Erratum: Molecular identification of the causal locus for the petaloid phenotype in Daucus carota.

[This corrects the article on p. 186 in vol. 69, PMID: 31086497.].

Molecular identification of the causal locus for the petaloid phenotype in Daucus carota.

Homeotic alteration phenotype of the flowers in Daucus carota are widely used for hybrid breeding, consequently molecular markers tightly-linked to such phenotype are in demand. Here we report the identification of a gene locus responsible for the phenotypic expression of stamen conversion into a petal-like structure, or petaloid. Using a segregating population and sequencing analysis of two bulked populations, we discovered a large contributing peak on the long arm of chromosome 4. DcMADS2, a homolog of the B-class floral homeotic gene PISTILLATA, found at the center of the peak region, was considered the strongest candidate causal gene. We established PCR primers that could be used to distinguish the two DcMADS2 alleles linked to each petaloid- and non-petaloid-phenotype.

MYB transcription factor gene involved in sex determination in Asparagus officinalis.

Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A. officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A. officinalis.

The Restorer-of-fertility-like 2 pentatricopeptide repeat protein and RNase P are required for the processing of mitochondrial orf291 RNA in Arabidopsis.

Eukaryotes harbor mitochondria obtained via ancient symbiosis events. The successful evolution of energy production in mitochondria has been dependent on the control of mitochondrial gene expression by the nucleus. In flowering plants, the nuclear-encoded pentatricopeptide repeat (PPR) superfamily proteins are widely involved in mitochondrial RNA metabolism. Here, we show that an Arabidopsis nuclear-encoded RNA-binding protein, Restorer-of-fertility-like PPR protein 2 (RFL2), is required for RNA degradation of the mitochondrial orf291 transcript via endonucleolytic cleavage of the transcript in the middle of its reading frame. Both in vivo and in vitro, this RNA cleavage requires the activity of mitochondrial proteinaceous RNase P, which is possibly recruited to the site by RFL2. The site of RNase P cleavage likely forms a tRNA-like structure in the orf291 transcript. This study presents an example of functional collaboration between a PPR protein and an endonuclease in RNA cleavage. Furthermore, we show that the RFL2-binding region within the orf291 gene is hypervariable in the family Brassicaceae, possibly correlated with the rapid evolution of the RNA-recognition interfaces of the RFL proteins.

Cullin1-P is an Essential Component of Non-Self Recognition System in Self-Incompatibility in Petunia.

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCFSLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCFSLF, and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S7-SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCFSLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI.

Mutagenesis of individual pentatricopeptide repeat motifs affects RNA binding activity and reveals functional partitioning of Arabidopsis PROTON gradient regulation3.

Pentatricopeptide repeat (PPR) proteins bind RNA and act in multiple eukaryotic processes, including RNA editing, RNA stability, and translation. Here, we investigated the mechanism underlying the functional versatility of Arabidopsis thaliana proton gradient regulation3 (PGR3), a chloroplast protein harboring 27 PPR motifs. Previous studies suggested that PGR3 acts in (1) stabilization of photosynthetic electron transport L (petL) operon RNA, (2) translation of petL, and (3) translation of ndhA. We showed here that replacement of the 4th amino acid of the 12th PPR with nonpolar or charged amino acids abolished functions (1) and (2) but not (3) of PGR3 by compromising the function of this specific PPR. This discovery enabled us to knock out the RNA binding ability of individual PPR motifs. Consequently, we showed that the 16 N-terminal PPRs were sufficient for function (1) via sequence-specific RNA binding, whereas the 11 C-terminal motifs were essential for functions (2) and (3) by activating translation. We also clarified that the 14th amino acid of the 12th PPR should be positively charged to make the PPR functionally active. Our finding opens up the possibility of selectively manipulating the functions of PPR proteins.

A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins.

The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA-binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA-protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.

The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing.

RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of pentatricopeptide repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the C terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue. Here, we describe two mutants, otp71 and otp72, disrupted in genes encoding mitochondrial E-type PPR proteins with single editing defects in ccmFN 2 and rpl16 transcripts, respectively. Comparisons between the E domains of these proteins and previously reported editing factors from chloroplasts suggested that there are characteristic differences in the proteins between the two organelles. To test this, we swapped E domains between two mitochondrial and two chloroplast editing factors. In all cases investigated, E domains from the same organelle (chloroplast or mitochondria) were found to be exchangeable; however, swapping the E domain between organelles led to non-functional editing factors. We conclude that the E domains of mitochondrial and plastid PPR proteins are not functionally equivalent, and therefore that an important component of the putative editing complexes in the two organelles may be different.

Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution.

Eukaryotic cells have harbored mitochondria for at least 1.5 billion years in an apparently mutually beneficial symbiosis. Studies on the agronomically important crop trait cytoplasmic male sterility (CMS) have suggested the semblance of a host-parasite relationship between the nuclear and mitochondrial genomes, but molecular evidence for this is lacking. Key players in CMS systems are the fertility restorer (Rf) genes required for the development of a functional male gametophyte in plants carrying a mitochondrial CMS gene. In the majority of cases, Rf genes encode pentatricopeptide repeat (PPR) proteins. We show that most angiosperms for which extensive genomic sequence data exist contain multiple PPR genes related to Rf genes. These Rf-like genes show a number of characteristic features compared with other PPR genes, including chromosomal clustering and unique patterns of evolution, notably high rates of nonsynonymous to synonymous substitutions, suggesting diversifying selection. The highest probabilities of diversifying selection were seen for amino acid residues 1, 3, and 6 within the PPR motif. PPR proteins are involved in RNA processing, and mapping the selection data to a predicted consensus structure of an array of PPR motifs suggests that these residues are likely to form base-specific contacts to the RNA ligand. We suggest that the selection patterns on Rf-like genes reveal a molecular "arms-race" between the nuclear and mitochondrial genomes that has persisted throughout most of the evolutionary history of angiosperms.

Rice MPR25 encodes a pentatricopeptide repeat protein and is essential for RNA editing of nad5 transcripts in mitochondria.

Pentatricopeptide repeat (PPR) proteins are involved in the modification of organelle transcripts. In this study, we investigated the molecular function in rice of the mitochondrial PPR-encoding gene MITOCHONDRIAL PPR25 (MPR25), which belongs to the E subgroup of the PPR family. A Tos17 knockout mutant of MPR25 exhibited growth retardation and pale-green leaves with reduced chlorophyll content during the early stages of plant development. The photosynthetic rate in the mpr25 mutant was significantly decreased, especially under strong light conditions, although the respiration rate did not differ from that of wild-type plants. MPR25 was preferentially expressed in leaves. FLAG-tagged MPR25 accumulated in mitochondria but not in chloroplasts. Direct sequencing revealed that the mpr25 mutant fails to edit a C-U RNA editing site at nucleotide 1580 of nad5, which encodes a subunit of complex I (NADH dehydrogenase) of the respiratory chain in mitochondria. RNA editing of this site is responsible for a change in amino acid from serine to leucine. Recombinant MPR25 directly interacted with the proximal region of the editing site of nad5 transcripts. However, the NADH dehydrogenase activity of complex I was not affected in the mutant. By contrast, genes encoding alternative NADH dehydrogenases and alternative oxidase were up-regulated. The mpr25 mutant may therefore provide new information on the coordinated interaction between mitochondria and chloroplasts.

Function of PPR proteins in plastid gene expression.

PPR proteins form a huge family in flowering plants and are involved in RNA maturation in plastids and mitochondria. These proteins are sequence-specific RNA-binding proteins that recruit the machinery of RNA processing. We summarize progress in the research on the functional mechanisms of divergent RNA maturation and on the mechanism by which RNA sequences are recognized. We further focus on two topics. RNA editing is an enigmatic process of RNA maturation in organelles, in which members of the PLS subfamily contribute to target site recognition. As the first topic, we speculate on why the PLS subfamily was selected by the RNA editing machinery. Second, we discuss how the regulation of plastid gene expression contributes to efficient photosynthesis. Although the molecular functions of PPR proteins have been studied extensively, information on the physiological significance of regulation by these proteins remains very limited.

SHI family transcription factors regulate an interspecific barrier.

Pre-zygotic interspecies incompatibility in angiosperms is an important mechanism to prevent unfavourable hybrids between species. Here we report our identification of STIGMATIC PRIVACY 2 (SPRI2), a transcription factor that has a zinc-finger domain and regulates interspecies barriers in Arabidopsis thaliana, via genome-wide association study. Knockout analysis of SPRI2/SRS7 and its paralogue SPRI2-like/SRS5 demonstrated their necessity in rejecting male pollen from other species within female pistils. Additionally, they govern mRNA transcription of xylan O-acetyltransferases (TBL45 and TBL40) related to cell wall modification, alongside SPRI1, a pivotal transmembrane protein for interspecific pollen rejection. SPRI2/SRS7 is localized as condensed structures in the nucleus formed via liquid-liquid phase separation (LLPS), and a prion-like sequence in its amino-terminal region was found to be responsible for the formation of the condensates. The LLPS-regulated SPRI2/SRS7 discovered in this study may contribute to the establishment of interspecific reproductive barriers through the transcriptional regulation of cell wall modification genes and SPRI1.

The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein.

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.

OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1.

Over 20 proteins of the pentatricopeptide repeat (PPR) family have been demonstrated to be involved in RNA editing in plant mitochondria and chloroplasts. All of these editing factors contain a so-called 'E' domain that has been shown to be essential for editing to occur. The presumption has been that this domain recruits the (unknown) editing enzyme to the RNA. In this report, we show that not all putative E-class PPR proteins are directly involved in RNA editing. Disruption of the OTP70 gene leads to a strong defect in splicing of the plastid transcript rpoC1, leading to a virescent phenotype. The mutant has a chloroplast transcript pattern characteristic of a reduction in plastid-encoded RNA polymerase activity. The E domain of OTP70 is not required for splicing, and can be deleted or replaced by the E domain from the known editing factor CRR4 without loss of rpoC1 splicing. Furthermore, the E domain of OTP70 is incapable of inducing RNA editing when fused to the RNA binding domain of CRR4. We conclude that the truncated E domain of OTP70 is no longer functional in RNA editing, and that the protein has acquired a new function in promoting RNA splicing.

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights evolutionary constraints on plastid genomes.

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.

Suppressed expression of Retrograde-Regulated Male Sterility restores pollen fertility in cytoplasmic male sterile rice plants.

Conflict/reconciliation between mitochondria and nuclei in plants is manifested by the fate of pollen (viable or nonviable) in the cytoplasmic male sterility (CMS)/fertility restoration (Rf) system. Through positional cloning, we identified a nuclear candidate gene, RETROGRADE-REGULATED MALE STERILITY (RMS) for Rf17, a fertility restorer gene for Chinese wild rice (CW)-type CMS in rice (Oryza sativa L.). RNA interference-mediated gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is a candidate gene to be regulated via retrograde signaling. We found that a reduced-expression allele of the RMS gene restored fertility in haploid pollen, whereas a normal-expression allele caused pollen to die in the CW-type CMS. RMS encodes a mitochondrial protein, 178 aa in length, of unknown function, unlike the majority of other Rf genes cloned thus far, which encode pentatricopeptide repeat proteins. The unique features of RMS provide novel insights into retrograde signaling and CMS.

Plant physiology: ATP at the center of self-recognition.

ATP acts as the common currency of metabolic activity in all life forms. A recent study uses inter-specific transfer of the self-recognition module in plants to enable live monitoring of the cellular status in vivo, revealing the pivotal role of ATP in signaling.

The selfing syndrome and beyond: diverse evolutionary consequences of mating system transitions in plants.

The shift from outcrossing to self-fertilization (selfing) is considered one of the most prevalent evolutionary transitions in flowering plants. Selfing species tend to share similar reproductive traits in morphology and function, and such a set of traits is called the 'selfing syndrome'. Although the genetic basis of the selfing syndrome has been of great interest to evolutionary biologists, knowledge of the causative genes or mutations was limited until recently. Thanks to advances in population genomic methodologies combined with high-throughput sequencing technologies, several studies have successfully unravelled the molecular and genetic basis for evolution of the selfing syndrome in Capsella, Arabidopsis, Solanum and other genera. Here we first introduce recent research examples that have explored the loci, genes and mutations responsible for the selfing syndrome traits, such as reductions in petal size or in pollen production, that are mainly relevant to pre-pollination processes. Second, we review the relationship between the evolution of selfing and interspecific pollen transfer, highlighting the findings of post-pollination reproductive barriers at the molecular level. We then discuss the emerging view of patterns in evolution of the selfing syndrome, such as the pervasive involvement of loss-of-function mutations and the relative importance of selection versus neutral degradation. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Transcriptome map of plant mitochondria reveals islands of unexpected transcribed regions.

BACKGROUND: Plant mitochondria contain a relatively large amount of genetic information, suggesting that their functional regulation may not be as straightforward as that of metazoans. We used a genomic tiling array to draw a transcriptomic atlas of Oryza sativa japonica (rice) mitochondria, which was predicted to be approximately 490-kb long. RESULTS: Whereas statistical analysis verified the transcription of all previously known functional genes such as the ones related to oxidative phosphorylation, a similar extent of RNA expression was frequently observed in the inter-genic regions where none of the previously annotated genes are located. The newly identified open reading frames (ORFs) predicted in these transcribed inter-genic regions were generally not conserved among flowering plant species, suggesting that these ORFs did not play a role in mitochondrial principal functions. We also identified two partial fragments of retrotransposon sequences as being transcribed in rice mitochondria. CONCLUSION: The present study indicated the previously unexpected complexity of plant mitochondrial RNA metabolism. Our transcriptomic data (Oryza sativa Mitochondrial rna Expression Server: OsMES) is publicly accessible at [http://bioinf.mind.meiji.ac.jp/cgi-bin/gbrowse/OsMes/#search].

The evolution of RNA editing and pentatricopeptide repeat genes.

The pentatricopeptide repeat (PPR) is a degenerate 35-amino-acid structural motif identified from analysis of the sequenced genome of the model plant Arabidopsis thaliana. From the wealth of sequence information now available from plant genomes, the PPR protein family is now known to be one of the largest families in angiosperm species, as most genomes encode 400-600 members. As the number of PPR genes is generally only c. 10-20 in other eukaryotic organisms, including green algae, the family has obviously greatly expanded during land plant evolution. This provides a rare opportunity to study selection pressures driving a 50-fold expansion of a single gene family. PPR proteins are sequence-specific RNA-binding proteins involved in many aspects of RNA processing in organelles. In this review, we will summarize our current knowledge about the evolution of PPR genes, and will discuss the relevance of the dramatic expansion in the family to the functional diversification of plant organelles, focusing primarily on RNA editing.