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.

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'.

Spatiogenetic characterization of S receptor kinase (SRK) alleles in naturalized populations of Raphanus sativus L. var. raphanistroides on Yakushima island.

In various coastal areas of Japan, naturalized radish populations are observed. Radish is a cruciferous plant and exhibits self-incompatibility, involving a system controlled by a single locus with multiple S alleles. Although the S allele diversity of radish cultivars and wild radishes has been characterized, the S allele distribution in naturalized populations has not yet been analyzed in relation to the positions of the plants in situ. Here, we show the S allele distribution in naturalized radish populations of Yakushima, a small island in the East China Sea, with positions of the plants. Radish plants were sampled in coastal areas in Yakushima, and their S alleles were detected and characterized. Most of the S alleles had been previously identified in radish cultivars. However, four novel S alleles, which may be unique to Yakushima, were also found. Moreover, seeds in siliques from plants growing in the study areas were sampled, and S allele determination in DNA extracted from these seeds suggested that the plants had exchanged their pollen among their close neighbors. There was also a problem in that the PCR amplification of some SRK alleles was difficult because of their sequence diversity in the naturalized populations, as occurs in cultivars. Our results suggest that the exchange of S alleles between cultivars and naturalized populations occurs and that S alleles in naturalized populations are highly diverse. The methodology established in our study should be applicable to other self-incompatible species to dissect the diversity of S allele distribution in naturalized populations.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Parallel evolution of dominant pistil-side self-incompatibility suppressors in Arabidopsis.

Selfing is a frequent evolutionary trend in angiosperms, and is a suitable model for studying the recurrent patterns underlying adaptive evolution. Many plants avoid self-fertilization by physiological processes referred to as self-incompatibility (SI). In the Brassicaceae, direct and specific interactions between the male ligand SP11/SCR and the female receptor kinase SRK are required for the SI response. Although Arabidopsis thaliana acquired autogamy through loss of these genes, molecular evolution contributed to the spread of self-compatibility alleles requires further investigation. We show here that in this species, dominant SRK silencing genes have evolved at least twice. Different inverted repeat sequences were found in the relic SRK region of the Col-0 and C24 strains. Both types of inverted repeats suppress the functional SRK sequence in a dominant fashion with different target specificities. It is possible that these dominant suppressors of SI contributed to the rapid fixation of self-compatibility in A. thaliana.

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.].

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

A stigmatic gene confers interspecies incompatibility in the Brassicaceae.

Pre-zygotic interspecies incompatibility in angiosperms is a male-female relationship that inhibits the formation of hybrids between two species. Here, we report on the identification of STIGMATIC PRIVACY 1 (SPRI1), an interspecies barrier gene in Arabidopsis thaliana. We show that the rejection activity of this stigma-specific plasma membrane protein is effective against distantly related Brassicaceae pollen tubes and is independent of self-incompatibility. Point-mutation experiments and functional tests of synthesized hypothetical ancestral forms of SPRI1 suggest evolutionary decay of SPRI1-controlled interspecies incompatibility in self-compatible A. thaliana. Hetero-pollination experiments indicate that SPRI1 ensures intraspecific fertilization in the pistil when pollen from other species are present. Our study supports the idea that SPRI1 functions as a barrier mechanism that permits entrance of pollen with an intrinsic signal from self species.

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.

Divergence of RNA editing among Arabidopsis species.

RNA editing altered the RNA sequence by replacing the C nucleotide to U in the organellar genomes of plants. RNA editing status sometimes differed among distant species. The pattern of conservation and variation of RNA editing status made it possible to evaluate evolutionary mechanisms impacting functional aspects of RNA editing. In this study, divergence of RNA editing in the chloroplast genome among Arabidopsis species was analyzed to determine 9 losses and 1 gain in RNA editing. All changes in A. thaliana lineage resulted from changes to the chloroplast genome sequence, whereas changes in the A. lyrata / halleri lineage were possibly due to exclusive changes in the nuclear editing factors. One loss of RNA editing in A. lyrata was caused by a deficiency in the PPR gene OTP80. The changes in RNA editing occurred approximately every two million years and were not observed at functionally important sites. These results highlight the conserved nature of RNA editing status suggesting the importance of RNA editing during evolution.

Multilayered dominance hierarchy in plant self-incompatibility.

KEY MESSAGE: Epigenetic dominance modifier. In polymorphic loci, complex genetic dominance relationships between alleles are often observed. In plants, control of self-incompatibility (SI) expression via allelic interactions in the Brassicaceae is the best-known example of such mechanisms. Here, with emphasis on two recently published papers, we review the progress toward understanding the dominance regulatory mechanism of SI in the Brassicaceae. Multiple small RNA genes linked to the Self-incompatibility (S) locus were found in both Brassica and Arabidopsis genera. Mono-allelic gene expression of the male determinant of SI, SP11/SCR, from a dominant S-allele is under epigenetic control by such small RNA genes. Possible evolutionary trajectories leading to the formation of multilayered dominance hierarchy in Brassicaceae are discussed. We also identify some remaining questions for future studies.

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.

A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy1-3. Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S44 > S60 > S40 > S29). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study4, we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

Non-self- and self-recognition models in plant self-incompatibility.

The mechanisms by which flowering plants choose their mating partners have interested researchers for a long time. Recent findings on the molecular mechanisms of non-self-recognition in some plant species have provided new insights into self-incompatibility (SI), the trait used by a wide range of plant species to avoid self-fertilization and promote outcrossing. In this Review, we compare the known SI systems, which can be largely classified into non-self- or self-recognition systems with respect to their molecular mechanisms, their evolutionary histories and their modes of evolution. We review previous controversies on haplotype evolution in the gametophytic SI system of Solanaceae species in light of a recently elucidated non-self-recognition model. In non-self-recognition SI systems, the transition from self-compatibility (SC) to SI may be more common than previously thought. Reversible transition between SI and SC in plants may have contributed to their adaptation to diverse and fluctuating environments.

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.

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.