Analysis of organellar RNA editing from leaf and floral transcriptomes of Phalaenopsis aphrodite subsp formosana

Abstract

SUMMARY Previously 44 plastid RNA editing sites have been identified from 24 protein-coding transcripts of leaf by using Sanger sequencing in Phalaenopsis aphrodite subsp formosana In this study we analyzed the organelle-enriched transcriptomes from leaf and floral tissues of moth orchid by next-generation sequencing (NGS) RNA editing sites were determined with the threshold of more than 5% of C-to-U or U-to-C conversion after mapping the sequencing reads to the reference plastid and mitochondrial DNA templates In total 137 and 1 032 edits of C-to-U and/or U-to-C conversions were identified in plastid and mitochondrial transcriptomes of P aphrodite respectively the highest number reported so far in monocots In plastid 110 and 106 edits were present in leaf and flower respectively with 91 edits in common As well 79 edits were involved in protein-coding transcripts and the 58 edits caused the non-synonymous substitution In mitochondria 978 and 898 edits were present in leaf and flower respectively with 847 edits in common As well 790 edits were involved in protein-coding transcripts and the 621 edits caused the non-synonymous substitution Furthermore at least 31 and 142 edits showed significantly (≧20%) differential editing between leaf and floral tissues in either plastid or mitochondria respectively which it suggested that some unidentified tissue-specific factors might be required for regulating RNA editing in moth orchid Finally RNA editing in plastid trnM is required for the formation of a standard cloverleaf structure INTRODUCTION RNA editing refers to a post-transcriptional modification of nucleotides at the RNA level which the nucleotide sequence of the transcripts did not consistently reflect the sequence of the corresponding template DNA In land plants the C-to-U RNA editing event was first found in mitochondrial transcripts (Covello and Gray 1989; Gualberto et al 1989; Hiesel et al 1989) The amino acid changed after RNA editing results in more similar to other orthologous proteins in other plants at the corresponding position (Gualberto et al 1989) Editing in both plastid and mitochondrion were subsequently reported in different lineages of land plant including angiosperms gymnosperms ferns lycophytes and bryophytes Phalaenopsis aphrodite subsp formosana is one of the two endemic moth orchids in Taiwan Previously using Sanger sequencing 42 editing sites were found in the 24 chloroplast transcripts of P aphrodite and two editing sites were found in the non-protein coding region (Zeng et al 2007) However the RNA editing status of the whole organelle transcriptome have not been fully understood so in this study by using high-throughput sequencing the transcripts of the chloroplast and mitochondrion from leaves and flowers of P aphrodite were analyzed to obtain the detailed RNA editing status MATERIALS AND METHODS The Phalaenopsis aphrodite subsp formosana cultivar TS97 used in this study was purchased from Taiwan Sugar Corporation (Tainan Taiwan) Plastid and mitochondrion were isolated from leaf and flower by using Percoll step gradient The genomic DNA were purified by using Tri-Plant Genomic DNA Reagent Kit (Geneaid Taiwan) The mtDNA was sequenced by using both Roche 454 pyrosequencing and Illumina HiSeq2500 platforms Sequencing reads from two different platforms of Illumina and Roche 454 were assembled by SPAdes For gene annotation of mitochondrial gene the ORF Finder and BlastX were used to search protein-coding gene In addition the tRNAscan-SE was used to search for potential tRNA genes and Ribosomal RNA Blast was used to search the rRNA sequence The putative editing sites were predicted by using the PREP-chloroplast and PREP-mitochondrion search program with the plastid and mitochondrial genome sequence The RNA from leaf or floral tissue were purified by using Trizol reagent (Omic Bio Taiwan) RNA from leaf and floral tissues underwent NGS with Ion Proton and Illumina Hiseq 2000 platforms respectively Transcriptome analysis involved the use of CLC Genomic Workbench 7 5 1 (CLC Bio Aarhus Denmark) The RNA sequence reads were mapped to the cpDNA (Accession number: AY916449) and mtDNA template of P aphrodite to determine the location of the RNA editing site the total read count and depth of coverage were calculated The degree of nucleotide conversion at each site was based on the reading of nucleotide conversion divided by the total reads The frequency of C-to-U or U-to-C conversion at a specific nucleotide position must be greater than 5% which is defined as the RNA editing site In addition we manually deleted more than five RNA edits in the homopolymer region which was considered a potential sequencing error The reads per kilobase of exon model per million mapped reads (RPKM) values was measured to estimate the gene expression profile RESULTS AND DISCUSSION In plastid transcripts we identified total 137 edits with 126 C-to-U and 11 U-to-C conversions representing an average of 0 09% of the nucleotides examined in moth orchid Compared with previous study 93 sites were newly discovered The 137 RNA edits revealing an average of 0 09% of the plastid nucleotides (149 kb) In leaf and floral tissues we found 110 and 106 RNA edits respectively There are 79 edits commonly present in the two tissues and 31 and 27 edits are specific in the leaf and floral tissues respectively Among 137 edits 79 edits were involved in protein?coding transcripts and the 58 nucleotide conversions caused the non?synonymous substitution (Table 1) In the trnM (cau) transcript both leaf and floral tissues showed efficient RNA editing (>60%) at 52 826 genomic position The result of C-to-U conversion in the trnM (cau) of Phalaenopsis resulted in increased nucleotide conservation among plant species and prompted the standard clover?leaf structure of trnM (cau) The mtDNA of P aphrodite was sequenced and assembled into 77 contigs After selection 44 contigs with a total length of 576 203 bp that are considered mtDNA After annotation the mtDNA of Phalaenopsis encodes 38 protein-coding 3 rRNA and 9 tRNA genes In addition among sequence derived from plastid DNA we found 31 coding genes including 19 protein-coding genes (7 have complete ORF and the other 12 contain only partial gene sequences) and 12 tRNA genes Among the 21 tRNA genes in mtDNA 9 are mitochondrial origin and 12 are plastid origin Analysis of the codons conferred by tRNA genes revealed that 21 tRNA genes may only recognize 45 of the 61 codons which it is not sufficient for translation Therefore for translation in the mitochondria of Phalaenopsis tRNA imported from the nucleus may be needed In mitochondrial transcripts we identified a total of 1032 RNA editing sites including 1 020 C-to-U and 12 U-to-C conversions on average representing 0 18% of detected nucleotides In all editing sites 978 and 898 edits were found in the leaf and floral tissues respectively A total of 844 edits were commonly identified both in the two tissues while the leaf or floral tissues have 33 and 53 specific edits respectively The leaf and floral tissues contained 894 and 837 edits respectively with a total of 790 edits in common CONCLUSION By high-throughput sequencing 137 and 1 032 RNA edits were identified in plastid and mitochondrial transcriptomes of P aphrodite respectively the highest number reported so far in monocots In intron IGS 3’ and 5’ UTR we identified several RNA editing sites and the secondary structure might change after RNA editing in plastid and mitochondrial transcripts These results suggested that editing in some cis-elements of primary transcripts might be important for splicing in moth orchid Additionally RNA editing occurs in trnM in plastid plays an important role in formation of standard clover-leaf tRNA structure

Date of Award	2020
Original language	English
Supervisor	Ching-Chun Chang (Supervisor)