Introduction

The chloroplast has its own genetic system consisting of the chloroplast genome and transcriptional/translational apparatus. Multi-protein complexes in chloroplasts consist of plastid-encoded core subunits and nuclear-encoded peripheral subunits, and chloroplast differentiation, development, function and maintenance depend on tightly coordinated expression of chloroplast and nuclear genes (Steiner et al. 2011; Sun et al. 2011).

There are at least two types of RNA polymerase in plastids. One is a bacteriophage-type, single-subunit, nuclear-encoded plastid polymerase (NEP; RPOTp in monocotyledons, RPOTp and RPOTmp in dicotyledons), and the other is a bacterial-type, multi-subunit, plastid-encoded plastid polymerase (PEP; Hu and Bogorad 1990; Lysenko and Kuznetsov 2005; Shiina et al. 2005; Börner et al. 2015). Based on promoter structure, plastid-encoded genes can be defined into three classes (class I, II and III) and transcription depends on the NEP and PEP (Hajdukiewicz et al. 1997; Shiina et al. 2005). NEP is lower in abundance than PEP in mature chloroplasts and cannot be co-purified with PEP by biochemical techniques (Majeran et al. 2012; Krupinska et al. 2013; Pfalz and Pfannschmidt 2013; Yu et al. 2014).

PEP is the dominating transcription apparatus and the source of not <80 % of all primary transcripts in mature chloroplasts (Zhelyazkova et al. 2012; Kindgren and Strand 2015). Many biochemical purification protocols have been developed to identify the actual subunits involved in the PEP complex. PEP is a large, dynamic complex with many transiently attached peripheral subunits (such as sigma factors) (Steiner et al. 2011; Yu et al. 2014). This adds obvious difficulties in identifying which protein is the actual and essential subunit for function and structure of the PEP complex (Reiss and Link 1985; Pfannschmidt and Link 1994; Suck et al. 1996). There are two types of preparations of the PEP complex: insoluble transcriptionally active chromosome (TAC) and soluble RNA polymerase (sRNAP; Briat et al. 1979; Rushlow and Hallick 1982; Reiss and Link 1985). Thirty-five components of the TAC complex were isolated from Arabidopsis and mustard (Sinapis alba). Eighteen of them had not been reported previously, and were named as plastid TAC proteins (pTACs; Pfalz et al. 2006). Shortly afterwards, ten non-rpo subunits were identified as essential components of the PEP complex, and were named as PEP-associated proteins (PAPs; Garcia et al. 2008; Steiner et al. 2011; Yu et al. 2013).

The activity of PEP is required for normal chloroplast development, any change in the PEP complex could affect chloroplast development. In Arabidopsis, ten mutants lacking in PAPs exhibited chlorotic phenotypes (Pfalz et al. 2006; Steiner et al. 2011; Pfalz and Pfannschmidt 2013; Börner et al. 2015; Pfannschmidt et al. 2015). Based on dramatic albinic phenotypes of Arabidopsis and maize mutants, MurE-like and pTAC7 were added to PAPs as PAP11 and PAP12, respectively (Garcia et al. 2008; Yu et al. 2013; Williams-Carrier et al. 2014). These abnormal phenotypes in pap mutants indicated that PAPs are essential for PEP activity. Interestingly, all 12 PAPs are TAC members. It has been demonstrated that these nuclear-encoded peripheral subunits are associated with the PEP complex and are essential for PEP activity. However, the relationships between the various peripheral subunits remain unclear.

Adult leaves in rice undergo a similar developmental course that can be defined into a range of successive stages from P0 to P6 (Itoh et al. 2005; Kusumi et al. 2010). Previous studies indicated that the first step of chloroplast development occurs at the early leaf developmental stages of P1–P3, the second step occurs at stage P4, and the third step takes place at the later stages of P5 and P6 (Kusumi et al. 1997). Several chloroplast-deficient rice mutants exhibit developmental stage-specific chlorotic phenotypes; these include v3 and st1 (Yoo et al. 2009), ysa (Su et al. 2012), lta1 (Peng et al. 2012), wsl (Tan et al. 2014), ylc1 (Zhou et al. 2013) and ygl2 (Chen et al. 2013).

In this study we isolated a rice chloroplast-deficient mutant white stripe leaf 3 (wsl3) with a developmental stage-specific chlorotic phenotype. We isolated the WSL3 gene encoding an essential peripheral subunit of the rice PEP complex, OsPAP1/OspTAC3, by map-based cloning and demonstrated that WSL3 not only interacts with itself but also with other subunits of the PEP complex. WSL3 did not directly bind to specific chloroplast DNA in the promoters of PEP-dependent genes in cpDNA IP and Y1H assays. We provide evidence to suggest that WSL3, an essential subunit of the PEP complex, is required for normal plastid-encoded gene expression, early chloroplast development, and plant growth by interacting with PAP2/pTAC2, PAP3/pTAC10, PAP4/FSD3, PAP5/pTAC12, PAP7/pTAC14 and PAP10/TRXz.

Materials and methods

Plant materials and growth conditions

The rice wsl3 mutant was isolated from 60Co-irradiated indica rice cultivar (cv.) 93-11. The youngest fully expanded leaves at the three-leaf stage were used for analyses unless otherwise noted. Wild-type and wsl3 mutant plants were grown in a paddy field during the rice growing season or in a growth chamber. Plants for temperature treatment were grown in a growth chamber at C25 (12 h of light at 25 °C/12 h of darkness at 25 °C) or C30 (12 h of light at 30 °C/12 h of darkness at 30 °C). Wild-type seedlings for light treatment were grown in a growth chamber in continuous darkness at 30 °C for 6 days prior to exposure to continuous illumination. To map the WSL3 locus we produced an F2 population from a cross between wsl3 mutant and japonica cv. DJY.

Chlorophyll contents measurement and transmission electron microscopy

Fresh leaves were collected and used to determine chlorophyll contents using a spectrophotometer according to the method described previously (Arnon 1949; Zhou et al. 2013). Leaves were cut and weighed and then immersed in 2 or 5 mL of 95 % ethanol for 48 h in darkness, Supernatants were collected following centrifugation and analyzed with a DU800 UV/Vis spectrophotometer (Beckman Coulter).

Transmission electron microscopy was performed as described by Dong et al. (2013). Plants were grown in a growth chamber at C25 or C30, and fresh leaves were collected and cut into small pieces, fixed in 2.5 % glutaraldehyde in phosphate buffer at 4 °C for 4 h, then rinsed and incubated in 1 % OsO4 overnight at 4 °C before dehydration in an ethanol series, and finally embedded in Spurr’s medium prior to thin sectioning. Samples were stained again and examined with a Hitachi H-7650 transmission electron microscope.

Map-based cloning and complementation testing of wsl3

Genetic analysis was performed on F2 populations from reciprocal crosses 93-11/wsl3 and wsl3/93-11. An F2 mapping population was constructed from the cross wsl3 mutant/DJY. The WSL3 locus was narrowed to a 66-kb genomic region delimited by InDel markers on Chr 10L (Table S1), and cDNA of the candidate gene LOC_Os10g32540 were amplified from both wild-type and wsl3 mutant total cDNA using primers wsl3F (5′-ATGGCCACCCCTACCCCCAC-3′) and wsl3R (5′-TTACTCCTCTGCAGGTGGCG-3′). The PCR products were confirmed by sequencing.

For complementation testing of wsl3 mutant a pWSL3Pro::WSL3cDNA vector was introduced into selected wsl3wsl3 homozygotes selected from F5 plants in the wsl3 mutant/DJY cross by Agrobacterium tumefaciens-mediated transformation as described previously (Jeon et al. 2000) and 25 independent positive transgenic plants were obtained.

Subcellular localization of WSL3 protein and isolation of chloroplasts, thylakoid membranes and stroma proteins

For subcellular localization of WSL3 protein in rice protoplasts, the coding sequence of WSL3 was amplified and inserted into the pA7 vector to form a translational fusion with the N-terminus of GFP. A PEND-CFP (cyan fluorescent protein) vector was constructed, and the DNA fragment encoding the N-terminal region of AtPEND (88 amino acids) was amplified and inserted into the pAN579 vector following reported protocols (Sato et al. 1993; Arsova et al. 2010). Transient expression constructs were transformed into rice protoplasts according to the published protocol (Chen et al. 2006). For subcellular localization of WSL3 protein in tobacco (Nicotiana benthamiana) leaves, the coding sequence of WSL3 was amplified and inserted into the pCAMBIA1305.1-GFP vector to fuse to the N-terminus of GFP. The recombinant vectors were co-transformed with P19 into 5–6-week-old tobacco leaves by A. tumefaciens-mediated transformation as previously described (Waadt and Kudla 2008). An empty pCAMBIA1305.1-GFP vector was used as the control. GFP fluorescence were observed with a confocal laser scanning microscope (Leica TCS SP5). Primers used in subcellular localization assays are listed in Table S2.

Chloroplasts, thylakoid membranes and stroma proteins were isolated from wild-type seedlings following a previously described protocol (Kauss et al. 2012).

Yeast two-hybrid and bimolecular fluorescence complementation assays

For yeast two-hybrid (Y2H) assays, the coding sequences of rpoA, rpoB, rpoC 1 , rpoC 2 , native and mutated WSL3 alleles and a series of WSL3 truncations were amplified using gene-specific primers and cloned into the pGBKT7 vector (BD) as a bait. WSL3, and OsPAP2–OsPAP12 were amplified using gene-specific primers and inserted into the pGADT7 vector (AD) as a prey. The primers used in Y2H assay are listed in Table S2. Prospective interacting partners were co-transformed into yeast strains AH109 as described in the Yeast Protocols Handbook (Clontech). Transformants were selected on a solid medium lacking tryptophan (Trp) and leucine (Leu), and interactions were assayed on a solid medium lacking tryptophan (Trp), leucine (Leu), histidine (His) and adenine (Ade).

For bimolecular fluorescence complementation (BiFC) assays, the amplified coding sequence of WSL3 was cloned into the pSPYNE173 vector to form YFPN-WSL3. The coding sequences of WSL3, OsPAP4/OsFSD3 and OsPAP7/OspTAC14 were amplified and inserted into the pSPYCE(M) vector to form YFPC-WSL3, YFPC-OsPAP4 and YFPC-OsPAP7, respectively. The recombinant BiFC vectors for transient expression were then co-transformed with P19 into 5–6-week-old tobacco leaves by A. tumefaciens-mediated transformation as described previously (Waadt and Kudla 2008; Lin et al. 2012). Primers used in BiFC assays are listed in Table S2.

RNA preparation and gene expression analysis

Total rice RNAs were extracted using a RNA Prep Plant Kit (Tiangen, Beijing) as instructed by the manufacturer. First-strand cDNA was synthesized from 2 μg of total RNA with SuperScriptI (TakaRa) and oligo(dT)18 for nuclear-encoded genes or random primers for plastid-encoded genes according to the instructions of the manufacturer. Quantitative real-time PCR was performed using a SYBR® Premix Ex Taq™ Kit (TaKaRa) on an ABI prism 7900 Real-Time PCR System and then analyzed as relative changes in gene expression using the 2− ∆∆CT method (Livak and Schmittgen 2001). Gene-specific primers used in real-time PCR are listed in Table S3 and the rice ubiquitin gene (LOC_Os03g13170) was used as a reference (primer pair Ubq).

Antibody preparation, protein extraction and immunoblot analysis

The coding sequences corresponding to amino acid residues 441–607 and 317–600 were amplified using gene-specific primers (Table S2), and cloned into the pET-28a(+) vector to form His-WSL3∆6(WT/MU) and His-WSL3∆7(WT/MU), respectively. These fused proteins were expressed in Transetta (DE3) cells (TransGen Biotech), and affinity purified with a Ni–NTA column. The purified recombinant protein His-WSL3∆6(WT) was injected into rabbits to produce polyclonal antibody against WSL3 (anti-WSL3). The purified recombinant proteins His-WSL3∆6(WT/MU) and His-WSL3∆7(WT/MU) were used to test the binding efficiency of anti-WSL3 and WSL3(WT)/WSL3(MU).

Protein extraction and immunoblot analysis were performed as described previously (Wang et al. 2010; Ren et al. 2014). Plant samples in liquid nitrogen were ground into powder in a mortar and pestle, and the powder was incubated with an appropriate volume (2 mL g−1) of NB1 buffer (50 mM Tris, 1 mM MgCl2, 500 mM sucrose, 10 mM EDTA, 5 mM DTT, protease inhibitor and adjusted to pH 8.0 using MES) for 30 min in ice. The supernatant was collected by centrifuging at 4 °C, 12,000 rpm for 20 min, mixed with 5 × SDS protein loading buffer and denatured at about 100 °C for 10 min, and components were finally separated by SDS–PAGE. Polyclonal antibodies against the photosynthetic proteins and Rubisco were obtained from BGI (http://www.genomics.cn/index).

Pull-down and cell-free degradation assays

OsPAP4 cDNA was cloned into the pMAL-c2x vector to form MBP-OsPAP4. Total proteins were extracted from the leaves of wild-type seedlings by NB1 buffer. For the pull-down assay, the total proteins and anti-WSL3 were co-incubated with protein A magnetic beads (Millipore) for about 6 h before being washed five times with NB1 (sucrose free) buffer and divided into two aliquots. Each aliquot was incubated overnight with roughly equal amounts of either free MBP or MBP-OsPAP4. Bound proteins were washed at least five times with NB1 (sucrose free) buffer and eventually re-suspended in NB1 (sucrose free) buffer prior to immunoblotting with anti-MBP and anti-WSL3 antibodies. The primers used in the pull-down assay are listed in Table S2.

Total proteins were extracted using degradation buffer (25 mM Tris–HCl pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT, 10 mM ATP), and a cell-free degradation assay was performed as described previously (Wang et al. 2009; Lin et al. 2012).

Chloroplast DNA immunoprecipitation and yeast one-hybrid assays

Rice chloroplast DNA immunoprecipitation (cpDNA IP) was performed following protocols described previously (Yagi et al. 2012; Zhong et al. 2013); primers used in cpDNA IP assays are listed in Table S4. For yeast one-hybrid (Y1H) assays, WSL3(WT), WSL3(MU), WSL3(WT)∆1, WSL3(MU)∆1 and the promoters of plastid-encoded genes were amplified using gene-specific primers (Table S2), and the PCR products were inserted into the pB42AD vector and reporter plasmid pLacZi, respectively. Plasmids were co-transformed into yeast strain EGY48 as described in the Yeast Protocols Handbook (Clontech). Transformants were grown on a solid medium lacking tryptophan (Trp) and uracil (Ura), and then transferred onto selection plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) in the medium for blue color development.

Results

Phenotypic characteristics of the wsl3 mutant

To understand the mechanism of chloroplast development in rice we used a white stripe leaf mutant identified in 60Co-irradiated indica cv. 93-11. We previously identified two white stripe leaf mutants named wsl and wsl2 (Tan et al. 2014; Jia Lyu unpublished) and herein name the new white stripe leaf mutant as wsl3. The wsl3 mutant is sensitive to low temperature, showing a lessened chlorotic phenotype when grown at C30 compared to C25 (Fig. 1a–d). When grown at C25 the wsl3 mutant seedlings displayed a severe albinic phenotype, eventually withering and dying (Fig. 1a). When grown at C30, the wsl3 mutant developed leaves with white stripes up to the three-leaf stage, and particularly at the third real leaf (L3) (Fig. 1c). Newly developed leaves starting from the four-leaf stage displayed normal green color (Fig. 1d). To determine whether the chlorotic phenotype was accompanied by reduced chlorophyll concentration, we measured the chlorophyll contents of wild-type and wsl3 mutant. Consistent with these observations, wsl3 mutant leaves contained less chlorophyll than wild type prior to the fourth-leaf stage, but similar levels were measured thereafter (Fig. 1e–h). The chlorotic leaves of wsl3 mutant seedlings did not recover to the normal green color at the four-leaf stage at C30 (Fig. 1d, h). Morphological and structural analyses of chloroplasts by transmission electron microscopy (Supplementary Fig. S1) showed that chloroplasts from wild-type and green sectors of L3 of wsl3 mutant grown at C30 and wild-type at C25 were normal and contained well-developed lamellar structures equipped with normally stacked grana and thylakoid membranes (Supplementary Fig. S1a–f, j–l). By contrast, chloroplasts from white sectors of L3 of wsl3 mutant grown at C30 were non-differentiated, smaller and less in number (Supplementary Fig. S1g–i). A large number of leaf cells in wsl3 mutant in C25 contained no chloroplasts (Supplementary Fig. S1m–o). These observations suggested that wsl3 is a temperature-sensitive mutant, and that WSL3 could have an important role in early chloroplast development.

Fig. 1
figure 1

Phenotypic characteristics of the wsl3 mutant. a Phenotypes of wild-type (WT) and wsl3 mutant seedlings at the three-leaf stage at C25. b–d Phenotypes of wild-type and wsl3 mutant seedlings at the two- (b), three- (c), and four- (d) leaf stages at C30, respectively. The white box in each image shows magnified mid-leaf sections. e Chlorophyll contents in wild-type and wsl3 mutant seedlings at the three-leaf stage at C25. f–h Chlorophyll contents in wild-type and wsl3 mutant seedlings at the two- (f), three- (g), and four- (h) real leaf stages at C30. Bars 2 cm in (a–d), L2 the second real leaf, L3 the third real leaf, L4 the fourth real leaf, Chl a chlorophyll a, Chl b chlorophyll b. Mean and SD values in (e–h) represent three independent experiments; FW fresh weight

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Map-based cloning of the WSL3 gene

For genetic analysis of the WSL3 locus, we constructed two reciprocal cross F2 populations. F1 plants from both the 93-11/wsl3 and wsl3/93-11 crosses developed normal green leaves and the F2 populations segregated in 3 normal: 1 mutant ratios (Table S5), indicating that the chlorotic phenotype in the wsl3 mutant was caused by a single recessive allele.

Genetic mapping of the WSL3 locus using the F2 population from wsl3 mutant/DJY delimited the WSL3 locus to a 66-kb genomic region flanked by insertion–deletion polymorphic (InDel) markers LY10-40 and LY10-48 on Chr 10L. Nine open reading frames (ORFs) were predicted in the region (http://www.gramene.org/; Fig. 2a). We sequenced the entire region and found a 9 bp deletion in the ninth exon of the fourth ORF (LOC_Os10g32540) at position 1498–1506 bp from the ATG start codon. The deletion caused a loss of three amino acids residues (Fig. 2b).

Fig. 2
figure 2

Map-based cloning of the WSL3 gene. a The WSL3 locus was mapped to a 66-kb region between InDel markers LY10-40 and LY10-48 on Chr 10L. Black arrows represent nine putative genes in the region, Candidate gene WSL3 (LOC_Os10g32540) is shown as the red arrow. b Structural model of the WSL3 (LOC_Os10g32540) gene. ATG and TAA represent the start and stop codons, respectively. Black boxes indicate exons, and lines between boxes indicate introns. c Phenotypes of wild-type, three complemented transgenic plants, and wsl3 mutant seedlings. d Chlorophyll contents in wild-type, complemented transgenic plants (Line1, Line2, and Line3) and wsl3 mutant seedlings at the three-leaf stage in C30. e Transmission electron microscopy images of chloroplasts from fully emerged third leaves of complemented transgenic plant grown at C30

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To confirm that the 9 bp deletion was responsible for the chlorotic phenotype in wsl3 mutant, we performed a complementation analysis. The complementation vector pWSL3Pro::WSL3cDNA containing a 1.6-kb upstream sequence and the entire cDNA sequence of wild-type WSL3 was constructed and introduced into selected wsl3wsl3 homozygotes. Positive transgenic plants had fully complemented normal phenotypes, and their chlorophyll contents were similar to those of wild-type plants (Fig. 2c, d). The ultrastructural analyses showed that the morphology and structure of chloroplasts from mesophyll cells of complemented wsl3 transgenic plants grown at C30 were normal (Fig. 2e).

To further confirm that disruption of the LOC_Os10g32540 gene function was responsible for the wsl3 mutant phenotype we constructed an RNAi vector of WSL3 and transformed it into a japonica cv. Kita-ake. However, we failed to obtain any positive transgenic plant, probably due to the low level of WSL3 protein in RNAi transgenic plants. These results thus confirm that LOC_Os10g32540 corresponds to the WSL3 gene.

WSL3 encodes a SAP protein targeted to the chloroplast

The full-length mRNA of WSL3 contains 2700 bp and 13 exons. WSL3 encodes 899 amino acids with an estimated molecular mass of 100 KD. A phylogenic tree was constructed to investigate the evolutionary relationships among WSL3 homologs (Supplementary Fig. S2). Rice WSL3 bears high sequence similarity to several proteins in monocots. The three amino acid residues (LLI), deleted in the wsl3 mutant, were highly conserved among monocots, suggesting an essential role of that site for functional integrity of the WSL3 protein. WSL3 contains two domains (Supplementary Fig. S3). It was previously reported that the integrated PLN03218 domain contains more than ten PPR motifs (Johnson et al. 2010). We found that amino acids 104–316 define the truncated PLN03218 domain, which in WSL3 contains five predicted PPR motifs. Amino acids 544–578 comprise a SAP (after SAF-A/B, Acinus and PIAS) domain, which is a putative DNA binding motif involved in chromosomal organization (Aravind and Koonin 2000; Pfannschmidt et al. 2015).

TagetP and ChloroP analysis predicted that WSL3 contains a chloroplast-target peptide (CTP) of 43 amino acids residues at the N terminus. Resembling its homologous protein, AtpTAC3, WSL3 has two nuclear localization signals (NLS) located in amino acids 28–57 and 771–793, respectively (Pfannschmidt et al. 2015; http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). We constructed two different WSL3-GFP fusion expression vectors to verify the subcellular location of WSL3. The vectors were transformed into rice protoplasts and tobacco leaves. Confocal microscopy showed that green fluorescent signals of WSL3-GFP exclusively co-localized with the autofluorescent signals of chlorophyll (Fig. 3a, b). The fluorescence patterns of WSL3-GFP as small dot-like structures co-localized with PEND-CFP in chloroplasts suggested that WSL3 was localized in the chloroplast nucleoids (Sato et al. 1993; Arsova et al. 2010). To confirm whether WSL3 is associated with the thylakoids, we firstly obtained a polyclonal antibody against WSL3 by immunizing rabbits and identified its specificity and efficiency by immunoblot analysis (Fig. 3c; Supplementary Fig. S4a). As shown in supplementary Fig. S4a, there was no significant difference in binding efficiency of anti-WSL3 to WSL3(WT) or to WSL3(MU). The distribution of WSL3 in intact chloroplast stromal and thylakoid membranes was measured by immunoblot analysis. Most of the WSL3 protein was present in the thylakoid membrane fractions, and only a miniscule amount was detected in the stromal fractions (Fig. 3d). Immunoblot analysis thus demonstrated that WSL3 was primarily associated with the thylakoid membranes. These results suggest that WSL3 was targeted to the chloroplast and associated with the nucleoids.

Fig. 3
figure 3

Subcellular localization of WSL3 protein. a GFP signals of WSL3-GFP fusion proteins localized in chloroplasts of rice protoplasts. b GFP signals of WSL3-GFP fusion proteins in chloroplasts of tobacco mesophyll cells. eGFP, empty GFP vector control. GFP, fluorescence of WSL3-GFP or eGFP; Chloroplast, chloroplast autofluorescence; Merged, merged image of GFP and chloroplast. c Specificity analysis of the polyclonal antibody against WSL3. d WSL3 localized mainly in the thylakoid membrane fractions. Intact chloroplasts were isolated from the leaves of wild-type seedlings at the three-leaf stage grown at C30 and then separated into stromal and thylakoid membrane fractions. Polyclonal antibodies were used against WSL3, membrane protein D1, and abundant stromal protein RbcL

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Analyses of the WSL3 gene and WSL3 protein

Quantitative real-time reverse transcription (qRT)-PCR and immunoblot analyses were performed to examine the accumulations of WSL3 transcript and WSL3 protein in the wsl3 mutant. The WSL3 allele in the wsl3 mutant was up-regulated at both temperatures, but higher amounts of transcript accumulated at the lower temperature (Fig. 4a). However, the accumulation of WSL3 protein was decreased in the wsl3 mutant at both C25 and C30 (Fig. 4b), and less WSL3 protein was present in the wsl3 mutant grown at C25 than at C30 (Fig. 4b). We measured the stability of native and mutated WSL3 by a cell-free degradation assay and found that the stabilities of the proteins were not significantly different (Supplementary Fig. S4b).

Fig. 4
figure 4

Expression analysis of the WSL3 gene. a qRT-PCR expression analysis of the WSL3 gene in wild-type and wsl3 mutant seedlings at the three-leaf stage at C25 and L3 of wild-type and wsl3 mutant seedlings at the three-leaf stage at C30. b Immunoblot analysis of WSL3 in wild-type and wsl3 mutant seedlings at the three-leaf stage at C25 and in L3 of wild-type and wsl3 mutant seedlings at the three-leaf stage at C30 (left) and quantification of the data (right). c Schematic of a rice seedling with fully expanded third leaf and qRT-PCR analysis of WSL3 transcripts in SB, L2, L3 and L4 of wild-type and wsl3 mutant seedlings at the three-leaf stage at C30. SB (shoot base) corresponds to a 4 mm piece from the bottom of the shoot. L2, L3 and L4 denote the second, third and fourth real leaves, respectively. P0–P6 indicate the seven successive developmental stages of the rice leaf. d qRT-PCR analysis of the WSL3 gene during greening of etiolated seedlings. Wild-type seedlings were grown in continuous darkness for 6 days after germination, and then the etiolated seedlings were illuminated for varying periods. Expression of the VYL gene is induced by light. The ubiquitin gene was used as a reference. Error bars indicate SD (n = 3)

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We measured both WSL3 transcript and WSL3 protein levels in different sections of leaves at various leaf developmental stages by qRT-PCR and immunoblot analyses. WSL3 was highly expressed in L4 of wild-type and wsl3 mutant seedlings at the three-leaf stage at C30, whereas the amount of transcript decreased in mature leaves (Fig. 4c). The WSL3 protein was highly accumulated in L4. The level of WSL3 protein in L3 of wsl3 mutant seedlings was lower than in the wild-type at the three-leaf stage at C30 (Supplementary Fig. S4c). These results suggested that WSL3 fully functions at the P4 stage and plays an important role in chloroplast development. To test whether its expression is induced by light we assayed the WSL3 transcript during greening of etiolated seedlings. qRT-PCR results showed that WSL3 was weakly expressed in etiolated seedlings, gradually increased after 3 h of illumination, peaked after 9 h, and then decreased to a stable level after 18 h (Fig. 4d). Changes in WSL3 transcript paralleled that of VYL, which is also light-induced during greening of etiolated seedlings (Dong et al. 2013). These observations suggest that the expression of the WSL3 gene is induced by light and is essential for the photomorphogenesis in rice.

Altered expression of plastid-encoded genes and reduced accumulation of photosynthetic proteins in wsl3 mutant

qRT-PCR was performed to examine whether the expression pattern of plastid-encoded genes was changed in wsl3 mutant. psaA, psbA and rbcL were selected as class I genes, rrn16 and clpP were selected as class II genes, rpoA, rpoB, rpoC 1 and rpoC 2 were chosen as class III genes, and CAB1R, CAB2R, rbcS and HEMA1 were chosen as nuclear-encoded genes. Transcripts of class I genes in wsl3 mutant plants grown at C25 were dramatically decreased, whereas transcripts of class III genes were elevated to varying degrees. Among class II genes, rrn16, the principal gene transcribed by PEP, was down-regulated, whereas the clpP, the principal gene transcribed by NEP, was up-regulated in the wsl3 mutant (Swiatecka-Hagenbruch et al. 2007; Hübschmann and Börner 1998; Zoschke et al. 2007). Transcripts of nuclear-encoded genes whose gene products are localized in the chloroplast were decreased as well (Fig. 5a). Similarly, we found parallel trends in variation of these plastid- and nuclear-encoded genes in the L3 of wsl3 mutant compared to wild-type when grown at C30 (Fig. 5b). These results are consistent with their phenotypes suggesting that PEP activity is deficient in wsl3 mutant.

Fig. 5
figure 5

Accumulation of plastid- and nuclear-encoded gene transcripts and photosynthetic proteins in wild-type and wsl3 mutant seedlings. qRT-PCR analysis of gene expression of plastid- and nuclear-encoded genes in wild-type and wsl3 mutant seedlings at the three-leaf stage at C25 (a) and in L3 of wild-type and wsl3 mutant seedlings at the three-leaf stage at C30 (b). The ubiquitin gene was used as a reference. Mean values and SDs of three independent experiments are shown. Immunoblot analysis of photosynthetic proteins in wild-type and wsl3 mutant seedlings at the three-leaf stage at C25 (c) and in L3 of wild-type and wsl3 mutant at the three-leaf stage at C30 (d). Heat-shock protein 82 (HSP82) was used as an internal control, and the relative band intensity was calculated by comparison with HSP82 band intensity. Images shown in (c) and (d) are representative of three independent experiments

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We next examined the accumulation of photosynthetic proteins in the wsl3 mutant at the three-leaf stage by immunoblot assays. These photosynthetic proteins, including the core components of photosystem I (A1 and A2), the core component of photosystem II (D2), the RuBisCO large subunit (RbcL) and small subunit (RbcS), were reduced in the wsl3 mutant at both C25 and C30 compared to wild-type (Fig. 5c, d). We found a negative correlation between the accumulation of these photosynthetic proteins and the severity of chlorosis phenotype in the wsl3 mutant (Figs. 1, 5c, d). These observations indicated that accumulation of photosynthetic proteins was reduced in the first few leaves of wsl3 mutant and that photosynthetic activity was inadequate.

WSL3 directly interacts with OsPAPs

To further investigate the role of WSL3 in the rice PEP complex we performed a Y2H assay to identify its interacting proteins. Firstly, we fused the coding sequence of the WSL3 in-frame into pGBKT7 (BD) and pGADT7 (AD), respectively, to examine whether WSL3 interacts with itself. The Y2H assay showed that WSL3 interacts with itself and this interaction was verified by BiFC assays (Supplementary Fig. S5). Secondly, we examined the interaction between WSL3 and other PEP subunits, including peripheral subunits and core subunits, by Y2H assays. As shown in Fig. 6a both the native and mutated WSL3 interacted with several PEP peripheral subunits and the binding strength appeared to be influenced by LLI-deletion (Fig. 6a; Supplementary Fig. S6b). Previous studies demonstrated that the interacting proteins were located in the chloroplast (Pfalz et al. 2006; Myouga et al. 2008; Chen et al. 2010; Gao et al. 2011; Steiner et al. 2011). Here we found that WSL3 did not directly interact with the PEP core subunits although its homologous protein AtpTAC3 co-precipitated with the PEP complex (Fig. 6b; Yagi et al. 2012). We then validated interactions of WSL3 with OsPAP4/OsFSD3 and OsPAP7/OspTAC14 by BiFC and pull-down assays (Fig. 6c, d). To determine which domain of WSL3 is responsible for interaction with PEP peripheral subunits, we constructed a series of WSL3 truncations (WSL3∆1–WSL3∆5) and examined their interactions with the PEP peripheral subunits in yeast stain AH109 cells (Supplementary Fig. S6a, b). As shown in Supplementary Fig. S6b, the N-terminal portion (WSL3∆4; amino acid residues 44–330) was sufficient for interactions with OsPAP2/OspTAC2, OsPAP3/OspTAC10, OsPAP4/OsFSD3, OsPAP5/OspTAC12, OsPAP7/OspTAC14 and OsPAP10/OsTRXz. However, deletion of amino acid residues 90–330 (∆241aa) and amino acid residues 104–316 (∆213aa) completely abolished this interaction, indicating that the 213 aa region, which predicts a truncated PLN03218 domain of WSL3, is essential for the interaction with PEP peripheral subunits. The truncated PLN03218 domain (WSL3∆2) was not sufficient for the interactions with OsPAP2/OspTAC2, OsPAP3/OspTAC10 and OsPAP7/OspTAC14 (Supplementary Fig. S6b). These results suggest that WSL3 is an essential subunit of the rice PEP complex and interacts with OsPAPs.

Fig. 6
figure 6

WSL3 interacts with other OsPAPs. a Y2H assay showing that WSL3 interacts with OsPAP2/OspTAC2, OsPAP3/OspTAC10, OsPAP4/OsFSD3, OsPAP5/OspTAC12, OsPAP7/OspTAC14 and OsPAP10/OsTRXz. b Y2H assay shows that WSL3 does not interact with the core subunits of PEP α, β, β′ and β″. c BiFC assay showing that WSL3 interacts with OsPAP4/OsFSD3 and OsPAP7/OspTAC14 in chloroplasts from leaf cells of tobacco. d The WSL3 complex interacts with OsPAP4/OsFSD3. In vitro pull-down assay shows that MBP-OsPAP4, but not MBP alone, was pulled down by WSL3 from plant extracts. Asterisk indicates the full-length MBP-OsPAP4 protein

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Association of WSL3 with chloroplast DNA

We performed a cpDNA IP assay to identify whether WSL3 is associated with chloroplast DNA in rice. Immunoprecipitated DNA was purified and analyzed by quantitative PCR. As shown in Fig. 7a, the cpDNA IP assay showed that WSL3/PEP (WSL3 or a WSL3 containing complex, PEP complex) preferentially associated with the promoter regions of PEP-dependent genes, but not NEP-dependent genes. WSL3/PEP also associated with the coding regions of the PEP-dependent genes. The relative amounts of chloroplast DNA that associated with WSL3/PEP such as the promoters and coding regions of PEP genes, decreased in the wsl3 mutant (Fig. 7a) suggesting that the function of WSL3/PEP association with chloroplast DNA was affected in the wsl3 mutant. We further examined the local patterns of spatial association of WSL3/PEP with the psbA transcription unit (Fig. 7b) and observed that WSL3/PEP was associated with chloroplast DNA along the psbA transcription unit, including the promoter, coding, and termination regions, during transcription. The relative amount of chloroplast DNA that associated with WSL3/PEP was also decreased in the wsl3 mutant. The association of WSL3/PEP with chloroplast DNA in rice is similar to the association of chloroplast DNA with pTAC3 in wheat and pTAC5 in Arabidopsis (Yagi et al. 2012; Zhong et al. 2013). A Y1H assay to investigate whether WSL3 directly or indirectly binds to chloroplast DNA as a peripheral subunit of the PEP complex showed that WSL3 did not directly bind to the promoter regions of PEP-dependent genes (Supplementary Fig. S7). Our results suggested that WSL3 participates in the whole transcription process as an essential subunit of the PEP complex in rice.

Fig. 7
figure 7

Association of WSL3 with chloroplast DNA. a Association of WSL3 with chloroplast DNA in wild-type and wsl3 mutant at the three-leaf stage in C30. Association of WSL3 with the PEP promoter regions (PpsaA, PpsbA, PrbcL, and Prrn16), a PEP coding sequence region (rbcL), a NEP promoter region (PrpoB), a NEP coding region (rpoA), and a noncoding spacer region located between rps12 and rrn16 (Spacer) were analyzed by cpDNA IP assay. Chloroplasts were prepared from wild-type and wsl3 mutant seedlings at the three-leaf stage in C30, and then subjected to cpDNA IP assay using antibody against WSL3. NoAb, no antibody control. The amount of immunoprecipitated DNA in each sample is presented as a percentage of the total input chromatin. Mean values and SDs of three independent experiments are shown. b Spatial association of WSL3 along the psbA transcription unit in wild-type and wsl3 mutant at the three-leaf stage in C30. The schematic gene map of the matK-psbA region is shown below. Arrow indicates the transcription start site of the psbA gene and the direction of transcription. P DNA regions corresponding to the psbA promoter; C DNA regions corresponding to the psbA coding region; T DNA regions corresponding to the psbA terminator; a and b DNA regions corresponding to two units in loci upstream of psbA. Mean values and SDs of three independent experiments are shown

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Discussion

wsl3 is a novel rice chloroplast-deficient mutant lacking OspTAC3

Chlorotic phenotypes produced by pap mutants in Arabidopsis indicated that PAPs are essential for PEP activity and chloroplast development in dicotyledons (Steiner et al. 2011; Pfalz and Pfannschmidt 2013; Börner et al. 2015; Pfannschmidt et al. 2015). Studies on mutants lacking Whirly and pTAC12 in Arabidopsis and maize (Zea mays) implied that the component of PEP is discrepant to some extent between dicotyledons and monocotyledons (Prikryl et al. 2008; Krause and Krupinska 2009; Marechal et al. 2009; Chen et al. 2010; Pfalz et al. 2015). Consequently, the component of PEP and function of the single PEP subunits in monocotyledons require further investigation.

In this study we characterized a new rice chloroplast-deficient mutant wsl3 (Fig. 1). The wsl3 mutant allele has a 9 bp deletion in the OspTAC3 gene, resulting in loss of three conserved amino acids (Fig. 2a, b; Supplementary Figs. S2 and S3). Chloroplast development can be divided into three steps. The second is a key step for the genetic system of the chloroplast, including the PEP complex that is established during this stage. In rice, the first and second steps of chloroplast development that occur during the early stages of leaf development are temperature-sensitive (Mullet 1993; Kusumi et al. 1997, 2011; Sugimoto et al. 2004). The WSL3 gene was most highly expressed at the P4 (L4) stage corresponding to the second step of chloroplast development (Fig. 4c). Previous studies indicated that these wsl3-like features may arise from retardation or impairment in early chloroplast development. YSA and WSL encode two PPR proteins that target the chloroplast and involve RNA processing (Su et al. 2012; Tan et al. 2014). In addition, V3 and ST1 encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1 and RNRS1, respectively, and the activity of RNR is essential in the first step of chloroplast development (Yoo et al. 2009). Like ysa, wsl, v3 and st1, wsl3 exhibit severe chlorotic phenotype at low temperature and display lessened chlorotic phenotype at permissive temperature, suggesting that WSL3 is essential in rice during the second step of chloroplast development. Moreover, we observed that the WSL3 gene is essential for photomorphogenesis since it was highly expressed during transformation from etioplast to chloroplast. These results revealed that wsl3 is a novel chloroplast-deficient mutant lacking OspTAC3 and has striking characteristics in rice.

WSL3 plays an important role in plastid-encoded gene expression

The WSL3 protein co-localized with the chloroplast nucleoid and mainly accumulated in L4 (Fig. 3; Supplementary Fig. S4c). As shown in Supplementary Fig. S4c, the amount of WSL3 protein in L3 of wsl3 mutant was markedly decreased, whereas the amount in L4 of both wsl3 mutant and wild-type were not significantly different. L3 of wsl3 mutant displayed white stripes but L4 was normal (Fig. 1c, d). We speculated that reduction in WSL3 protein is an important factor in causing the chlorotic phenotype during growth of the first three leaves of wsl3 mutant. We also found that the expression of the WSL3 gene was up-regulated in the wsl3 mutant. It is likely that the up-regulation of WSL3 transcript was a response to down-regulation of WSL3 protein. We failed to obtain transgenic knock-out plants by RNA interference (RNAi) probably due to the low level of WSL3 protein in transgene lines. These results suggest that WSL3 is essential for chloroplast development in wsl3 mutant.

Accumulation of WSL3 protein in the wsl3 mutant is decreased and WSL3 function is impaired. These changes could lead to a decline in PEP activity and subsequently arrest early chloroplast development. Analysis of plastid-encoded gene expression in the wsl3 mutant revealed that transcripts of PEP-dependent genes were decreased, whereas those of NEP-dependent genes were elevated to varying degrees (Fig. 5a, b). The typical variation in plastid-encoded gene expression suggested that PEP activity was defective in the wsl3 mutant and that WSL3 has an essential role in plastid-encoded gene expression. We therefore, deduced that retardation or impairment of early chloroplast development in the wsl3 mutant was caused by reduced PEP activity.

Previous studies on a v2 mutant demonstrated that normal expression of nuclear-encoded genes for chloroplast proteins at a later stage of chloroplast development is regulated by developmental plastid-to-nucleus signals and generation of these signals conveying information on plastid developmental state is accompanied by establishment of functional transcription/translation machinery (Sugimoto et al. 2004). Thus, any change affecting the activity of the transcription/translation apparatus during early chloroplast development prevents the generation of developmental plastid-to-nucleus signals, thereby altering the expression pattern of nuclear-encoded genes for later chloroplast development. Like ysa and v2, the transcripts of nuclear-encoded genes whose gene products are localized to the chloroplast were also reduced in the wsl3 mutant due to the impaired PEP activity (Fig. 5a, b). We concluded that WSL3 plays an essential role in plastid gene expression and chloroplast development in rice.

WSL3 is essential for the PEP activity by interaction with PEP peripheral subunits in rice

Null mutants or transgenically silenced plants lacking PAPs or most of the TACs exhibited very similar phenotypes and similar changes in plastid gene expression (Pfalz et al. 2006; Steiner et al. 2011; Yagi et al. 2012; Pfalz and Pfannschmidt 2013). However, the relationships involving each peripheral subunit are still uncertain. It was demonstrated that both PAP6/FLN1 and FLN2 interact with PAP10/TRXz (Arsova et al. 2010), PAP7/pTAC14 interacts with PAP5/pTAC12 (Gao et al. 2011), PAP4/FSD3 interacts with PAP9/FSD2 (Myouga et al. 2008), and PAP12/pTAC7 interacts with PAP6/FLN1, PAP3/pTAC10, PAP5/pTAC12 and PAP7/pTAC14 (Yu et al. 2013) in Arabidopsis. Previous studies on an Arabidopsis ptac3 mutant revealed that AtpTAC3 co-precipitates with the PEP complex and has an essential role in PEP activity and chloroplast development, but evidence for direct interaction between pTAC3 and other PEP peripheral subunits was missing (Kindgren and Strand 2015). In this study, we showed that WSL3 directly interacts with PAP2/pTAC2, PAP3/pTAC10, PAP4/FSD3, PAP5/pTAC12, PAP7/pTAC14, PAP10/TRXz, and itself (Fig. 6; Supplementary Figs. S5 and S6). Our results also demonstrated that WSL3 did not interact with PEP core subunits (Fig. 6b) and provided evidence suggesting that WSL3 is an essential subunit of the rice PEP complex.

The wsl3 mutant displayed a chlorotic phenotype, but the LLI deletion in WSL3 is outside of the truncated PLN03218 and SAP domains (Fig. 1; Supplementary Fig. S3). This implies that the deleted LLI affects the folding of WSL3, but not the folding of the truncated PLN03218 (including the PPR motif) and the SAP domains. Indeed, we found that the α-helix around the mutation site converted to a random coil in the secondary structure of mutated WSL3 using Psipred and I-TASSER, thus suggesting that the LLI residues are essential for correct folding of WSL3 and that their loss affects the stability and function of WSL3. Cell-free degradation assays showed no difference between the stabilities of native and mutated WSL3 (Supplementary Fig. S4b), but the interaction features between PEP peripheral subunits with native and mutated WSL3 were changed (Fig. 6a; Supplementary Fig. S6b). Thus, incorrect folding of WSL3 in the wsl3 mutant could contribute to the chlorotic phenotype.

We further analyzed the relationship between WSL3 and chloroplast DNA because there was a SAP domain in WSL3. However, we suggest that WSL3 plays an important role in PEP activity to stabilize the structure rather than participate in recognition of specific promoters (Fig. 7; Supplementary Fig. S7). We concluded that WSL3 is a peripheral subunit of PEP that is essential for PEP activity in rice by interacting with PAP2/pTAC2, PAP3/pTAC10, PAP4/FSD3, PAP5/pTAC12, PAP7/pTAC14 and PAP10/TRXz.

In conclusion, we report the novel chloroplast-deficient mutant wsl3 in rice. We demonstrate that the WSL3 gene encodes a member of the PEP complex and plays an important role in the plastid gene expression. We also demonstrate that the wild-type WSL3 protein interacts with subunits of the PEP complex and is essential for chloroplast development in rice seedlings.