposon protein, putative, unclassifiedLOC_Os12g10604N/A Others0.248

Out of the 211 proteins, > 70% (148) were downregulated and the remaining (63) were upregulated in the rice RNAi mutant. As the rice RNAi mutants showed a strongly chlorotic phenotype, we were particularly interested in the chloroplast proteome changes. Chloroplast proteins (128) were assigned according to the TargetP prediction, the Plant Plastid Proteome Database (http://ppdb.tc.cornell.edu) annotations for the closest Arabidopsis homologs, and manual examinations of their annotated functions. A total of 119 chloroplast proteins were found to be significantly downregulated (M : W average 0.394, range 0.070–0.691), including 19 chloroplast-encoded proteins which showed more drastically reduced expression (M : W average 0.250, range 0.103–0.456). Most of these downregulated proteins are subunits of the different light-reaction complexes, such as PSII, PSI, cytochrome b6f, ATP synthase, and NADP reductase, which are all localized in the thylakoid membranes. The abundances of 28 chloroplast ribosomal proteins were also reduced significantly (M : W average 0.404, range 0.225–0.623). Other downregulated chloroplast proteins included enzymes in all three stages of the Calvin cycle, some enzymes in amino acid metabolism, a number of redox-related proteins (thioredoxins, ascorbate peroxidase and peroxiredoxins), RNA-binding proteins, translation-related factors, one enzyme in tetrapyrrole biosynthesis, etc. (Table 1). By contrast, only nine chloroplast proteins were found to be upregulated significantly (M : W average 1.39, range 1.31–1.48) and they are all stromal proteins.

Outside the chloroplasts, there were a total of 83 proteins (Table 2) showing our specified fold-changes in the RNAi mutant; some of them were targeted to the secretory pathway or the mitochondria while the others were mainly cytosolic (predictions by the TargetP algorithm). The majority of these proteins (54) were found to have increased expression levels (M : W average 1.530, range 1.301–2.987) and the remaining (29) were reduced in abundances (M : W average 0.449, range 0.174–0.682). Unlike the situation in chloroplasts, eight cytosolic ribosomal proteins were upregulated in the RNAi mutant (M : W average 1.438, range 1.340–1.599) and the same is true for two translation-related factors. Other upregulated proteins included enzymes in lipid, amino acid and secondary metabolism, heat shock proteins, chaperonins, actin, etc. The glycolytic pathway appeared to be have enhanced activity in the RNAi plants as enzymes in at least four reaction steps showed increased expression levels, including fructose-bisphosphate aldolase (FBA, two isoforms), glyceraldehyde 3-phosphate dehydrogenase, enolase and pyruvate kinase (two isoforms). By contrast, another FBA isoform and a triose phosphate isomerase were down-regulated. Reduce abundances in the RNAi mutant were also detected for enzymes in photorespiration, gluconeogenesis, amino acid metabolism, abiotic stress-related proteins, tubulin, etc. Different classes of redox-related proteins were either upregulated (thioredoxin, ascorbate peroxidase, glutathionine S-transferase, catalase) or downregulated (catalases) in the RNAi mutant (Table 2).

Discussion

Chloroplasts are generally believed to descend from free-living photosynthetic bacteria that became internal inhabitants of early eukaryotic cells (Dyall et al., 2004). During the course of chloroplast evolution, a large amount of genetic information has been transferred to the nuclear genome, resulting in the increased control of the endosymbiont by the host cell (Gray et al., 2002). AtNOA1/RIF1 was recently reported to be a chloroplast-targeted protein homologous to B. subtilis YqeH (Flores-Pérez et al., 2008), a cGTPase required for the correct formation of the bacterial 70S ribosome and the assembly or stability of the 30S subunit (Uicker et al., 2007). Chloroplast-targeted chimeric YqeH proteins are able to complement Arabidopsis rif1 mutants (Flores-Pérez et al., 2008; Sudhamsu et al., 2008) which were shown to have decreased levels of a few chloroplast-encoded proteins (Flores-Pérez et al., 2008). Both the Arabidopsis and bacterial proteins bind and hydrolyse GTP, and the enzyme activities were proposed to be coupled to RNA and/or protein binding that is essential for ribosome biogenesis and/or functions (Moreau et al., 2008; Sudhamsu et al., 2008). In the present study, OsNOA1/RIF1 and other plant homologs were found to possess all the structural domains (ZBD, CPG and CTD, Fig. 1a) that are necessary for the functional expression of AtNOA1 and G. stearothermophilus YqeH (Moreau et al., 2008; Sudhamsu et al., 2008). Expression of OsNOA1/RIF1-EYFP fusion in transgenic Arabidopsis demonstrated that the rice homolog is also targeted to chloroplasts (Fig. 3). In addition, OsNOA1/RIF1 complemented most mutant phenotypes in the Arabidopsis rif1-2 mutant (Fig. 4), further indicating that it represents a functional AtNOA1/RIF1 homolog. The occurrence of gymnosperm (Sitka spruce), monocot (rice, maize and sorghum) and dicot (castor bean, grape and tobacco) NOA1/RIF1 homologs (Fig. 1) with predicted chloroplast location strongly suggested the conservation of an essential nuclear-encoded plant protein required for proper chloroplast functions. It also highlights the significance of our independent investigation on the functional role of OsNOA1/RIF1 in the monocot model plant.

Chloroplast biogenesis is a light-regulated process involving the translation of both chloroplast and nuclear-encoded proteins. In accordance with its expected role for proper ribosomal function in chloroplasts, RNAi-mediated gene suppression of OsNOA1/RIF1 resulted in a chlorotic phenotype with impaired photosynthetic capacity in rice seedlings (Fig. 5). In maize, pigmentation deficiencies are typical phenotypes of mutants with ribosome-deficient chloroplasts or having defects in the chloroplast translation machinery (Williams & Barkan, 2003). For example, the ivory mutant crs2 (chloroplast RNA splicing 2) does not contain chloroplast ribosomes and CRS2 is a nuclear-encoded protein required for group II intron splicing in chloroplasts (Jenkins et al., 1997). Similarly, the albino ppr2 (pentatricopeptide repeat 2) mutant lacks plastid rRNA and translation products, and PPR2 is a chloroplast-localized protein found in large macromolecular complexes in the stroma (Williams & Barkan, 2003). In addition, decreases in the maximum quantum yield and operating efficiency of PSII was previously recorded in Arabidopsis mutants with defects in chloroplast translation (Pesaresi et al., 2001, 2006). Our iTRAQ–LC-MS/MS experiments further provided comprehensive proteome information strongly supporting the functional role of OsNOA1/RIF1 in rice chloroplasts. A total of 28 chloroplast ribosomal proteins belonging to either the 50S or 30S subunits were substantially reduced in abundances in the mutant (Table 1). Together with the low levels of chloroplast rRNAs detected in the RNAi seedlings (Fig. 6c), these are strong indications of reduced ribosome accumulation and protein synthesis in chloroplasts. The downregulation of methionyl-tRNA synthetase and several elongation factors (TS, Tu and P) was also in accordance with potential decreases in translation efficiency. Most of the quantified chloroplast-encoded proteins were consistently found to have drastically reduced expression levels in the OsNOA1/RIF1 RNAi mutant (Table 1).

The quantitative proteomics data also revealed that large numbers of nuclear-encoded chloroplast proteins were downregulated in the rice mutant. Many of chloroplast-localized proteins are subunits of the different photosynthetic enzyme complexes, such as PSI, PSII, cytochrome b6f, ATP synthase and RUBISCO, which all contain components derived from the chloroplast genome. Their reduced abundances may represent consequences of protein degradation and/or repression of nuclear gene expression. For example, reduced synthesis of chloroplast-encoded subunits is believed to result in proteolytic degradation of the unassembled nuclear-encoded components (Williams & Barkan, 2003). However, accumulating evidence is now available for the presence of retrograde signals derived from chloroplasts to regulate the expression of nuclear photosynthetic genes (Fernández & Strand, 2008; Woodson & Chory, 2008). Thus, the expression of nuclear genes encoding the different light reaction complexes and stromal photosynthetic enzymes were downregulated in albino mutant plants that lack plastid ribosomes or in plants treated with chloroplast translation inhibitors (Gray et al., 2003). Similar to the barley albostrians mutant which contains ribosome-deficient chloroplasts (Hess et al., 1991, 1994), our rice RNAi mutant seedlings were found to have reduced levels of enzymes involved in different stages of the Calvin cycle as well as a photorespiration enzyme (Table 1).

A small number of chloroplast proteins showed significantly increased expression levels in the rice mutant (Table 1), including the stromal enzymes for the biosynthesis of fatty acid and amino acids. Similarly, elevated levels of DXS were detected by immunoblot analysis (Fig. 6c). Interestingly, FSM-treated wild-type rice seedlings showed the same degree of pale phenotype as observed in the mutant seedlings which remained unchanged in coloration after the FSM treatment (data not shown). The upregulation of MEP pathway enzymes (e.g. DXS) in the Arabidopsis rif1-1 mutant has been attributed to the decreased amounts of the chloroplast ATP-dependent Clp protease, which targets nuclear-encoded proteins (Flores-Pérez et al., 2008). The abundances of two protein annotated as the ClpA subunit were reduced by < 30% in the rice RNAi mutant (M : W ratios of 0.71 and 0.78, Table S1), presumably also resulting in lower Clp protease activities in chloroplasts. However, the possibility that MEP pathway enzymes are direct substrates for Clp proteolysis has been disputed in two recent reports (Kim et al., 2009; Zybailov et al., 2009). Nevertheless, it is still plausible that the other enhanced stromal proteins in the rice RNAi mutant are potential substrates for the Clp proteins which represent the largest chloroplast protease family.

Nuclear-encoded proteins other than the chloroplast-targeted proteins also showed altered expression levels in the rice mutant (Table 2). The apparently enhanced translational activities in the cytoplasm may reflect some compensatory responses to the dramatic changes in chloroplast metabolism. For example, the apparent increases in glycolytic activity may represent a mechanism to generate more ATP in the rice mutant as the chloroplast ATP synthase complex is substantially depleted. Similarly, the enhanced level of a pentose phosphate enzyme may serve to provide more reducing power in response to the decreased generation of NADPH in the mutant thylakoid membranes. Moreover, several enzymes of the phenylpropanoid pathways were upregulated in the RNAi mutant and this could be explained as an indirect effect of increasing light stress caused by the reduction of light-harvesting pigmentation. Similar patterns of enhanced glycolytic enzyme and chalcone synthase expression have also been observed in the barley albostrains mutant (Hess et al., 1994).

In conclusion, our work provides independent evidence from the monocot model plant that a highly conserved plant cGTPase of likely prokaryotic origin is essential for normal ribosome biogenesis and translational activities in chloroplasts. Thus, it becomes increasingly evident that the reduced NO production originally detected in the Arabidopsis rif1 mutants (Guo et al., 2003) was a secondary effect resulting from perturbed chloroplast metabolism (Zemojtel et al., 2006; Gas et al., 2009).

Acknowledgements

This work was supported by the National High Techno-logy Research and Development Program of China (2006AA10A102) to Yuezhi Tao as well as the Research Grants Council of Hong Kong (HKU7527/06M) and HKU Seed Funding Programme (200811159080) to Clive Lo. We are grateful to Professor Jirong Huang (National Key Laboratory of Plant Molecular Genetics, Shanghai Institute for Biological Sciences) for his helpful discussions on our investigations. We also thank Dr P. Leon (Instituto de Biotechnologia – UNAM) for providing the DXS antibodies.

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