The chloroplast serves as the primary site of photosynthesis, and its development plays a crucial role in regulating plant growth and morphogenesis. The PentatricopeptideRepeat Sequence (PPR) proteins constitute a vast protein family that function in the post modification of RNA within plant organelles. In this study, we characterized a rice pale green leaves (pgl3) mutant. The chlorophyll content of pgl3 at the seedling stage was significantly reduced compared to the wild type (WT). Transmission electron microscopy (TEM) and quantitative PCR analysis revealed that the pgl3 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 single base deletion occurred in the coding region of Os03g0136700 in pgl3. By employing CRISPR/Cas9 mediated gene editing, two homozygous cr-pgl3 mutant were generated and exhibited similar phenotype to pgl3, thereby confirming that Os03g0136700was responsible for pgl3 and subsequently designating it as PGL3. PGL3belong to the DYW-type PPR protein family and is localized in chloroplasts. Moreover, we showed that RNA editing efficiency of rps8-182 and rpoC2-4106, and splicing of ycf3-1 are significantly decreased in pgl3 mutants compared to WT. Collectively, these result indicate that PGL3 plays a crucial role in the process of chloroplast development via regulating the editing and splicing of chloroplast genes in rice.
Research Article
Rice PALE GREEN LEAVES, encoding a DYW-type pentatricopeptide repeat protein, is involved in chloroplast RNA editing and splicing, and regulate chloroplast development
https://doi.org/10.21203/rs.3.rs-3800019/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 26 Mar, 2024
You are reading this latest preprint version
Oryza sativa
chloroplast development
pentatricopeptide repeat (PPR) protein
RNA editing
RNA splicing
The chloroplasts serve as the primary photosynthetic organelles and play a pivotal role in facilitating plant growth and development(Ruuska et al. 2004). The formation of mature chloroplasts from proplastids requires coordinated actions between the plastid and nuclear genomes(Yu et al. 2014). This intricate process involves two RNA polymerases, nucleo-coded polymerase (NEP) and plastid-encoded polymerase (PEP), which can be further divided into three stages(Jarvis and Lopez-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 transcription of genes involved in photosynthesis such as psbA, psbD, and rbcL(Lerbs-Mache 2011). The generation of mature mRNA for chloroplast genes undergoes multiple post-transcriptional processes, encompassing 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, have 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 have 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).
Accumulating evidence has revealed that PPR proteins are involved in chloroplast formation and development. 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 in RNA editing or splicing of specific chloroplast genes. Consequently, this led to abnormalities in chloroplast development and defects in photosynthetic function(Lv et al. 2020; Lin et al. 2015; Lv et al. 2022; Wang et al. 2018; Huang et al. 2020; Wang et al. 2022; Tang et al. 2017; Wang et al. 2016). Another PPR family gene mutants exhibited white striped leaves during early leaf development, such as rice white striped leaf mutants wsl, wsl4 and ylws which were characterized by reduced chlorophyll content and abnormalities in chloroplast structure (Tan et al. 2014; Wang et al. 2017; Lan et al. 2023). Additionally, some 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, involved in chloroplast development are temperature-regulated, and 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 elusive.
In this study, we characterize a pale green leaf (pgl3) mutant showing decreased chlorophyll content and stunted growth. Genetic analysis indicated that PGL3 encodes a DYW-type PPR protein, which localizes within chloroplasts. Mechanism analysis demonstrate that PGL3 function in regulating the RNA editing at rps8-182 and rpoC2-4106, as well as splicing of ycf3-1, in the chloroplast genome.
2.1 Plant materials and growing conditions
The pgl3 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 pgl3 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.
2.2 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-hour 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 spectotometer (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 pgl3 mutant 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.
2.3 Cloning of the PGL3 gene and generation of pgl mutant
plg3 was crossed with ZH11, and developed the F2 generation population. After approximately 6 weeks of transplanting, 30 plants resembling ZH11 and plg3 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 15G. The Mutmap analysis was using 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 PGL3, we designed two targeted sequences within the exon region of PGL3. The target site sequences were cloned into a single guideRNA 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 in 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.
2.4 The subcellular localization analysis of PGL3.
In order to determine the subcellular localization of the PGL3 protein, we cloned the full-length cDNA (without a stop codon) of PGL3 into pHBT-AvrRpm1-GFP vector driven by a 35S promoter. Subsequently, we transformed the 35S::PGL3-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 in Table S2.
2.5 RNA extraction and Quantitative real-time 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 (qRT-PCR) analysis was carried out a LightCycler 96 Instrument (Roche, Germany) utilizing the TransStart Top Green qPCR SuperMix kit (TransTstsrt, China). The primer pairs used for qRT- PCR are listed in Tables S2.
2.6 Analysis of chloroplast RNA editing /splicing sites.
The primers were designed based on the reported chloroplast RNA editing sites. Specific products of ZH11 and pgl3 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 PGL3 in RNA splicing, we amplified chloroplast genes harboring at least one intron by employing primers specifically designed flanking the intronic region. Following this, RT-PCR analysis was conducted to discern any differential banding patterns between WT and pgl3 samples. The primer pairs used for RNA editing/ splicing analysis are listed in Tables S2.
3.1 The pgl3 mutant displays pale green leaves and stunted growth.
The pgl3 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 reached maturity, the chlorophyll content gradually increased and the leaves regained their green color (Fig. S1). Additionally, pgl3 plants exhibited delayed growth and development, reduced tillering capacity, lower seed setting rate, and incomplete grain filling resulting in decreased 1000-grain weight (Fig. 1A, D-G, S1A). The growth of pgl3 seedlings was significantly inhibited under conditions of insufficient light or unfavorable environmental factors, resulting in a failure to recover leaf color and ultimately leading to the mortality of the seedlings.
Due to the pale green color of pgl3 leaf and the decrease in chlorophyll content, we hypothesized that there might be a disruption in the structure of chloroplasts. To investigate this, we employed transmission electron microscopy (TEM) to examine the ultrastructure of chloroplasts. The WT leaf cells contained well-developed chloroplasts with regular and distinct thylakoids and stacked grana (Fig. 1H, I). In contrast, the pgl3 leaf cells contained indistinct thylakoids and less abundant grana stacks. (Fig. 1E, G). These results demonstrated that the lower chlorophyll content and pale green leaf phenotype of the pgl3 mutant is caused by abnormal chloroplast development.
3.2 Mapping of the PGL3 gene
To map the PGL3 locus, we crossed pgl3 mutant with ZH11. All heterozygous F1 plants exhibits normal green phenotype and segregation occurred in F2 plants. A total of 245 F2 generation plants were obtained, comprising 192 normal plants and 53 pale green plants with delayed development. The segregation ratio of approximated 3:1 (χ2 = 1.48 < χ2 0.05 = 3.84) suggests that a single gene recessive mutation resulted in the pgl3 phenotype. A notable mutation peaks within the 1.5-3M interval on chromosome 3 was unveiled by the Mutmap analysis (Fig. 2A). Four candidate SNPs were found with the SNP index. To identify the best candidate SNP, we checked all the four SNPs in 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 2016949 position (Table S1). Genomic DNA sequence analysis of WT and pgl3 revealed that there was a single nucleotide deletion of 1374G in the CDS of Os03g0136700, leading to a frameshift mutation. Thus, we speculated that Os03g0136700 may be the potential candidate gene for the pale green leaf phenotype. To prove it, three individual plants displaying the pgl3 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 pgl3 showed that all alleles were the same as that of the pgl3 mutant at the 1374G in the CDS of Os03g0136700, confirming the candidate SNP (Fig. 2B).
To further verify that the mutation of Os03g0136700 is responsible for the pale green leaf phenotype in pgl3, the CRISPR/Cas9 system was used to knock out Os03g0136700 in wild type (ZH11). Two distinct homozygous mutant variants, cr-pgl3-1 and cr-pgl3-2, were successfully generated, exhibiting a pale green leaf phenotype with decreased chlorophyll content (Fig. 2C-F). These results suggest that the phenotype of pgl3 is caused by a mutation in the Os03g0136700 gene, which is designated as PGL3.
3.3 The PGL3 gene encodes a DYW - type PPR protein belonging to the PLS subfamily
The sequence analysis revealed that PGL3 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 782 amino acids. A search of the plant PPR database (http://ppr.plantenergy.uwa.edu.au/) revealed that PGL3 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 PGL3 amino acid sequence identified very similar homologs in many other species. However, the functional characterization of these homologs remains largely unexplored. Phylogenetic tree analysis showed that proteins homologous to PGL3 are widely distributed across land plants and showed significant distinctions between monocotyledonous and dicotyledonous plants (Fig. 3B). These findings suggest that PGL3 encodes a novel DYW - type member of the rice PPR protein .
3.4 The expression pattern and subcellar localization of PGL3
To analysis the expression pattern of PGL3, we performed the qRT-PCR to analyze the relative expression levels of PGL3 in different tissues including roots, stems, leaves, leaf sheath, and young panicles (white) of WT rice. The results showed that PGL3 was expressed in all tissues with the highest expression in leaves(Fig. 4A), which is consistent with data from the Rice Expression Profile Database (RiceXPro) (https://ricexpro.dna.affrc.go.jp).
PGL3 was predicted to localize to the chloroplast by the Wolf website (https://www.genscript.com/wolf-psort.html). To experimentally verify this prediction, a transformation vector expressing an PGL3-GFP fusion protein was constructed and introduced into rice protoplasts. Analysis of transformed protoplasts by fluorescence microscopy showed that the green fluorescence of PGL3-GFP exclusively co-localized with chloroplast auto-fluorescence (Fig. 4B). The results showed that PGL3 was localized in the chloroplast.
3.5 PGL3 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 the possibility that the impaired chloroplasts in pgl3 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 pgl3 mutant and WT plant by qRT-PCR analysis. The expression levels of PEP-dependent genes, rbcL and rbcS, which encode the large and small subunits of ribosomes respectively, were significantly reduced in pgl3, cr-pgl3-1 and cr-pgl3-2 compared to the WT (Fig. 5A). In contrast, the expression levels of NEP-dependent genes, such as the RNA polymerase subunit genes (rpoC1, rpoC2, and rpoTP), was elevated in the pgl3 mutants (Fig. 5A). Down-regulation of PEP-dependent genes and up-regulation of NEP-associated genes usually imply defects in PEP complex activity. These findings suggested that PEP complex activity was disrupted in the pgl3 mutant.
We also investigated the transcript levels of nuclear-encoded genes associated with chlorophyll biosynthesis and photosynthesis. The expression levels of photosynthetic chlorophyll a/b synthesis gene Cab2R, chlorophyll synthase coding gene YGL1, and gene V2 regulating chloroplast rRNA and nucleotide metabolism were significantly reduced in pgl3 mutants compared to the WT (Fig. 5B). Furthermore, the expression levels of HEMA1 and CAO1 were significantly lower in cr-pgl3-1 and cr-pgl3-2 than those observed in the WT (Fig. 5B). Overall, the observations indicated that the pgl3 mutation affects the transcriptional expressions of genes associated with not only photosynthesis but also the early chloroplast development.
3.6 PGL3 regulates the chloroplast RNA editing and splicing.
Many characterized PPR proteins participate in chloroplast RNA editing and splicing. To address whether PGL3 influences RNA editing, we sequenced the transcripts containing 17 chloroplast editing sites in pgl3 and WT plants (Table S2). The editing of nucleotide 182 in the rps8 gene (rps8-182) and 4106 in the rpoC2 (rpoC2-4106) was strongly reduced in pgl3 plants (Table 1). In wild-type plants, 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 pgl3. The editing efficiency at these two sites in both CRISPR plants is comparable to that of pgl3, ranging from 20–40% (Table 1). The editing of both rps8-182 and rpoC2-4106 all generate a Ser-to-Leu amino acid change in the RPS8 and RPOC2 protein. These conserved sites are critical for protein function, and a decrease in editing efficiency could have implications for chloroplast development and function.
Next, we verified whether PGL3 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. The splicing events of chloroplast genes with introns were assessed in the pgl3 and the WT using reverse - transcription PCR (RT-PCR) with primers flanking the introns. The spliced RNA of ycf3-1 was significantly decreased in the pgl3 mutant compared to WT plants (Fig. 6). Similarly, spliced transcripts of ycf3-1 in both CRISPR plants were significantly lower than that of the WT. Taken together, these results indicate that PGL3 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.
4.1 The DYW type PPR protein PGL3 plays a pivotal role in the development of chloroplasts.
The pgl3 mutant exhibited a pale green leaves with delayed growth and development, as well as reduced tillering. The Chlorophyll accumulation and chloroplast ultrastructure of pgl3 plants were defective at seeding stage (Fig. 1). Positional cloning and subcellular localization showed that PGL3 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. across the entire genome, revealing a total of 491 members, with the majority being intronless. The sequence analysis revealed that the PGL3 gene is composed of 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 have been characterized. The available literature suggests that mutations in some chloroplast- localized PPR members are associated with impairments in chloroplast development or function, leading to 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; Lin et al. 2015; Huang et al. 2020; Lv et al. 2022; Wang et al. 2018; Gothandam et al. 2005; Tang et al. 2017; Wang et al. 2016). These mutations result in aberrant chloroplast development and thylakoid membrane structure that significantly impairs photosynthetic function. Differ from these mutant, the pgl3 mutant gradually reverts to wild-type leaf color as plants grow under optimal conditions of light, temperature, and water availability (Fig. S1). Chloroplast development in pgl3 seedlings exhibited aberrations with incomplete formation of thylakoid structures; however, complete degradation was not observed. (Fig. 1D-G). Some PPR mutant seedings 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 pgl3 seedlings exhibit pale green leaves and stunted growth even under normal temperatures (Fig. 1A, B). There are also reports indicating that the mutation of PPR family genes exhibits a phenotypic similarity to pgl3. 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.
4.2 Repression of PGL3 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 and 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 genes encoding large/small subunits of ribosomes (rbcL and rbcS) in pgl3 seedings compared to the WT (Fig. 5A). Conversely, there was a notable increase in NEP-dependent RNA polymerase genes (rpoC1, rpoC2 and rpoTP) in pgl3 seedings (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 pgl3 (Fig. 5B). 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 mutant, down-regulation of PEP-dependent plastid gene expression was evident, accompanied by very low accumulation levels of plastid rRNA and translation products (Tan et al. 2014). Additionally, abnormal transcript accumulation and reduced translation products were observed due to chloroplast transcript rpl2 splicing defects in the mutant (Tan et al. 2014). These findings suggest that maintaining optimal functionality of PGL3 is crucial for both chloroplast development and photosynthetic function.
4.3 PGL3 is involved in editing and splicing of chloroplast RNA
PPR proteins have the ability to interact with RNA in organelles and play a role in various post-transcriptional processes, including editing, splicing, stabilization, 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. 2014). 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 chloroplast-localized DYW-type PPR proteins 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 PGL3 governs 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 the plastosomal ribosomal protein, which plays an indispensable role in the 30S subunit of the plastid ribosome. The rpoC2 gene is responsible for encoding RNA polymerase subunits and actively participates in transcription of genes located on plasmids. It is noteworthy that there is no significant difference in the transcriptional level of rps8 between WT and pgl3, while the expression level of rpoC2 in pgl3 is significantly higher than that in WT (Fig. 5A). This observation suggests that PGL3 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 PGL3 as it contributes to the process of intron splicing within the chloroplast gene ycf3-1 (Fig. 6). 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; thereby playing a crucial role during early stages chloroplast development (Wang et al. 2018). Furthermore, OsPPR6 participates not only in transcript editing specifically at the ndhB-737 site but also governs splicing of ycf introns (Tang et al. 2017).
In conclusion, this study has identified a novel DYW - type PPR protein, PGL3, which plays a crucial role in early chloroplast development. PGL3 exhibits regulatory functions in transcription editing of rps8-182 and ropC2-4106, splicing of ycf3-1, as well as modulation of transcription levels of genes associated with chloroplast development and photosynthesis.
Acknowledgments
We thank Prof. Chunming Wang for the assistance in Mutmap data analysis.
Funding
This study was supported by the National Natural Science Foundation of China (32171989 and U20A2025), Heilongjiang Key Research and Development Program (2022ZX02B03).
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 draf. 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.
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.
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