Chlorophyll contents were measured according to a method described by Wu et al. (2007). Briefly, fresh leaves of WT and wsl4 mutant plants were collected at the two-, three-, four-, and five-leaf stages, and then cut and marinated in 5 ml of 95% ethanol for 48 h in darkness. After centrifugation, the residual plant debris was removed. The supernatants were analyzed with a DU 800 UV/Vis Spectrophotometer (Beckman Coulter) at 665, 649, and 470 nm, respectively.

For TEM analyses, third leaves from WT and green and white sectors of wsl4 mutant were cut into slices of 0.5 cm and three slices of each tissue were fixed in a solution of 2.5% glutaraldehyde in phosphate buffer at 4°C for 4 h, and incubated overnight at 4°C in 1% OsO4. The tissues were subsequently dehydrated in an ethanol series, infiltrated in a gradient series of epoxy resin, and finally embedded in Spurr’s medium prior to thin sectioning (50–80 nm). Samples were stained again and examined with a Hitachi H-7650 transmission electron microscope.

Sequence polymorphisms between Nipponbare (japonica) and 93-11 (indica) were identified from a public database¹ and used to develop Indel markers for fine mapping. Primer pairs were designed with Primer Premier 3.0. Newly developed PCR-based molecular markers used in this study are listed in Supplementary Table S1. The PCR procedure was as follows: 94°C for 5 min, followed by 34 cycles of 94°C for 30 s, annealing for 30 s, 72°C for 30 s, and a final elongation step at 72°C for 7 min.

For complementation of the wsl4 mutation a 5.7 kb WT genomic fragment (primer pairs PPR-G, Supplementary Table S1) containing a 2.2 kb upstream sequence, the entire coding region of WSL4, and a 2.0 kb downstream sequence was amplified from RX69 with Prime STAR HS DNA Polymerase (TaKaRa) and cloned into the binary vector pCAMBIA1305 to generate the vector pCAMBIA1305-GWSL4. This vector was introduced into Agrobacterium tumefaciens EHA105, which was then used to infect wsl4 mutant calli (Hiei et al., 1994).

For RNAi test, the construct pCUbi1390-DFAD2 (ubiquitin promoter and a FAD2 intron inserted into pCAMBIA1390) was used as an RNAi vector (Stoutjesdijk et al., 2002; Wu et al., 2007). Both anti-sense and sense versions of a specific 332 bp fragment from the coding region of the WSL4 5′-end were amplified (primer pairs WSL4-RNAi-SacI-InF and WSL4-RNAi-BamHI-InR, Supplementary Table S1), and successively inserted into pCUbi1390-DFAD2, to form the RNAi construct pUbi-dsRNAiWSL4. The construct was then used to infect the calli produced from RX69. Transformation was conducted according to the method described above.

Gene prediction was performed using the GRAMENE database². Homologous sequences of WSL4 were identified using the Blastp search program of the National Center for Biotechnology Information (NCBI³). A chloroplast transit peptide at the N-terminus of WSL4 was predicted by ChloroP⁴ and TargetP⁵. A phylogenetic tree was constructed using MEGA v4.1 software⁶ by the bootstrap method with 1,000 replicates. Multiple sequence alignments were conducted with BioEdit software (Borland Company).

For subcellular localization of WSL4 and wsl4 proteins in rice the coding sequences of WSL4 and wsl4 were amplified using specific primer pairs (listed in Supplementary Table S1) and cloned into the transient expression vector pA7-GFP to generate the fusion genes WSL4-GFP and wsl4-GFP driven by the CaMV 35S promoter. All transient expression constructs were separately transformed into rice protoplasts and incubated in darkness at 28°C for 16 h before examination according to the protocols described previously (Chiu et al., 1996; Chen et al., 2006). Fluorescence of GFP in transformed protoplasts was visualized using a confocal laser scanning microscope (Leica TCS SP5).

Total RNA was extracted from flag leaves, culms, young panicles, and leaf sheaths at the booting stage, as well as shoot bases, leaves, and seedling roots using an RNA Prep Pure Plant kit (Tiangen Co., Beijing). Each RNA sample (about 3 μg) was reverse transcribed using primer Script I (TaKaRa) and an oligo(dT)18 primer for nuclear-encoded genes or random primers for plastid-encoded genes since mRNA from most plastid-encoded genes carried no poly-A tail. The RT-PCR procedure was as follows: 94°C for 5 min, followed by 33 cycles of 94°C for 30 s, annealing for 30 s, 72°C for 1 kb/s, and a final elongation step at 72°C for 7 min. The primer pairs are listed in Supplementary Table S1. For analysis of RNA splicing we sequenced full-length RT-PCR products with special primers flanking the introns (Supplementary Table S1). For Real-Time RT-PCR analysis of RNA splicing efficiency, specific primers (Supplementary Table S1) were designed for intron-exon (unspliced forms) and exon–exon (spliced forms) links of each gene (de Longevialle et al., 2007; Koprivova et al., 2010; Hsieh et al., 2015; Yap et al., 2015).

Real-Time RT-PCR was performed using a SYBR Premix Ex Taq^TM Kit (TaKaRa) on an ABI prism 7500 Real-Time PCR System. The program was as follows: initial polymerase activation for 30 s at 95°C followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The 2^-ΔΔCT method was used to analyze relative transcript levels of genes (Livak and Schmittgen, 2001). The primer sequences for qRT-PCR are listed in Supplementary Table S1. The rice Ubiquitin gene (LOC_Os03g13170) was used as a reference in qRT–PCR (primer pairs Ubq).

Total RNA from leaves (L3-3) at the seedling stage was isolated using TransZol Up (TransGen) following recommendations by the manufacturer. About 2 μg of RNA was fractionated in a 1.2% (w/v) denaturing formaldehyde agarose gel and transferred onto Hybond N+ nylon membranes (GE Healthcare Biosciences). Digoxigenin-labeled probes were obtained by PCR using a DIG Northern Starter Kit (Roche). The primers are listed in Supplementary Table S1. Prehybridization of the membrane was carried out for 1 h at 68°C in PerfectHyb Plus Hybridization Buffer (SIGMA). Hybridization was performed overnight in the same buffer at 68°C. Signal was detected using the DIG Wash and Block Buffer Set (Roche), and eventually read in an imaging system (Tanon 5200).

Fresh leaves (L3-3) were standardized by fresh weight and ground into fine powder. Total plant proteins were extracted with an appropriate volume (2 mL g^-1) of NB1 buffer [50 mM Tris-MES, 1 mM MgCl₂, 0.5 M sucrose, 10 mM EDTA, 5 mM DTT, and protease inhibitor cocktail (Roche, Cat. No. 11 836-153-001; the used concentration was 1 tablet for a volume of 50 ml solution), pH 8.0]. The protein samples were resolved in 10% SDS–polyacrylamide gel electrophoresis (PAGE) gels, and then transferred onto PVDF membranes (0.45 μm, Millipore), followed by incubation with antibodies. Signals were detected using a Luminata^TM Forte Western HRP Substrate (Millipore) and visualized by an imaging system (ChemiDocTMX- RS; Bio-Rad). The polyclonal antibodies used in this study were obtained from BGI⁷. The intensities of chloroplast proteins were quantified by “ImageJ” software.

All statistical analyses were performed using Student’s t-test. The number of biological replicates (n) in each experiment is indicated in the corresponding figure legends. Values were considered statistically significant at P < 0.05, and very significant at P < 0.01.

To characterize the wsl4 mutant in detail, we conducted a time-course examination of the mutant phenotype from the two to five-leaf stages. The leaves of seedlings at the 3–4 leaf stage exhibited a bleached appearance with the most extreme symptoms observed in the third leaf and distal half of the fourth leaf (Figures 1A–C). All leaves after the 5-leaf stage were as green as WT plants (Figure 1D). Young leaves on newly emerging tillers of the wsl4 mutant also exhibited the green/white leaf color defect (Figure 1E). No difference was observed between WT and wsl4 mutant after the heading (Figure 1F). Consistent with these observations, levels of Chl a and b in striped leaves of wsl4 plants were much lower than those in WT (Figures 1G,H) Compared with WT plants, wsl4 mutants showed no statistically significant differences in plant height, number of tillers, number of branches per panicle, number of spikelets per panicle, and 1000-grain weight (Table 1).

In contrast to the moderate striped phenotype and Chl accumulations under optimum temperature (L30/D25), the wsl4 mutant exhibited less color deficiency and accumulated much higher Chl contents when grown at a constant 30°C (C30) (Figure 2). At a constant 20°C the wsl4 mutant showed more extreme symptoms and Chl content that were barely detectable (Figure 2). Our results show that the wsl4 mutant is sensitive to low temperatures.

To determine whether the color deficiency resulting from the decreased photosynthetic pigment accumulation in the wsl4 mutant were associated with ultrastructural damages in chloroplasts, we compared the ultrastructural features of chloroplasts from WT and wsl4 mutant leaves using TEM. As shown in Figures 1I–L, the lamellar structures of WT chloroplasts were well developed and were equipped with normally stacked grana and thylakoid membranes (Figure 1I). While green sectors of wsl4 mutant seedlings were still able to develop normal chloroplasts (Figure 1J), the white striped regions had abnormal chloroplasts that lacked organized lamellar structures (Figures 1K,L). Thus the stripe leaf phenotype and the reduced Chl contents in the wsl4 mutant leaves were probably due to developmentally defective chloroplasts.

Genetic analyses of reciprocal crosses between the WT and wsl4 mutant showed that the wsl4 phenotype was inherited by a single recessive nuclear mutation (Table 2). Using the F₂ population derived from the wsl4/Yue13 cross, the wsl4 locus was initially mapped to the long arm of rice chromosome 2, between the insertion/deletion markers L-3 and L-9 (Figure 3A). Based on 978 F₂ recessive homozygous wsl4 background individuals, the WSL4 locus was ultimately narrowed down to an 86-kb genomic region flanked by the insertion/deletion markers L-37 and L-26 on the BAC clone P0020C11, in which nine putative open reading frames (ORFs) were predicted (Figure 3B). Comparison with WT cDNA sequences revealed a 2 bp deletion of positions 1,330 and 1,331 bp from the ATG start codon in the 7th ORF (LOC_Os02g35750) (Figure 3C), generating a premature stop codon. Sequence comparison between genomic and cDNA showed that the WSL4 gene contained a single exon, which was predicted to encode a 554 amino acid residue polypeptide with a calculated molecular mass of approximately 60 kDa. By contrast, the wsl4 gene, if translated, would encode a truncated protein, named wsl4, and missing the C-terminal 111 amino acid residues, but with five added unrelated amino acid residues resulting from translation of the frame-shift (Supplementary Figure S1).

To test whether the 2 bp deletion conferred the wsl4 mutant phenotype, a 5.7 kb WT genomic fragment harboring the transcriptional regulation elements and putative full-length coding sequence of LOC_Os02g35750 was introduced into the wsl4 mutant. All six positive transgenic lines exhibited green leaves, and had comparable levels of Chl a and b to the WT (Figures 3D,E). To further confirm that disruption of LOC_Os02g35750 function was responsible for the wsl4 mutant phenotype, we generated RNAi transgenic lines in WT background and obtained four independent transgenic lines, that phenocopied the wsl4 mutant with respect to leaf color and Chl contents (Figures 3D,E). Thus LOC_Os02g35750 corresponds to the WSL4 gene.

An analysis of WSL4 protein in the Pfam database (Finn et al., 2010) showed that it contained a tandem repeat of 12 PPR motifs and belonged to a member of the P subfamily (Figure 4A). The 12 PPR motifs in WSL4 protein were sequentially arranged, with an average 31.5% sequence identity among them (Figure 4B). The 2 bp deletion in wsl4 mutant occurred at the middle of the 10th PPR motif site, resulting in loss of the last three PPR repeats (Figure 4A and Supplementary Figure S1). The fact that wsl4 mutant exhibited the same or a slightly weaker phenotype (Figures 3D–F) and splicing defects (see below) as the WSL4 knock-down transgenic lines suggests that the 2 bp deletion in wsl4 mutant causes the partial loss-of-function mutation.

A BLAST search of the NCBI database showed that WSL4 is a single-copy gene in the rice genome, and homologs can be found in many other plant genomes. We performed phylogenetic analyses of WSL4 and these homologs to determine their evolutionary relatedness. The WSL4 protein shared high sequence homology with homologs in Brachypodium distachyon, Hordeum vulgare, Setaria italic, Zea mays, Sorghum bicolor, and Aegilops tauschii (Supplementary Figures S2, S3); however, the functions of these genes including the Arabidopsis homolog were unknown. The WSL4 protein thus defined a new member of the P-superfamily of PPR proteins.

The prediction of a chloroplast transit peptide at the N-terminus of WSL4 by ChloroP⁸ and TargetP⁹, together with the Chl-deficient phenotype of wsl4 mutant, suggests that WSL4 is localized to chloroplasts. To determine its actual localization, we transiently expressed p35S:WSL4-GFP in rice protoplasts. As shown in Figure 4C confocal microscopy observations showed that WSL4-GFP was localized to punctate compartments associated with Chl autofluorescence. Moreover, we noted that wsl4-GFP was still targeted to the chloroplast, suggesting that the 111 missing amino acid residues at the C-terminus of WSL4 were not necessary for chloroplast localization (Figure 4C). To further determine the nature of these small dot-like structures, we coexpressed WSL4-GFP and CFP-tagged plastid binding protein PEND-CFP known to be a fluorescent marker protein for plastid nucleoids (Terasawa and Sato, 2005). As expected, the GFP signals colocalized with the CFP signals, indicating that WSL4 was localized to the chloroplast nucleoids (Figure 4D). These results show that WSL is indeed a chloroplast nucleoid-localized protein.

A survey of the rice expression profile database^10,11 revealed that WSL4 was constitutively expressed in all tissues examined, with the strongest expression in the young leaves. For validation, expression analyses performed by quantitative RT-PCR (qRT-PCR) using RNA samples prepared from culms (C), flag leaves (FL), young leaves (YL), shoot bases (SB), young panicles (P), young roots (R), and leaf sheaths (LS) showed that the abundance of WSL4 transcript was significantly higher in young leaves compared to other tissues (Figure 5A). These expression data together with the observed wsl4 phenotype support the hypothesis that WSL4 has an important role at chloroplast development stage during the early leaf development in rice. Interestingly, accumulation of WSL4 transcript was enhanced under low temperature conditions (Figure 5B).

Multiple chloroplast-associated genes were investigated to further determine whether the defective chloroplast development was associated with the altered gene expression. As shown in Figure 6A, key photosynthesis-associated nuclear genes (cab1R, cab2R, and rbcS) were significantly down-regulated in wsl4 mutant, suggesting that photosynthesis in wsl4 mutant was compromised. Plastidic genes can be grouped into three types. Class I genes are mainly transcribed by PEP, whereas Class III genes are absolutely transcribed by NEP. In contrast, NEP functions cooperatively with PEP to regulate the transcription of Class II genes (Hedtke et al., 1997; Yu et al., 2014). We next examined expression levels of Classes I and plastid genes. Expression of PEP-dependent Class I genes (e.g., psaA, psbA, and rbcL) was significantly down-regulated in the wsl4 mutant, whereas expression of NEP-dependent Class III genes (e.g., rpoB, rpoC1, and rpoC2) was significantly up-regulated (Figure 6A). These results suggest that PEP complex activity was also compromised in the wsl4 mutant as well. Consistent with the reduced Chl content, expression of Chl synthesis genes CHLD, CHLI, CHLH, and PORA, but not YGL1, was significantly inhibited in wsl4 mutant (Supplementary Figure S4). We therefore propose that WSL4 might coordinate the expression of numerous nuclear- and plastid-encoded genes to regulate chloroplast development at the early leaf development stage.

We finally assessed the accumulation of core subunits of photosynthetic enzyme complexes in WT and the wsl4 mutant, including photosystem I (PsaA and PsaB) and photosystem II (PsbA and PsbD) subunits, RNA polymerase subunits (RpoB), ATP synthase CF1 β subunit (AtpB), and cytochrome b6f (PetD). All assayed proteins in bleached tissues, except RpoB, were barely detectable in the wsl4 mutant compared to WT (Figures 6B,C).

Accumulating evidence showed that a large group of nuclear-encoded PPR proteins required for RNA editing, splicing, stability, and translation were critical for chloroplast development (Hattori et al., 2007; Beick et al., 2008; Schmitz-Linneweber and Small, 2008; Barkan et al., 2012; Tan et al., 2014; Hsieh et al., 2015; Hammani et al., 2016; Xie et al., 2016). Firstly, we determined whether loss of WSL4 function affected editing at 21 identified RNA editing sites in chloroplast RNA (Corneille et al., 2000). The results showed that the editing efficiency of rpoB at C545 and C560 exhibited a significant increase in wsl4 mutant compared with WT (Figure 7), while the rest 11 genes and the corresponding 19 editing sites were normally edited in wsl4 mutant (Supplementary Figure S5). Furthermore, we analyzed the editing efficiency of rpoB at C545 and C560 in complemented and RNAi transgenic plants. As expected, the editing efficiency of rpoB at C545 and C560 showed a markedly reduction in complemented plant, while the RNAi plant mimicked a similar increase in RNA editing as the wsl4 mutant (Figure 7). These data further suggest that the mutation in WSL4 affects the editing efficiency of rpoB gene.

Accumulating evidence shows that a large group of nuclear-encoded PPR proteins required for RNA editing, splicing, stability, maturation, and translation are critical for chloroplast development (Hattori et al., 2007; Beick et al., 2008; Barkan et al., 2012; Tan et al., 2014; Hsieh et al., 2015; Hammani et al., 2016; Wu et al., 2016; Xie et al., 2016; Zoschke et al., 2016; Zhou et al., 2017). To determine whether loss of WSL4 function affected RNA splicing of chloroplast genes containing 17 group II introns and one group I intron (Hiratsuka et al., 1989), we carried out RT-PCR analyses using primers situated in exons flanking intron 1, and then compared the lengths of the amplified products between WT and wsl4 mutant plants. Four chloroplast transcripts containing group II introns were spliced with much reduced efficiency in wsl4 mutant compared to WT (Figures 8, 9A). The splicing defects were largely rescued in complemented transgenic plants and were mimicked in RNAi transgenic plants (Figure 9A). As a control, the splicing efficiencies of atpF, ndhA, rpl2, and rps12 intron 2 were not significantly affected in the wsl3 mutant (Figure 9A), a rice chloroplast translation-defective mutant lacking an essential peripheral subunit of the PEP complex (Wang L. et al., 2016). Splicing of other chloroplast transcripts was not significantly impeded in wsl4 mutant (Figure 8).

Quantitative RT-PCR was performed to quantify spliced (using primers situated in exons 1 and 2) and unspliced transcripts (using primers situated in exon 1 and intron 1) to further determine the extent of the splicing defects, Compared with wsl3, wsl4 and RNAi transgenic plants showed strong splicing defects in atpF, ndhA, rpl2, and rps12 transcripts (Figure 9B). Consistent with phenotypic expression, the splicing defects of atpF, ndhA, rpl2, and rps12 in wsl4 mutant appeared to be worse in plants frown at 20°C (Supplementary Figure S6) suggesting that loss of WSL4 function caused the temperature-sensitive leaf bleaching and splicing defects at the early leaf developmental stage. The defective splicing of these four chloroplast transcripts was confirmed by RNA gel blot hybridizations using specific probes (Figures 9C,D). As a means of further investigating the effects of impaired splicing of atpF, ndhA, and rpl2 transcripts on post-processing, western blots were performed to examine protein accumulations of atpF, ndhA, and rpl2 in wsl4 mutant. As shown in Figures 9E,F, atpF, ndhA, and rpl2 were present at lower levels in wsl4 mutant compared with WT. We propose that WSL4 functions in the splicing of chloroplast group II introns.