Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

Umakanta Ngangkham, Swarup Kumar Parida, Ashok Kumar Singh, Trilochan Mohapatra

Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

Umakanta Ngangkham1, 2, Swarup Kumar Parida1, 3, Ashok Kumar Singh4, Trilochan Mohapatra1, 5

()

RNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins. This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other. DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10,wild abortive (WA) cytoplasm based male sterile (A linePusa6A), maintainer (B line Pusa6B) and restorer (R line PRR78) lines. Pusa6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines. The genomic and cDNA amplicons for eight mitochondrial genes (,,,,,,and) were sequenced and compared. Differences in genomic and cDNA sequences were considered as editing. Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in(5.74%) and the lowest in(0.60%). The highest editing sites were observed in fertile maintainer Pusa6B followed by PRR78 and Pusa6A,of which random five editing sites in five different rice mitochondrial transcripts namely,,,andwere chosen and validated through cleaved amplified polymorphism sequence (CAPS) analysis and found to be partially edited in four genes. The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.

cleaved amplified polymorphism sequence; cytoplasmic male sterility; hybrid rice; RNA editing; mitochondrial gene; wild abortive; nucleotide

RNA editing is an important posttranscriptional modification process that increases the diversities of transcriptomes and proteomes in eukaryotic organisms(Wu et al, 2017). In the mitochondria of flowering plants, the primary transcripts undergo ‘editing’ that involves predominantly C to U substitutions(Covello and Gray, 1989). Besides substitutions, insertions and/or deletions of other nucleotides have also been reported (Hanson et al, 1996; Smith et al, 1997; Stuart et al, 1997). The consequences of RNA editing are either modification of the coded information for some amino acids or generation of new initiation and/or termination codons of the proteins. The first report of RNA editing was described in 1986 which involved addition or deletion of uridine residues in the RNAs of kinetoplastid, the trypanosomal mitochondria (Benne et al, 1986). Since then, it has been reported as a frequent phenomenon of both monocotyledonous and dicotyledonous higher plant species (Hiesel et al, 1989; Gray et al, 1992), abundantly in mitochondria as compared to chloroplast (Chateigner-Boutinet al, 2008). The first evidence of RNA editing in the plant mitochondria comes from comparison of the protein sequence of subunit 9 () of wheat mitochondrial ATP synthase (ATPase) and its corresponding gene sequence (Graves et al, 1990). Specific RNA editing data for plant mitochondrialmRNA are able to illustrate the basic features of RNA editing in plant mitochondria (Cattaneo, 1991). Higher degree of conservation editing sites in different plant species has indicated sharing of editing sites by both monocots and dicots. However, species specific RNA editing patterns have also been reported in several plant mitochondrial genes (Covello and Gray, 1990).

Cytoplasmic male sterility (CMS), a maternally inherited trait, is widely observed in more than 150 plant species (Liu et al, 2007), where the male organ is sterile/non-functional but having functional female organ leading to compulsory cross-pollination. In many crops, the fertility of male sterile line is restored by fertility restorer () gene(s), which are encoded by nuclear genome. Such systems have been exploited in many crops for commercial production of hybrid seeds and to understand the nuclear-cytoplasmic organelles interaction. The best studied case of a nucleo-cytoplasmic incompatibility leading to the expression of CMS phenotype is the CMS-T of maize. In this case, mitochondrial dysfunction and male-sterility are associated with mitochondrial DNA rearrangements creating a new ORF (), which is transcribed and translated into a chimeric polypeptide (Dewey et al, 1986). Other examples are the chimeric genes-of petunia (Young and Hanson, 1987), andandof the ‘Polima’ CMS in rapeseed (Handa et al, 1995). The transcripts of all these chimeric genes, with the exception of, were found to be edited. Correlation of RNA editing in the plant mitochondrial gene with CMS has also been observed forin transgenic tobacco (Hernould et al, 1993). Transgenic tobacco plants showed male sterility when the unedited(non-functional) was introduced, while all the transgenic plants carrying the edited (functional) form ofwere found to be fertile. Cell type specific RNA editing inmitochondrial gene is involved in CMS in(Howad and Kempken, 1997). The complete editing of angene that might restore the fertility of BT-type CMS has also been reported in rice (Iwabuchi et al, 1993). Das et al(2010) and Chakraborty et al (2015) have reported that the sterility of wild abortive (WA) cytoplasm is due to competition between translated products of edited and uneditedtranscripts, which leads to impaired biogenesis of ATPase complex in rice mitochondria. Most of the mitochondrial DNA rearrangements in CMS identified in plants are associated with the genes encoding subunits of ATP synthase(Ji et al, 2014).

The present study was carried out to gain insight into RNA editing taking place in eight mitochondrial genes from WA-cytoplasm which involves in vital mitochondrial processes and are widely used in rice hybrid breeding, and to examine possible relationship of any editing sites with expression of CMS.

Materials and Methods

Rice materials

Rice materials included three lines of hybrid rice (L.), Pusa Rice Hybrid10 (PRH10) seed production viz. CMS line Pusa6A, maintainer line Pusa6B and restorer line Pusa Rice Restorer78 (PRR78). These lines were grown in the research field of the Indian Agricultural Research Institute, New Delhi, India, following standard agronomic practices.

Isolation of total genomic DNA and cellular RNA

Total cellular DNA was isolated from young panicles of Pusa6A, Pusa6B and PRR78 following Doyle and Doyle (1990). DNA was purified, quantified and diluted to 50ng/µL for PCR reaction. Total cellular RNA was isolated from young panicles using SV Total RNA Isolation System (www.promega.com) following the manufacturer’s protocol.

Primer designing, PCR and RT-PCR amplification

The primer sequences of eight rice mitochondrial genes namely,,,,,,andwere taken from Ngangkham et al (2010) (Table 1). The PCR constituents and cycling conditions with varying annealing temperature were carried out according to Ngangkham et al (2010). For reverse transcript PCR (RT-PCR), RNA was convertedto cDNA using ImProm-IITMReverse Transcription System (www.promega.com) following the manufacturer’s protocol and cDNAs were directly used for gene specific PCR amplification. The amplicons were purified using E.Z.N.A. Gel Extraction Kit (www.Omegabiotek.com).

Cloning and sequencing

Purified PCR and RT-PCR products were cloned into pGEM-T Easy Vector System (www.promega.com),following the manufacturer’s instruction. Recombinant white colonies were selected and used for plasmid isolation using AxyPrep Easy-96 plasmid DNA Kit (Axygen Biosciences, USA). Purified recombinant plasmids were sequenced with universal primers in both forward and reverse directions using a capillary-based Automated DNA Sequencer (MegaBACE 4000, Amersham Biosciences, USA). The vector sequences from the sequencing data were cleaned using VecScreen of NCBI online tools (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html).

Detection of RNA editing sites through multiple sequence alignment and cleaved amplified polymorphism sequence (CAPS) analysis

For discovering of RNA editing sites, the high quality sequences obtained from genomic and cDNA of each rice mitochondrial gene were aligned and compared using BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/bioedit/bioedit.html).RNA editing site is considered only when a specific nucleotide base alteration was detected in the cDNA sequences, as compared to the corresponding genomic sequences of a rice line. Frequency of RNA editing for each rice mitochondrial genes was also estimated. To validate the presence of editing sites identified through sequencing allignment, SNP2CAPS software tool (Thiel et al, 2004) was used for identification of appropriate restriction enzymes whose target sites were altered or introduced due to editing. The restriction enzymes predicted by SNP2CAPS were used to validate the SNP in the enzyme target sites in the mitochondrial genes. PCR amplicon of each gene was quantified on 1% agarose gel along with different known quantity of uncut lambda DNA as standard for comparison. Digestion of amplified products was carried out according to the manufacturer’s protocol and the digested products were resolved on 1.5% agarose gel using 0.5× TBE buffer. Fragment size of the digested products was determined using 100bp DNA ladder plus (Fermentas, Lithuania) and further compared with that of the undigested control to infer editing site.

Results

Editing in Pusa 6A, Pusa 6B and PRR78

Comparison of the cDNA sequence of eight rice mitochondrial genes for sterile (Pusa6A), maintainer (Pusa6B) and restorer (PRR78) lines individually, with their corresponding genomic DNA sequences, led to identification of 230 nucleotides being edited, which consisted of substitution and insertion/deletion of nucleotides, from a total of 6765 nucleotides, with an overall editing frequency of 3.39% (Table 2). Maximum edited nucleotides (52 sites) were observed in, followed by(41),(36),(35),(29),(23),(8) and(6). The frequency of RNA editing ranged from 0.60% () to 5.74% () among the eight mitochondrial genes studied. Overall, there were 196 substitutions (74.24%), 37 insertions (14.02%) and 31 deletions (11.74%) in the three rice lines(Table 2).

In the CMS line Pusa6A, there were 42 nucleotides edited (Table 2), which was the lowest among the three lines studied. Of these, 1 was deletion, 13 were insertions and 28 were substitutions. The maximum of 11 edited nucleotides were found intranscripts (1.36%), of which 3 were insertions, 1 was deletion and 7 were substitutions. This was followed by 10 edited nucleotides intranscript, 9 in, 8 in,2 inand 1 each inand, while in, there was no editing.

Table 2. Statistics of eight mitochondrial genes in Pusa 6A, Pusa 6B and PRR78.

NS, Number of substitution sites; NI, Number ofinsertion sites; ND, Number ofdeletion sites; NT, Number of total sites edited; F, Frequency; TPPE, Total physical position edited; TF, Total editing frequency; ‘–’, None.

In the case of PRR78, there were 109 edited bases which included 82 substitutions, 14 insertions and 13 deletions. The maximum edited nucleotides were observed in(36), followed by(32),(14),(12),(11),(3) and(1) with no editing intranscript. When compared to iso-cytoplasmic Pusa6A line, there were 67 additional edited nucleotides in four genes viz.,,andin PRR78 (Table 2).

In contrast to the above two parental lines carrying WA cytoplasm that has male sterilization effect, the frequency of editing was much higher in the maintainer line, Pusa6B carrying normal fertile cytoplasm. There were a total of 113 edited nucleotides distributed across all the eight genes studied. Of these, 86 were base substitutions, 10 were insertions and 17 were deletions. The majority of these edited bases were different from those in the other two rice lines. The maximum editing took place intranscript (36), followed by(24),(19),(15),(8),(6),(3) and(2). As compared to isonuclear CMS line Pusa6A, Pusa6B had 71 new edited sites distributed across the eight mitochondrial genes.

CAPS analysis of editing sites

The RNA editing sites detected in the transcripts of eight rice mitochondrial genes were validated through CAPS analysis using appropriate restriction enzymes as predicted by SNP2CAPS. The editing sites in eight rice mitochondrial genes were predicted to have target sites for 18 different tetra-cutter restriction enzymes. Of these, five different rice mitochondrial transcripts (,,,and) were chosen and examined through CAPS analysis.

In case of, a 406 bp amplicon was obtained from both mitochondrial DNA and cDNA in all the three lines. When digested withI, the amplicons from genomic DNA were not affected in any of the three lines, while the cDNA got digested only in Pusa6B giving a fragment of 390 bp, but not in the other two lines. This validated the T/G substitution in theI site GATC in Pusa6B (Fig. 1-A).

PCR amplification using primers specific togene gave 810 bp fragments for the mitochondrial DNA templates of Pusa6A, Pusa6B and PRR78, which weren’t digested byCH4IV. The same 810 bp RT-PCR product forcould not be digested in maintainer line Pusa6B. In contrast, cDNAs from Pusa6A and PRR78 were digested into fragments of 350 bp and 300 bp, and even lesser size (Fig.1-B), revealing post transcriptional creation of two or more ACGT target sites for the enzymeCH4IV including a C/A substitution in the sequenced region. This result again validated editing intranscript in Pusa6A and PRR78 but not in Pusa6B.

In case ofgene, a 1001 bp fragment was amplified in all the three lines when mitochondrial DNA was used as a template, which could not be digested by509I. The 1001 bp fragments from cDNA of Pusa6A and PRR78 were digested in where there was no digestion for cDNA of Pusa6B (Fig.1-C). In this digestion, cDNA amplicon from Pusa6A and PRR78 gave a multiple fragments including four distinct and detectable fragments with different sizes of 701, 551, 450 and 300 bp. The combination of two digested fragment pairs such as 700 with 300 bp and 550 with 450 bp might be constituted the pre-digested amplicon. This result indicated the occurrence of two sites edited in sterile cytoplasms which wasn’t edited completely and cDNAs of Pusa6A and PRR78 consistedof both the partial edited transcripts.

Gene specific primers forgene gave a PCR amplicon of size 1002bp in all the three lines, which was cleaved by enzymeI only in Pusa6B to give two fragments (Fig.1-D), whereas, the amplicons in Pusa6A and PRR78 remained uncut. This revealed a target site for the enzyme existing in the mtDNA only in Pusa6B. When the RT-PCR products of 1002 bp were digested using the same enzyme, digestion pattern in Pusa6B was identical to that observed for PCR product suggesting that no further change had occurred post transcriptionally. However, in case of Pusa6A and PRR78, the RT-PCR products for thegot digested to produce multiple fragments in contrast to PCR product. This indicated that multiple editing led to creation of target site CTAG for the enzymeI.

Fig. 1. Validation of RNA editing in mitochondrialgenes by cleaved amplified polymorphism sequence analysis.

M, 100 bp DNA ladder plus; 6A, CMS line Pusa6A; 6B, Maintainer line Pusa6B; PRR78, Restorer line PRR78.

The recognition sites ofI (GATC) for(A),CH4IV (ACGT) for(B),509I (AATT) for(C),I (CTAG) for(D) andI (AGCT) for(E) were affected by SNPs T/G, C/A, T/A, G/C and A/T as predicted by SNP2CAPS, respectively. The digested PCR fragments were separated in 1.2% agarose gel.

Similar to thegene patterns, the amplicon of 749 bp obtained from thegene was digested byI in Pusa6B giving two fragments of 400 bp and 349bp but not in Pusa6A and PRR78, revealing thereby presence of one target site AGCT for the enzyme in Pusa6B only. In RT-PCR using RNA templates, a 749bp amplicon was obtained in all the three lines. This fragment got digested to give multiple fragments in Pusa6A and PRR78 unlike to the PCR products, while the restriction fragment size remained identical to that from mtDNA template in Pusa6B (Fig.1-E). This result also revealed that editing took place at multiple sites in thetranscript of Pusa6A and PRR78.

Discussion

RNA editing, a post transcriptional process, and CMS are two important phenomena associated with higher plant mitochondria where inadequate or defective RNA editing in specific mitochondrial genes causes male sterility (Hu et al, 2013). RNA editing is reported to regulate expression of several CMS-related mitochondrial genes (Howad and Kempken, 1997). Presence of edited and unedited forms of mRNA may give rise to biologically active proteins having different physical and/or functional properties (Gray and Covello, 1993). In some cases, RNA editing is an absolute requirement, which restores critical amino acid residue(s), in absence of which non-functional proteins might get synthesized as in case of wheatmRNA (Covello and Gray, 1989).

Effect of isonuclear-alloplasmic interaction on RNA editing

Alloplasmic lines are created by exchanging the cytoplasm of one species line with another through substitution backcrossing while maintaining the nuclear genome, which leads to disruption of the interactions between the nucleus and cytoplasm that have co-evolved (Kianian and Kianian, 2014). It provides an opportunity to understand the interactions between the cytoplasmic organelles and the nucleus. The fertile maintainer line Pusa6B with totally different mitochondrial genome from the sterile line Pusa6A having WA sterile cytoplasm had identified 113 editing sites in eight mitochondrial genes, which is expected according to the hypothesis that decreased RNA editing frequency contributes to male sterility (Howad and Kempken, 1997; Yu et al, 2013). Presence of additional 71 (63.71%) edited nucleotides in Pusa6B as compared to Pusa6A covering all the eight mitochondrial genes might indicate occurrences of higher and different mode of post-transcriptional editing in the mitochondrial genome of Pusa6B, since it has male fertile cytoplasm compared to male sterile cytoplasm Pusa6A (Table 2). While compared to Pusa6A, the maximum additional edited nucleotides in Pusa6B were observed in, followed bywith the minimum inandalthough nuclear background were identical, and therefore the same trans-factors for editing process. Since most of the maximum additional edited nucleotides were observed in genes involved in ATP synthesis, this shows the important role of editing in vital mitochondrial processes. These results also showed that the cytoplasm may play a vital role in the editing process. Similar results are also reported by Hu et al (2013), in which five isonuclear-alloplasmic male sterile lines, when compared to their corresponding CMS lines of rice, display different editing efficiencies at the conserved sites of,andgenes.

Effect of allonuclear-isoplasmic interaction on RNA editing

The male sterile line Pusa6A and the restorer line PRR78 are allonuclear-isoplasmic parental lines since both having the same WA based sterile cytoplasm and thus the same mitochondrial genome constitution. Unlike in Pusa6A line, there were 67 additional edited nucleotides in four genes viz.,,andgenes in PRR78. However, there was a decrease in number of total sites edited for. Out of the total 12 edited nucleotides in the remaining three genes viz.,and, six editing sites had similar editing patterns in both iso-cytoplasmic Pusa6A and PRR78 lines, which couldn’t be correlated to male sterility. However, a total of 35 editing sites in six genes (,,,,and) observed in Pusa6A but not in PRR78, indicating that RNA editing isn’t specific to the restorer lines. However, 67 new editing sites in PRR78 compared to its iso-cytoplasmic Pusa6A, pointed to difference in nuclear-cytoplasmic interaction, which can be ascribedto the presence of fertility restorer () gene in PRR78.

Varying degree of editing in different mitochondrial transcripts in different lines

Comparison of the transcript sequence of eight rice mitochondrial genes in the parental lines of PRH10, viz. sterile (Pusa6A), maintainer (Pusa6B) and restorer (PRR78) lines revealed a total of 3.39% edited nucleotide, ranging from 0.60% to 5.74%, which consisted of substitutions and insertions/deletions. The frequency of RNA editing ranged from 0.60% () to 5.47% () in the eight mitochondrial genes studied, which are agreeable to those reported earlier (Giege and Brennicke, 1999; Picardi et al, 2010), and similar in-consistency of editing frequency across these eight genes as a function of gene length, which is also reported in earlier studies in,and(Mower and Palmer, 2006). Abundance of base substitution (74.2%) type of post transcriptional editing at multiple positions in transcripts of the rice mitochondria is consistent with the earlier observations in several monocotyledonous and dicotyledonous plant mitochondrial genes (Schuster et al, 1990). This result has suggested the existence of apparent coding anomalies in rice mitochondria. When we compared the RNA editing sites between Pusa6B and PRR78, there were 21 editing sites in four genes namely,(11),(6),(3) and(1), which were identical in both the fertile lines but absent in sterile line Pusa6A, for which further analysis is required to validate this editing sites with the male sterility mechanism. Since, alteration of RNA editing in CMS lines compared to other normal cytoplasm is not always necessarily the mechanism of CMS in plants (Horn et al, 2014). A relevant example in rice is that the presence ofgene, whose protein interacts with the nuclear encoded mitochondrial protein COXII, triggers premature tapetal programmed cell death and consequent pollen abortion (Luo et al, 2013) and hence male sterility in the WA CMS system, which is in contradict to Das et al (2010) who had earlier showed that an uneditedtranscript as molecular cause for this CMS system in rice and co-segregated with the male sterility. RNA editing is also influenced by many factors, such as tissue type, organ type, developmental stage, nucleo-cytoplasmic interactions and nuclear background (Grosskopf and Mulligan, 1996; Zehrmann et al, 2008) as well as by environmental stress (Liew et al, 2017). Such kinds of phenomena are also reported by Hu et al (2013),which indicated that nucleo-cytoplasmic interactions affect the RNA editing of,andtranscripts. Therefore, further analysis and characterization of these potential RNA editing sites in candidate mitochondrial genes is required to identify the mitochondrial gene responsible for male sterility.

Partial/incomplete editing in mitochondrial transcripts

Presence of multiple digested fragments of RT-PCR products of five mitochondrial genes namely,,,andduring the CAPS analysis, showed multiple sites of RNA editing. However, there was discrepancy between the number of editing sites revealed by sequencing and that based on CAPS analysis. For instance, in case ofgene, one edited sites corresponding to restriction enzyme target site (AATT) for restriction enzyme,509I based on sequence analysis was predicted. However, digestion of the same amplicon using the same restriction enzyme509I leads to four fragments and mixed of smaller fragments as compared to two digested fragments expected. This might be due to occurrence of incomplete/partial editing of gene transcripts and sequencing only one cloned RT-PCR product that was sequenced which do not represent the whole populations of differentially edited transcripts. An editing site of a particular gene is considered as partially/incompletely edited when the site is not edited in all the transcript pool. It is well known that partially/incompletely edited transcripts do exist in plant mitochondria (Schuster et al, 1990;Mower and Palmer, 2006; Zehrmannet al, 2008),which were represented in the RT-PCR products that were digested. While sequence comparison of genomic and multiple cDNA clones ofgene inmitochondrion, Schuster et al (1990) found none of the cDNA clones were completely edited for all potential editing sites, suggesting that such completely edited transcripts might be rare as compared to abundant of mixture transcripts edited partially. Abundance of partial editing process is further shown by sequence comparison ofmitochondrion through RNAseq which detects 76% of editing sites as partial editing, of which 28% is found to be tissue specific(Picardi et al, 2010). They further suggested that partialediting was derived from incomplete editing transcripts as well as partial edited from mixed tissue sample. Therefore, the discrepancy of editing sites in these studies might be due to failure of potential partial editing site detection because of restriction of single cDNA clone sequencing thereby precluding multiple partially edited transcripts from the transcript pool and assessment of tissue specificity of editing sites.

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19 July 2018;

10 October 2018

s:Trilochan Mohapatra(tmnrcpb@gmail.com); Umakanta Ngangkham (ukbiotech@gmail.com)

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