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. 2024 Mar 26;44(4):29. doi: 10.1007/s11032-024-01468-7

OsPGL3A encodes a DYW-type pentatricopeptide repeat protein involved in chloroplast RNA processing and regulated chloroplast development

Min Xu 1,2, Xinying Zhang 3, Jinzhe Cao 4, Jiali Liu 4, Yiyuan He 1,2, Qingjie Guan 4, Xiaojie Tian 1, Jiaqi Tang 1, Xiufeng Li 1, Deyong Ren 5, Qingyun Bu 1,✉, Zhenyu Wang 1,✉
PMCID: PMC10965880  PMID: 38549701

Abstract

The chloroplast serves as the primary site of photosynthesis, and its development plays a crucial role in regulating plant growth and morphogenesis. The Pentatricopeptide Repeat Sequence (PPR) proteins constitute a vast protein family that function in the post-transcriptional modification of RNA within plant organelles. In this study, we characterized mutant of rice with pale green leaves (pgl3a). The chlorophyll content of pgl3a at the seedling stage was significantly reduced compared to the wild type (WT). Transmission electron microscopy (TEM) and quantitative PCR analysis revealed that pgl3a exhibited aberrant chloroplast development compared to the wild type (WT), accompanied by significant alterations in gene expression levels associated with chloroplast development and photosynthesis. The Mutmap analysis revealed that a single base deletionin the coding region of Os03g0136700 in pgl3a. By employing CRISPR/Cas9 mediated gene editing, two homozygous cr-pgl3a mutants were generated and exhibited a similar phenotype to pgl3a, thereby confirming that Os03g0136700 was responsible for pgl3a. Consequently, it was designated as OsPGL3A. OsPGL3A belongs to the DYW-type PPR protein family and is localized in chloroplasts. Furthermore, we demonstrated that the RNA editing efficiency of rps8-182 and rpoC2-4106, and the splicing efficiency of ycf3-1 were significantly decreased in pgl3a mutants compared to WT. Collectively, these results indicate that OsPGL3A plays a crucial role in chloroplast development by regulating the editing and splicing of chloroplast genes in rice.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11032-024-01468-7.

Keywords: Oryza sativa, Chloroplast development, Pentatricopeptide repeat (PPR) protein, RNA editing, RNA splicing

Introduction

The chloroplast, as the primary photosynthetic organelle, plays a pivotal role in facilitating plant growth and development (Ruuska et al. 2004). Coordinated actions between the plastid and nuclear genomes are required for proplastids to develop into mature chloroplasts (Yu et al. 2014). This intricate process involves two RNA polymerases, the nuclear-encoded polymerase (NEP) and the plastid-encoded polymerase (PEP), which can be further divided into three stages (Jarvis and López-Juez 2013; Kusumi and Iba 2014). Initially, plastid DNA is synthesized and replicated by DNA polymerase. Subsequently, NEP translocates into the plastids to transcribe housekeeping genes such as rpoC1, rpoC2, rpoA, and rpoB that encode PEP core subunits responsible for regulating early-stage plastid development. Finally, PEP is assembled within the chloroplasts to facilitate the transcription of genes involved in photosynthesis, such as psbA, psbD, and rbcL (Lerbs-Mache 2011). The generation of mature mRNA for chloroplast genes undergoes various post-transcriptional processes such as intron splicing, editing, processing, trimming, and stabilization (Shikanai and Fujii 2013).

The Pentatricopeptide repeat (PPR) family, one of the largest gene families in higher plants, has been identified in numerous terrestrial plant species (O'Toole et al. 2008). The PPR family proteins are characterized by tandem structures consisting of multiple repeat motifs, each composed of 30 to 40 amino acid residues (Manna 2015). Based on the motif structure, members of the PPR family can be classified into two distinct subfamilies: P and PLS. The PLS subfamily of PPR proteins can be further classified into E1, E2, and DYW subtypes based on the presence of additional domains downstream of the PPR motifs, which may occur individually or in combination (Shen et al. 2016). PPR proteins play multiple roles in various post-transcriptional processes, including RNA editing, splicing, stabilization, cleavage, degradation, and translation of mitochondrial and chloroplast (Hayes et al. 2015; Okuda et al. 2007; Lurin et al. 2004).

Evidence has revealed that PPR proteins are involved in formation and development of chloroplast. Some mutants of PPR proteins exhibit phenotypic similarities to chloroplast dysfunction (Wang et al. 2021). The most severe phenotype observed in these mutants was seedling chlorosis, followed by subsequent mortality. The functional loss of rice PPR proteins, such as OsSLC1, ASL3, WAL3, OsSLA4, OsPPR16, SSA1, OsPPR6 and OspTAC2 resulted in alterations to RNA editing or splicing of specific chloroplast genes. Consequently, this led to abnormalities in the development of chloroplasts and defects in photosynthetic function (Lv et al. 2020, 2022; Lin et al. 2015; Wang et al. 2018, 2022, 2016; Huang et al. 2020; Tang et al. 2017). Other mutants of the PPR family genes exhibited white striped leaves during early leaf development, such as rice white striped leaf mutants wsl, wsl4 and ylws. These mutants were characterized by reduced chlorophyll content and abnormalities in chloroplast structure (Tan et al. 2014; Wang et al. 2017; Lan et al. 2023). Additionally, certain PPR family gene mutations, such as pgl12, mpr25, ysa, and ospgl1 mutants, display a light green leaf phenotype during the seedling stage. However, this yellowish-green color gradually reverts to normal green as the plant matures (Chen et al. 2019; Toda et al. 2012; Su et al. 2012; Xiao et al. 2018). Some PPR genes, such as CDE4, TCD10, OsATP4 and DUA1, which are involved in chloroplast development are temperature-regulated. The corresponding mutants exhibit reduced chlorophyll content and abnormal chloroplast development under low-temperature conditions while displaying normal development under high-temperature conditions (Liu et al. 2021; Wu et al. 2016; Zhang et al. 2020; Cui et al. 2019). Chloroplast biogenesis is a complex process governed by intricate molecular mechanisms that have yet to be fully elucidated. Although numerous studies have demonstrated the direct or indirect impact of PPR proteins on chloroplast biogenesis and development, the precise mechanism underlying PPR-mediated regulation of chloroplast development and function remains unclear.

In this study, we characterized a rice pale green leaf (pgl3a) mutant that showed decreased chlorophyll content and stunted growth. Genetic analysis indicated that OsPGL3A encodes a DYW-type PPR protein, which localizes within chloroplasts. Mechanism analysis demonstrates that OsPGL3A functions in regulating the RNA editing at rps8-182 and rpoC2-4106, as well as the splicing of ycf3-1, in the chloroplast genome.

Materials and methods

Plant materials and growing conditions

The pgl3a was identified from a mutant library derived from Zhonghua 11 (ZH11), a japonica rice variety. The causal gene was mapped using the F2 population between pgl3a and ZH11. All genetic materials were cultivated in farms at the Center for Agricultural Technology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences during the normal growing season.

Detection of chlorophyll content and chloroplast structure

The chlorophyll content was quantified using a spectrophotometric method. In brief, fresh leaves were harvested from seedlings, finely chopped, weighed, and then subjected to 48-h dark extraction with equal volume (w/v) of 95% ethanol. The resulting extract was centrifuged and the supernatant was analyzed for absorbance at wavelengths of 665 nm, 649 nm, and 470 nm using a Thermo Fisher Scientific BIOMATE3 spectrometer (Wu et al. 2007). Finally, the cytochrome content was calculated.

The chloroplast microstructure of the leaves at the trileaf stage in both wild type and pgl3a was examined. The leaf samples were fixed with 2.5% glutaraldehyde and 1% OsO4, dehydrated using a series of ethanol concentrations, and embedded in Spurr resin. The sections were stained and observed under an H-7650 (Hitachi, Japan) transmission electron microscope.

Cloning of the OsPGL3A gene and generation of OsPGL3A deficient mutant

Osplg3 was crossed with ZH11, and developed the F2 generation population. After approximately 6 weeks of transplanting, 30 plants resembling ZH11 or osplg3 were chosen and pooled as two bulks, respectively. Genomic DNA of two bulks and ZH11 leaves were extracted and sent to BGI for resequencing at a depth of 15G. The Mutmap analysis used the International Rice Genome Sequencing Project (IRGSP) database as a reference for the rice genome. Subsequently, PCR sequencing was conducted to validate the obtained outcomes by employing primers designed on both flanks of the potential mutation sites.

In order to generate a CRISPR-mediated mutant with dysfunctional OsPGL3A, we designed two targeted sequences within the exon region of OsPGL3A. The target site sequences were cloned into a single guide RNA expression vector pYLgRNA and subsequently inserted into the target vector pYLCRISPR/Cas9-MH. The CRISPR/Cas9 vector system for simultaneous targeting of multiple plant gene loci was provided by Yaoguang Liu (South China Agricultural University), and its application method was described by Ma et al. (Ma et al. 2015). Rice cultivar ZH11 transformation was achieved through Bacillus tumefaciens-mediated delivery. All primers used for vector construction and detection are listed in Table S2.

The subcellular localization analysis of OsPGL3A

In order to determine the subcellular localization of the OsPGL3A protein, we cloned the full-length cDNA (without a stop codon) of OsPGL3A into a pHBT-AvrRpm1-GFP vector driven by a 35S promoter. Subsequently, we transformed the 35S::OsPGL3A-GFP vector into rice protoplasts using PEG induction followed by overnight culture at 28℃ (Chen et al. 2006). The green fluorescence signal was visualized using a Leica Microsystems CMS GmbH Ernst-Leitz-Str (Germany), while red fluorescence indicated chloroplast autofluorescence. The primers used for vector construction are in Table S2.

RNA extraction and Real-time fluorescent quantitative PCR

The total RNA extraction from rice was conducted following the manufacturer's instructions for extraction and purification using the RNA preparation pure plant kit (Tiangen, China). The RNA samples were reverse-transcribed using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) from TaKaRa. Real-time fluorescent quantitative PCR (RT-qPCR) analysis was carried out in a LightCycler 96 Instrument (Roche, Germany) utilizing the TransStart Top Green qPCR SuperMix kit (TransTstsrt, China). The primer pairs used for RT-qPCR are listed in Table S2.

Analysis of chloroplast RNA editing /splicing sites and efficiency

The primers were designed based on the reported chloroplast RNA editing sites. Specific products of ZH11 and pgl3a mutants were amplified, sequenced, and compared using BioEdit software to determine C-T changes. Editing efficiency was assessed by sequencing at least 10 positive PCR clones individually.

To explore the potential role of OsPGL3A in RNA splicing, we amplified chloroplast genes harboring at least one intron by employing primers specifically designed to flank the intronic region. The splicing differential and efficiency of chloroplast RNA introns in ZH11 and pgl3a mutants were analyzed by RT-PCR and RT-qPCR. The primers used for splicing efficiency analysis were obtained from a previous study (Lv et al. 2020).

Results

The pgl3a mutant displays pale green leaves and stunted growth

The pgl3a mutant, exhibiting a pale green leaf color, was identified from a pool of mutants generated using the rice cultivar ZH11 as the background. The mutant significantly reduced the chlorophyll content compared to the WT at the seedling stage (Fig. 1A-C). However, as the plants matured, the chlorophyll content gradually increased, and the leaves regained their green color (Fig. S1). Furthermore, pgl3a plants exhibited delayed growth and development, reduced tillering capacity, lower seed setting rate, and incomplete grain filling, resulting in a decreas in 1000-grain weight (Fig. 1A, D-G, S1A). Insufficient light or unfavorable environmental factors significantly inhibited the growth of pgl3a seedlings, resulting in a failure to recover leaf color and ultimately leading to the mortality of the seedlings. Compared to the wild type, the pgl3a also exhibited a significantly reduced root system, shorter root length, and slender grains (Fig. S2).

Fig. 1.

Fig. 1

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Phenotypic characterization of mutant. (A, B) Plant and leaves of ZH11 and the pgl3a at seedling stage. Scale bars, 8 cm in (A) and 3 cm in (B). (C) chlorophyll contents in leaves of ZH11 and the pgl3a at seedling stage. (D-G) Comparison of major agronomic traits affecting plant yield between ZH11 and pgl3a. Plant height (cm) (D), tiller number (E), seed setting rate (%) (F) and 1000-grain weight (g) (G) **, P < 0.01 (Student’s t-test); error bars represent SD (n = 3 in C, n = 10 in D-G). (H–K) The chloroplast ultrastructure of seedlings of ZH11 (H, I) and pgl3a (J, K). I and K are enlarged view of the red rectangle in H and J, respectively. Scale bars, 2 μm in

Due to the pale green color of pgl3a in the leaf and the decrease in chlorophyll content, we hypothesized that there might be a disruption in chloroplasts structure. To investigate this, we employed transmission electron microscopy (TEM) for examining the ultrastructure of chloroplasts. The leaf cells of ZH11 contained well-developed chloroplasts with regular and distinct thylakoids, as well as stacked grana (Fig. 1H, I). In contrast, the pgl3a leaf cells contained indistinct thylakoids and fewer grana stacks (Fig. 1E, G). These results demonstrated that the lower chlorophyll content and pale green leaf phenotype of the pgl3a mutant were caused by abnormal chloroplast development.

Mapping of the OsPGL3A gene

To map the OsPGL3A locus, we crossed pgl3a with ZH11. All heterozygous F1 plants exhibited normal green phenotypes, and segregation occured in F2 plants. A total of 245 plants were obtained from F2 generation, comprising 192 normal plants and 53 pale green plants with delayed development. The segregation ratio of approximately 3:1 (χ2 = 1.48 < χ2 0.05 = 3.84) suggested that the pgl3a phenotype resulted from a single gene recessive mutation. The Mutmap analysis revealed a notable mutation peak within the 1.5-3 M interval on chromosome 3 (Fig. 2A). Four candidate SNPs were were identified using the SNP index. To identify the best candidate SNP, we cross-referenced all four SNPs with the International Rice Genome Sequencing Project (IRGSP) database (https://rgp.dna.affrc.go.jp/E/IRGSP/index.html). However, no SNP was identified in the coding region except Os03g0136700 on chromosome 3 at position 2,016,949 position (Table S1). Genomic DNA sequence analysis of ZH11 and pgl3a revealed a single nucleotide deletion at position 1374G in the CDS of Os03g0136700, resulting in a frameshift mutation. Therefore, we speculate that Os03g0136700 may be the potential candidate gene for the pale green leaf phenotype. To prove it, three individual plants displaying the pgl3a mutant phenotype were selected from F2 populations, and PCR was performed to confirm the point mutation. The sequence alignment of three individuals, together with WT and pgl3a, showed that all alleles were the same as that of the pgl3a mutant at the 1374G in the CDS of Os03g0136700, confirming the candidate SNP (Fig. 2B).

Fig. 2.

Fig. 2

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Mutmap-based mapping of the OsPGL3A gene. (A) Mutmap analysis unveiled a notable mutation peaks within the 1.5- 3 M interval on chromosome 3. (B) Sequencing analysis of the mutation site of Os03g0136700 from ZH11 together with pgl3a and three F2 individual plants. (C) Selection of knockout target sites and sequence alignment between WT and transgenic plants. (D-F) The chlorophyll content (D) and phenotypes (E, F) of ZH11, cr-pgl3a-1 and cr-pgl3a-2 at the seedling stage. Scale bars, 8 cm in (E, F). **, P < 0.01 (Student’s t-test); error bars represent SD (n = 3)

To further comfirm that the mutation of Os03g0136700 is responsible for the pale green leaf phenotype in pgl3a, we utilized the CRISPR/Cas9 system to knock out Os03g0136700 in ZH11. Two distinct homozygous mutant variants, cr-pgl3a-1 and cr-pgl3a-2, were successfully generated. These variants exhibited a pale green leaf phenotype with decreased chlorophyll content (Fig. 2C-F). These results suggest that the phenotype of pgl3a is caused by a mutation in the Os03g0136700 gene, which is designated as OsPGL3A.

The OsPGL3A gene encodes a DYW-type PPR protein belonging to the PLS subfamily

The sequence analysis revealed that OsPGL3A possessed a single exon without introns (Fig. 2C), with a full-length coding DNA sequence (CDS) spanning 2346 bases and encoding a protein consisting of 781 amino acids. A search of the plant PPR database (http://ppr.plantenergy.uwa.edu.au/) revealed that OsPGL3A contained a total of 15 PPR motifs with varying lengths, two E motifs (E1 and E2), and a DYW domain at the C-terminal region, which belongs to the DYW group of the PLS subfamily (Fig. 3A). Blastp searches of the OsPGL3A amino acid sequence identified highly similar homologs in many other species; however, the functional characterization of these homologs remains largely unexplored. Phylogenetic tree analysis revealed that proteins homologous to OsPGL3A are widely distributed among land plants and exhibit significant distinctions between monocotyledonous and dicotyledonous plants (Fig. 3B). These findings suggest that OsPGL3A encodes a novel member of the DYW-type rice PPR protein.

Fig. 3.

Fig. 3

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Structure and phylogenetic analysis of OsPGL3A. (A) Structure prediction of OsPGL3A. OsPGL3A is composed of 15 PPR motifs with varying lengths (P, L, S), 2 E motif (E1, E2) and an atypical DYW-like motif. (B) phylogenetic tree of OsPGL3A homologs. All of the protein sequences were downloaded from NCBI

The expression pattern and subcellar localization of OsPGL3A

To analyze the expression pattern of OsPGL3A, we performed the RT-qPCR to analyze the relative expression levels of OsPGL3A in different tissues, including roots, stems, leaves, leaf sheaths, and young panicles (white) of WT rice. The results showed that OsPGL3A was expressed in all tissues, with the highest expression observed in leaves (Fig. 4A), which is consistent with the data from the Rice Expression Profile Database (RiceXPro, https://ricexpro.dna.affrc.go.jp).

Fig. 4.

Fig. 4

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Expression profile of OsPGL3A in different tissues and subcellular localization of OsPGL3A in rice protoplasts. (A) Expression analysis of OsPGL3A in roots (R), stems (S), sheaths (SH), leaves (L), young panicles (white, YP). (B) Free GFP and OsPGL3A-GFP fusion protein was expressed in rice protoplasts. Scale bars, 2 μm

According to the Wolf website (https://www.genscript.com/wolf-psort.html), OsPGL3A was predicted to localize in the chloroplast. To experimentally verify this prediction, a transformation vector expressing an OsPGL3A-GFP fusion protein was constructed and introduced into rice protoplasts. Analysis of transformed protoplasts by fluorescence microscopy showed that the green fluorescence of OsPGL3A-GFP exclusively co-localized with chloroplast auto-fluorescence (Fig. 4B). The results indicated that OsPGL3A was localized in the chloroplast.

OsPGL3A modulates the expression of genes related to chloroplast development

The development of chloroplasts is regulated by the transcription of plastid-encoded genes, which are controlled by RNA polymerase complexes NEP and PEP. To assess whether the impaired chloroplasts in the pgl3a mutant may be reflected at the level of related gene expression, we examined the transcription levels of genes associated with photosynthesis and chloroplast development both in the pgl3a mutants and ZH11 plant using RT-qPCR analysis. The expression levels of the PEP-dependent gene, rbcL, which encode the large subunits of ribosomes, was significantly reduced in pgl3a, cr-pgl3a-1 and cr-pgl3a-2 compared to the ZH11 (Fig. 5A). In contrast, the expression levels of NEP-dependent genes, such as the RNA polymerase subunit genes (rpoC1, rpoC2, and rpoTP), were elevated in the pgl3a mutants (Fig. 5A). Down-regulation of PEP-dependent genes and up-regulation of NEP-associated genes usually imply defects in PEP complex activity (Pfalz et al. 2006; Tan et al. 2014; Wang et al. 2016). These findings suggested that PEP complex activity was disrupted in the pgl3a mutant.

Fig. 5.

Fig. 5

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Transcript analysis of genes associated with chloroplast rRNAs and chloroplast development- and photosynthesisrelated genes in WT and pgl3a mutants. (A) The relative expression levels of PEP-dependent genes and NEP-dependent genes in WT and pgl3a mutants. (B) The relative expression levels of chloroplast development-and photosynthesis- associative genes in WT and pgl3a mutants. Means ± SD of three independent replicates (Student’s t-test, ** P < 0.01). OsActin was selected as control

We also investigated the transcript levels of nuclear-encoded genes associated with chlorophyll biosynthesis and photosynthetic processes. The expression levels of the photosynthetic chlorophyll a/b synthesis gene Cab2R, the chlorophyll synthase coding gene YGL1, and the gene V2 regulating chloroplast rRNA and nucleotide metabolism were significantly reduced in pgl3a mutants compared to ZH11 (Fig. 5B). Furthermore, the expression levels of HEMA1 and CAO1 were significantly lower in cr-pgl3a-1 and cr-pgl3a-2 than those observed in the ZH11 (Fig. 5B). Overall, these observations indicated that the pgl3a mutation affects not only the transcriptional expressions of genes associated with photosynthesis but also early chloroplast development.

OsPGL3A regulates the chloroplast RNA editing and splicing

Many PPR proteins are involved in chloroplast RNA editing and splicing. To investigate the impact of OsPGL3A on RNA editing, we sequenced transcripts containing 17 chloroplast editing sites in pgl3a mutants and ZH11 (Table S2). The editing of nucleotide 182 in the rps8 gene (rps8-182) and 4106 in the rpoC2 gene (rpoC2-4106) was strongly reduced in pgl3a plants (Table 1). In ZH11, the editing efficiencies of rps8-182 and rpoC2-C4106 reached approximately 90% and 80%, respectively. However, these efficiencies dropped to only 50% and 40%, respectively, in pgl3a. The editing efficiency at both of these sites in CRISPR plants is comparable to that of pgl3a, ranging from 20 to 40% (Table 1). The editing of both rps8-182 and rpoC2-4106 results in a substitution of serine with leucine in the RPS8 and RPOC2 protein. This alteration potentially affects the stability of the plastid ribosomal protein ribosome, as well as the assembly and function of the PEP complex, thereby influencing chloroplast development and function.

Table 1.

Analysis of chloroplast RNA editing efficiency (C to U) in the wild type and pgl3a mutants

Gene Editing position Edited codon Amino acid change WT Editing efficiency(U/C%)
pgl3a cr-pgl3a-1 Cr-pgl3a-2
rps8 C182 UCA S → L 90 50 30 40
rpoC2 C4106 UCA S → L 80 50 20 30
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Next, we verified whether OsPGL3A affects the RNA splicing of chloroplast-encoded transcripts. The rice chloroplast genome has 18 introns, comprising one group-I intron and 17 group-II introns. The splicing events of chloroplast genes with introns were assessed in the pgl3a mutants and the ZH11 using semi-quantitative reverse-transcription PCR (RT‐PCR) with primers flanking the introns. The level of spliced RNA for ycf3-1 was significantly decreased in the pgl3a mutants compared to ZH11 (Fig. 6A). RT-qPCR analysis furthere revealed a significantly reduced splicing efficiency of ycf3-1 in the pgl3a mutants compared to ZH11 (Fig. 6B). YCF3 is one of the crucial factors involved in the assembly of PSI sub-units (Nellaepalli et al 2018). The abnormal splicing of ycf3 transcripts negatively affects the assembly and function of the PSI complex. Taken together, these results indicate that OsPGL3A is involved not only in the editing of rps8-182 and rpoC2-4106 but also in the regulation of ycf3-1 intron splicing, thereby exerting control over chloroplast development and function.

Fig. 6.

Fig. 6

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OsPGL3A participates in splicing of ycf3-1. (A) Semi-quantitative RT-PCR analyses of rice chloroplast intron-containing transcripts in WT and pgl3a munuts. U and S indicate unspliced and spliced transcripts, espectively. (B) Splicing efficiency analysis of chloroplast intron-containing transcripts in the wild type and pgl3a mutants. Histogram showing the log2 ratio of spliced to unspliced transcripts in the pgl3a mutants compared with the corresponding values in the wild type. Three biological replicates were performed. Error bars represent SD (n = 3)

Discussion

The DYW-type PPR protein OsPGL3A plays a pivotal role in the development of chloroplasts

The pgl3a mutant exhibited pale green leaves, delayed growth and development, and reduced tillering. The chlorophyll accumulation and chloroplast ultrastructure of pgl3a plants were defective at the seedling stage (Fig. 1). Positional cloning and subcellular localization revealed that OsPGL3A encodes a DYW‐type PPR protein localized in chloroplasts (Fig. 2, 3, 4B). The rice PPR family members were comprehensively analyzed by Chen et al. (2018) across the entire genome, revealing a total of 491 members, with the majority being intronless. Sequence analysis revealed that the OsPGL3A gene comprises a single exon without any introns. (Fig. 2C).

Although the PPR family is extensively distributed across plant species, only a limited subset of PPR proteins has been characterized. The available literature suggests that mutations in some chloroplast-localized PPR members are associated with impairments in chloroplast development or function, resulting in phenotypes resembling chloroplast dysfunction. Some mutations in PPR family genes, such as OsSLC1, ASL3, OsPPR16, WAL3, OsSLA4, OsPPR1, OsPPR6, OspTAC2, and SSA1 can lead to seedling mortality in rice (Lv et al. 2020, 2022; Lin et al. 2015; Huang et al. 2020; Wang et al. 2018, 2016; Gothandam et al. 2005; Tang et al. 2017). These mutations result in aberrant chloroplast development and thylakoid membrane structure significantly impairs photosynthetic function. Some PPR mutant seedlings showed an albino phenotype under low temperature conditions. For instance, the mutants cde4, tcd10, osatp4, and dua1 displayed an albino phenotype during early leaf development, reduced chlorophyll content, and abnormal chloroplast development at 20℃; however, they exhibited a normal phenotype at 32℃ (Liu et al. 2021; Wu et al 2016; Zhang et al. 2020; Cui et al. 2019). Unlike these mutants, the pgl3a seedlings exhibit pale green leaves and stunted growth even at normal temperatures (Fig. 1A, B). Additionally, when compared to ZH11, the pgl3a also exhibited a significantly reduced root system with shorter root length, inadequate seed filling, and narrower grain width (Fig. S2). These observed phenotypes may result from insufficient photosynthesis in pgl3a, leading to inadequate nutrient accumulation for the optimal development of organs such as roots and seeds. The leaf color of pgl3a mutant gradually reverts to wild-type as plants grow under optimal conditions of light, temperature, and water availability (Fig. S1A-C). No significant difference was observed in the expression levels of OsPGL3A between the seedling and filling stages in WT (Fig. S1D). Chloroplast development in pgl3a seedlings exhibited aberrations with incomplete formation of thylakoid structures; however, complete degradation was not observed. (Fig. 1D-G). It is hypothesized that other genes with analogous functions are involved in this process. There are also reports indicating that the mutation of PPR family genes exhibits phenotypic similarity to pgl3a. For instance, the leaf color of the pgl12 mutant appears yellowish green during the seedling stage and gradually transitions to light green as the plant grows (Chen et al. 2019). The mpr25 mutant displays slow growth and reduced chlorophyll content in its early developmental stages, resulting in light green leaves (Toda et al. 2012). Similarly, ysa mutants exhibit albino leaves prior to reaching the three-leaf stage, after which they progressively turn green until attaining normal pigmentation at the six-leaf stage (Su et al. 2012). However, mechanisms underlying how these PPR family proteins regulate rice chloroplast development and function have remained elusive.

Repression of OsPGL3A function results in alterations in the expression levels of genes associated with chloroplast development and photosynthetic functionality

The formation of a functional chloroplast necessitates the coordinated expression of NEP- and PEP- transcribed genes (Swiatecka-Hagenbruch et al. 2008). The PEP complex consists of four core subunits encoded by plastid genes, as well as several proteins encoded by nuclear genes (Steiner et al. 2011; Pfannschmidt et al. 2015). The functional defects of the PEP complex in chloroplasts can result in the upregulation of NEP-associated genes and the downregulation of PEP-dependent genes (Pfalz et al. 2006; Tan et al. 2014; Wang et al. 2016). In this study, we observed a significant reduction in the expression level of PEP-dependent gene encoding large subunits of ribosomes (rbcL) in pgl3a seedings compared to ZH11 (Fig. 5A). Conversely, there was a notable increase in NEP-dependent RNA polymerase genes (rpoC1, rpoC2 and rpoTP) in pgl3a seedlings (Fig. 5A). Furthermore, a significant decrease was observed in the expression levels of genes involved in chlorophyll synthesis (Cab2R and YGL1), as well as the gene associated with chloroplast development (V2) in pgl3a (Fig. 5B). The significant decrease in the expression levels of certain genes involved in chloroplast development and photosynthesis, observed in pgl3a, could be attributed to impaired chloroplast development and compromised PSI function. This is consistent with previous reports, such as the significant reduction in transcription levels of all PEP-dependent genes observed in the seedling lethal mutant osptac2, while transcription levels of NEP-dependent genes were found to be increased (Wang et al. 2016). In the rice-striated leaf mutant wsl, there was evidence of down-regulation of PEP-dependent plastid gene expression, accompanied by significantly low levels of plastid rRNA and translation products (Tan et al. 2014). Additionally, the mutant exhibited abnormal accumulation of transcripts and reduced translation products due to defects in chloroplast transcript rpl2 splicing (Tan et al. 2014). These findings suggest that maintaining the optimal functionality of OsPGL3A is crucial for both chloroplast development and photosynthetic function.

OsPGL3A is involved in editing and splicing of chloroplast RNA

PPR proteins possess the ability to interact with RNA in organelles and play a role in various post-transcriptional processes, including editing, splicing, stabilizing, cleavage, degradation, and translation of mitochondrial, chloroplast or nuclear RNA. RNA editing involves the site-specific conversion of cytidine (C) to uridine (U) in mitochondrial or plasmid transcripts, thereby modifying the amino acid sequence corresponding to the DNA template (Okuda et al. 2007; Takenaka et al. 2010; Hammani et al. 2009). The majority of DYW-type PPR proteins have been reported to regulate RNA editing processes in plastids or mitochondria (Colcombet et al. 2013). For example, the DYW protein PPS1 localized in mitochondria regulates rice pollen development by exerting control over RNA editing at five adjacent conserved sites (Xiao et al. 2021). Similarly, OsPPR16, OsPPR6, PGL1, and DUA1 are DYW-type PPR proteins localized in chloroplast that modulate chloroplast development through transcriptional editing of RpoB545, ndhB-737, ndhD-878, and rps8-182, respectively (Huang et al. 2020; Tang et al. 2017; Xiao et al. 2018; Cui et al. 2019). Our findings also reveal that OsPGL3A governs the RNA editing of rps8-182 and ropC2-4106, leading to the conversion of serine to leucine at these two amino acid positions (Table 1). The rps8 gene encodes plastosomal ribosomal protein S8 (RPS8), which plays an indispensable role in the 30S subunit of the plastid ribosome. The rpoC2 gene is one of the plastid genes that encod PEP core subunits. The alteration of RNA editing at critical sites in rps8 or rpoC2 results in an amino acid substitution, changing a hydrophilic serine to a hydrophobic leucine. This substitution potentially affects the stability of the plastid ribosomal protein and the function of the PEP complex (Cui et al. 2019; Chen et al. 2023). The dysfunction of the PEP complex leads to aberrant transcription of plastid genes, resulting in abnormal chloroplast and plant growth. For example, in osppr16 mutants, the lack of rpoB editing results in reduced PEP activity, which restricts chloroplast biogenesis during early leaf development (Huang et al. 2020). It is noteworthy that there is no significant difference in the transcriptional level of rps8 between ZH11 and pgl3a mutants, while the expression level of rpoC2 in pgl3a mutants is significantly higher than that in WT (Fig. 5A). This observation suggests that OsPGL3A does not directly influence the transcription of these genes but rather affects their products at the post-transcriptional level.

Besides its involvement in RNA editing processes, our findings reveal an additional function for OsPGL3A as it contributes to the process of intron splicing within the chloroplast gene ycf3-1 (Fig. 6). The PSI complex is a large protein assembly located on the thylakoid membrane of chloroplasts, consisting of multiple subunits, cofactors, and pigment molecules. YCF3 is one of the crucial factors involved in assembling PSI subunits (Nellaepalli et al 2018). The abnormal splicing of ycf3 transcripts negatively affects the assembly and function of the PSI complex. The Arabidopsis PPR protein PBF2 is specifically required for the splicing of intron 1 in ycf3. Loss of PBF2 results in a seedling lethal phenotype, loss of PSI activity, and disintegration of PSI complex due to a dramatic decrease in the level of ycf3 transcripts in the pbf2 mutants (Wang et al 2020). Previous studies have reported the participation of DYW-type PPR proteins in splicing plastid group II introns. For instance, OsSLA4, a DYW-type PPR protein localized in chloroplasts, exerts regulatory control over the splicing of multiple introns, including atpF, ndhA, petB, rpl2, rpl16, rps12-2 and trnG; thus playing a crucial role during early stages of chloroplast development (Wang et al. 2018). Furthermore, OsPPR6 not only participates in transcript editing specifically at the ndhB-737 site but also governs the splicing of ycf introns (Tang et al. 2017).

In conclusion, this study has identified a novel DYW-type PPR protein, OsPGL3A, which plays a crucial role in early chloroplast development. By influencing the editing of rps8 and rpoC2, OsPGL3A may impact the stability of chloroplast ribosomal proteins as well as the function of the PEP complex, ultimately affecting chloroplast biogenesis and function. Additionally, the OsPGL3A protein regulates the splicing of the ycf3-1 intron, thereby influencing PSI assembly at the chloroplast thylakoid membrane and impacting photosynthesis. Consequently, the pgl3a mutant exhibits defective chloroplast development and impaired PSI function, resulting in a significant reduction in the expression levels of certain genes involved in chloroplast development and photosynthesis.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 717 KB) (716.9KB, pdf)

Acknowledgements

We thank Prof. Chunming Wang for the assistance in Mutmap data analysis.

Author contribution

Min Xu: performed the subcellular localization, expression analysis and the RNA editing. Xinying Zhang: performed the RNA splicing. Jinzhe Cao and Jiali Liu: analyzed the phenotypes and cloned PGL3. Deyong Ren: examined the ultrastructure of chloroplasts. Yiyuan He: performed some of the experiments and provided technical assistance. Qingjie Guan, Xiaojie Tian, Jiaqi Tang, and Xiufeng Li: helped with the discussion of the work. Zhenyu Wang: conceived and supervised the entire project, analyzed the data, and wrote the original draft. Qingyun Bu: conceived and supervised the entire project, refined preliminary manuscripts. All authors commented on previous versions of the manuscript, and all authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32171989 and U20A2025), Heilongjiang Key Research and Development Program (2022ZX02B03).

Data availability

All data are enclosed either in the main text or as supplementary materials. Other data can be requested from the corresponding author.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Min Xu and Xinying Zhang contributed equally to this work.

Contributor Information

Qingyun Bu, Email: buqingyun@iga.ac.cn.

Zhenyu Wang, Email: wangzhenyu@iga.ac.cn.

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Supplementary Materials

Supplementary file1 (PDF 717 KB) (716.9KB, pdf)

Data Availability Statement

All data are enclosed either in the main text or as supplementary materials. Other data can be requested from the corresponding author.

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