PPR

Arnal N, Quadrado M, Simon M & Mireau H 2014 A restorer-of-fertility like pentatricopeptide repeat gene directs ribonucleolytic processing within the coding sequence of rps3-rpl16 and orf240a mitochondrial transcripts in Arabidopsis thaliana. Plant J 78:134-145.

  • we describe the function of the RFL9 gene from Arabidopsis thaliana
  • it is associated with ribonucleolytic cleavages within the coding sequences of rps3-rpl16 and orf240a mitochondrial transcripts in the Col-0 accession
  • RFL9 is active in only a few Arabidopsis accessions genetically close to Col-0
  • Rf genes are grouped in clusters together with highly homologous RFL genes and are obviously subject to active local duplications
  • RFL genes also tend to be poorly conserved across species
  • Rf and RFL genes are under positive selective pressures that drive alteration and multiplication of their sequences, unlike other PPR genes for which natural selection acts negatively to maintain the same function
  • the subgroup of RFL genes in Arabidopsis thaliana comprises 26 genes that encode PPR proteins strongly predicted to target mitochondria
  • very little is known about the function of these genes
  • none of them has been associated with the suppression of a CMS so far
  • three members of the RFL subgroup have been the subjects of recent functional characterization in Arabidopsis
  • the concerned RFL proteins are all involved in 5'-end maturation of different mitochondrial mRNAs
  • RFL9 is involved in processing events located within the coding sequences of mitochondrial transcripts
  • these concern the rps3 transcript, encoding an essential ribosomal protein, and orf240a mRNA, which has been suspected to be a CMS-causing gene in Arabidopsis
  • while analyzing mitochondrial mRNA processing in a collection of Arabidopsis ppr mutants, we noticed that differential processing occurred for the rps3-rpl16 transcript between the Columbia (Col-0) and Wassileskija (Ws) Arabidopsis accessions
  • the rps3-rpl16 mitochondrial mRNA is a di-cistronic transcript carrying both the rps3 and rpl16 open reading frames (ORFs)
  • circular (c)RT-PCR performed with primers specific to the rps3-rpl16 mRNA produces two distinct bands from Col-0 RNA
  • a single amplification product is generated from Ws RNA
  • cloning and sequencing of the shorter cRT-PCR product indicated that it was heterogeneous and corresponded to truncated rps3-rpl16 transcripts bearing predominantly a 5' end mapping between 15 and 17 nucleotides downstream of the rps3 AUG
  • rps3-rpl16 di-cistronic mRNAs accumulate to much higher levels in Col-0 plants
  • a second and slightly smaller signal was systematically detected in Col-0 but never in Ws
  • both di-cistronic and potentially mono-cistronic rps3-rpl16 messenger RNAs accumulate in the mitochondria of Col-0 plants
  • the mono-cistronic transcripts are significantly less abundant than the di-cistronic ones
  • no amplification product could be generated for the RFL5, RFL9, RFL12, RFL15 or RFL26 genes from Ws DNA, indicating that these genes were either absent or profoundly different in this accession compared with the Col-0 sequence
  • T-DNA insertion mutants could be identified for the RFL5, RFL9 and RFL12 genes in the Col-0 genetic background from the public mutant collections
  • rfl5, rfl9 and rfl12 homozygous mutant plants were selected and then tested for their ability to process the rps3-rpl16 RNA between positions +15 to +17
  • both rfl9-1 and rfl9-2 mutants gave a single amplification product corresponding to the longest rps3-rpl16 transcript and have lost the ability to perform cleavage at the +15 to +17 positions within the rps3 ORF
  • these results clearly indicate that RFL9 (At1g62910) is the gene that specifies rps3 mRNA processing 15–17 nucleotides downstream of the AUG in Col-0 plants
  • from these 48 accessions, only the Mh-1 accession showed the same pattern as Col-0
  • the identified cleavage within the rps3 ORF is a rather rare phenomenon in Arabidopsis
  • both Col-0 and Mh-1 Arabidopsis accessions originated from Poland
  • the presence of this gene could be restricted to accessions sharing a recent evolution history with Col-0
  • this large-scale phylogeny allowed us to select a group of 25 Arabidopsis accessions classified as evolutionarily highly related to Col-0 on the basis of the 341 SNP markers selected
  • out of these 25 accessions, only five showed the same profile as the Col-0 plants
  • this further proved that the maturation trait directed by RFL9 is poorly represented in A. thaliana
  • two pairs of specific primers were designed to amplify the RFL9 coding sequence and parts of its 3' and 5' non-coding regions
  • these PCR amplifications were conducted on the 25 Arabidopsis accessions forming the Col-0 cluster
  • amongst the accessions that do not process the rps3 RNA at the +15 to +17 positions, the RFL9 amplification was successful only for the accessions Pr-0, Mz-0b, Or-0 and El-0
  • four nucleotides within the coding sequence differed from the RFL9 sequence in Col-0
  • one difference (corresponding to T to C replacement on the third position of codon 377) does not cause any change in the RFL9 protein
  • the other three concern the same codon and transform the aspartic acid at position 152 (GAT) into a serine (AGC)
  • the position of this aspartic acid corresponds to the first amino acid of the fourth PPR repeat of the RFL9 protein
  • rfl9-2 mutants were transformed with a construct allowing the expression of a modified version of RFL9 bearing these three corresponding nucleotide changes
  • the generated transformants were analyzed by cRT-PCR and showed a complete inability to process the rps3-rpl16 transcript between the +15 and +17 positions
  • the presence of an aspartic residue at position 152 is essential for RFL9 activity
  • we investigated the possibility that RFL9 could be also involved in the processing of a transcript derived from one of the numerous non-conserved ORFs encoded on the Arabidopsis mitochondrial genome
  • this led us to identify that a 554-bp DNA segment of the rps3 5' region and comprising the cleavage site associated with RFL9 is duplicated and constitutes part of the upstream region of the orf240a mitochondrial gene
  • orf240a is differentially processed between Col-0 and Ws accessions
  • analysis of the smaller amplification product, which is specific to Col-0 plants, showed that it derived from truncated orf240a transcripts bearing an identical 3' end but with a 5' end situated 152 bp downstream of the predicted orf240a AUG codon
  • both identified orf240a 5' extremities (−28 and +152) were identical to the 5' ends (−163 and +17) found for rps3-rpl16 transcripts in Col-0 plants
  • Northern blot analysis detected a single orf240a mRNA species in both accessions, suggesting strongly that 5'-truncated orf240a transcripts accumulate to very low levels in Col-0
  • RFL9 also specifies the orf240a mRNA processing in Col-0 plants at a position that is identical to the +15/+17 cleavage site identified in the case of the rps3-rpl16 transcripts
  • rfl9 mutants do not show any improved growing capacity compared with wild-type plants
  • RFL9 does not significantly limit mitochondrial translation in Col-0 plants
  • this lack of an apparent negative effect of RFL9 is very likely due the fact that RFL9-associated cleavage concerns a fraction of rps3-rpl16 transcripts that is insufficient to have a significant impact on the production of the RPS3 protein
  • we noticed that rps3-rpl16 di-cistronic mRNAs accumulate at a much higher level in Col-0 compared with Ws plants
  • this increase might have evolved to compensate for the potential negative effect of RFL9 on rps3 expression
  • orf240a has been suspected of being the gene causing an alloplasmic CMS involving Arabidopsis as the cytoplasmic donor
  • the protein it encodes shares significant homology with the ORF224 responsible for the polima CMS in rapeseed
  • rfl9 mutant plants were not found to be male sterile
  • this result may indicate that RFL9 is not a restorer of this CMS as suggested by its very partial effect on orf240a transcript accumulation
  • alternatively, one cannot exclude that RFL9 may have contributed to the establishment of a restoration against the orf240a CMS and that today another genetically fixed and more active restorer gene is present in Arabidopsis, masking all effects that RFL9 might have on ORF240a production
  • the presence of an active RFL9 copy appears to be limited to the accessions genetically close to Col-0, which supports a recent emergence of this gene
  • constant sequence reshuffling, associated with other types of sequence variations, contribute to the emergence of new RFL gene copies that may sometimes coincide with the creation of true Rf genes
  • new copies of RFL genes may have a negative impact on the expression of essential mitochondrial genes if the associated cleavage occurs within ORFs and could become detrimental for plant fitness if they were expressed to a sufficient level
  • several members of the RFL subfamily are under tight control by different classes of short regulatory RNAs
  • RFL9 (At1g62910) was described as likely to be under the double control of MIR161 and TAS2
  • the function associated with RFL9 provides a potential explanation for this short RNA-mediated control, which may be associated with the necessity to limit its detrimental influence on rps3 expression
  • Asp152Ser modification is responsible for the loss of rps3 and orf240a processing in Pr-0, Mz-0b, Or-0 and El-0 Arabidopsis accessions