A chloroplast-targeted pentatricopeptide repeat protein PPR287 is crucial for chloroplast function and Arabidopsis development
Even though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis (http://ppr.plantenergy.uwa.edu.au).
The homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants.
These results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.
KeywordsArabidopsis thaliana Chloroplast Development PPR RNA metabolism
quantum yield of photosystem II
Green fluorescent protein
Murashige and Skoog
Reactive oxygen species
The chloroplast genome encodes approximately 120–130 proteins necessary for photosynthesis and plastid biogenesis [1, 2, 3]. However, more than 3000 nucleus-encoded proteins are transported into the chloroplast, many of which are needed for chloroplast gene expression [4, 5, 6, 7]. Chloroplast gene expression is modulated and regulated by posttranscriptional processes, such as mRNA and tRNA splicing, mRNA editing, RNA stability, and translational control, during which many RNA-binding proteins (RBPs) play essential roles [8, 9, 10, 11, 12]. In particular, most of the RBPs involved in chloroplast RNA metabolism are nucleus-encoded and are transported into chloroplasts [5, 6, 7, 13, 14].
The pentatricopeptide repeat (PPR) proteins are among the nucleus-encoded chloroplast RBPs. Notably, PPR proteins are particularly abundant in land plants [15, 16, 17]. The Arabidopsis genome encodes more than 450 PPR proteins, whereas less than 10 PPR proteins are found in humans [15, 18], suggesting their plant-specific functions. PPR proteins are divided into several subfamilies in terms of their tandem repeat motifs and additional domains. In general, the P-class PPR proteins that harbor only the PPR motifs are involved in intercistronic processing, splicing of group II introns, and RNA stabilization, whereas the PLS-class PPR proteins that contain additional C-terminal domains, such as E, E+, and DYW, are required for C to U RNA editing [17, 19, 20].
In chloroplasts of several plant species, including Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays), the functions of many PPR proteins have been determined, and most of the chloroplast PPR proteins characterized so far participate mainly in the stabilization of mRNAs. Examples include CRP1 for stabilizing the 5′ and 3′ ends of the petB-petD intergenic region in maize [21, 22], PPR10 for stabilizing the 5′ and 3′ ends of the atpI-atpH and psaJ-rpl33 intergenic regions in maize [23, 24, 25], HCF152 for stabilizing the 5′ and 3′ ends of the psbH-petB intergenic region in Arabidopsis thaliana [26, 27], MRL1 for stabilizing the rbcL 5′ end in Arabidopsis , and PGR3 for stabilizing the petL 5′ end in Arabidopsis [29, 30]. Similarly, mitochondrial PPR19 and MTSF1 are needed for stabilizing the nad1 intron 3′ end and nad4 3′ end in Arabidopsis [31, 32]. Analysis of loss-of-function mutants demonstrated that these aforementioned PPR proteins are critical for normal growth and development of plants. Although these previous studies clearly show that PPR proteins are essential for organellar functions and plant development, the cellular functions of many PPR proteins still remain to be characterized.
In this study, we determined the function of a chloroplast-targeted PPR protein possessing 10 PPR motifs (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis (http://ppr.plantenergy.uwa.edu.au) , thus designated PPR287. We show that PPR287 affects the level of chloroplast rRNAs, which is essential for chloroplast biogenesis and function as well as for the normal growth and development of Arabidopsis.
Cellular localization and expression patterns of PPR287
PPR287 is essential for normal growth and development of Arabidopsis
All CS814021 mutant seeds that were germinated on MS medium containing sucrose were heterozygotes. Many CS814021 mutant seeds were not germinated on sucrose-containing MS medium, which are supposed to be homozygotes. These results suggest that PPR287 is essential for embryogenesis. In contrast, a homozygous mutant of the SALK_041236C line, in which T-DNA was inserted at the position encoding 572th amino acid near the stop codon in the last exon of PPR287 (Fig. 2a), could be obtained. No band corresponding to PPR287 was detected in the ppr287 mutant by RT-PCR using the primer pair amplifying the PPR287 transcript spanning the T-DNA insertion site (Fig. 2b), suggesting knockout mutant. However, because the T-DNA is inserted downstream of the PPR motif (Fig. 2a), it is possible that partially truncated PPR287 protein containing entire PPR motifs can be expressed in the ppr287 mutant, suggesting the ppr287 mutant is not a genuine loss-of-function mutant. To examine whether T-DNA insertion affects the transcript level of PPR287 in the mutant, the level of PPR287 in the mutant was analyzed by RT-PCR and quantitative real-time RT-PCR using the primer pair amplifying the PPR287 transcript upstream of the T-DNA insertion site (Fig. 2a). Cleary, the PPR287 level was decreased down to approximately 50% of the wild-type level (Fig. 2c), confirming that the ppr287 is a knockdown mutant.
PPR287 affects photosynthetic activity and chloroplast biogenesis
We next examined whether PPR287 is involved in chloroplast biogenesis by observing chloroplast structures using a transmission electron microscope. Chloroplast morphology was abnormal in ppr287 mutants, displaying a few and loosely stacked thylakoids, whereas the wild type and the complementation line had normal chloroplasts (Fig. 4d and Additional file 5). To identify whether this abnormal chloroplast structure affects chloroplast function, we analyzed the levels of reactive oxygen species (ROS), which are an indicator of chloroplast function. Among ROS, such as superoxide (O2−), hydrogen peroxide (H2O2), and nitric oxide (NO), the amount of H2O2 was measured using a 3,3′-diaminobenzidine (DAB) staining. Evidently, ppr287 mutants had much stronger DAB staining than the wild type and complementation line (Fig. 4e). These results were further supported by quantifying H2O2 levels in the leaves of each plant. The levels of H2O2 were much higher in ppr287 mutants than the wild type and complementation line (Fig. 4e). Collectively, these results imply that PPR287 is important for photosynthesis and chloroplast biogenesis and function.
PPR287 affects transcript levels of chloroplast rRNAs
Contrary to the increasing understanding of the distribution and organellar targeting of PPR proteins in higher plants, the function and cellular role of only a few PPR proteins have been determined until recently. Our current results demonstrate that PPR287 plays a crucial role in Arabidopsis development by affecting the level of chloroplast rRNAs. No loss-of-function homozygous mutants of Arabidopsis PPR287 could be obtained (Fig. 2), suggesting that PPR287 is essential for plant development. In contrast, the homozygous mutant of PPR287 that has T-DNA inserted near the stop codon in the last exon of PPR287 (Fig. 2) could be obtained, which showed pale-green phenotypes but developed normal seeds (Fig. 2). This mutant presumably behaves like a knockdown mutant because it can synthesize partially truncated functional proteins (Fig. 2). This hypothesis was further supported by the observation that ppr287 knockdown mutants generated by an amiRNA knockdown method exhibited similar pale-green phenotypes. Both knockdown mutants displayed severe defects in leaf greening, photosynthesis, chlorophyll biosynthesis, and chloroplast biogenesis (Figs. 2, 3 and 4).
Notably, PPR287 affects transcript levels of chloroplast rRNAs, which then affects chloroplast biogenesis and function. When the processing of rRNAs is impaired, the mature rRNAs are decreased, with concomitant increase in the precursor rRNAs, as exemplified in other PPR mutants, such as svr7 and sot1 [34, 35]. Because our northern blotting analysis revealed that the intensities of precursor rRNAs were not increased in ppr287 mutants, but both the precursor and mature products of all chloroplast rRNAs were decreased in the mutants (Fig. 5a), we propose that PPR287 affects the stability of chloroplast rRNAs. Although we cannot rule out the possibility that the lower amount of rRNAs in ppr287 mutants is due to the lower transcription of rRNA genes, it is unlikely that PPR287, as an RNA-binding protein, affects transcription of rRNA genes. Contrary to many reports demonstrating the roles of PPR proteins in the stabilization of chloroplast mRNAs, the function of PPR proteins involved in the stabilization of rRNAs is largely unknown. A previous study has shown that maize PPR53, which is orthologous to the Arabidopsis PPR protein SOT1, enhances the stability of chloroplast 23S rRNA . Our current results add PPR287 as another PPR protein possibly involved in the stabilization of chloroplast rRNAs. However, we do not know at present the mechanistic role of PPR287 affecting the level of chloroplast rRNAs. Given that plastid rRNA operon encodes all plastid rRNAs and two tRNAs (trnI and trnA) as a single transcript unit (Fig. 5), which is then processed to each mature transcript, it is likely that PPR287 binds to the primary or precursor rRNA transcript and thereby stabilizes all chloroplast rRNAs (Fig. 5). More analysis is required to identify the binding sites of PPR287 and the effects of PPR287 binding on the stabilization of chloroplast rRNAs. In particular, it would be necessary to determine whether PPR287 binds to the specific sequence in the 5′ end or 3′ end of plastid primary rRNA transcript and thereby affects the stability of all rRNAs or whether PPR287 stabilizes all chloroplast rRNAs by binding to the conserved sequences in each rRNA transcript. In addition, it would be interesting to investigate whether PPR287 interacts with other proteins, which affects the level and stability of chloroplast rRNAs. These key experiments will greatly contribute to fully understand the cellular role of PPR287 in the stabilization of chloroplast rRNAs.
Our results demonstrate that the chloroplast-transported PPR287 affects transcript levels of chloroplast rRNAs, which is crucial for chloroplast biogenesis and function during plant growth and development. Given that the role of PPR proteins in the stabilization and processing of chloroplast rRNAs has been identified in only a few cases, our results demonstrating that PPR287 affects the level of all chloroplast rRNAs are intriguing. Characterizing the effect of PPR287 binding on the stabilization of chloroplast rRNAs should be a next important experiment. Moreover, further research is needed to identify the functions of many as-yet uncharacterized PPR proteins and their coordinated roles in the splicing, processing, and stability of chloroplast transcripts.
Plant materials, mutants, and growth conditions
The wild-type and mutant A. thaliana were Col-0 ecotype. The Arabidopsis T-DNA insertion mutants, CS814021 and SALK_ 041236C, were obtained from the Arabidopsis Biological Resources Center. The ppr287 knockdown mutants were generated using an artificial microRNA-mediated knockdown method [37, 38]. The amiRNA constructs targeting the first exon of PPR287 were designed using the Web MicroRNA Designer program (http://wmd3.weigelworld.org) and were cloned into the pBI121 vector. Arabidopsis transformation was performed by means of vacuum infiltration using Agrobacteruim tumefaciens GV3101 . Complementation lines were generated by expressing the full-length PPR287 under the control of the cauliflower mosaic virus 35S promoter in the ppr287 mutant background. The T3 or T4 homozygote transgenic lines were selected for phenotypic analysis. The expression levels of PPR287 in knockdown mutants and complementation lines were analyzed by RT-PCR with gene-specific primers listed in Additional file 8. All plants were grown in soil or half-strength Murashige and Skoog (MS) medium containing 1% sucrose at 23 ± 2 °C under long-day conditions (16 h-light / 8 h-dark cycle).
Analysis of subcellular localization of PPR287
The cDNA encoding full-length PPR287 was cloned into the XbaI/EcoRI site of CsV-GFP3-PA vector using the primers listed in Additional file 8, which expresses the PPR287-GFP fusion protein under the control of cassava vein mosaic virus promoter. Transgenic Arabidopsis plants that express the PPR287-GFP fusion protein were generated by means of vacuum infiltration using Agrobacteruim tumefaciens GV3101 , and GFP signals were detected using a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss Inc. Thornwood, NY, USA). The excitation and emission wavelengths were 488 and 505 nm, respectively.
Chlorophyll content and chlorophyll fluorescence measurement
Chlorophyll content was measured using ethanol extraction and quantification method as previously described . Briefly, leaves of one-week-old wild-type, mutants, and complementation lines were ground in liquid nitrogen, and chlorophyll was extracted with 96% ethanol. The samples were kept overnight at room temperature in the dark. After centrifugation, the absorbance of the supernatant was measured at 648 nm and 664 nm. Chlorophyll fluorescence (Fv/Fm) was measured with a Handy PEA chlorophyll fluorimeter according to the manufacturer’s instructions (Hansatech Instruments Ltd., Norfolk, UK).
Transmission electron microscopy
Chloroplast structures were analyzed using TEM as previously described [41, 42]. Briefly, two-week-old seedlings were fixed with a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 50 mM cacodylate buffer, pH 7.2, at room temperature for 4 h. The samples were embedded in LR White (London Resin Co., London, UK) at 50 °C for 24 h, and thin sections (80–100 nm thickness) were prepared using an ultra-microtome with a diamond knife. The thin sections were stained with uranyl acetate and lead citrate and then examined using a transmission electron microscope JEM-1400 (Jeol, Tokyo, Japan).
RNA extraction, RT-PCR, and northern blot analysis
Total RNA was isolated from the frozen samples using the Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). The splicing pattern of the intron-containing genes was analyzed by RT-PCR using the gene-specific primers listed in Additional file 8 as previously described . Splicing efficiency was measured by real-time RT-PCR using the gene-specific primers listed in Additional file 9 as previously described [32, 41]. The levels of chloroplast transcripts were measured by quantitative RT-PCR using the gene-specific primers listed in Additional file 10. Real-time RT-PCR was carried out on a Rotor-Gene Q thermal cycler (Qiagen) using a SYBR Green RT-PCR kit (Qiagen). For northern blot analysis, four or five micrograms of total RNA were separated on a 1.2% formaldehyde-agarose gel and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences, Parsippany, NJ, USA). The [α-32P]-labeled probes were synthesized using a random primer DNA labeling kit (TaKaRa Bio., Shiga, Japan). Hybridization, washing, and detection of signals were performed essentially as described previously .
GUS staining, DAB staining, and H2O2 measurement
To examine the tissue-specific expression patterns of PPR287, an approximately 1.5-kb fragment of the genomic DNA harboring the putative promoter of PPR287 was cloned into the SphI/BamHI site in front of a GUS reporter gene in pBI121 vector using the primers listed in Additional file 8, and the resulting PPR287PRO::GUS construct was introduced into Arabidopsis by means of vacuum infiltration using Agrobacteruim tumefaciens GV3101 . The transgenic plants were stained in a 0.5 M potassium phosphate buffer (pH 7.0) solution containing 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, 1 mg/ml 5-bromo-4-chloro-3-indole-β-D-glucuronide, 20% methanol, and 0.05% Triton X-100 at 37 °C for 24 h in the dark. After washing the samples with ethanol, GUS images were observed using a Zeiss Axioplan microscope (Carl Zeiss, Inc.). In situ detection of H2O2 was carried out by 3,3′-diaminobenzidine (DAB) staining as previously described . Briefly, 2-week-old leaves were immersed in a solution (0.1% DAB, 0.01 M Na2HPO4, pH 3.8, 0.05% Tween-20), vacuum-infiltrated for 10 min, and incubated overnight at room temperature in the dark. Photographs were taken after bleaching out chlorophylls in an 80% ethanol solution. The level of H2O2 was measured as previously described . Briefly, approximately 200 mg of tissue samples were treated with 0.1% trichloroacetic acid at 4 °C, and the extract was mixed with 100 mM potassium phosphate buffer (pH 7.0) and 1 M KI solution. The reaction mixture was placed in the dark at room temperature for 1 h, and the H2O2 content was determined by measuring the absorbance at 410 nm.
The differences in growth parameters, chlorophyll contents, and photosynthetic activity between the wild type, mutants, and complementation lines were compared by t test (p ≤ 0.05) using the SigmaPlot 10 program (Systat Software, Inc., San Jose, CA, USA).
We thank The Arabidopsis Biological Resource Center for providing the T-DNA mutant seeds.
HK designed the experiments; KL, SJP, JHH, and YJ conducted most of research and analyzed the data together with HK and HSP; KL and HK contributed to the writing of the manuscript. All authors read and approved the final manuscript.
This work was financially supported from the Next-Generation BioGreen21 Program (PJ01314701), Rural Development Administration, Republic of Korea, and from the Mid-career Researcher Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2016R1A2B4009172), Republic of Korea. The funding agencies provided funding to the research projects, but played no role in the design of the study, collection, analysis, the interpretation of data and in the writing of this manuscript. These were the sole responsibilities of the authors.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- 10.del Campo EM. Post-transcriptional control of chloroplast gene expression. Gene Regul Syst Biol. 2009;3:31.Google Scholar
- 15.Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette ML, Mireau H, Peeters N, Renou JP, Szurek B, Taconnat L, Small I. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell. 2004;16:2089–103.CrossRefGoogle Scholar
- 32.Lee K, Han JH, Park YI. Colas des Francs-Small C, Small I, Kang H. the mitochondrial pentatricopeptide repeat protein PPR19 is involved in the stabilization of NADH dehydrogenase 1 transcripts and is crucial for mitochondrial function and Arabidopsis thaliana development. New Phytol. 2017;215:202–16.CrossRefGoogle Scholar
- 33.Cheng S, Gutmann B, Zhong X, Ye Y, Fisher MF, Bai F, Castleden I, Song Y, Song B, Huang J, Liu X, Xu X, Lim BL, Bond CS, Yiu SM, Small I. Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J. 2016;85:532–47.CrossRefGoogle Scholar
- 35.Wu W, Liu S, Ruwe H, Zhang D, Melonek J, Zhu Y, Hu X, Gusewski S, Yin P, Small ID, Howell KA, Huang J. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana. Plant J. 2016;85:607–21.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.