Arabidopsis leucine-rich repeat malectin receptor-like kinases regulate pollen-stigma interactions.

Flowering plants contain tightly controlled pollen-pistil interactions required for promoting intraspecific fertilization and preventing interspecific hybridizations. In Arabidopsis (Arabidopsis thaliana), several receptor kinases (RKs) are known to regulate the later stages of intraspecific pollen tube growth and ovular reception in the pistil, but less is known about RK regulation of the earlier stages. The Arabidopsis RECEPTOR-LIKE KINASE IN FLOWERS1 (RKF1)/RKF1-LIKE (RKFL) 1-3 cluster of 4 leucine-rich repeat malectin (LRR-MAL) RKs was previously found to function in the stigma to promote intraspecific pollen hydration. In this study, we tested additional combinations of up to 7 Arabidopsis LRR-MAL RK knockout mutants, including RKF1, RKFL1-3, LysM RLK1-INTERACTING KINASE1, REMORIN-INTERACTING RECEPTOR1, and NEMATODE-INDUCED LRR-RLK2. These LRR-MAL RKs were discovered to function in the female stigma to support intraspecific Arabidopsis pollen tube growth and to establish a prezygotic interspecific barrier against Capsella rubella pollen. Thus, this study uncovered additional biological functions for this poorly understood group of RKs in regulating the early stages of Arabidopsis sexual reproduction.

Genetic basis and timing of a major mating system shift in Capsella.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C. orientalis occurred < 2.6 Mya and maps as a dominant trait to the S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude.

Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the Qinghai-Tibet Plateau (QTP) about 4,000 m above sea level and represents an attractive model system for studying speciation and ecological adaptation in extreme environments. We assembled a draft genome sequence of 234.72 Mb encoding 27,019 genes and investigated its origin and adaptive evolutionary mechanisms. Phylogenomic analyses based on 4,586 single-copy genes revealed that C. himalaica is most closely related to Capsella (estimated divergence 8.8 to 12.2 Mya), whereas both species form a sister clade to Arabidopsis thaliana and Arabidopsis lyrata, from which they diverged between 12.7 and 17.2 Mya. LTR retrotransposons in C. himalaica proliferated shortly after the dramatic uplift and climatic change of the Himalayas from the Late Pliocene to Pleistocene. Compared with closely related species, C. himalaica showed significant contraction and pseudogenization in gene families associated with disease resistance and also significant expansion in gene families associated with ubiquitin-mediated proteolysis and DNA repair. We identified hundreds of genes involved in DNA repair, ubiquitin-mediated proteolysis, and reproductive processes with signs of positive selection. Gene families showing dramatic changes in size and genes showing signs of positive selection are likely candidates for C. himalaica's adaptation to intense radiation, low temperature, and pathogen-depauperate environments in the QTP. Loss of function at the S-locus, the reason for the transition to self-fertilization of C. himalaica, might have enabled its QTP occupation. Overall, the genome sequence of C. himalaica provides insights into the mechanisms of plant adaptation to extreme environments.

Evidence for facultative protocarnivory in Capsella bursa-pastoris seeds.

Many plants derive nutrients by attracting, ensnaring and killing invertebrates, a process that is described as "protocarnivory". This has been observed in seeds of the weed Capsella bursa-pastoris, but it is unclear as to whether it confers any material benefit in terms of germination, establishment and development. In the present study, seeds were germinated in zero, low, medium and high nutrient soils in both the presence and absence of nematodes (Steinernema feltiae). Nematodes were attracted to the seeds, with many dying within three days. Germination rates and seedling fresh masses were higher at all nutrient levels, and seedling fresh lengths were higher in all but the zero nutrient treatment, in the presence of nematodes. After transplantation, young plant fresh root lengths and dried leaf and root masses were generally higher in plants that had been germinated in the presence of nematodes across all nutrient levels, with the majority of significant differences being observed in the low-nutrient treatment. Our findings suggest that protocarnivory may play a role in the germination, establishment and early development of C. bursa-pastoris, and that this process may be facultative, since differences between nematode and non-nematode treatments were generally more pronounced in soils with low nutrient levels.

Parallel Evolution of Common Allelic Variants Confers Flowering Diversity in Capsella rubella.

Flowering time is an adaptive life history trait. Capsella rubella, a close relative of Arabidopsis thaliana and a young species, displays extensive variation for flowering time but low standing genetic variation due to an extreme bottleneck event, providing an excellent opportunity to understand how phenotypic diversity can occur with a limited initial gene pool. Here, we demonstrate that common allelic variation and parallel evolution at the FLC locus confer variation in flowering time in C. rubella. We show that two overlapping deletions in the 5' untranslated region (UTR) of C. rubella FLC, which are associated with local changes in chromatin conformation and histone modifications, reduce its expression levels and promote flowering. We further show that these two pervasive variants originated independently in natural C. rubella populations after speciation and spread to an intermediate frequency, suggesting a role of this parallel cis-regulatory change in adaptive evolution. Our results provide an example of how parallel mutations in the same 5' UTR region can shape phenotypic evolution in plants.

Competitive ability of Capsella species with different mating systems and ploidy levels.

Background and Aims: Capsella is a model genus for studying the transition from outcrossing to selfing, with or without change in ploidy levels. The genomic consequences and changes in reproductive traits (selfing syndrome) associated with these shifts have been studied in depth. However, potential ecological divergence among species of the genus has not been determined. Among ecological traits, competitive ability could be relevant for selfing evolution, as selfing has been shown to be statistically associated with reduced competitiveness in a recent meta-analysis. Methods: We assessed the effect of competition on three Capsella species differing in their mating system and ploidy level. We used an experimental design where fitness related traits were measured in focal individuals with and without competitors. Key Results: The diploid selfer (C. rubella) was most sensitive to competition, whereas the tetraploid selfer (C. bursa-pastoris) performed the best, with the diploid outcrosser (C. grandiflora) being intermediate. Conclusions: These results add to the detailed characterization of Capsella species and highlight the possible roles of ecological context and ploidy in the evolutionary trajectories of selfing species.

Long-term balancing selection contributes to adaptation in Arabidopsis and its relatives.

BACKGROUND: In contrast to positive selection, which reduces genetic variation by fixing beneficial alleles, balancing selection maintains genetic variation within a population or species and plays crucial roles in adaptation in diverse organisms. However, which genes, genome-wide, are under balancing selection and the extent to which these genes are involved in adaptation are largely unknown. RESULTS: We performed a genome-wide scan for genes under balancing selection across two plant species, Arabidopsis thaliana and its relative Capsella rubella, which diverged about 8 million generations ago. Among hundreds of genes with shared coding-region polymorphisms, we find evidence for long-term balancing selection in five genes: AT1G35220, AT2G16570, AT4G29360, AT5G38460, and AT5G44000. These genes are involved in the response to biotic and abiotic stress and other fundamental biochemical processes. More intriguingly, for these genes, we detected significant ecological diversification between the two haplotype groups, suggesting that balancing selection has been very important for adaptation. CONCLUSIONS: Our results indicate that beyond the well-known S-locus genes and resistance genes, many loci are under balancing selection. These genes are mostly correlated with resistance to stress or other fundamental functions and likely play a more important role in adaptation to diverse habitats than previously thought.

Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis.

Directional control of tip-growing cells is essential for proper tissue organization and cell-to-cell communication in animals and plants. In the sexual reproduction of flowering plants, the tip growth of the male gametophyte, the pollen tube, is precisely guided by female cues to achieve fertilization. Several female-secreted peptides have recently been identified as species-specific attractants that directly control the direction of pollen tube growth. However, the method by which pollen tubes precisely and promptly respond to the guidance signal from their own species is unknown. Here we show that tip-localized pollen-specific receptor-like kinase 6 (PRK6) with an extracellular leucine-rich repeat domain is an essential receptor for sensing of the LURE1 attractant peptide in Arabidopsis thaliana under semi-in-vivo conditions, and is important for ovule targeting in the pistil. PRK6 interacted with pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which are important for pollen tube growth through activation of the signalling switch Rho GTPase ROP1 (refs 7, 8). PRK6 conferred responsiveness to AtLURE1 in pollen tubes of the related species Capsella rubella. Furthermore, our genetic and physiological data suggest that PRK6 signalling through ROPGEFs and sensing of AtLURE1 are achieved in cooperation with the other PRK family receptors, PRK1, PRK3 and PRK8. Notably, the tip-focused PRK6 accumulated asymmetrically towards an external AtLURE1 source before reorientation of pollen tube tip growth. These results demonstrate that PRK6 acts as a key membrane receptor for external AtLURE1 attractants, and recruits the core tip-growth machinery, including ROP signalling proteins. This work provides insights into the orchestration of efficient pollen tube growth and species-specific pollen tube attraction by multiple receptors during male-female communication.

A receptor heteromer mediates the male perception of female attractants in plants.

Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth. Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube, the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1-MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana. MDIS1, MIK1 and MIK2 are plasma-membrane-localized receptor-like kinases with extracellular leucine-rich repeats and an intracellular kinase domain. LURE1 specifically binds the extracellular domains of MDIS1, MIK1 and MIK2, whereas mdis1 and mik1 mik2 mutant pollen tubes respond less sensitively to LURE1. Furthermore, LURE1 triggers dimerization of the receptors and activates the kinase activity of MIK1. Importantly, transformation of AtMDIS1 to the sister species Capsella rubella can partially break down the reproductive isolation barrier. Our findings reveal a new mechanism of the male perception of the female attracting signals.

The influence of population structure on gene expression and flowering time variation in the ubiquitous weed Capsella bursa-pastoris (Brassicaceae).

Population structure is a potential problem when testing for adaptive phenotypic differences among populations. The observed phenotypic differences among populations can simply be due to genetic drift, and if the genetic distance between them is not considered, the differentiation may be falsely interpreted as adaptive. Conversely, adaptive and demographic processes might have been tightly associated and correcting for the population structure may lead to false negatives. Here, we evaluated this problem in the cosmopolitan weed Capsella bursa-pastoris. We used RNA-Seq to analyse gene expression differences among 24 accessions, which belonged to a much larger group that had been previously characterized for flowering time and circadian rhythm and were genotyped using genotyping-by-sequencing (GBS) technique. We found that clustering of accessions for gene expression retrieved the same three clusters that were obtained with GBS data previously, namely Europe, the Middle East and Asia. Moreover, the three groups were also differentiated for both flowering time and circadian rhythm variation. Correction for population genetic structure when analysing differential gene expression analysis removed all differences among the three groups. This may suggest that most differences are neutral and simply reflect population history. However, geographical variation in flowering time and circadian rhythm indicated that the distribution of adaptive traits might be confounded by population structure. To bypass this confounding effect, we compared gene expression differentiation between flowering ecotypes within the genetic groups. Among the differentially expressed genes, FLOWERING LOCUS C was the strongest candidate for local adaptation in regulation of flowering time.

Cis-Regulatory Changes Associated with a Recent Mating System Shift and Floral Adaptation in Capsella.

The selfing syndrome constitutes a suite of floral and reproductive trait changes that have evolved repeatedly across many evolutionary lineages in response to the shift to selfing. Convergent evolution of the selfing syndrome suggests that these changes are adaptive, yet our understanding of the detailed molecular genetic basis of the selfing syndrome remains limited. Here, we investigate the role of cis-regulatory changes during the recent evolution of the selfing syndrome in Capsella rubella, which split from the outcrosser Capsella grandiflora less than 200 ka. We assess allele-specific expression (ASE) in leaves and flower buds at a total of 18,452 genes in three interspecific F1 C. grandiflora x C. rubella hybrids. Using a hierarchical Bayesian approach that accounts for technical variation using genomic reads, we find evidence for extensive cis-regulatory changes. On average, 44% of the assayed genes show evidence of ASE; however, only 6% show strong allelic expression biases. Flower buds, but not leaves, show an enrichment of cis-regulatory changes in genomic regions responsible for floral and reproductive trait divergence between C. rubella and C. grandiflora. We further detected an excess of heterozygous transposable element (TE) insertions near genes with ASE, and TE insertions targeted by uniquely mapping 24-nt small RNAs were associated with reduced expression of nearby genes. Our results suggest that cis-regulatory changes have been important during the recent adaptive floral evolution in Capsella and that differences in TE dynamics between selfing and outcrossing species could be important for rapid regulatory divergence in association with mating system shifts.

Beyond the thale: comparative genomics and genetics of Arabidopsis relatives.

For decades a small number of model species have rightly occupied a privileged position in laboratory experiments, but it is becoming increasingly clear that our knowledge of biology is greatly improved when informed by a broader diversity of species and evolutionary context. Arabidopsis thaliana has been the primary model organism for plants, benefiting from a high-quality reference genome sequence and resources for reverse genetics. However, recent studies have made a group of species also in the Brassicaceae family and closely related to A. thaliana a focal point for comparative molecular, genomic, phenotypic and evolutionary studies. In this Review, we emphasize how such studies complement continued study of the model plant itself, provide an evolutionary perspective and summarize our current understanding of genetic and phenotypic diversity in plants.

Frequent introgressions from diploid species contribute to the adaptation of the tetraploid Shepherd's purse (Capsella bursa-pastoris).

Adaptation is the most important ability for organisms to survive in diverse habitats. Animals have the option to escape from stressful environments, but plants do not. In plants, polyploids consist of about 30%-70% angiosperms and 95% ferns, of which some are important crops such as cotton and wheat. How polyploid plants adapt to various habitats has been a fundamental question remained largely unanswered. The tetraploid Shepherd's purse (Capsella bursa-pastoris) is one of the most successful plants on earth and has been distributed across the world, thus being an ideal model system for studying the adaptation of polyploids. We found that there are frequent introgressions from congeneric diploids to Shepherd's purse. Ecological niche modeling suggests that ecological differentiation is evident between the introgressed and non-introgressed C. bursa-pastoris, and the introgressions are a source of adaptation. This result links an evolutionary process to the adaptation of polyploids, and sheds light on the breeding strategy of polyploids as well. We conclude that frequent introgressions from congeneric diploids contributed to the acquisition of adequate genetic variations, thereby allowing C. bursa-pastoris to adapt to various habitats across the world. Our results highlight how a polyploid could have successfully established after it originated.

Root secreted metabolites and proteins are involved in the early events of plant-plant recognition prior to competition.

The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually (Col-0, Ler and Cap grown alone) and the plants co-cultured with two homozygous individuals (Col-Col, Ler-Ler and Cap-Cap) or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress-related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions.

Characterization of the variable 3' UTR and expression of the two intron-containing KIN transcripts from Capsella bursa-pastoris.

KIN genes are crucial members of the cold-regulated (COR) gene family, and are exclusively involved in normal developmental processes in many organs and respond to a variety of abiotic stresses in plants. Here, we cloned and sequenced not only two completely-spliced KIN transcripts (CbKIN1-S and CbKIN2-S), but also two intron-containing KIN transcripts (CbKIN1-U and CbKIN2-U), from Capsella bursa-pastoris, a widespread plant of the Brassicaceae family. The CbKIN1-U and CbKIN2-U transcripts each contained one additional intron in the coding region compared to the corresponding CbKIN1-S and CbKIN2-S transcripts. In addition, the two intron-containing KIN transcripts were found by rapid amplification of cDNA 3' ends (3' RACE) analysis with specific primers to have variable 3' untranslated regions (3' UTRs). We also analyzed CbKIN1-U and CbKIN2-U levels in different organs and embryonic stages by quantitative polymerase chain reaction (qPCR). They were found to be expressed in middle-stage embryos and flowers. After abscisic acid (ABA) treatment, CbKIN1-U and CbKIN2-U showed strong responses in young leaves and weak responses in flowers. Levels of the two intron-containing KIN transcripts were markedly increased in young leaves when plants were exposed to cold and heat stress. Both of them showed stronger responses to ABA treatment and cold stress than that to heat stress. CbKIN1-U and CbKIN2-U share similar gene expression profiles in development and in response to exposure to different stresses, suggesting that they probably play similar biological roles in C. bursa-pastoris.

Genetic architecture and adaptive significance of the selfing syndrome in Capsella.

The transition from outcrossing to predominant self-fertilization is one of the most common evolutionary transitions in flowering plants. This shift is often accompanied by a suite of changes in floral and reproductive characters termed the selfing syndrome. Here, we characterize the genetic architecture and evolutionary forces underlying evolution of the selfing syndrome in Capsella rubella following its recent divergence from the outcrossing ancestor C. grandiflora. We conduct genotyping by multiplexed shotgun sequencing and map floral and reproductive traits in a large (N= 550) F2 population. Our results suggest that in contrast to previous studies of the selfing syndrome, changes at a few loci, some with major effects, have shaped the evolution of the selfing syndrome in Capsella. The directionality of QTL effects, as well as population genetic patterns of polymorphism and divergence at 318 loci, is consistent with a history of directional selection on the selfing syndrome. Our study is an important step toward characterizing the genetic basis and evolutionary forces underlying the evolution of the selfing syndrome in a genetically accessible model system.

Flowering time and transcriptome variation in Capsella bursa-pastoris (Brassicaceae).

• Flowering is a major developmental transition and its timing in relation to environmental conditions is of crucial importance to plant fitness. Understanding the genetic basis of flowering time variation is important to determining how plants adapt locally. • Here, we investigated flowering time variation of Capsella bursa-pastoris collected from different latitudes in China. We also used a digital gene expression (DGE) system to generate partial gene expression profiles for 12 selected samples. • We found that flowering time was highly variable and most strongly correlated with day length and winter temperature. Significant differences in gene expression between early- and late-flowering samples were detected for 72 candidate genes for flowering time. Genes related to circadian rhythms were significantly overrepresented among the differentially expressed genes. • Our data suggest that circadian rhythms and circadian clock genes play an important role in the evolution of flowering time, and C. bursa-pastoris plants exhibit expression differences for candidate genes likely to affect flowering time across the broad range of environments they face in China.

Expression analysis and functional characterization of a novel cold-responsive gene CbCOR15a from Capsella bursa-pastoris.

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd's purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA(3) accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA(3) regulation and confers enhanced cold acclimation in transgenic plants.

Genome-wide evidence for efficient positive and purifying selection in Capsella grandiflora, a plant species with a large effective population size.

Recent studies comparing genome-wide polymorphism and divergence in Drosophila have found evidence for a surprisingly high proportion of adaptive amino acid fixations, but results for other taxa are mixed. In particular, few studies have found convincing evidence for adaptive amino acid substitution in plants. To assess the generality of this finding, we have sequenced 257 loci in the outcrossing crucifer Capsella grandiflora, which has a large effective population size and low population structure. Using a new method that jointly infers selective and demographic effects, we estimate that 40% of amino acid substitutions were fixed by positive selection in this species, and we also infer a low proportion of slightly deleterious amino acid mutations. We contrast these estimates with those for a similar data set from the closely related Arabidopsis thaliana and find significantly higher rates of adaptive evolution and fewer nearly neutral mutations in C. grandiflora. In agreement with results for other taxa, genes involved in reproduction show the strongest evidence for positive selection in C. grandiflora. Taken together, these results imply that both positive and purifying selection are more effective in C. grandiflora than in A. thaliana, consistent with the contrasting demographic history and effective population sizes of these species.

Floral visitation and reproductive traits of Stamenoid petals, a naturally occurring floral homeotic variant of Capsella bursa-pastoris (Brassicaceae).

Homeotic changes played a considerable role during the evolution of flowers, but how floral homeotic mutants initially survive in nature has remained enigmatic. To better understand the evolutionary potential of floral homeotic mutants, we established as a model system Stamenoid petals (Spe), a natural variant of Capsella bursa-pastoris (Brassicaceae). In the flowers of Spe plants, petals are transformed into stamens, whereas all other floral organs are unaffected. In contrast with most other homeotic mutants, the Spe variant occurs in relatively stable populations in the wild. In order to determine how the profound change in floral architecture influences plant performance in the wild, we performed common garden experiments running over 3 years. Here, we show that Spe and wild-type plants attract the same assemblage of floral visitors: mainly hoverflies, wild bees and thrips. However, floral visitation is about twice as frequent in wild-type plants as in Spe plants. Nevertheless, the numbers of seeds per fruit were about the same in both variants. Wild-type plants produced more flowers, fruits and seeds per plant than Spe plants, whereas the germination capacity of Spe seeds was higher than that of the wild-type. Determination of volatile composition revealed monoterpenes and 3,4-dimethylbenzaldehyde, which were detected only in wild-type flowers, presumably because they are produced only by petals. Our data indicate that the similar fitness of Spe and wild-type C. bursa-pastoris in the field results from complex compensation between plant architecture and germination capacity. In contrast, flower structure and floral visitation are only of minor importance, possibly because C. bursa-pastoris is mainly self-pollinating.