OTP970 Is Required for RNA Editing of Chloroplast ndhB Transcripts in Arabidopsis thaliana

RNA editing is essential for compensating for defects or mutations in haploid organelle genomes and is regulated by numerous trans-factors. Pentatricopeptide repeat (PPR) proteins are the prime factors that are involved in RNA editing; however, many have not yet been identified. Here, we screened the plastid-targeted PLS-DYW subfamily of PPR proteins belonging to Arabidopsis thaliana and identified ORGANELLE TRANSCRIPT PROCESSING 970 (OTP970) as a key player in RNA editing in plastids. A loss-of-function otp970 mutant was impaired in RNA editing of ndhB transcripts at site 149 (ndhB-C149). RNA-immunoprecipitation analysis indicated that OTP970 was associated with the ndhB-C149 site. The complementation of the otp970 mutant with OTP970 lacking the DYW domain (OTP970∆DYW) failed to restore the RNA editing of ndhB-C149. ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex; however, neither NDH activity and stability nor NDH-PSI supercomplex formation were affected in otp970 mutant compared to the wild type, indicating that alteration in amino acid sequence is not necessary for NdhB function. Together, these results suggest that OTP970 is involved in the RNA editing of ndhB-C149 and that the DYW domain is essential for its function.


Introduction
RNA editing is considered an indirect repair strategy that is required for the functional expression of organelle genomes [1]. RNA editing was first discovered in trypanosomes, where four Us were inserted into the mature mRNA of the cytochrome c oxidase II (coxII) in mitochondria (kinetoplasts), thus restoring the original function of the protein [2]. Subsequently, cytidine (C)-to-uridine (U) RNA editing in apolipoprotein-B48 mRNA in human and rabbit intestines [3] and adenine (A)-to-inosine (I) RNA editing in pre-mRNAs in other animals have been reported [4]. In terms of the conversion of C to U, the protein product of mature RNA following RNA-editing is different from that encoded by the genomic DNA, which may be attributed to the fact that C residues undergoing RNA editing are predominantly located at the first or second positions of codons. It is usually possible for C-to-U RNA editing to generate a new start codon by altering ACG to AUG [5] or to introduce a termination codon by altering CGA/CAA to UGA/UAA [6], thus extending or shortening the open reading frames. 2 of 14 In the plant kingdom, C-to-U RNA editing was first discovered in mitochondria [7,8]. Two years later, the same type of RNAA editing was identified in chloroplasts [5]. In contrast, RNA editing has not been reported in the cytoplasmic RNAs of plants. Organelle RNA editing has been documented in almost all land plants, including bryophytes, ferns, gymnosperms, and angiosperms [9][10][11][12]. RNA editing usually occurs in the coding regions of mRNAs and occasionally in the non-coding regions of mRNAs, such as in the introns, and in transfer RNAs (tRNAs) [13][14][15].
In Arabidopsis, more than 40 and more than 600 C-to-U editing sites have been detected in RNAs transcribed from the chloroplast and mitochondrial genomes, respectively [16,17]. In vivo, in vitro, and organelle studies indicate that the nucleotide that is targeted for editing is recognized via 20-25 nt cis-elements located upstream (5 ) of the editing site [18][19][20]. To date, a large number of trans-factors have been identified in the RNA editing process in plastids, including those involved in the editing of ndhB transcripts; the ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex that is involved in cyclic electron transport (CET) around the PSI. For example, CHLORORESPIRA-TORY REDUCTION 28 (CRR28), CRR22, EDITING LACKING INSERTIONAL MUTANT 1 (ELI1), ORGANELLE TRANSCRIPT PROCESSING 82 (OTP82), QUINTUPLE EDITING FACTOR 1 (QED1), CHLOROPLAST RNA EDITING FACTOR 7 (CREF7), and OTP84 are required for ndhB editing at sites 467, 746, 830, 836, 872, 1255, and 1481, respectively [21][22][23][24][25][26]. However, the trans-factors that are specifically required for the RNA editing of ndhB site 149 have not yet been identified.
Pentatricopeptide repeat (PPR) proteins comprise an extraordinarily large protein family in land plants [27], with 450 members belonging to Arabidopsis and 477 in rice (Oryza sativa) [28]. The PPR proteins are defined by a tandem array (2 to 27 repeats) of the PPR motif, each of which contains 35 degenerate amino acids [29]. The PPR family members can be divided into the P and PLS subfamilies; the P proteins are composed of P motifs only, whereas the PLS proteins comprise the triplets of P-L-S motifs. A large number of recent studies show that PLS proteins are mainly involved in RNA editing. The PLS proteins are further divided into the PLS, E, and DYW subfamilies according to the differences in their amino acid sequence at the C-terminus [30]. Analysis of the crystal structure of these proteins reveals that each PPR motif folds into a pair of antiparallel α helices, with tandem arrays of PPR motifs forming a superhelix, which is the RNA-binding face [31][32][33]. The E motif is involved in protein-protein interactions [34,35], and the DYW domain contains a conserved zinc-binding motif (HxE(x)nPCxxC) that is similar to the cytidine deaminase domains, which have recently been shown to be able to crystallize, and its cytidine interim deaminase activity has been clearly proven [26,36,37]. The body of work described above shows that PLS proteins are crucial for RNA editing. However, the functions of most PPR proteins belonging to the PLS subfamily remain unidentified. In this study, we characterized the role of the OTP970 protein in the RNA editing of ndhB; An otp970 T-DNA insertion mutant in Arabidopsis did not accumulate OTP970 and exhibited defective RNA editing at the ndhB-C419 site. Additionally, the OTP970 protein was found to bind to a cis-element surrounding ndhB-C149 in vivo.

Plant Material and Growth Conditions
The Arabidopsis ecotype Columbia (Col-0) was used as the wild type in this study. Seeds were sown on half-strength Murashige and Skoog (MS) medium containing 2% sucrose and were allowed to synchronize their germination at 4 • C for 3 days. Plants were grown at 22 • C under a 12 h light/12 h dark cycle. The otp970 mutant (SALK_150217) was obtained from the Arabidopsis Biological Resource Center, OH, USA. The T-DNA insertion site in the otp970 mutant was identified via the PCR amplification of genomic DNA and T-DNA borders using gene-specific primers (Table S1).

RNA Isolation, RT-PCR, and RT-Quantitative PCR(QPCR)
Total RNA was extracted from the otp970 mutant and wild-type plants using the Plant RNA Kit (Magen), and 5 µg total RNA was used for first strand cDNA synthesis, which was conducted using the PrimeScript RT Reagent Kit (TaKaRa). For the RNA editing analyses, 34 published RNA editing sites were amplified and sequenced using specific primers that spanned the editing site [38]. The level of RNA editing was calculated by comparing the relative heights of the nucleotide peaks at the editing site. For RT-PCR, otp970 was amplified using gene-specific primers. RT-qPCR was carried out using the SYBR Premix Ex Taq™ (TaKaRa) on a real-time RT-PCR System (LC480; Roche). Actin 2 was used as a reference gene. The primers used for RNA editing, RT-PCR, and RT-qPCR analyses are listed in Table S1.

RNA Immunoprecipitation (RIP)
RIP assay was performed using 7-day-old 35S:OTP970-FLAG transgenic and wild-type plants, as previously described [39]. Primers used in the analysis are listed in Table S1.

Chlorophyll Fluorescence Analysis
Chlorophyll fluorescence was detected with the MAXI version of the Imaging-PAMM-Series chlorophyll fluorescence system. Before measurement, plants were adapted to the dark for 30 min, and light-response curves were then measured as previously reported [42]. Transient increases in chlorophyll fluorescence were measured with dural-PAM 2000 after turning off the actinic light (AL), as previously described [43].

Complementation of otp970 Mutation
The wild-type At1g18485 genomic fragment (2910 bp) was PCR-amplified using genespecific primers (Table S1) and was cloned into the pCAMBIA1301 binary vector. Transgenic lines were screened on half-strength Murashige and Skoog agar plates supplemented with 50 µg/mL hygromycin. Plants resistant to hygromycin were transferred to the soil to produce seeds. The complementation of otp970 mutation was further confirmed by immunoblot analyses and by sequencing the editing site.

OTP970 Protein Subcellular Localization
The OTP970 protein was fused with the green fluorescent protein (GFP) as previously described [44] but with slight modifications. A fragment encoding the first 100 N-terminal amino acids of OTP970 was amplified by RT-PCR using the primers listed in Table S1, and the fragment was cloned into the pUC18 under the control of 35S promoter to construct a fusion protein with GFP.

RNA Editing at the ndhB-C149 Site Is Impaired in otp970 Mutant Plant
Chlorophyll fluorescence changes are often observed after plants have experienced AL illumination, which is indicative of NDH activity. Therefore, we used AL illumination to identify the mutants involved in NDH activity. However, this method cannot identify all of the possible NDH RNA editing mutants, as it may or may not affect NDH activity.
The PLS subfamily members of the PPR proteins function in terms of the site recognition of the RNA editing. The DYW domain has been a strong candidate for the C deaminase activity required for C-to-U conversion in RNA editing [26]. To identify the additional PPR proteins that are involved in the RNA editing of ndhB in the chloroplasts, we focused on the phylogenetic analyses of the PPR proteins that belong to the PLS-DYW subfamily, which are predicted to be targeted to the plastids because previous research studies showed that the trans-factors involved in ndhB RNA editing all belong to the PLS-DYW subfamily. Because functionally related genes are believed to undergo co-evolution, we analyzed the co-evolution of the genes encoding PLS-DYW PPR proteins in Arabidopsis by means of hierarchical clustering. The results showed that several of the PPR genes that are involved in ndhB RNA editing were clustered together ( Figure 1). We focused on a single co-evolution cluster comprising the following PPR proteins found to be involved in ndhB RNA editing: QED1/OTP81 (AT2g29760), CREF7 (At5g66520), OTP82 (At1g08070), ELI1 (At4g37380), and CRR28 (At1g59720). Among these, OTP970 was present, which is an intron-less gene that encodes a putative DYW PPR protein with 970 amino acids. OTP970 contains 23 characteristic PPR motifs, including PPR-related motifs in the E and DYW domains, as predicted by a web portal (https://ppr.plantenergy.uwa.edu.au/ accessed on 12 December 2021). We obtained an otp970 mutant, the T-DNA was inserted in the coding sequence of At1g18485 (Figure 2A), and the T-DNA insertion site in the otp970 mutant (Salk_150217) was confirmed by PCR ( Figure 2B); We detected no OTP970 transcripts in otp970 homozygous mutant plants, as determined by RT-qPCR ( Figure 2C); However, we observed no visible phenotypic differences between the mutant and wild-type plants.
Several PPR proteins belonging to the PLS subfamily are involved in RNA editing in the organelles of land plants. To determine whether RNA editing was impaired in otp970 mutant, we detected the editing status of the chloroplast transcripts. The RT-PCR products encompassing the 34 editing sites present in the Arabidopsis chloroplast transcripts [45] were directly sequenced. The results showed defective RNA editing at the ndhB-C149 site in the otp970 mutant; this site was completely edited in wild-type plants but not in otp970 mutants ( Figure 2D). However, other RNA editing sites were not altered in transcripts from the otp970 mutant. To further confirm that the defect in RNA editing at the ndhB-C149 site in the otp970 mutant was due to the disruption of At1g18485, complementation was conducted by expressing OTP970 from the 35S promoter. Two complemented lines showed the 70% and 80% restoration of RNA editing at the ndhB-C149 site compared to the wild type, respectively ( Figure 2D). Protein expression of the complemented lines were detected by immunoblot ( Figure S1). Thus, At1g18485 successfully complemented the defective RNA-editing phenotype of the otp970 mutant, suggesting that At1g18485 is the OTP970 gene.
Aberrant RNA processing can be a secondary cause of defective RNA editing [46]. To determine whether the defect in the RNA editing of ndhB was due to abnormal intron splicing, we performed RT-qPCR to examine the expression of ndhB in the otp970 mutant. The abundance of ndhB transcripts was similar in the mutant and wild type ( Figure S2A). Next, we examined intron splicing in the wild-type and otp970 mutant plants by RT-qPCR and RT-PCR. ndhB showed no difference in terms of intron-splicing efficiency in the otp970 mutant compared to in wild-type plants ( Figure S2B,C). Taken together, our results indicated that the defective RNA editing of ndhB in the otp970 mutant was not caused by aberrant intron splicing but was due to the loss of OTP970 function.

Tissue-Specific Expression Pattern of OTP970
Genevestigator was used to predict the expression of OTP970, and the expression data showed that OTP970 mRNA is ubiquitously expressed in various Arabidopsis tissues [47]. To provide experimental evidence for the OTP970 expression patterns, we extracted RNA from the root, stem, rosette, flower, and silique of a wild-type Arabidopsis plant and detected the expression of OTP970 by RT-PCR and RT-qPCR with gene-specific primers. The results of RT-PCR analysis showed that OTP970 was expressed in all vegetative and reproductive tissues ( Figure 3A). Moreover, RT-qPCR data showed that the expression of OTP970 was relatively highest in the rosette, intermediate in the flower, silique, and stem, and the lowest in the root ( Figure 3B).

OTP970 Is Localized to Plastids and Associates with ndhB Transcripts at Site 149
According to the prediction of TargetP 1.1, 37 amino acids at the N-terminus o OTP970 are targeted to chloroplasts [48]. To validate this prediction experimentally, fragment spanning the 1-100 N-terminal amino acids of OTP970 was fused to GF (100AA-GFP) under the control of the Cauliflower Mosaic Virus 35S promoter (Figur 3C), and the 100 AA-GFP fusion protein was transiently expressed in the Arabidopsis pro

OTP970 Is Localized to Plastids and Associates with ndhB Transcripts at Site 149
According to the prediction of TargetP 1.1, 37 amino acids at the N-terminus of OTP970 are targeted to chloroplasts [48]. To validate this prediction experimentally, a fragment spanning the 1-100 N-terminal amino acids of OTP970 was fused to GFP (100AA-GFP) under the control of the Cauliflower Mosaic Virus 35S promoter (Figure 3C), and the 100 AA-GFP fusion protein was transiently expressed in the Arabidopsis protoplasts. Analysis of the subcellular localization of the 100 AA-GFP fusion proteins by confocal laser scanning microscopy revealed that OTP970 is localized to the chloroplast ( Figure 3D), which is consistent with the inference that OTP970 is involved in the RNA editing of plastid transcripts. We showed that OTP970 was involved in RNA editing at the ndhB-C149 site. To detect whether OTP970 is associated with these transcripts, we performed the RIP assay. The protein extracts of OTP970-FLAG transgenic and wild-type plants were immunoprecipitated using agarose beads coated with anti-FLAG antibody. Our results showed that OTP970-FLAG is enriched in the editing site of the ndhB transcript, while the enrichment of OTP970-FLAG at the psbF editing site is not apparent (Figure 3E,F). These results suggest that OTP970 is directly associated with the ndhB transcripts at site 149, which is consistent with a previous prediction [49].

DYW Motif of OTP970 Is Essential for RNA Editing
The DYW motif is suggested to be responsible for RNA editing, and its cytidine interim deaminase activity has been clearly proven [26,36,37]. To identify the role of the DYW motif of OTP970 in the otp970 mutant, we expressed the OTP970 lacking the DYW motif (OTP970 ∆DYW ) fused with two tandem FLAG tags in the otp970 mutant under the control of the 35S promoter. The RNA editing efficiency at the ndhB-C149 site in the transgenic plants was similar to that in the otp970 mutant ( Figure 2D). Meanwhile, we detected the protein expression of complemented lines ( Figure S3). These data suggest that the DYW motif of OTP970 is essential for RNA editing.

NDH Function Is Not Impaired in otp970 Mutant Plant
RNA editing at the ndhB-C149 site results in a Ser-to-Leu substitution at amino acid position 50. Therefore, a defect in RNA editing at the ndhB-C149 site in the otp970 mutant may change the amino acid and may destabilize the NdhB protein in vivo. To detect this possibility, protein blots were immunodetected using the anti-NdhB antibody. The NDH complex consists of at least 29 subunits and may be destabilized by the loss of either NdhB or NdhD [50]. The NdhB protein level in the otp970 mutant was similar to that in the wild type ( Figure 4A), indicating that NdhB protein accumulation was not affected in otp970. Because RNA editing at the ndhB-C149 site in the otp970 mutant was below the detection limit ( Figure 2D), it is possible that the NdhB protein in the otp970 mutant was translated from unedited RNA. This pattern is similar to other trans-factors, such as CRR22, which is involved in RNA editing at the ndhB-C746 and ndhD-C887 sites; CRR28, which is involved in RNA editing at the ndhB-C467 and ndhD-C878 sites; and CRR21, which is involved in RNA editing at the ndhD-C383 site [21,35]. Thus, we conclude that Ser-50 in NdhB (ndhB-50) is not essential for stabilizing the NDH complex.
We also determined the accumulation of PsaB, Cytf, PsbO, and ATPB, which are representative subunits of PSI, cytochrome (Cyt) b 6 f complex, PSII, and chloroplast F 0 F 1 -ATPase, respectively. The results showed that all of these photosynthetic complexes were not affected in the otp970 mutant ( Figure 4A). Consistently, there are no obvious defects were observed during photosynthetic electron transport in otp970 ( Figure S4). To assess whether RNA editing in otp970 destabilizes the NDH-PSI supercomplex, BN-PAGE was used to analyze the accumulation of this supercomplex, and the crr2-2 mutant was used as a PSI-NDH supercomplex-less control [50]. The abundance of the photosystem complexes corresponding to the NDH-PSI supercomplex in the opt970 mutant was similar to that in the wild type but was reduced compared to the crr2-2 mutant ( Figure 4B, band I). This result suggests that the Ser-50 of NdhB is dispensable for the interaction between the NDH and PSI complexes.
Because RNA editing at the ndhB-C149 site in the otp970 mutant was below the detection limit ( Figure 2D), it is possible that the NdhB protein in the otp970 mutant was translated from unedited RNA. This pattern is similar to other trans-factors, such as CRR22, which is involved in RNA editing at the ndhB-C746 and ndhD-C887 sites; CRR28, which is involved in RNA editing at the ndhB-C467 and ndhD-C878 sites; and CRR21, which is involved in RNA editing at the ndhD-C383 site [21,35]. Thus, we conclude that Ser-50 in NdhB (ndhB-50) is not essential for stabilizing the NDH complex. Although CRR22, CRR28, and CRR21 are involved in the alteration of NDH activity, these proteins are not essential for stabilizing the NDH-PSI supercomplex and NDH complex [21,35]. To determine whether the function of OTP970 was similar to that of these CRR proteins, we analyzed the NDH activity in opt970 by detecting the transient increase in chlorophyll fluorescence after turning off AL. The results showed no differences in chlorophyll fluorescence and post-illumination between the wild type and otp970 mutant ( Figure 4C), demonstrating that NDH activity was not affected in otp970. Taken together, these data suggest that the conversion of Ser-50 to Leu-50 in NdhB is not essential for the function of the NDH complex.

The Extent of RNA Editing at the ndhB-C149 Site Varies among Tissues
The extent of RNA editing varies among different tissues during plant development [51,52]. To detect whether the RNA editing that takes place at the ndhB-C149 site varied among different tissues, we conducted RT-PCR on the root, stem, rosettes, flower, and silique of wild-type plants during the reproductive phase. The RT-PCR sequencing products showed that RNA editing at the ndhB-C149 site was dramatically reduced in the root compared to in other tissues ( Figure 5). Moreover, the rate of RNA editing at ndhB-C467 and ndhB-C1255 sites were also dramatically reduced in the root, whereas that at the ndhB-C586 site showed a slight reduction in the root compared to other tissues ( Figure 5). Figure 5. Analysis of the extent of RNA editing at nine ndhB sites in different tissues. RT-PCR products amplified from different tissues of wild-type (Col-0) were directly sequenced. All nine ndhB editing sites are indicated with asterisks above the corresponding peaks.

Discussion
RNA editing is an important post-transcriptional regulation in plastids. Although some regulators have been identified in the RNA editing process in plastids [53], there are still regulators that are specifically required at some RNA editing sites in the plastids that have not yet been identified. In this study, we identified and characterized the role of OTP970 encoding a PLS-DYW subfamily of PPR proteins in RNA editing at the ndhB-C149 site that has not been identified in Arabidopsis plastids.
The chloroplast NDH complex is divided into four subcomplexes, including the membrane, lumen, and stroma-exposed A and B subcomplexes, based on the characterization of the eubacterial NDH complex and Arabidopsis mutants lacking NDH subunits [54]. The membrane subcomplex contains seven plastid-encoded subunits (NdhA-NdhG), whereas the stroma-exposed A subcomplex includes four plastid-encoded subunits (NdhH-NdhK) and four nuclear-encoded subunits (NdhM-NdhO) [54]. The activity and stability of the NDH complex and its ability to form a supercomplex with PSI are impaired in mutants lacking the proteins required for the assembly of the stroma-exposed subcomplexes [55,56].
The C-to-U editing of the ndh transcripts alters the amino acid sequence of the encoded subunit proteins at certain sites; thus, the functional importance of amino acids varies with site changes. For example, defects in RNA editing at the ndhB-C746 and ndhD-C887 sites in crr22, ndhB-C467, and ndhD-C878 sites in crr28 and at the ndhD-C383 site in crr21 can impair NDH activity [21,35]; however, these alterations do not affect the stability of the NDH complex. In this study, we also observed that the otp970 mutant was defective in RNA editing at ndhB-C149; nevertheless, NDH activity and stability of the NDH complex were unaffected compared to in the wild type ( Figure 4). This is similar to the otp82 mutant, in which the RNA editing defects at the ndhB-C836 and ndhG-C50 sites did not affect NDH function [23].
NDH-dependent CET plays a crucial role in response to various stresses. For example, tobacco mutant NDH complex defects show no differences in photosynthetic activity when compared to wild-type [43,57]. However, NDH-defective mutants are sensitive to various stress, such as high light intensity [58], low humidity [57], drought [59], and high/low temperature [60,61], indicating that the NDH complex protects the photosynthetic activity in the chloroplasts from oxidative stress. Thus, these editing sites that do not affect NDH function under normal conditions but probably play important roles under certain stress conditions, or mediate NDH function maintenance during certain evolutionary periods.
Previous studies have shown that RNA editing at individual sites occurs in some but not all species [62]. Additionally, the extent at which RNA editing occurs at the same site varies across different developmental stages and/or among different tissues [51,52]. In this study, our results showed that the degree of C-to-U editing at the ndhB-C149 site was 100% in the stem, rosette, flower, and silique tissues, but only~10% in the root sample ( Figure 5). Notably, the ndhB-149 in the stem is fully edited, but the expression patterns indicated that OTP970 was expressed at very low levels in stems ( Figure 3A), implying that differences in editing rates among these tissues probably resulted from both the expression and binding activity of their corresponding trans-acting factors.

Conclusions
In this paper, we identified OTP970, a gene encoding the PLS-type PPR protein, which is required for RNA editing in plastids. The sequencing results of RT-PCR products showed that otp970 mutant was impaired in RNA editing at the ndhB-C149 site. RIP analysis demonstrated that OTP970 was associated with ndhB-C149 site. Furthermore, we discovered that the DYW domain of OTP970 is required for RNA editing at the ndhB-C149 site. These findings increased our understanding of the mechanism in plastid RNA editing.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13010139/s1, Figure S1: Western blot analysis of otp970 complemented 35:OTP970/otp970 plants, Figure S2: Transcript profiles of genes with the editing defects in otp970 mutant, Figure S3: Western blot analysis of the OTP970 lacking the DYW motif (OTP970 ∆DYW ), Figure S4: In vivo analysis of electron transport activity in wild-type and otp970 Arabidopsis mutant plants, Table S1: List of primers used in this study.