PALE-GREEN LEAF12 Encodes a Novel Pentatricopeptide Repeat Protein Required for Chloroplast Development and 16S rRNA Processing in Rice
Pentatricopeptide repeat (PPR) proteins regulate organellar gene expression in plants, through their involvement in organellar RNA metabolism. In rice (Oryza sativa), 477 genes are predicted to encode PPR proteins; however, the majority of their functions remain unknown. In this study, we identified and characterized a rice mutant, pale-green leaf12 (pgl12); at the seedling stage, pgl12 mutants had yellow-green leaves, which gradually turned pale green as the plants grew. The pgl12 mutant had significantly reduced Chl contents and increased sensitivity to changes in temperature. A genetic analysis revealed that the pgl12 mutation is recessive and located within a single nuclear gene. Map-based cloning of PGL12, including a transgenic complementation test, confirmed the presence of a base substitution (C to T), generating a stop codon, within LOC_Os12g10184 in the pgl12 mutant. LOC_Os12g10184 encodes a novel PLS-type PPR protein containing 17 PPR motifs and targeted to the chloroplasts. A quantitative real-time PCR analysis showed that PGL12 was expressed in various tissues, especially the leaves. We also showed that the transcript levels of several nuclear- and plastid-encoded genes associated with chloroplast development and photosynthesis were significantly altered in pgl12 mutants. The mutant exhibited defects in the 16S rRNA processing and splicing of the plastid transcript ndhA. Our results indicate that PGL12 is a new PLS-type PPR protein required for proper chloroplast development and 16S rRNA processing in rice.
Keywords: Chloroplast; Oryza sativa; PGL12; PPR protein; Rice; RNA splicing
Introduction
Chloroplasts, which are deemed to be derived from cyanobacteria via endosymbiosis, are the site of photosynthesis in land plants and algae, and are indispensable for plant growth and development because they control carbon assimilation and other important physiological processes ([29], [19]). Chloroplast reproduction and function are precisely regulated by the co-ordinated expression of nuclear and chloroplastic genes. Two types of RNA polymerase regulate chloroplast gene expression: nucleus-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP) ([15], [37]). PEP transcribes the chloroplast genes that encode proteins involved in photosynthesis, and NEP largely transcribes housekeeping genes through the recognition of specific promoter types ([24], [37]). The plastid genome is very small (about 135 kb, containing 154 genes), but around 3,000 proteins are present in the chloroplast, indicating that nuclear genes encode >95% of chloroplast proteins ([33]). This suggests that chloroplast development is under the control of nuclear genes; therefore, isolating and characterizing these genes should further our understanding of the mechanisms of chloroplast development.
The pentatricopeptide repeat (PPR) protein family was discovered during a bioinformatic analysis of the Arabidopsis thaliana genome ([38]). PPR proteins generally include tandem arrays comprising 2–26 copies of a 35 amino acid motif (defined as the PPR motif), which is predicted to form a pair of antiparallel α-helices ([27], [4]). Based on the size and sequence variations of these repeats, the PPR motifs have been classified into three types: P, L and S ([27]). Based on the presence of these different motifs, the PPR proteins are separated into the P and PLS subfamilies ([27]). Most eukaryotes have around 5–30 PPR family members; however, this family is greatly expanded in higher plants, with 450 members in A. thaliana and 477 in rice (Oryza sativa), only 20 of which have been characterized ([35]). The majority of PPR proteins are predicted to target either the chloroplasts or the mitochondria ([4]).
Post-transcriptional modulation has an important impact on regulating gene expression in organelles. So far, many genes encoding PPR proteins have been reported. The functions of these genes involved in diverse aspects of RNA metabolism, include RNA editing ([54], [12], [1], [23]), splicing ([41], [55], [5], [8]), stability ([31], [13], C. [46], Y.F. [60]), translation ([36], [43], [64]), processing ([28], [14], W. [51]) and maturation ([63]). There seems to be little or no redundancy between the few PPR proteins that have been characterized in the monocots and dicots ([4]). In rice, OGR1 and MPR25 (PPR proteins containing a DYW motif) are targeted to the mitochondria and implicated in RNA editing ([21], [45], [57]). Moreover, Rf5 and Rf6, two fertility-restoring (Rf) genes, encode proteins that physically interact with the PRR proteins GRP162 and OsHXK6, respectively, during mitochondrial RNA regulation ([16], [17]). In addition to these four mitochondria-targeting PPR proteins, other chloroplast-localized PPR proteins have been reported in rice. These include OsPPR676, a PPR-SMR protein confirmed to interact with Osj10gBTF3, which is indispensable for pollen development and plant growth ([26]). Other chloroplast-targeted PPR proteins include OsPPR1 ([11]), OsPPR4 ([3]), OsPPR6 ([42]), YSA ([39]), WSL ([41]), WSL4 (Y. [48]), OsV4 ([10]), ALS3 ([25]), OsOTP51 ([58]), OspTAC2 ([47]) and TCD10 (L. [50]), all of which are essential for normal chloroplast development at the seedling stage. Recently, OsPGL1, a dual-localized PPR protein, was revealed to be essential for RNA editing in the chloroplasts and mitochondria ([53]). Despite the important advances mentioned above, however, the functions of the majority of PPR proteins in the control of the chloroplast post-transcriptional modifications in rice are largely unknown.
In this study, we identified and characterized a rice mutant, pale-green leaf12 (pgl12), which displays pale-green leaves throughout its life cycle. We successfully isolated the PGL12 gene using map-based cloning, revealing that it encodes a new PPR protein belonging to the PLS subgroup. Further investigation indicated that PGL12 greatly influences the processing of 16S rRNA and splicing of the chloroplast transcript ndhA.
Results
Characterization of pgl12 mutants
The pgl12 mutant was identified from a large pool of mutants generated from the ethyl methanesulfonate (EMS)-based mutagenesis of 'ZhongHua 11' (ZH11; japonica variety). The mutant exhibited paler leaves than the wild type (WT) throughout its entire life cycle, with a more obvious phenotype at the seedling stage than at the tillering and mature stages (Fig. 1A–E). A photosynthesis pigment content analysis revealed that the Chl a and Chl b contents were significantly decreased in pgl12 compared with the WT at the seedling, tillering and mature stages, although no differences in carotenoid content were observed (Fig. 1F). Moreover, the Chl contents of the pale-green leaves of pgl12 had partially recovered by the mature stage.
Graph: Fig. 1 Visual phenotypes and photosynthetic pigment contents of the wild-type (WT) and pgl12 plants. (A–E) Phenotype of the WT (cultivar 'ZH11'; left) and the pgl12 mutant (right) at the seedling (A), tillering (B, C) and mature (D, E) stages. (C, E) WT (left) and pgl12 (right) leaves, corresponding to B (C) and D (E), respectively. Scale bars = 5 cm. (F) Photosynthetic pigment contents of the WT and pgl12 plants at the seedling, tillering and mature stages. Car, carotenoid. (G) Comparison of the WT and pgl12 photosynthetic rates. Values are means ± SD of three biological replicates. *P < 0.05; **P < 0.01 (Student's t-test).
Chl plays an important role in plant photosynthesis. To determine whether the significant decrease in Chl content affected photosynthesis in pgl12, we measured its rate of photosynthesis at the heading stage. As expected, the rate of photosynthesis in pgl12 was significantly decreased compared with the WT (Fig. 1G). These characteristics suggest that PGL12 is required for Chl metabolism and photosynthesis.
We used transmission electron microscopy (TEM) to observe the cytological morphology of the WT and pgl12 leaves under natural conditions. The WT leaf cells contained typical chloroplasts with stacked thylakoid membranes (grana; Fig. 2A, B); however, the pgl12 grana were less abundant and thinner than those of the WT, with some in a complete state of disorder (Fig. 2C, D). These observations suggest that PGL12 plays an important role in chloroplast development.
Graph: Fig. 2 Chloroplast ultrastructure in wild-type (WT) and pgl12 seedlings. (A–D) Transmission electron microscopy images of WT ('ZH11'; A and B) and pgl12 mutant chloroplasts (C and D). T, thylakoid membrane system.
Reduced grain yield in the pgl12 mutants
The significant difference in the rate of photosynthesis of the pgl12 mutant led us to investigate several major agronomic traits related to grain yield. The plant height and panicle length were reduced in pgl12 mutants, which produced significantly fewer tillers, primary branches, secondary branches and grains per panicle than the WT (Fig. 3). In contrast, pgl12 produced larger grains than the WT (Supplementary Fig. S1), resulting in a heavier 1,000-grain weight (Supplementary Table S1). In addition to these characteristics, the heading date of pgl12 was delayed by approximately 1 week (Supplementary Table S1). These results indicated that the PGL12 mutation caused an overall reduction in grain yield.
Graph: Fig. 3 Major agronomic traits of the wild-type (WT) and pgl12 plants. (A–C) Phenotypes of the WT ('ZH11'; left) and pgl12 (right) plants; scale bars = 0.3 cm. In (B) and (C), scale bars = 5 cm. (D–I) Comparison of major agronomic traits affecting plant yield. PBN, primary branch number; SBN, secondary branch number; GNPP, grain number per panicle. **P < 0.01 (Student's t-test).
Map-based cloning of PGL12
Map-based cloning was carried out to identify the PGL12 locus and clarify the molecular mechanisms influencing the pgl12 phenotype. The genetic analysis revealed that the pgl12 phenotype was conferred by a single recessive nuclear locus, as the cross between NJ06/pgl12 and 93-11/pgl12 resulted in an F2 population with a 3:1 phenotypic segregation of normal to pale-green leaves. The initial analysis of 100 mutant individuals led to the PGL12 locus being mapped to the short arm of chromosome 12 (Chr. 12), between the markers RM27625 and B12-7 (Fig. 4A). Five polymorphic markers were developed between RM27625 and B12-7, enabling the PGL12 locus to be more finely mapped to a 100 kb genomic region between the insertion–deletion (InDel) markers YS22 and YS24 on the short arm of Chr. 12 (Fig. 4B). A total of 14 open reading frames (ORFs) were identified in the target region using the Rice Genome Annotation Project (Fig. 4B). All predicted ORFs were sequenced, and a single base change (C to T) was identified 1,681 bp along the eighth ORF (LOC_Os12g10184), generating a premature stop codon (Fig. 4C).
Graph: Fig. 4 Molecular cloning of PGL12. (A) The PGL12 locus was initially mapped to the RM27625–B12-7 region on chromosome 12 (Chr. 12). This region was spanned by the BAC clones AL713901, AL713947, AL731879, BX664711, BX649218, BX000560, AL954853 and AL713952. The number of recombinants identified from an F2 population of 576 plants with the recessive mutant phenotype is shown below each marker. (B) The PGL12 locus was fine-mapped to a 189 kb region between markers YS22 and YS24. (C) The region contained 14 open reading frames (ORFs), and a single nucleotide polymorphism was detected in ORF8, resulting in a premature stop codon. (D) Complementation of the pgl12 mutant. A wild-type (WT) plant (left), pgl12 plant (middle) and complemented pgl12 mutant transformed with the PGL12 vector (com1; right) are presented. Scale bar = 10 cm. (E) Chl content of the WT, pgl12 mutant and complemented pgl12 mutant plants. **P < 0.01 (Student's t-test).
To confirm that the mutation in LOC_Os12g10184 caused the pgl12 mutant phenotype, we introduced a 5.4 kb genomic fragment, containing the entire ORF of LOC_Os12g10184, an approximately 2,200 bp 5′-upstream region and the 1,100 bp 3′-downstream region, into the pgl12 mutant using Agrobacterium tumefaciens-mediated transformation. A total of 20 transgenic plants were obtained, all of which had recovered the WT phenotype (Fig. 4D, E). It was therefore concluded that LOC_Os12g10184 is PGL12, and that its mutation causes the pgl12 phenotype.
PGL12 encodes a PPR protein
A sequence analysis of PGL12 revealed that it is an intronless gene. The full-length PGL12 cDNA comprises 2,049 bp, and encodes a protein containing 683 amino acids. A search of the plant PPR database (http://ppr.plantenergy.uwa.edu.au/) ([7]) revealed that PGL12 is a PPR protein containing 17 PPR motifs of different lengths, and is a member of the PLS subfamily (Fig. 5A, B). The premature stop codon identified in the pgl12 mutant is located in the 15th PPR motif, resulting in the loss of 122 amino acids from its C-terminal region. The pgl12 mutant phenotype suggests that these 122 absent amino acids play an important role in the functional integrity of the PGL12 protein.
Graph: Fig. 5 Sequence and phylogenetic analysis of the PGL12 protein. (A) The PGL12 protein has 17 pentatricopeptide repeat (PPR) motifs. The chloroplast transit peptide (CTP) in the N-terminus and the pgl12 mutation in the 15th PPR motif are shown. (B) Comparison of the PPR motifs of PGL12. Fully or partially conserved amino acids are shaded black and gray, respectively. The amino acid outlined in black represents the mutation. (C) Alignment of amino acid sequences from other species with the highest similarity to the PGL12 protein. GenBank accession numbers: Arabidopsis thaliana (NP_177302.1); Sorghum bicolor (XP_002441982.2); Brachypodium distachyon (XP_014758302.1); Populus trichocarpa (XP_002325518); and Medicago truncatula (XP_003604235). Conserved amino acids are highlighted in gray and black. The mutant amino acid in pgl12 is outlined in black. PPR motifs are indicated by double-headed arrows above the sequences.
BLAST searches of the PGL12 protein sequence revealed close homologs in A. thaliana, Sorghum bicolor, Brachypodium distachyon and Medicago truncatula; PGL12 shared a high degree of identity with S. bicolor (82.11%) and B. distachyon (80.35%) but a low degree of identity with A. thaliana (46.35%) and M. truncatula (45.58%) (Fig. 5C). A phylogenetic analysis of PGL12 and these closely related proteins was carried out to evaluate their evolutionary relatedness. The result showed that there is a clear divergence between monocots and dicots (Supplementary Fig. S2). However, very little is known about the functions of these homologous proteins. The above findings indicated that PGL12 encodes a novel member of the PPR protein superfamily.
Subcellular localization and expression pattern of PGL12
Most PPR proteins are predicted to be targeted to the chloroplasts or mitochondria ([27]). Our analysis of the PGL12 protein sequence using ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) and TargetP (http://www.cbs.dtu.dk/services/TargetP/) predicted that the PGL12 protein is localized to the chloroplasts, with a probable chloroplast transit peptide (CTP) on its N-terminus. To determine the actual subcellular localization of PGL12, a p35S: PGL12-GFP (green fluorescent protein) construct was generated using the full-length PGL12 CDS (coding sequence) without the stop codon. Plasmids containing the fusion gene or an empty p35S:GFP vector (control) were introduced into rice protoplasts using the polyethylene glycol (PEG)-mediated method. Confocal microscopy was used to detect the fluorescent signals, revealing that the PGL12–GFP fusion protein co-localized with the autofluorescence signals of the chloroplasts in the rice protoplasts (Fig. 6A–D). Using the control vector, the GFP signals were observed in the plasma membrane, cytoplasm and nucleus (Fig. 6E–H). These findings show that PGL12 targets the chloroplasts.
Graph: Fig. 6 Subcellular localization and expression analysis of PGL12. (A–H) Subcellular localization of PGL12–GFP or the GFP control. The fluorescence signals were detected by confocal microscopy. (A, E) Green fluorescence of PGL12–GFP (A) and GFP (E). (B, F) Red chloroplast autofluorescence. (C, G) Bright-field image of the p35S:PGL12-GFP (C) and p35S:GFP (G) rice protoplasts. (D) Merged image of (A), (B) and (C). (H) Merged image of (E), (F) and (G). Scale bars = 5 µm. (I) Wild-type ('ZH11') plants at the five-leaf stage were used for the qRT-PCR analysis in (J). L2–L5, second to fifth leaves; R, root; S, stem; ML, mature leaf; P, panicle; SH, sheath. (J) Transcript levels of PGL12 in various tissues (values are means ± SD, n = 3).
Data submitted to the microarray transcript-profiling database (www.genevestigator.ethz.ch) showed that PGL12 was transcribed in all tissues throughout plant development. To confirm these data, we carried out an expression analysis of PGL12 using quantitative real-time PCR (qRT-PCR) with RNA extracted from different tissues of the WT plants. Our results indicated that PGL12 was expressed in all tissues examined, with low expression levels in the roots, stem and panicle tissues, and high levels in the older leaves, especially the second mature leaf (L2; Fig. 6I, J). These results, along with the chloroplast localization and the observed pgl12 phenotype, support the hypothesis that PGL12 plays a significant role in chloroplast development in rice leaves.
Transcript expression was altered in pgl12
The development of chloroplasts is closely associated with the expression levels of the chloroplast-encoded genes, which are regulated by the two classes of RNA polymerase, NEP and PEP ([32]). Many previous studies have shown that PPR proteins are involved in RNA processing ([4]), so we investigated the expression levels of the plastid genes in pgl12. The relative expression levels of the PEP-dependent genes PsbA and rbcL were significantly reduced, while the expression of PsaA was not significantly different in the pgl12 mutant compared with the WT (Fig. 7A). The transcript abundance of the NEP-dependent genes (rpoA, rpoB, RpoC1 and RpoC2) was significantly increased (Fig. 7A). The expression levels of genes associated with ribosome development (16S rRNA, 23S rRNA, rps2, rps4, rps11, rps12, rps14, rps16, rps18, rps19, rpl16, rpl20 and rpl23) were also investigated in the mutant, and were almost all found to be significantly up-regulated, with the exception of 16S rRNA and rpl16 (Fig. 7B). The expression of rpl16 was not significantly different in pgl12 compared with the WT, while the relative expression of 16S rRNA was dramatically decreased in the pgl12 mutant (Fig. 7B).
Graph: Fig. 7 The expression analysis of genes associated with chloroplast development in wild-type (WT) and pgl12 seedlings. (A) The relative expression levels of plastid-encoded plastid RNA polymerase (PEP)-dependent genes (PsaA, PsbA and rbcL), nucleus-encoded plastid RNA polymerase (NEP)-dependent genes (rpoA, rpoB, RpoC1 and RpoC2), and chloroplast development and photosynthesis genes in 5-day-old WT ('ZH11') and pgl12 plants. (B) The relative expression levels of genes related to ribosome development in 5-day-old WT and pgl12 plants. Values are the mean ± SD of three biological replicates. **P < 0.01 (Student's t-test).
In addition to chloroplast-encoded genes, we also studied the expression levels of several nuclear-encoded genes associated with Chl biosynthesis, photosynthesis and chloroplast development in the WT and pgl12 plants. These genes included psaD (encoding a PSI subunit), psbP (encoding PSII subunit P), Lhcb2 (encoding the PSII light-harvesting complex protein), rbcS (encoding the small subunit of Rubisco), RpoTp (encoding the NEP core subunits), CAO1 (encoding Chl a oxygenase 1), DVR (encoding divinyl reductase), Cab1R (encoding light-harvesting Chl a/b-binding proteins of PSII) and YGL1 (encoding a Chl synthetase). Our qRT-PCR analysis indicated that the expression levels of RpoTp, DVR and CAO1 were not significantly altered in the pgl12 mutant compared with the WT, but the expression levels of psaD, psbP, Lhcb2, rbcS and Cab1R were significantly down-regulated in the pgl12 mutant compared with the WT, and the YGL1 transcripts were significantly increased (Fig. 7A). Taken together, these results revealed that the pgl12 mutation caused the abnormal expression of genes related to Chl biosynthesis, photosynthesis and chloroplast development, which resulted in the pale-green phenotype observed in the pgl12 mutant.
PGL12 affects the processing of 16S rRNA
To determine further whether the processing of 16S rRNA was affected, RNA gel blot hybridization with specific probes against the 16S rRNA was carried out. The result revealed that the processing of 16S rRNA was significantly affected in the pgl12 mutant (Fig. 8B). The 16S rRNA was virtually only present in its mature form (1.5 kb) in the WT; however, large amounts of unprocessed precursor (1.7 kb) accumulated in the pgl12 mutant (Fig. 8B). Moreover, the accumulated amount of the full-length rRNA precursor (7.4 kb) was higher in the pgl12 mutant compared with the WT (Fig. 8B). These findings revealed that PGL12 affects the processing of 16S rRNA.
MAP: Fig. 8 Analysis of 16S rRNA processing in pgl12 mutant plants. (A) Physical map and transcript pattern of 16S rRNA. The position of hybridization probes for 16S rRNA was indicated. (B) Analysis of 16S rRNA accumulation in WT and pgl12 mutants. As a loading control, rRNAs on the ethidium bromide-stained gel are shown.
PGL12 affects the splicing of the plastid-encoded ndhA transcripts
The PPR proteins are mainly thought to be involved in RNA processing, including RNA editing, splicing, stability and translation ([4]). Since PGL12 belongs to the PLS subgroup and lacks any special functional domains such as E, E+ or DYW at its C-terminus, it is implicated in all transcript processing activities. To date, 23 editing sites have been reported in the chloroplast RNA of plants. To determine which of these sites are edited in the WT and pgl12 plants, we directly sequenced all 23 sites in their chloroplast cDNA using RNA extracted from the leaves of seedlings. The sequence analysis indicated that editing events occurring in the pgl12 mutant were analogous to those in the WT, suggesting that the PGL12 protein is not required for the editing of the known RNA editing sites (Supplementary Fig. S4; Supplementary Table S2).
Next, we verified whether PGL12 affects the RNA splicing of chloroplast-encoded transcripts. In the rice chloroplast genome, there are 18 introns, comprising one group-I intron and 17 group-II introns. All chloroplast genes that contain introns were amplified using reverse transcription–PCR (RT–PCR) with primers flanking the introns, then the lengths of the amplified RT–PCR products was compared between the WT and pgl12 samples. Only one chloroplast transcript, ndhA, was spliced at a very low efficiency in pgl12 relative to the WT (Fig. 9A, B). This defect in ndhA splicing was rescued in the complemented transgenic rice plants (Fig. 9C); therefore, our findings suggest that PGL12 is essential for splicing of the chloroplast transcript ndhA.
Graph: Fig. 9 Splicing analysis of all transcripts from rice chloroplast genes containing introns in the wild type (WT) and pgl12. (A) Splicing analysis of chloroplast genes in the leaves of 1-week-old seedlings, performed using RT–PCR. Spliced (S) and unspliced (U) transcript bands are indicated on the right. 23S rRNA was used as a reference. (B) Structure of the ndhA gene and the primers used in (C). (C) RT–PCR analysis of the ndhA transcripts of the WT, pgl12 and complemented (CP) plants. The product of the spliced ndhA transcripts had an expected size of 1,089 bp; however, the unspliced transcripts were 2,076 bp. Molecular weight markers (M) are shown on the left; the amplification product from total DNA (gDNA) is presented in the center. The right side of the panel shows a lack of amplification using the RNA templates for reverse transcription, indicating that the 2,076 bp product of the cDNA sample resulted from an unspliced transcript rather than from RNA contamination of the genomic DNA.
Loss of PGL12 function alters rice seedling adaptability to temperature
Temperature is an important environmental factor affecting plant growth and development. Previous studies have indicated that some leaf mutants are sensitive to temperature, including wsl4 (Y. [48]) and osv4 ([10]). When grown at a constant elevated temperature of 35°C, the pgl12 mutants displayed a severely yellow phenotype (Fig. 10A, B), contained less Chl than the WT (Fig. 10D) and had abnormal chloroplast development (Fig. 10E, H). At a constant lower temperature of 20°C, the pgl12 mutant exhibited albino symptoms (Fig. 10C, B) and hardly any Chl could be detected in the plants (Fig. 10D). At this lower temperature, the cells of the pgl12 mutant contained only a few chloroplasts, many of which were disrupted and had an irregular arrangement with empty lamellae (Fig. 10G, J). These results indicate that PGL12 is implicated in regulating adaptability to temperature.
Graph: Fig. 10 Response of the pgl12 mutant to temperature. (A–C) Wild-type (WT; 'ZH11') and pgl12 mutant seedlings grown in a growth chamber under a 12 h photoperiod at a constant temperature of 35°C (C35; A), a light/dark temperature of 30°C/25°C (L30/D25; B) and a constant temperature of 20°C (C20; C). Scale bars = 2.5 cm. (D) Chl a and Chl b contents of the WT and pgl12 plants corresponding to the images in (A–C). **P < 0.01 (Student's t-test). (E–J) TEM images of chloroplasts from the WT cultivar (E–G) and the pgl12 plants (H–J) under different temperature treatments: L35 (E, H); L30/D25 (F, I); C20 (G, J).
Discussion
Previous studies have reported and characterized several leaf color mutants, which have been subdivided into multiple categories, including virescent, albino, zebra, chlorina and striped, according to their phenotype ([20]). In this study, we identified a rice mutant with pale-green leaves, named pgl12, which had a Chl-deficient phenotype throughout its entire life cycle. This phenotype is similar to other leaf color mutants, such as ygl1 ([52])and vyl ([9]); however, unlike these other mutants, pgl12 was also found to be sensitive to temperature. Under a lower growth temperature of 20°C, the pgl12 mutant displayed an albino phenotype with very low Chl levels, resembling other rice mutants including osv4 ([10]), v1 ([22]) and v2 ([40]); however, these mutants phenotypically resemble the WT under normal growth conditions. Low temperature is a key factor affecting chloroplast gene expression and particularly in exacerbating chloroplast translation defects caused by various mutations, such as RNA-binding proteins ([10], [41]). The expression levels of many genes related to the chloroplast system in the pgl12 mutant were lower than those of the WT (Supplementary Fig. S3). The down-regulation or abnormal expression of these genes may be a reason for the albino phenotype of the pgl12 mutant under low temperature.
The proper formation of chloroplasts is essential for the growth and development of plants, and is co-ordinately regulated by NEP and PEP ([32]). In our study, the relative expression of the NEP-dependent genes, such as RpoA, RpoB, RpoC1 and RpoC2, was significantly increased in the pgl12 mutant (Fig. 7A), which is probably linked to defects in the chloroplast gene expression machinery. The chloroplast ribosome is an important part of this machinery, consisting of the 50S large subunit (containing three types of rRNA, 23S, 5S and 4.5S rRNA, and 33 ribosome proteins) and the 30S small subunit (containing only one rRNA, 16S rRNA and 24 ribosome proteins) ([44]). Among these ribosome proteins, 12 and nine of the 30S and 50S proteins are encoded by plastid genes, respectively. We found that the transcript levels of almost all of the investigated genes related to ribosome development were significantly up-regulated in the pgl12 mutant; however, the expression of 16S rRNA was greatly reduced (Fig. 7B), which suggests that the mutation of PGL12 may affect 16S rRNA maturation. RNA gel blot experiments revealed that the processing of 16S rRNA was affected in the pgl12 mutant (Fig. 8). This defect may influence the translation efficiency of some chloroplast genes and cause a feedback regulation that alters the transcript levels of many genes, resulting in the phenotypes observed in the pgl12 mutant.
To date, several PPR genes have been reported to be involved in organellar RNA metabolism in rice. Depending on their structure, these proteins could be divided into two categories: P-class and PLS-class. P-class proteins include OsOTP51([58]), WSL ([41]), OsPPR4 ([3]), OsPPR6 ([42]) and WSL4 (Y. [48]). These proteins all target the chloroplast. OsOTP51 and WSL are implicated in RNA splicing; the former impacts the splicing of many plastid genes, particularly the ycf3 transcript, while the latter only affects the splicing of the rpl2 transcript ([58], [41]). OsPPR4 influences the splicing of many plastid genes, such as atpF, ndhA, rpl2, rps12, petB and rps16, and the editing of the ndhA transcript ([3]). OsPPR6 participates in the splicing of ycf3 and the editing of ndhB transcripts ([42]), while WSL4 is involved in the editing of rpoB and the splicing of ndhA, atpF, rpl2 and rps12 (Y. [48]). PLS-class proteins include OGR1 and MPR25 ([21], [45]). These two proteins have a special domain in the C-terminus and are involved in RNA editing. OGR1 is a mitochondrial-targeted PPR protein containing a DYW motif at the C-terminus, and is involved in editing seven specific sites in five mitochondrial transcripts ([21]). MPR25, a member of the E subgroup of the PPR family, accumulates in the mitochondria and is essential for the normal editing of the nad5 transcript ([45]). The C-terminus of PLS proteins almost always possesses special domains, such as E or DYW, which are involved in RNA editing ([4]). However, there are a few exceptions, such as PPR103 ([13]), OTP70 ([6]) and PDM1/SEL1([34]); these proteins either have no special domains at the C-terminus or are indirectly involved in RNA editing. PGL12, a chloroplast-targeted PPR protein without any special motifs at the C-terminus, belongs to PLS-class proteins, and is closest in structure to the PDM1/SEL1 in Arabidopsis ([34]). No differences in RNA editing were detected between pgl12 and the WT in an analysis of all chloroplast editing sites (Supplementary Fig. S4; Supplementary Table S2). Thus, PGL12 may be another exception in PLS proteins. A splicing analysis using RT–PCR revealed that the intron of ndhA was spliced inefficiently in the mutant (Fig. 9A), a phenotype that had also previously been reported in the pdm1/sel1 mutant in Arabidopsis. PDM1/SEL1 belongs to the PLS-PPR protein subfamily, and is involved in the splicing of rpoA and TrnK transcripts, in addition to ndhA ([59]). The splicing of ndhA transcripts is also severely affected in the PpPPR-66 knockout mutant in Physcomitrella patens, and its homologous gene (At2g35130) in A. thaliana is also involved in ndhA splicing ([18]). In addition to OsPPR4 and WSL4 affecting the splicing of the ndhA transcripts in rice, WSP1, a MORF family member, has also been implicated in the splicing of ndhA transcripts and the editing of ndhD, ndhG, rpoB and rps14 transcripts ([61]). Interestingly, our yeast two-hybrid analysis of the interaction of the proteins regulating ndhA splicing revealed that PGL12 interacted with WSP1 (Supplementary Fig. S5), suggesting that the splicing of ndhA may be mediated by a complex.
ndhA encodes a subunit of the chloroplast NADH dehydrogenase-like (NDH) complex ([30]). Previous studies have shown that plants with impaired chloroplast NDH complexes had no obvious phenotypic differences from the WT under normal growth conditions ([56], [18]); however, the pgl12 mutant displayed pale-green leaves throughout its life cycle, with significant differences in many important agronomic traits, including the number of tillers, plant height, number of spikelets per panicle and number of branches per panicle (Fig. 3). This suggests that the ndhA splicing defect in the pgl12 mutant did not cause its phenotypic differences.
Materials and Methods
Plant materials and growth conditions
The pgl12 mutant was identified from a library of EMS-mutagenized rice plants with a ZH11 japonica variety background. Genetic and phenotypic analyses were performed using the F1 and F2 generations of a cross between the pgl12 mutant and ZH11. The individuals displaying the mutant phenotype from the F2 generation of the cross between the pgl12 mutant and the indica cultivar NJ06 were used for fine mapping.
Plants used in this study were cultivated either under natural conditions in the experimental field of the China National Rice Research Institute, Hangzhou, Zhejiang province (119°6'E, 30°0'N) or in plant incubators (Versatile Environmental Test Chamber MLR 351; Sanyo) with a 12 h light (L)/dark (D) photoperiod and L/D temperatures of 30°C/25°C. The seedlings subjected to different temperature treatments were cultivated in plant incubators at a constant temperature of 20 or 35°C, and a 12 h photoperiod.
Measurement of photosynthetic pigments and major agronomic traits
Sections of fresh plant leaves (∼0.4 g) were placed in 4 ml of 80% acetone and incubated for 24 h in the dark at 28°C. The optical density of the extract was measured using an ultraviolet spectrophotometer (DU800; Beckman Coulter) at wavelengths of 470, 645 and 663 nm. Three biological repeats were performed per sample, and 80% acetone was used as a control. The concentrations of Chl a, Chl b and carotenoid were determined as described previously ([2], [49]).
The photosynthetic rate of the plants was measured at the grain-filling stage, while the tiller numbers were counted at the tillering stage. Other major agronomic traits, such as the number of grains per panicle, branch numbers and plant height, were measured when the plants reached maturity.
TEM
The fully expanded third leaves of the WT and pgl12 mutant plants cultivated under conditions of 10 h D/14 h L and at L/D temperatures of 30°C/25°C and 20°C were gathered for TEM. The leaf materials were cut into 0.5 cm sections, fixed in 2.5% glutaraldehyde and incubated in 1% OsO4. Subsequently, the samples were dehydrated in an ethanol series, infiltrated in epoxy resin and finally fixed in Spurr resin for sectioning. The specimens were stained with uranyl acetate and lead citrate, then examined using a JEM-1230 transmission electron microscope (JEOL).
Map-based cloning
Primers for the fine mapping of PGL12 were designed to target the InDel markers identified between the genomes of the 'Nipponbare' (japonica) and '9311' (indica) varieties using Primer Premier 6.0 (PREMIER Biosoft International). The sequences of the primer pairs are listed in Supplementary Table S3. The PCR settings used to amplify these InDel regions were as follows: denaturation at 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, annealing for 30 s and extension at 72°C for 1 min; with a final elongation step at 72°C for 5 min. The PCR products were then analyzed using PAGE or on a 5–6% agarose gel. Candidate genes within the 100 kb target region were amplified from the pgl12 mutant and sequenced by the Tsingke Biological Engineering Technology and Service Co. Ltd.
Vector construction and rice transformation
In order to complement the pgl12 mutant, a 5.5 kb WT genomic PGL12 fragment, containing the 2.2 kb upstream sequence, the entire PGL12 coding region and the 1.1 kb downstream sequence, was amplified from ZH11 using specific primers containing the HindIII restriction site: 10184-com-F (5′-GCAGGCATGCAAGCTTTCCAACCATACCGCTACAT-3′) and 10184-com-R (5′-GGCCAGTGCCAAGCTTTCTTCCCATCAAACTAACCC-3′). This fragment was then cloned into the pCAMBIA1300 vector to generate the fusion vector pCAMBIA1300-PGL12, and subsequently introduced into the pgl12 mutant using Agrobacterium tumefaciens-mediated transformation. The transgenic plants were detected using hygromycin.
Sequence and phylogenetic analysis
Gene prediction was carried out using the Rice Genome Annotation Project analysis tools (http://rice.plantbiology.msu.edu). Sequences homologous to the rice PGL12 protein were obtained using a BLASTp (NCBI) search. A chloroplast signal peptide was predicted at the N-terminus of the PGL12 protein using TargetP (http://www.cbs.dtu.dk/services/TargetP/) and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/). The homologous sequences were aligned using DNAMAN (https://www.lynnon.com), and the phylogenetic tree was constructed using MEGA 6.0 (https://www.megasoftware.net).
Subcellular localization of PGL12 protein
In order to determine the subcellular localization of the PGL12 protein, a GFP fusion vector was constructed, in which the expression of the PGL12-GFP fusion gene was driven by the 35S promoter. The coding region fragment, without the stop codon, was amplified from the ZH11 plants using gene-specific primers: 10184-GFP-F (5′-TGCTCACCATTCTAGAGCAATTCAAAGAGGGAGTTGATA-3′) and 10184-GFP-R (5′-CCGGGGATCCTCTAGAATGGCGATGGCGTCCTCC-3′). The construct was transformed into rice protoplasts, as previously described (Chiu et al. 1996). The blank GFP vector was used as a control. GFP fluorescence was visualized using a confocal laser scanning microscope (Zeiss LSM700; Carl Zeiss).
RNA extraction, RT–PCR analysis and RNA gel blot analysis
Total RNA was extracted from various organs, including the leaves, roots, stems, sheaths and panicles, using a QuickExtract RNA Extraction Kit (Axygen). The RNA samples were reverse-transcribed using a ReverTra Ace-α kit (FSK-101; Toyobo). RT–PCR was carried out using a Power SYBR Green PCR Master Mix kit (Thermo Fisher Scientific). The RT–PCR conditions were as follows: denaturation at 94°C for 4 min; followed by 40 cycles of 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min; with a final elongation at 72°C for 5 min. The primer sequences used for the RT–PCR are listed in Supplementary Table S4. The rice Actin gene was used as an internal reference. RNA gel blots were performed as described by [62]. The sequences of the probe primers were as follows: 16S rRNA-F (5'-TCGGAAAGAACACCAACG-3') and 16S rRNA-R (5'-TCGGAAAGAACACCAACG-3').
Analysis of RNA editing sites and RNA splicing
Specific cDNA fragments were obtained from the WT and pgl12 plants using RT–PCR with gene-specific primers. The cDNA sequences were compared to examine any C to T changes resulting from RNA editing. To analyze RNA splicing, the chloroplast genes with at least one intron were selected and amplified using RT–PCR, with primers flanking the introns. The primer pair sequences derived from the editing and splicing analyses are listed in Supplementary Tables S5 and S6, respectively.
Yeast two-hybrid analysis
The coding sequences of rice PPR proteins related to the splicing of ndhA were amplified from ZH11 plants using the respective primers and then cloned into the bait (pGBKT7) and prey (pGADT7) vectors. The yeast two-hybrid analysis was done in yeast cells (Y2H) according to the Matchmaker- GoldYeast Two-Hybrid System User Manual (Clontech Laboratories). The relevant primers are listed in Supplementary Table S3.
Funding
This work was supported by the National Natural Science Foundation of China [grant Nos. 31661143006 ND 91735303]; the Chinese Academy of Agriculture Sciences ['Science and technology innovation project']; and the Hangzhou Scientific and Technological Program [20170432B03].
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By Long Chen; Lichao Huang; Liping Dai; Yihong Gao; Weiwei Zou; Xueli Lu; Changjian Wang; Guangheng Zhang; Deyong Ren; Jiang Hu; Lan Shen; Guojun Dong; Zhenyu Gao; Guang Chen; Dawei Xue; Longbiao Guo; Yongzhong Xing; Qian Qian; Li Zhu and Dali Zeng
Reported by Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author
Long Chen and Lichao Huang authors contributed equally to this work
s: Qian Qian, E-mail, ; Fax, 0571-63370389; Li Zhu, E-mail, ; Fax, 0571-63370389; Dali Zeng, E-mail, ; Fax, 0571-63370389
s: Qian Qian, E-mail, ; Fax, 0571-63370389; Li Zhu, E-mail, ; Fax, 0571-63370389; Dali Zeng, E-mail, ; Fax, 0571-63370389
s: Qian Qian, E-mail, ; Fax, 0571-63370389; Li Zhu, E-mail, ; Fax, 0571-63370389; Dali Zeng, E-mail, ; Fax, 0571-63370389
