Rice TCM1 Encoding a Component of the TAC Complex is Required for Chloroplast Development under Cold Stress

Abstract

Transcriptionally active chromosome (TAC) is a component of protein–DNA complexes with RNA polymerase activity, expressed in the plastid. However, the function of rice TAC proteins is still poorly understood. In this paper, we first report the identification of a new rice (Oryza sativa L.) mutant (tcm1) in the gene encoding TAC. The tcm1 mutant displayed an albino phenotype and malformed chloroplasts before the three‐leaf stage when grown at low temperatures (20°C) and a normal phenotype at higher temperatures (>28°C). Map‐based cloning revealed that TCM1 encodes a novel chloroplast‐targeted TAC protein in rice. In addition, the transcript levels of all examined plastid‐encoded polymerase (PEP)‐dependent genes were clearly downregulated in tcm1 mutants at low temperatures, although partially recovering levels were obtained at high temperatures, comparable to wild‐type plants. Furthermore, the TCM1 transcripts were ubiquitously expressed in all examined tissues, with high expression levels in green tissues. The data suggest that the rice nuclear‐encoded TAC protein TCM1 is essential for proper chloroplast development and maintaining PEP activity under cold stress.

Abbreviations
• ΦPSII
• photosynthetic efficiency
• Chl
• chlorophyll
• F_v/F_m
• variable-to-maximum fluorescence ratio
• GFP
• green fluorescent protein
• PCR
• polymerase chain reaction
• PEP
• plastic-encoded polymerase
• PSII
• Photosystem II
• pTAC
• plastid TAC proteins
• RT-qPCR
• real-time quantitative polymerase chain reaction
• TAC
• transcriptionally active chromosome
• WT
• wild-type

Core Ideas

• Transcriptionally active chromosome (TAC) is a component of protein–DNA complexes with RNA polymerase activity, expressed in the plastid.
• Map-based cloning revealed that TCM1 encodes a novel chloroplast-targeted TAC protein in rice, in which a mutation leads to an albino phenotype and malformed chloroplasts before the three-leaf stage at low temperatures.
• TAC protein TCM1 is essential for proper chloroplast development and maintaining plastid-encoded polymerase activity under cold stress conditions.

Chloroplasts are semiautonomous organelles with limited coding information for components, including plastid machinery and photosystem genes (Sato et al., 1999; Leister 2003). Chloroplast development consists of a series of complex actions and can be classified into three phases, which are coordinately regulated by plastid and nuclear genes (Mullet 1993; Kusumi et al., 2011): (i) the activation of plastid replication and plastid DNA synthesis; (ii) the chloroplast “build-up”, characterized by the establishment of the chloroplast genetic system; and (iii) the plastid and nuclear genes encoding the photosynthetic apparatus being expressed at very high levels. In chloroplasts, transcription is principally executed by PEP and nucleus-encoded RNA polymerase (for reviews, see Liere and Maliga 2001; Pyke 2007; Schweer et al., 2010; Barkan 2011; Lerbs-Mache 2011). Plastid-encoded polymerase accounts for the transcription of photosynthetic genes, whereas nucleus-encoded RNA polymerase predominantly transcribes the housekeeping genes, including the rpo genes (rpoA, rpoB, rpoC1, and rpoC2) encoding the PEP core subunits (Baumgartner et al., 1993; Hajdukiewicz et al., 1997; Lopez-Juez and Pyke 2005; Demarsy et al., 2006; Schweer et al., 2010). The molecular mechanisms involved in altering the expression patterns during the chloroplast development in higher plants remain largely unknown (Pfalz and Pfannschmidt 2013).

Until now, research efforts have focused on the identification of nuclear-encoded proteins involved in plastid transcription and translation in plants. It is recognized that subunits of the PEP core are present in two plastid protein preparations, TAC and the soluble RNA polymerase (sRNAP) (Hallick et al.1976; Krause et al., 2000; Suzuki et al., 2004). The TAC and soluble RNA polymerase preparations from proplastids, chloroplasts, and etioplasts have different protein compositions, demonstrating their dynamics in response to environmental and developmental changes (Reiss and Link 1985; Pfannschmidt and Link 1994; Suck et al., 1996). Transcriptionally active chromosome is a type of chloroplast multisubunit complex enzyme which contains polypeptides playing a role in replication, transcription, translation, detoxification, protein modification, and plastid metabolism (Pfalz et al., 2006). Pfalz et al. (2006) isolated TACs from Arabidopsis thaliana (L.) Heynh. and mustard (Sinapis alba L.) chloroplasts and identified 35 of the components, with 18 components named plastid TAC proteins (pTACs; pTAC1–pTAC18). In addition, previous studies showed that the A. thaliana knock-out lines of the genes encoding the respective TACs all exhibited the albino, lethal, or defective chloroplast or pale-green phenotype under standard light conditions (Kroll et al., 2001; Pfalz et al., 2006; Aseeva et al., 2007; Garcia et al., 2008; Myouga et al., 2008; Alexandre et al., 2009; Arsova et al., 2010; Gao et al., 2011; Yu et al., 2013). More interestingly, Chen et al. (2010) reported that A. thaliana pTAC12/HEMERA had dual functions in the nucleus and in the chloroplast. In addition, Gao et al. (2011) found that A. thaliana pTAC14 interacts with pTAC12 and is essential for proper chloroplast development. Yagi et al. (2012) reported that TAC3 is essential for PEP activity and chloroplast development. More recently, Yu et al. (2013) found that A. thaliana pTAC7 interacts with fructokinase-like proteins, as well as pTAC10, pTAC12, and pTAC14. Furthermore, A. thaliana pTAC5, pTAC6, and pTAC10 were predicted to contain typical phosphorylation sites and to be involved in the phosphorylation processes (Reiland et al., 2009). Obviously, A. thaliana TACs have various functions, and cross-talk exists between chloroplast gene expression and environmental factors (Reiland et al., 2009). Nevertheless, to our knowledge, no genes coding rice TACs had have their function reported yet.

In this study, we first describe the mutant of TAC in rice, tcm1, which exhibited the albino phenotype before the three-leaf stage at low temperatures (20°C) but remained a normal green at high temperatures (>28°C). Furthermore, we report the cloning of the TCM1 gene encoding a novel TAC protein in rice. Additionally, the transcription levels of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development were severely affected in tcm1 mutants at low temperatures. Our work implies that rice TCM1 plays an important role in chloroplast development and maintenance of PEP activity under cold stress conditions.

MATERIALS AND METHODS Plant Materials and Growth Conditions

The thermosensitive chlorophyll-deficient mutant (tcm1) used in this study was isolated from the O. sativa japonica variety ‘Jiahua 1’(wild-type, WT), induced by 60Co γ-radiation in 2006. All rice plants were grown in a rice paddy field in Shanghai (31°11ʹ N, summer season, temperate climate) and Hainan (18°16¢N, winter season, subtropical climate) under local conditions or in growth chambers. The germinated rice seeds were grown in growth chambers at four temperatures (20, 24, 28, and 32°C) under a 12:12 h light/dark photoperiod and a light intensity of 120 μmol photons m-2 s-1.

Phenotype Characterization and Photosynthetic Pigment Measurements

Plant extracts were obtained from 100 mg of the third fresh leaf from seedlings at the three-leaf stage and homogenized in 10 mL of 100% acetone. Spectrophotometric quantification was performed with a Beckman Coulter DU-720 spectrophotometer (Beckman Coulter, Brea, CA). Total chlorophyll (Chl), Chl a, Chl b, and carotenoid concentrations were calculated as described by Arnon (1949) and Alan (1994).

In addition, WT and tcm1 plants were grown in the experimental station of Shanghai Normal University in 2010. Subsequently, leaf Chl content values (Supplemental Fig. S1) were obtained by using a chlorophyll meter (SPAD-502, Minolta Co. Ltd, Japan), which provides a simple, quick, portable and nondestructive tool for estimating leaf Chl content (Peng et al., 1993; Turner and Jund, 1991; Dwyer et al., 1991). These values were measured every week from the transplanting (summer) to heading (autumn) stages. Lastly, some yield-related traits in rice (Supplemental Fig. S2) were surveyed at maturity.

Chlorophyll Fluorescence Analysis and Transmission Electron Microscopy

Rice seedlings were planted at 20°C or 32°C in growth chambers. Chlorophyll fluorescence analyses for the third leaves at the three-leaf stage were performed with a PAM-2000 portable chlorophyll fluorimeter (MINI-PAM, Walz, Effeltrich, Germany). The variable-to-maximum fluorescence ratio (F_v/F_m) and photosynthetic efficiency (ΦPSII) representing the actual were measured and calculated according to Meurer et al. (1996) after plants were dark-adapted for 20 min. The F_v/F_m ratio was calculated as follows: F_v/F_m = (F_m – F₀) F_m^–1, where F₀ and F_m are the minimum and maximum Chl a fluorescence of dark-adapted leaves, respectively. The F_v/F_m ratio reflects the maximum potential capacity of the Photosystem II (PSII) photochemical reactions (Krause and Weis 1991).

For electron microscopy, transverse sections of the top leaves were obtained from the three-leaf-stage seedlings. The samples were fixed in 4% glutaraldehyde buffer, 2.5% glutaraldehyde, and 1% osmic acid phosphate buffer at 4°C for 5 h after vacuum extraction. After staining with uranyl acetate, tissues were further dehydrated in an ethanol series and ultimately embedded in Spurr's medium prior to ultrathin sectioning. Samples were stained again and observed with a Hitachi-7650 transmission electron microscope (Hitachi, Tokyo, Japan).

Map-based Cloning of the TCM1 Gene

For genetic analysis, a cross was made between the tcm1 mutant and the O. sativa indica variety ‘Peiai64S’. The F₂ population of 6982 plants with the mutant phenotype was used for fine mapping of the TCM1 locus. New simple sequence repeat and insertion–deletion markers (Supplemental Table S2) were developed from the entire genomic sequences of the O. sativa japonica ‘Nipponbare’ variety (Goff et al., 2002) and the O. sativa indica variety ‘9311’ (Yu et al., 2002). The polymerase chain reaction (PCR) procedure was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, annealing for 30 s, 72°C for 40 s, and a final elongation step at 72°C for 5 min. The candidate gene's predicted function was acquired via TIGR (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/, accessed 9 Aug. 2017) and full-length cDNAs were acquired from KOME (http://rapdb.dna.affrc.go.jp/download/irgsp1.html, accessed 14 Nov. 2017).

Genetic Complementation

For the complementation test, a cDNA clone (AK065563) in the plasmid vector (J013034F04) (http://rapdb.dna.affrc.go.jp/download/irgsp1.html, accessed 14 Nov. 2017) containing the full-length TCM1 (LOC_Os01g56350) cDNA sequence was purchased from National Institute of Agrobiological Sciences of Japan. Subsequently, the 1894-bp DNA fragment covering the full length coding sequence (1656 bp) were amplified using the primers 5’-AAAATCTAGAAGACCAAACCGGCTACCTGAA-3’ (XbaI) (forward) and pR: 5’-GGGCTGCAGTCATGAGCTTTTGGATGATTT-3’ (PstI) (reverse). The underlined sequences stand for cleavage sites of the restriction enzymes. The amplified fragment was cloned into the pMD18-T vector (TaKaRa, Tokyo, Japan). Following sequence verification, the fragments were cloned into the pCAMBIA1301 binary vector (CAMBIA, Canberra, ACT, Australia). Ultimately, the pCAMBIA1301:CaMV35S:TCM1-cDNA plasmids were transferred into Agrobacterium tumefaciens strain EHA105 and introduced into the tcm1 mutants via Agrobacterium-mediated transformation, as described previously (Hiei et al., 1994). The genotype of the transgenic plants was determined via the procedure of Jiang et al. (2014).

Sequence and Phylogenetic Analyses

Gene prediction and structure analysis were performed by using the GRAMENE database (www.gramene.org/, accessed 9 Aug. 2017). Homologous sequences of TCM1 were identified by using the BLASTP search program of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/, accessed 9 Aug. 2017). Multiple sequence alignments were conducted with DNAMAN version 6.0 (Lynnon Biosoft, San Ramon, CA). The signal peptide was predicted with SignalP version 2.0 (Nielsen and Krogh 1998). The chloroplast transit peptide was predicted via the ChloroP program. Polymerase chain reaction primers were designed with Primer Premier version 5 software (http://www.premierbiosoft.com/primerdesign/, accessed 14 Nov. 2017). A phylogenetic tree was constructed using MEGA version 6 software (www.megasoftware.net/, accessed 9 Aug. 2017).

RNA Preparation and Real-Time Quantitative PCR Analysis

Total rice RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad. CA). DNase I-treated RNA was obtained using an RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. First-strand cDNA was synthesized from 2 μg of total RNA using the SuperScript II kit (TaKaRa). OsActin was used as the internal control. The specific primers for real-time quantitative PCR (RT-qPCR) are listed in Supplemental Table S3. Real-time quantitative PCR was performed in an ABI7500 PCR system with the SYBR Premix Ex Taq (TaKaRa; RR041A). For each sample, RT-qPCR was performed in triplicate. The 2^–ΔΔCT method was used to analyze the relative transcript levels in gene expression (Livak and Schmittgen, 2001). The mRNA levels of the corresponding genes in WT plants were defined as 1.0.

Subcellular Localization of TCM1–Green Florescent Proteins

To explore the subcellular localization of TCM1 protein, the cDNA fragment encoding the N-terminal region (amino acids 1–98) of TCM1 without the stop codon was amplified using the primer pairs: 5’-AAAAGGTACCATGGCGTCGTGCTCCCGCACTT-3’ (forward) and 5’-AAAGGTACCCCCAGGTATGCTTGCCCGAG-3’ (reverse) (the underlined sequence represents cleavage sites of Kpn I enzyme) from the cDNA clone AK065563. This was then ligated into the pMON530-GFP vector, in frame with green fluorescent protein (GFP) under the control of the CaMV 35S promoter. The resultant pMON530-TCM1-GFP plasmids were introduced into tobacco (Nicotiana tabacum L.) leaves and cocultured at 25°C for 2 d. In the meantime, pMON530-GFP empty vector was used as a control. Green fluorescent protein fluorescence in tobacco cells were observed after 2 d with a Zeiss confocal laser scanning microscope (LSM 5 PASCAL; Zeiss, Oberkochen, Germany).

RESULTS Phenotypic Characteristics of the TCM1 Mutant

We isolated the tcm1 mutant from the mutant pool of japonica variety Jiahua 1, induced by γ-radiation. The phenotypes and photosynthetic pigment contents of the three-leaf-stage tcm1 mutant seedlings grown at four different temperatures (20, 24, 28, and 32°C) are shown in Fig. 1. The tcm1 mutants appeared as an albino or pale yellow phenotype when grown at 20°C and 24°C, but normal green like WT plants at 28 and 32°C (Fig. 1A). Corresponding with the mutant phenotype, the total Chl, Chl a, and Chl b contents in the third leaves were much lower than those in WT at 20 and 24°C (Fig. 1B,C); however, they were comparable between WT and tcm1 plants at 28 and 32°C (Fig. 1D,E). These data revealed the thermosensitivity of the chlorophyll-deficient phenotype in the tcm1 mutants.

Fig. 1. Characterization of the tcm1 mutants. (A) four-leaf stage seedlings of wild-type (WT) (right) and tcm1 mutants (left) grown at 20, 24, 28, and 32°C, respectively; indicate the pigment contents in the third leaves of WT and mutants grown at (B) 20°C, (C) 24°C, (D) 28°C, and € 32°C, respectively. Chl, total chlorophyll; Chl a, chlorophyll a; Chl b, chlorophyll b; Car, carotenoid; WT, wide type. Data are means ± SD (n = 3).

To verify whether the lack of photosynthetic pigments in the tcm1 mutants was accompanied by ultrastructural changes in the chloroplasts, we compared the ultrastructure of the chloroplasts in tcm1 and WT plants at 20 and 32°C via transmission electron microscopy. As expected, cells in WT leaves, irrespective of the temperature, contained normal chloroplasts displaying well-organized lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2 A,C). In contrast, cells in leaves from the tcm1 mutants grown at 20°C had no intact chloroplasts and fewer grana lamella stacks (Fig. 2B), but cells in leaves from the tcm1 mutants grown at 32°C contained apparently normal chloroplasts, similar to those in WT plants (Fig. 2D). These results implied that the tcm1 mutation led to malformed chloroplasts at an early seedling stage under cold stress.

Fig. 2. Transmission electron microscopic images of chloroplasts in wild-type (WT) and tcm1 mutants. (A, B) Chloroplast structure in WT (A) and tcm1 (B) cells at 20°C. (C, D) Chloroplast structure in WT (C) and tcm1 (D) cells at 32°C. CP, chloroplast; G, grana lamella stacks.

Furthermore, PSII activity of the three-leaf-stage tcm1 and WT plants was surveyed by measuring the F_v/F_m ratio. At 20°C, F_v/F_m was 0.79 ± 0.03SD (n = 3, the same as below) in WT plants, and 0.47 ± 0.02 in tcm1 mutants, indicating that the photochemical efficiency of PSII was greatly reduced in the tcm1 mutants. In addition, at 20°C, the ΦPSII value was 0.63 ± 0.04 in WT plants and 0.36 ± 0.04 in the mutants, showing that the photosynthetic efficiency decreased tremendously in tcm1 mutants. By contrast, at 32°C, all values in the tcm1 mutants (0.78 ± 0.03 for F_v/F_m, 0.63 ± 0.09 for ΦPSII) were similar to WT values (0.76 ± 0.02 for F_v/F_m, 0.64 ± 0.14 for ΦPSII). Consequently, our data indicate that the tcm1 mutation hampered PSII activity under cold stress.

Under field conditions, the leaf Chl contents in tcm1 plants were indistinguishable from those of WT plants (Supplemental Fig. S1). In addition, no obvious differences for yield-related traits were observed between tcm1 and WT plants, such as plant height, panicle number, panicle length, and seed-setting, except for a slight reduction in both 1000-grain weight and grain number (Supplemental Fig. S2), implying that the tcm1 mutation did not have obvious negative effects on the later growth stages of rice.

Map-based Cloning of the TCM1 Gene

To recognize the molecular mechanisms underlying the thermosensitive phenotype, we isolated the TCM1 gene via map-based cloning. Genetic analyses of a cross between the tcm1 mutant and indica rice cultivar Peiai64S showed that the mutant phenotype was caused by a single recessive nuclear mutation (Supplemental Table S1). Using 252 mutant individuals from the F₂ population, the TCM1 locus was initially located between RM297 and ID32935 on chromosome 1 (Fig. 3A) at genetic distances of 7.3 cM and 0.6 cM, respectively. Subsequently, five insertion-deletion polymorphic markers (P1–P5) were designed in the target region (Supplemental Table S2). Furthermore, when we used a larger population of 6982 F₂ mutant individuals, TCM1 was fine-mapped to a 69-kb interval between the markers P2 and P4 and cosegregated with marker P3 (Fig. 3B). Within this target region, seven candidate genes (LOC_Os01g56320, LOC_Os01g56330, LOC_Os01g56340, LOC_Os01g56350, LOC_Os01g56360, LOC_Os01g56370, and LOC_Os01g56380) were predicted with the program RGAP (http://rice.plantbiology.msu.edu/, accessed 9 Aug. 2017). We sequenced all candidate genes and found a 3-bp (CTC) deletion at 438 bp from the ATG start codon in LOC_Os01g56350 (Fig. 3D), which caused loss of an amino acid (serine) without a premature stop codon and a frame-shift mutation. Additionally, the significant downregulation of the LOC_Os01g56350 transcript at 20°C, compared with WT (Fig. 3E) also supported the existence of the LOC_Os01g56350 mutation in tcm1 mutants.

Fig. 3. Genetic analysis and cloning of the TCM1 gene. (A) The TCM1 gene was initially located between the markers RM297and ID32935 on chromosome 1 using 252 F2 mutant individuals. (B) TCM1 was narrowed to a 69-kb window between the P2 and P4 markers and cosegregated with P3 in a population of 6982 F2 mutant individuals. (C)The target region contains seven predicted candidate genes containing LOC_Os01g56350. (D) A 3-bp (CTC) deletion in the third exon of LOC_Os01g56350. (E) Transcript levels of TCM1 in WT and tcm1 mutants at the three-leaf stage grown at 20°C and 32°C, respectively, OsActin was used as a control for real-time quantitative polymerase chain reaction. (F) The plants complemented with pCAMBIA1301:CaMV35S:TCM1 (left) and transgenic T0 plants with the empty vector pCAMBIA1301 (right) grown at 20°C. (G) Chloroplast structure in complemented plants grown at 20°C.

To further prove that the loss of function in TCM1 was the cause of the mutant phenotype, genetic complementation was performed. As a result, all 23 complemented plants with pCAMBIA1301:CaMV35S:TCM1 were obtained and all showed normal WT behavior at 20°C (Fig. 3F,G). In contrast, 13 independent lines harboring the empty vector pCAMBIA1301 all failed to rescue the tcm1 mutants, demonstrating that deletion of serine caused the mutant phenotype under cold stress. These results confirmed that LOC_Os01g56350 is the TCM1 gene.

Characterization of TCM1

TCM1 consists of 10 exons and nine introns (Fig. 3D) and encodes a component of the TAC complex that has homology to A. thaliana AtTAC12 (Kawahara et al. 2013). TCM1 encodes a protein with 651 amino acids with a molecular mass of approximately 62.9 kDa. A database search with Pfam (Finn et al., 2010) revealed that TCM1 contains domains of unknown function (data not shown). Basic Local Alignment Search Tool searches of the available genome sequences with the TCM1 protein sequence revealed close homologs in A. thaliana, Brachypodium distachyon (L.) P.Beauv., Sorghum bicolor (L.) Moench, and Zea mays L. (Fig. 4A), showing that TCM1 is highly conserved within higher plants. However, the functions of these homologs largely remain unknown. It was noted that the deleted amino acid (serine) was highly conserved among higher plants (Fig. 4A), suggesting the vital role of this site for the functional integrity of the TCM1 protein. In addition, phylogenetic analysis showed that the evolutionary relationships of TCM1 homologs were consistent with the taxonomy (Fig. 4B).

Fig. 4. Sequence alignment and phylogenetic analysis of the TCM1 protein. (A) Amino acid sequence alignment of TCM1 with the five homologs. Amino acids that are fully or partially conserved are shaded black and gray, respectively. The red box shows mutation in the tcm1 mutant. (B) Phylogenic tree of TCM1 and homologs. Protein sequences are Aegilops tauschii Coss. (F775_28056), Amborella trichopoda Baill. (18427209), Arabidopsis thaliana (AT2G34640), Brachypodium distachyon (100840007), Brassica napus L. (106396188), Citrus sinensis (L.) Osbeck (102620035), Coffea canephora Pierre ex A.Froehner, Elaeis guineensis Jacq. (105058814), Erythranthe guttatus Spach, Malus domestica Borkh. (103435027), Musa acuminata Colla (103983535), Nelumbo nucifera Gaertn. (104610222), Nicotiana sylvestris Speg. & S. Comes, Nicotiana tomentosiformis Goodsp., Phoenix dactylifera L. (103703674), Physcomitrella patens (Hedw.) Bruch & Schimp. (PHYPADRAFT_99071), Picea sitchensis (Bong.) Carrière, Pyrus sp. (103951322), Setaria italica (L.) P.Beauv. (101762731), Sorghum bicolor (SORBI_03g035780), Triticum aestivum L., Vitis vinifera L. (100251522), and Zea mays (100275546). The rooted tree is based on a multiple sequence with the MAFFT alignment algorithm (neighbor-joining) generated with the program MEGA version 6. The scale represents the percentage of substitutions per site. Statistical support for the nodes is indicated.

Subcellular Localization and Expression Patterns of TCM1

The TCM1 protein was predicted to localize to chloroplasts with TargetP (Emanuelsson et al., 2000; http://www.cbs.dtu.dk/services/TargetP/, accessed 9 Aug. 2017) and iPSORT (http://psort.nibb.ac.jp/, accessed 9 Aug. 2017). To survey the actual subcellular localization of TCM1, a TCM1–GFP construct was generated and then introduced transiently into tobacco cells via Agrobacterium-mediated transformation. Green fluorescent signals were visualized using confocal microscopy. Green fluorescent signals of TCM1–GFP colocalized with the autofluorescent signals of chlorophyll (Fig. 5A), showing that TCM1 targeted the chloroplasts at least.

Fig. 5. Subcellular localization of TCM1 protein. (A) TCM1–green fluorescent protein (GFP) fusion; (B) empty GFP vector without a specific targeting sequence. The scale bar represents 20 μm.

Real-time PCR analyses with RNA from different tissues of WT plants demonstrated that TCM1 transcripts are preferentially expressed in all tissues investigated and that the mRNA levels are relatively low in roots and panicles (Fig. 6). These results, together with chloroplast localization and the mutant phenotype, supported the notion that TCM1 plays an important role in chloroplast development in rice. In addition, TCM1 transcript levels at 20°C were drastically reduced in the tcm1 mutants, but remained relatively unchanged at 32°C, compared with the WT plants (Fig. 3 E). Our data show that the functional product of TCM1 is indispensable for proper chloroplast function at low temperatures.

Fig. 6. Expression analysis of TCM1 by real-time polymerase chain reaction analysis. R, roots; S, stem; L, leaf; P, panicles. OsActin was used as a control (the cycle number for OsActin was 28; the cycle number for TCM1 was 35).

The Transcript Expressions of Chloroplast Development and Photosynthesis-related Genes in the tcm1 Mutants

We surveyed the transcript levels of genes associated with Chl biosynthesis, photosynthesis, and chloroplast development (PORA, HEMA1, CAO1, YGL1, Cab1R, rbcL, rbcS, psaA, psbA, LhcpII, ST1, V2, V3, OsRpoTp, OsRpoLp, rpoB, rps7, and FtsZ) in the tcm1 mutants via RT-qPCR. Under low temperatures, apart from YGL1, encoding Chl synthase (YGL1), all transcript levels of genes for Chl biosynthesis (Fig. 7A), such as glutamyl tRNA reductase (HEMA1), chlorophyllide A oxygenase (CAO1), and nicotinamide adenine dinucleotide phosphate (reduced):protochlorophyllide oxidoreductase (PORA), were drastically suppressed in tcm1 mutants, consistent with the reduced Chl contents (Fig. 1 B, C) and the Chl deficient phenotype (Fig. 1A). Without exception, all photosynthesis-related genes (Fig. 7B) [rbcL, encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase (Rubisco), rbcS encoding the small subunit of Rubisco, psaA and psbA for the reaction center polypeptides in photosystems, and LhcpII encoding light-harvesting complex protein] were expressed at low levels in the tcm1 mutants, which corresponded with the low photosynthesis efficiency under low temperatures.

Fig. 7. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the genes associated with chlorophyll (Chl) biosynthesis, photosynthesis, and chloroplast development at 20°C. Expression levels of genes related to Chl biosynthesis (A), photosynthesis (B), and chloroplast development (C) in the wild-type (WT) and tcm1 mutants in the third leaves. The relative expression level of each gene in WT and mutant was analyzed via RT-qPCR and normalized with OsActin as an internal control. Data are means ± SD (n = 3).

For chloroplast development-associated transcripts, we investigated the expressions of V3 (RNRL) and ST1 (RNRS), encoding the large and small subunits of ribonucleotide reductase, respectively (Yoo et al., 2009); V2, encoding plastid or mitochondrial guanylate kinase (Sugimoto et al., 2007); OsRpoTp, encoding PEP core subunits (Hiratsuka et al., 1989); rpoB encoding a PEP core subunit (Inada et al., 1996; Kusumi et al., 2011); OsPoLP, encoding a plastidial DNA polymerase (Vitha et al., 2001); and FtsZ, encoding a component of the plastid division machinery (Takeuchi et al., 2007). We also examined the mRNA levels of rps7, encoding the small subunit ribosomal protein. With the exception of OsPoLP1 and FtsZ, the expression of all the genes, especially OsRpoTp and rpoB, were severely decreased (Fig. 7C), which probably led to malformed chloroplasts at low temperatures (Fig. 2B). Collectively, the tcm1 mutation blocked the mRNA levels of certain genes for not only Chl biosynthesis and photosynthesis, but also for chloroplast development under low temperatures. By contrast, all transcripts of the genes affected at 20°C in the tcm1 seedlings partially recovered to WT levels when grown at 32°C (Fig. 8) (within twofold range), which corresponded with the thermosensitivity of the mutant phenotype.

Fig. 8. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the genes associated with chlorophyll (Chl) biosynthesis, photosynthesis, and chloroplast development at 32°C. Expression levels of genes related to Chl biosynthesis (A), photosynthesis (B), and chloroplast development (C) in the wild-type (WT) and tcm1 mutants in the third leaves. The relative expression level of each gene in the WT and mutants was analyzed via RT-qPCR and normalized with OsActin as an internal control. Data are means ± SD (n = 3).

DISCUSSION

In this study, we first identified and characterized a TAC protein, TCM1, in rice, which is required for normal chloroplast development under low temperatures. Its disruption led to malformed chloroplasts and a Chl-deficient phenotype, resulting from abnormal expression of the genes associated with Chl biosynthesis, photosynthesis, and chloroplast development under cold stress. Our results demonstrated that TCM1 plays an important role in chloroplast development under low temperatures.

TCM1 is Essential for Chloroplast Development under Low Temperatures

The chloroplast is a semiautonomous organelle that contains about 100 genes. More than 3000 proteins function within it (Leister 2003). The plastid and nuclear-encoded proteins are assembled to form photosynthetic and metabolic complexes in chloroplasts. The tightly coordinated gene expression between the plastid and nuclear genomes is essential for chloroplast development; in general, mutations of these genes might cause Chl-deficiencies or chloroplast defects in plants. Currently, more than 70 Chl-deficient or chloroplast-defective mutants in rice have been identified (for a review, see Kurata et al., 2005). Among these, the v1, v2, v3, tcd9, osv4, tcd5, tcd10, and tcd11 mutants (Iba et al., 1991; Kusumi et al., 1997; Sugimoto et al., 2007; Jiang et al., 2014; Gong et al., 2014; Wang et al. 2016, 2017; Wu et al., 2016) were reported to have the similar phenotypes to the tcm1 mutants. It is known that the V1 gene encodes a chloroplast protein that is involved in the regulation of chloroplast RNA metabolism and is essential for chloroplast differentiation during early leaf development at low temperatures (Kusumi et al., 2011). The V2 gene for plastid or mitochondrial guanylate kinase is required for the early stages of chloroplast differentiation at low temperatures (Sugimoto et al., 2007). The V3 gene, encoding the large subunits of ribonucleotide reductase, is also required for chloroplast development during early leaf growth (Yoo et al., 2009). More recently, another five genes, TCD9, encoding α subunit of chaperonin protein 60 (Jiang et al., 2014); OsV4 and TCD 10, both encoding PPR proteins (Gong et al., 2014; Wu et al., 2016); TCD5, encoding a monooxygenase (Wang et al., 2016); and TCD11, encoding plastid ribosomal protein S6 (Wang et al., 2017) were found to be essential for chloroplast development at low temperatures.

Based on this evidence, there is a strong molecular basis for chloroplast development in response to cold stress in rice. In this study, the impaired chloroplasts (Fig. 2B), the aberrant transcript levels of some genes for chloroplast development under cold stress, and the recovered levels (comparable to WT) at high temperatures (Fig. 8) supported the notion of thermosensitivity in the tcm1 mutants. In addition, the high expression levels in all green tissues (Fig. 6) or at least the chloroplast-localized protein (Fig. 5A) revealed the function of TCM1 in chloroplasts. These results strongly confirm the notion that TCM1 is needed for chloroplast development under cold stress. However, it is unclear yet why the aberrant chloroplast or albino phenotypes in tcm1 mutants occur under solely lower temperatures, which has also not been well documented. Interestingly, similar results were observed for v1, v2, v3, osv4, tcd5, tcd9, tcd10, and tcd11 rice mutants (Iba et al., 1991; Kusumi et al., 1997; Sugimoto et al., 2007; Gong et al., 2014; Jiang et al., 2014; Wang et al. 2016, 2017; Wu et al., 2016). A reasonable explanation in this study is that TCM1's function is possibly dispensable under higher temperatures but is essential under lower temperatures for chloroplast development. Alternatively, the cold sensitivity of tcm1 could be explained by the reduced TCM1 expression that we observe during cold stress.

As already described, the procedure of chloroplast development from proplastids to mature chloroplasts can be divided into three phases (Mullet 1993; Kusumi et al., 1997). Since those key genes for the second (OsRpoTp and rpoB) and third phases (PEP-dependent plastid genes, Cab1R, rbcL, psaA, psbA, and LhcpII) of chloroplast development were severely repressed at low temperatures in tcm1 mutants (Fig. 7), without obvious suppression of OsPOLP and FtsZ for the first phase of chloroplast development (Vitha et al., 2001; Takeuchi et al., 2007), we could conclude that TCM1 regulated the second phase, thereafter affecting the third phase during chloroplast development in rice.

TCM1 may be Involved in PEP Activity and PSII at Low Temperatures

Transcriptionally active chromosomes are involved in replication, transcription, translation, detoxification, protein modification, and plastid metabolism (Pfalz et al., 2006). Currently, the A. thaliana knock-out lines or mutants of the genes coding for TACs, including TAC12 (At2g34640, pTAC12), which is homologous to rice TCM1 in this study, have been reported to exhibit the albino, lethal, or defective chloroplast or the pale green phenotype under standard light conditions and not be affected by temperatures (Pfalz et al., 2006). In addition, subsequent studies showed that three A. thaliana TACs (pTAC2, pTAC6, and pTAC12) are required for proper function of the PEP transcription machinery and might also be involved in post-transcriptional processes in chloroplasts (Pfalz et al., 2006). Interestingly, the A. thaliana homozygote knock-out line ptac12 displayed the pale green phenotype but was lethal without exogenous C sources; moreover, the low-light-grown phenotype is caused by photooxidative damage caused by light stress (Pfalz et al., 2006). Likewise, the tcm1 mutants displayed abnormal chloroplast development (Fig. 2B) and reduced PEP activity, as determined by the expression of PEP-dependent chloroplast genes under low temperatures (Fig. 7B). Hence, TCM1 also may be involved in the regulation of PEP transcription machinery under low temperatures.

Furthermore, the reduced F_v/F_m ratio in the tcm1 mutants at low temperatures indicated smaller PSII antennae and/or fewer functional PSII centers (Meurer et al., 1998). In addition, the obvious decreases of ΦPSII at 20°C and the similar levels to WT plants at 32°C indicated that the malfunction of TCM1 barely blocked photosynthetic efficiency under cold stress. From these results, we proposed that TCM1 may play an important role in PSII function under cold stress. It is worth noting that A. thaliana pTAC12 had dual functions in the nucleus, where it is essential for phytochrome nuclear body formation, and in chloroplasts, where it directly regulates the expression of photosynthetic genes (Chen et al., 2010), unlike TCM1, which possibly only functions in rice chloroplasts. As to whether TCM1 has a similar nucleus localization to A. thaliana pTAC12, this will be verified in further studies.

Here, we report the first characterization of a TAC gene in rice. Our results should be helpful for understanding the functional role of TACs and chloroplast development in rice. Further work on TCM1 will involve an understanding of how it participates in chloroplast development and maintains PSII function under low temperatures.

Supplemental Information

Supplementary Figure S1. The change in chlorophyll contents (SPAD values) in tcm1 and WT plants from Week 1 (summer) to Week 12 (heading date, autumn) after transplanting (2010, Shanghai, China). Data are means ± SD (n = 3).

Supplementary Figure S2. Agronomic traits of tcm1 and wild-type of rice plants grown under field conditions (2010, Shanghai, China). PH, plant height; PN, panicle number per plant; GW: 1000-grain weight (g); GN, grains per panicle. Data are means ± SD (n = 3).

Supplemental Table S1. Genetic segregation analysis of tcm1 mutants in the F₂ population.

Supplemental Table S2. Polymerase chain reaction-based molecular markers designed for fine mapping.

Supplemental Table S3. Markers designed for RT-qPCR.

Conflict of Interest Disclosure

The authors declare that there is no conflict of interest.

Acknowledgments

We thank the DNA Bank of National Institute of Agrobiological Sciences, Japan, for kindly providing the rice cDNA clone (No. AK065563). We are grateful to Prof. Zhongnan Yang for kindly providing the pMON530-GFP vector and for his constructive comments and suggestions on this paper as well. The project was financially supported by the Natural Science Foundation of China (No. 30971552), Minister of Science and Technology of China (MOST) (2016YFD0100902), the Shanghai Municipal Science and Technology Commission (10DZ2271800, 16ZR14253000, and 16391900700), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00039), and the Scientific Program of Shanghai Normal University (SK20159). D. Lin, K. Zheng, and Z. Liu contributed equally to this work.