Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3. In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

KEYWORDS chloroplast development ribosome biogenesis rice (Oryza sativa L.) Spo0B GTP-binding protein (Obg) thermo-sensitive virescent The chloroplast is a semiautonomous organelle that contains many genes important for the metabolic pathways of photosynthesis (Mandel et al. 1996). Chloroplast development during leaf development consists of a series of processes associated with chloroplast differentiation, which can be divided into three steps coregulated by the plastid and nuclear genes (Mullet 1993;Kusumi et al. 2011). The first step involves the activation of plastid replication and plastid DNA synthesis. The second step is the chloroplast "build up," characterized by the establishment of the chloroplast genetic system. At this step, nuclear-encoded plastid RNA polymerase (NEP) preferentially transcribes plastid genes encoding plastid gene expression machineries (Hajdukiewicz et al. 1997), and the transcription and translation activities in the chloroplast are dramatically increased. In the third step, the plastid and nuclear genes that encode the photosynthetic apparatus are expressed at very high levels. The plastid genes for the photosynthetic apparatus are predominantly transcribed by plastid-encoded RNA polymerase (PEP) (Santis-Maciossek et al. 1999). Expression of all of these genes leads to chloroplast biosynthesis and assembly. In spite of this basic understanding of chloroplast development, the mechanisms of the major genes involved in this process in higher plants remain largely unknown (Pfalz and Pfannschmidt 2012). GTPases comprise a large family of enzymes that hydrolyze guanosine triphosphate (GTP). GTPases occur in all of the kingdoms of life and are known to play crucial roles in many cellular processes (Bourne et al. for the viability of nearly all bacteria (Maddock et al. 1997;Okamoto and Ochi 1998;Shepherd et al. 2002;Foti et al. 2005;Michel 2005). With the exception of the Escherichia coli homolog ObgE, which is involved in chromosome partitioning and is partially associated with the cell membrane (Kobayashi et al. 2001), the majority of the Obg proteins studied to date are associated with the ribosome. For example, Obgs from B. subtilis, Caulobacter crescentus, and E. coli have been reported to be associated with the 50S ribosomal subunit (Scott et al. 2000;Lin et al. 2004;Wout et al. 2004). Furthermore, mutations in the Obg proteins have been shown to affect ribosome assembly or maturation (Datta et al. 2004;Jiang et al. 2006). Recently, mutations in the Obg fold and the OCT region of B. subtilis Obg impaired its ability to associate with the ribosomes and to induce a stress response, suggesting that Obg plays dual functions in ribosome biogenesis and stress responses (Kuo et al. 2008). Among eukaryotes, an Arabidopsis Obg homolog, AtObgC/CPSAR1, which localizes both in the inner envelope and the stroma of chloroplasts, has been shown to be essential for the formation of thylakoid membranes (Garcia et al. 2010). However, others have suggested that AtObgC/CPSAR1 may play an important role in the biogenesis of chloroplast ribosomes (Bang et al. 2009). More recently, studies on Arabidopsis AtObgC/CPSAR1 and rice OsObgC led Bang et al. (2012) to conclude that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development.
Rice (Oryza sativa) mutants are ideal for explicating the function of chloroplast development in higher plants (Sachetto-Martins et al. 2000;Leister 2003). In this study, we identified a new rice thermo-sensitive virescent mutant tsv3, which exhibited an albino phenotype before the three-leaf stage at 20°and a normal green color at 32°or even at 20°a fter the four-leaf stage. Mutation of rice TSV3, encoding the Obg subfamily of small GTP-binding protein, was responsible for the mutant phenotype. Additionally, the transcript levels of genes associated with chlorophyll (Chl) biosynthesis and photosynthesis, and those associated with biogenesis of the chloroplast ribosome 50S subunit, were severely affected in the tsv3 mutants at low temperature. These findings suggest that rice TSV3 plays an important role in chloroplast biogenesis during leaf development under cold stress.

Plant materials and growth conditions
The rice thermo-sensitive virescent mutant, tsv3, was discovered in our mutant pool from the japonica cv. Jiahua 1 [wild type (WT)] irradiated with 60 Co gamma rays in 2006. The F 2 population for genetic mapping was generated from a cross between the indica cv. Pei'ai 64S and the tsv3 mutant. The thermo-sensitive virescent mutant phenotype of tsv3 could be distinguished from the normal (WT) green phenotype during the winter season at Hainan, China (subtropical climate) and the spring season in Shanghai, China (temperate climate) under local growing conditions. WT and tsv3 plants were grown in growth chambers under a controlled photoperiod (12 hr of light and 12 hr of dark) and at a constant temperature of either 20 or 32°for phenotypic characterization, pigment content measurement, and RNA extraction. Figure 1 Characterization of the tsv3 mutants. Seedlings of Jiahua 1 (WT, left) and tsv3 mutant (right) at the two-, three-, and four-leaf stage grown at (A) 20°and at (B) 32°. (C) Three-leaf-stage seedlings for WT (left) and tsv3 mutant (right) grown at 20°. Measurements of total Chl, Chl a, Chl b, and Car contents of the third leaf harvested from the three-leaf-stage seedlings of WT and tsv3 mutants grown at (D) 20°and (E) 32°. Pigment contents are expressed in milligrams per gram fresh weight of leaf tissue.
Chl and carotenoid content measurement Both Chl and carotenoid (Car) content were assessed using a spectrophotometer following the methods of Arnon (1949) and Wellburn (1994) with slight modifications. Briefly, 0.2 g of fresh leaves were harvested from the three-leaf-stage seedlings grown at 20 or 32°and were homogenized in a 5-ml solution of acetone:ethanol:H 2 O (5:4:1) for 18 hr under dark conditions. Residual debris was removed by centrifugation. The supernatants were analyzed using a UV5100 Spectrophotometer (Beckman Coulter) at 663, 645, and 470 nm.

Transmission electron microscopy
The tops of the third leaves from the three-leaf-stage seedlings grown at 20 or 32°were used for preparing transverse sections for transmission electron microscopy (TEM) analysis. Leaf samples were fixed in a solution of 2.5% glutaraldehyde followed by 1% OsO 4 buffer at 4°for 5 hr after vacuum. After staining with uranyl acetate, tissues were further dehydrated in an ethanol series and finally embedded in Spurr's medium prior to ultrathin sectioning. Samples were stained again and examined under a Hitachi (Tokyo, Japan) H-7650 Transmission Electron Microscope.
Mapping of the TSV3 gene Rice genomic DNA was extracted from fresh leaves using the modified CTAB method (Murray and Thompson 1980). A total of 1430 plants with the mutant phenotype were selected from the F 2 populations. Initially, we used 81 SSR primers based on the Gramene database (http://www.gramene.org). New SSR and InDel markers were devel-oped later based on the genomic sequences available for japonica cv. Nipponbare (Goff et al. 2002) and indica cv. 9311 (Yu et al. 2002). Details of the markers used for mapping are listed in Supplemental Material, Table S1. Genomic DNA fragments of the candidate genes from the mutant and WT plants were PCR amplified and sequenced (SinoGenoMax, Shanghai, China). The function and open reading frames of the candidate genes were obtained from TIGR (http://rice. plantbiology.msu.edu/cgi-bin/gbrowse/rice/). Conserved domain structures were predicted using SMART (http://smart.embl-heidelberg.de/).

RT-PCR and real-time PCR analyses
Expression of TSV3 was monitored in various tissues of Jiahua 1 (WT) plants including germinating buds, plumules, roots, stems, and young leaves before the five-leaf stage, and flag leaves and young panicles at the heading stage. Total RNA was isolated using an RNA Prep Pure Plant kit (TIANGEN, Beijing, China), reverse transcribed using ReverTra Ace (ToYoBo, Osaka, Japan) following the manufacturer's instructions, and subjected to RT-PCR analysis.

Subcellular localization
To investigate the subcellular localization of TSV3 protein, a cDNA fragment containing the N-terminal region of TSV3 (1-280 aa) was amplified from total RNA in WT plants using the primer pair 59-GAAGATCTATGCCGCTCCTCCTCCAC-39 and 59-GGGGTACCCCACCAACGTCTGCAACCAC-39 (where the underlined sequences represent BglII and KpnI restriction sites, respectively), and introduced into the pMON530-GFP vector. The resulting pMON530:CaMV35S:TSV3-GFP plasmid was sequence confirmed and introduced into the Agrobacterium strain EHA105. For subcellular localization of TSV3, transient expression assays were performed in tobacco (Nicotiana tabacum) as described . The GFP fluorescence images were obtained using an argon ion laser excitation of 488 nm with a 505-to 530-nm band pass filter.

Sequence and phylogenetic analyses
Gene prediction was performed using the Rice Genome Annotation Project (RGAP) database (http://rice.plantbiology.msu.edu/). The full-length amino acid sequence of TSV3 was used to identify orthologs using a BLASTP search. The orthologous protein sequences were aligned using the MUSCLE tool (Edgar 2004)

Data availability
The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.

RESULTS
Phenotypic characterization of the tsv3 mutant Leaves of the tsv3 mutant appeared albino only before the four-leaf stage when grown at 20° (Figure 1, A and C), gradually turned yellowish green, and finally a normal green after the four-leaf stage at 20°or a higher temperature (32°) ( Figure 1B). Consistent with the phenotype, Chl a, Chl b, and Car contents of the third leaves of the tsv3 mutant were drastically lower than those in the WT plants at 20°( Figure 1D), but were comparable to the WT and tsv3 plants at 32°( Figure 1E). These observations suggested that the virescent phenotype of the tsv3 mutant was thermosensitive at the early seedling stage.
To examine whether the lack of photosynthetic pigments in the tsv3 mutant at low temperatures was accompanied by ultrastructural  changes in the chloroplast, the tops of the third leaves from the threeleaf-stage seedlings were analyzed using TEM. The grana lamella stacks in the WT plants were dense and well structured, regardless of the temperature at which the plants were grown (Figure 2, A and C). The tsv3 mutant plants, on the other hand, exhibited abnormal chloroplast structure with fewer grana lamella stacks at 20°( Figure 2D). This abnormal phenotype of the tsv3 mutants was corrected at a warmer temperature of 32°, where the chloroplasts displayed well-developed lamella structures ( Figure 2B) similar to WT (Figure 2, A and C), suggesting the tsv3 mutation affected chloroplast biogenesis only before the four-leaf stage under cold stress (20°). Moreover, except for the slight reduction in plant height of the WT plants after transplanting ( Figure S1A), other yield-related traits such as panicle number, grains per panicle, and 1000-grain weight showed no significant differences between the WT and tsv3 mutant plants ( Figure S1B). This indicates that the tsv3 mutation would not have any negative effects on plant growth under field condition.
Identification of the gene underlying the tsv3 mutant phenotype To understand the molecular mechanism underlying the tsv3 mutant phenotype, a map-based cloning strategy was used. The indica cv. Pei'ai 64S was crossed to the tsv3 mutant. All plants of the F 1 progeny displayed a normal green phenotype, while individuals of the F 2 population displayed a 3:1 segregation ratio (green:albino phenotype of 453:132; x 2 = 3.45; P . 0.05), demonstrating that the albino phenotype is a recessive trait controlled by a single gene (tsv3). Using the PCRbased markers P1 and RM570 (Table S1) and 214 mutant F 2 individuals, the tsv3 locus was mapped to chromosome 3 ( Figure 3A). Fine mapping using additional markers narrowed the tsv3 locus down to a 36-kb interval between the markers P2 and P5 on the BAC clone AP104321; no recombinant was found between the markers P3 and P4 ( Figure 3A and Table S1). Within this target region, seven candidate genes were predicted using the program RGAP (http://rice.plantbiology.msu.edu) and verified by sequencing. Four discontinuous nucleotide deletions (CAA Ã G) in the tsv3 mutant were identified in the first exon of LOC_Os03g58540, which encodes an Obg subfamily of small GTP-binding protein (Figure 3A), causing a premature stop codon and consequently a frame-shift mutation. In addition, the significant upregulation of LOC_Os03g58540 transcript in the tsv3 mutant compared to the WT plants at 20°( Figure 3B) further suggested LOC_Os03g58540 as the most probable gene underlying the tsv3 locus.
To further confirm that the LOC_Os03g58540 mutation was responsible for the tsv3 mutant phenotype, a genetic complementation test was performed. The pCAMBIA1301 vector expressing LOC_Os03g58540 under the control of its own promoter was introduced into the tsv3 mutant plants. A total of 23 independent transgenic events were identified, all of which showed a complete reversion to the green leaf phenotype, comparable to the WT plants. Furthermore, the T 1 progeny showed the segregation of mutant plants at 20°( Figure 3C). These data affirmed LOC_Os03g58540 as the gene underlying the TSV3 locus.
Characterization of the TSV3 protein TSV3 was predicted to be a 504-aa polypeptide with a calculated molecular mass of 54.5 kDa. Conserved domain analysis using pfam (http://pfam.janelia.org) showed that TSV3 encoded a Spo0B-associated GTP-binding protein (the Obg subfamily of small GTP-binding protein) containing a 50S ribosomal subunit-binding domain.

Subcellular localization of TSV3
The protein subcellular localization prediction tools, TargetP (Emanuelsson et al. 2000; http://www.cbs.dtu.dk/services/TargetP/) and iPSORT (http://ipsort.hgc.jp/), predicted TSV3 to be localized in the chloroplasts. To verify this prediction, the pMON530:CaMV35S:TSV3-GFP plasmid was introduced into tobacco cells in a transient expression assay. The pMON530:CaMV35-GFP vector was used as a control. The GFP fluorescence signal in the tobacco mesophyll cells transformed with the pMON530:CaMV35S:TSV3-GFP plasmid perfectly overlapped with the chloroplast autofluorescence ( Figure 5B), while the empty GFP vector without a specific targeting sequence showed green fluorescence in both the cytoplasm and the nucleus ( Figure 5A). These results confirmed that TSV3 is localized in the chloroplast.

(B) Transcript levels of TSV3 relative to
OsActin in the top-most leaves sampled from the plumule, and seedlings at the two-, three-, four-, and five-leaf stage. Relative TSV3 transcript levels at all stages were normalized to the two-leaf stage.

Expression analysis of TSV3
To examine the expression pattern of TSV3, RT-PCR was performed on various tissues of WT plants, including germinating buds, plumules, roots, stems, and young leaves before five-leaf stage, and flag leaves and young panicles at the heading stage. Among all tissues examined, expression level of TSV3 was highest in the leaves, with the young seedling leaves showing greater expression than the flag leaves ( Figure 6A). The expression in the roots and stems of young seedlings was considerably lower and was almost undetectable in the germinating buds and young panicles ( Figure 6A). This was consistent with the data from rice gene expression profiling in the RiceXPro database ( Figure S3). In addition, the expression level of TSV3 increased along with leaf development from the plumule to the five-leaf stage ( Figure 6B). These results together with the phenotypic characterization of the tsv3 mutants ( Figure  1 and Figure 2) suggest that TSV3 might play an important role in leaf chloroplast development, especially during the early seedling stage.

TSV3 regulates the expression of chloroplast-associated genes
The transcript levels of genes involved in Chl biosynthesis, photosynthesis, and chloroplast development were examined in the tsv3 mutant and WT plants at 20 and 32°. Genes involved in Chl biosynthesis (Wu et al. 2007); including CAO1 (CHLOROPHYLLIDE A OXYGENASE1), HEMA1 (encoding glutamyl tRNA reductase), YGL1 (encoding a Chl synthetase), and PORA (encoding NADPH-dependent protochlorophyllide oxidoreductase); were downregulated in the tsv3 mutant at 20°( Figure 7A), consistent with its decreased Chl content ( Figure 1D) and albino phenotype (Figure 1, A and C). Genes associated with photosynthesis, including the plastid genes Cab1R (encoding the light harvesting Chla/b-binding protein of PSII), PsaA and PsbA (encoding two reaction center polypeptides), RbcL (encoding the large subunit of Rubisco), and the nuclear gene RbcS (encoding the small subunit of Rubisco) (Kyozuka et al. 1993), were greatly suppressed in the mutant at 20°( Figure 7B). However, at 32°, transcript levels of nearly all the above-mentioned affected genes were recovered to WT or slightly higher levels (Figure 8, A and B).
Among the genes associated with chloroplast development, we investigated nuclear-encoded genes such as RNRS and RNRL (encoding the large and small subunits of ribonucleotide reductase; Yoo et al. 2009); V2 (encoding plastidial guanylate kinase; Sugimoto et al. 2007); OsRpoTp (encoding NEP core subunits; Hiratsuka et al. 1989); OsPoLP1 (encoding one plastidial DNA polymerase; Vitha et al. 2001); Rpl21 (encoding the ribosomal protein L21); FtsZ (encoding a component of the plastid division machinery; Takeuchi et al. 2007); and plastid-encoded genes including 23S ribosomal RNA (rRNA), 16S rRNA, Rps7 (encoding one ribosomal protein; Kusumi et al. 2011), Rps20 (encoding ribosomal protein S20; Gong et al. 2013), and RpoA and RpoB (encoding the PEP core a and b subunits; respectively; Kusumi et al. 2011). Relative expression levels of FtsZ, 23S rRNA, and Rpl21 were remarkably reduced in the tsv3 mutant, while the other genes displayed WT levels or slightly higher levels at 20°( Figure 7C). Interestingly, all affected genes were recovered to WT levels at 32°F igure 7 Quantitative expression analysis of genes related to (A) Chl biosynthesis, (B) photosynthesis, and (C) chloroplast development in the tsv3 mutant and WT plants at 20°. Expression level of each gene was analyzed by qPCR and normalized relative to OsActin. Error bars represent SD (n = 3).
( Figure 8C). It is possible that the abnormal expression of these key genes (FtsZ, 23S rRNA, and Rpl21) led to the mutant phenotype under cold stress.

DISCUSSION
In this study, we cloned a novel rice gene, TSV3, which encoded a small GTP-binding protein of the Obg subfamily, using a map-based cloning strategy. Deletion of four nucleotides in the N-terminal region of TSV3 resulted in a thermo-sensitive virescent phenotype and drastically affected the expression levels of certain genes associated with Chl biosynthesis, photosynthesis, and chloroplast development at low temperature ( Figure 7). TSV3 is needed for early chloroplast development under cold stress At a low temperature (20°), expression levels of genes associated with Chl biosynthesis and photosynthesis (Figure 7, A and B) as well as those of a few key genes involved in chloroplast development, such as FtsZ, Rpl21, and 23SrRNA, were greatly reduced ( Figure 7C) in the tsv3 mutant compared to the WT. However, increasing the temperature to 32°upregulated the expression of these genes to levels that were either comparable or slightly higher than the WT plants ( Figure  8). Additionally, the tsv3 mutants exhibited an albino phenotype and abnormal chloroplasts at 20°before the three-leaf stage. Both of these phenotypes were corrected either with continued plant development ( Figure 1) or with an increase in temperature (Figure 2). Together, these data suggested that TSV3 was essential for early chloroplast development under cold stress. The finding that abnormal chloroplasts occur only in the early leaves of rice tsv3 mutants under cold stress suggests that the function of TSV3 may not be a prerequisite at higher temperatures. Regarding the recovery of chloroplast development after the four-leaf stage under cold stress, TSV3 paralogs might perform the same or similar function. In addition, the FtsZ gene is known to be involved in the first step of chloroplast development (Takeuchi et al. 2007) and a reduced expression level of FtsZ in the tsv3 mutant under cold stress (20°) might affect plastid division, which was consistent with the chloroplast development in the mutant (data not shown).
TSV3 may regulate biogenesis of the chloroplast 50S large ribosomal subunit under cold stress The chloroplast 50S large subunit consists of three rRNAs (23S, 4.5S, and 5S) and 30S ribosomal proteins, such as RPL21. Although much work has been undertaken to elucidate the composition of the chloroplast ribosome, the molecular basis of its assembly in higher plants remains elusive. Previous studies on bacteria showed that the majority of Obg proteins are associated with the 50S ribosomal subunit, ribosome assembly, and stress responses (Scott et al. 2000;Datta et al. 2004;Lin et al. 2004;Wout et al. 2004;Jiang et al. 2006;Kuo et al. 2008). In view of these results, rice TSV3 may have similar functions in higher plants. Except for the OCT region, the Obg homologs in Arabidopsis, AtObgC/CPSAR1 (At5g18570), and in rice, OsObgC1 (LOC_Os07g47300), share high structural similarity ( Figure S2). AtObgC/CPSAR1 was shown to be essential for the formation of normal thylakoid membranes (Garcia et al. 2010) and was suggested to play an important role in the biogenesis of chloroplast ribosomes (Bang et al. 2009). More recently, based on the results of previous studies on rice OsObgC1 (LOC_Os07g47300) and Arabidopsis AtObgC (At5g18570), Bang et al. (2012) reported that plant ObgC is a light-induced gene and its protein is translocated to chloroplast where it may be involved in the biogenesis of the large (50S) ribosomal subunit, thus influencing the PEP-related plastid gene transcription. The authors also proposed a hypothetical model of three ObgC domains (OCT, Obg fold, and G domain) resulting in ObgC-mediated regulation of chloroplast ppGpp signaling, association of ObgC with the 50S ribosomal subunit, and the action of ObgC depending on its GTP-/GDPbound states, respectively ( Figure S2).
In this study, Rpl21 and 23SrRNA genes encoding components of the chloroplast large ribosomal subunit (50S) were greatly downregulated in the tsv3 mutant at 20°( Figure 7C); but the expression levels of genes encoding components of the chloroplast small ribosomal subunit (30S), including Rps7, Rps20, and 16SrRNA, did not appear to be affected by temperature ( Figure 7C and Figure 8C). Additionally, the severely reduced transcription levels of PEP-dependent plastid genes (RbcL, psaA, and psbA) at 20°( Figure 7B) suggested that the tsv3 mutation affected the PEP transcription. These data suggest that, like AtObgC (Bang et al. 2012), rice TSV3 might be involved in biogenesis of the large (50S), but not the small (30S), subunit of chloroplast ribosomes. This hypothesis was further supported by RNA gel blot analysis data, where the accumulation of the mature 23S rRNA was greatly reduced in the tsv3 mutant only under cold stress, but nearly reached WT levels at high temperatures ( Figure S3). Interestingly, only under cold stress, TSV3 affected the 50S ribosome assembly and, in turn, produced the albino phenotype. Accordingly, the loss of TSV3-mediated activation of the Rpl21-23S rRNA mRNA regulation might produce a thermo-sensitive virescent phenotype under cold stress ( Figure S4 and File S1).
As in the case of AtObgC RNAi Arabidopsis lines exhibiting chlorotic phenotypes, null mutants (obgc1-d1 and obgc1-t) and the OsObgC1 knockdown mutant (obgc1-d2) of OsObgC1 (LOC_Os07g47300) in rice were reported to exhibit a severe or partially chlorotic phenotype during early leaf development regardless of the temperature (Bang et al. 2012). Additionally, the OsObgC1 mutation has little effect on the expression of other rice Obg homologs such as OsObgC2/TSV3 and OsObgM (Bang et al. 2009). In addition to its structural similarity to both OsObgM and AtObgM in mitochondria, the obvious differences between TSV3/OsObgC2 and AtObgC1/OsObgC1 in their OCT regions ( Figure S2) and their clustering in distinct subgroups of group I ( Figure 4B) strongly support the existence of differences in their response to environmental changes (e.g., temperature vs. light) and regulating pathways. These findings highlight the notion that even highly conserved genes within or across species might play more diverse and complex roles than previously recognized. Also, our observations provide evidence for versatile roles for ObgCs in plant development.
TSV3 may be important for recovery from cold stress In this study, the tsv3 mutant is a typical thermo-sensitive virescent mutant, similar to the previously described thermo-sensitive virescent/ albino rice mutants such as v1 (Kusumi et al. 1997), v2 (Sugimoto et al. 2004(Sugimoto et al. , 2007, v3, st1 (Yoo et al. 2009), wlp1 (Song et al. 2014), osv4 , and tcd9 . Despite the similar phenotypes in v1, v2, v3, st1, wlp1, osv4, and tcd9 mutants, the mechanisms and regulations affecting the chloroplast development are possibly different. Briefly, the V1 (NUS1) gene encoding the chloroplast-localized protein NUS1 regulates rRNA transcription under low temperature (Kusumi et al. 2011), and the v1 mutation severely blocks the accumulation of PEP subunits . The V2 gene, which encodes plastid/mitochondrial guanylate kinase, regulates guanine nucleotide pools (Sugimoto et al. 2007) under low temperature, and the v2 mutation blocks the formation of functional PEP (Sugimoto et al. 2004). The V3 and St1 genes, encoding the large and small subunits of ribonucleotide reductase (RNR), respectively, are required for Figure 9 A functional model of AtObgC in Arabidopsis (Bang et al. 2012) and the possible model of TSV3 in developing chloroplast. Two types of RNA polymerases (NEP and PEP) have been identified in higher plant chloroplasts. TSV3 interacts with the rice chloroplast 50S ribosome subunit, which functions in the translation of protein encoded by the chloroplast gene and also regulates the transcription of photosynthetic or some housekeeping genes by affecting PEP synthesis. In the absence of TSV3, PEP activity stays low, which couples biosynthesis of Chl and proteins, and is significantly reduced at the seedling stage under cold stress, leading to the virescent phenotype. chloroplast biogenesis during early leaf development; and the v3 and st1 mutants withered to death at $30 d after germination at 20° (Yoo et al. 2009). In the wlp1 mutant, the mutation of the rice large subunit protein L13 led to abnormal chloroplast development only under cold stress (Song et al. 2014). Additionally, mutation of rice TCD9, encoding the a subunit of chaperonin protein 60 (Cpn60a), hinders FtsZ transcription/translation and, in turn, influences plastid division and finally leads to abnormal chloroplasts under cold stress . The mutation of OsV4, encoding a novel chloroplast-targeted PPR protein, leads to dramatically reduced transcription levels of some ribosomal components and PEP-dependent genes under cold stress .
Clearly, the loss of TSV3 function produced the low-temperaturesensitive virescent phenotype before the four-leaf stage, indicating that TSV3 was involved in a pathway that may be required only under cold stress. This was strongly supported by the high expression level of TSV3 at 20°, regardless of the genotype (WT or mutant) ( Figure 3B). It has been reported that cold stress disrupts protein biosynthesis in plastids by delaying translational elongation (Grennan and Ort 2007), and virescence/thermo-sensitivity are important for protection against photo-oxidative damage before healthy chloroplasts are developed (Zhou et al. 2009). Previous reports also confirmed that the deficiency of plastid translation often leads to a cold-sensitive phenotype (Tokuhisa et al. 1998;Ahlert et al. 2003;Rogalski et al. 2008;Liu et al. 2010). Taken together, TSV3 might be involved in a protective mechanism under cold stress and the reduction of TSV3 would lead to a cold-sensitive chloroplast deficiency.
In conclusion, our data clearly indicated that TSV3 was fundamentally involved in the biogenesis of plastid ribosomes under cold stress during chloroplast development in early leaves. A hypothetical model for TSV3 function is shown in Figure 9. In this model, analogous to AtObgC/OsObgC1 which is translocated into the chloroplast under light-induced conditions (Bang et al. 2012), TSV3 was translocated into the chloroplast under cold stress to interact with the chloroplast ribosomal 50S subunit to produce active PEP capable of transcribing photosynthetic or housekeeping genes. Gaining a better understanding of TSV3 function and whether it might control expression of active PEP, according to cell type, developmental stage, or environmental conditions merits further investigation.