Dominance in self-compatibility between subgenomes of allopolyploid Arabidopsis kamchatica shown by transgenic restoration of self-incompatibility.

The evolutionary transition to self-compatibility facilitates polyploid speciation. In Arabidopsis relatives, the self-incompatibility system is characterized by epigenetic dominance modifiers, among which small RNAs suppress the expression of a recessive SCR/SP11 haplogroup. Although the contribution of dominance to polyploid self-compatibility is speculated, little functional evidence has been reported. Here we employ transgenic techniques to the allotetraploid plant A. kamchatica. We find that when the dominant SCR-B is repaired by removing a transposable element insertion, self-incompatibility is restored. This suggests that SCR was responsible for the evolution of self-compatibility. By contrast, the reconstruction of recessive SCR-D cannot restore self-incompatibility. These data indicate that the insertion in SCR-B conferred dominant self-compatibility to A. kamchatica. Dominant self-compatibility supports the prediction that dominant mutations increasing selfing rate can pass through Haldane's sieve against recessive mutations. The dominance regulation between subgenomes inherited from progenitors contrasts with previous studies on novel epigenetic mutations at polyploidization termed genome shock.

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

Expression of Brassica napus GLO1 is sufficient to breakdown artificial self-incompatibility in Arabidopsis thaliana.

Members of the Brassicaceae family have the ability to regulate pollination events occurring on the stigma surface. In Brassica species, self-pollination leads to an allele-specific interaction between the pollen small cysteine-rich peptide ligand (SCR/SP11) and the stigmatic S-receptor kinase (SRK) that activates the E3 ubiquitin ligase ARC1 (Armadillo repeat-containing 1), resulting in proteasomal degradation of various compatibility factors including glyoxalase I (GLO1) which is necessary for successful pollination. In Brassica napus, the suppression of GLO1 was sufficient to reduce compatibility, and overexpression of GLO1 in self-incompatible Brassica napus stigmas resulted in partial breakdown of the self-incompatibility response. Here, we verified if BnGLO1 could function as a compatibility factor in the artificial self-incompatibility system of Arabidopsis thaliana expressing AlSCRb, AlSRKb and AlARC1 proteins from A. lyrata. Overexpression of BnGLO1 is sufficient to breakdown self-incompatibility response in A. thaliana stigmas. Therefore, GLO1 has an indisputable role as a compatibility factor in the stigma in regulating pollen attachment and pollen tube growth. Lastly, this study demonstrates the usefulness of an artificial self-incompatibility system in A. thaliana for interspecific self-incompatibility studies.

TMT-Based Quantitative Proteomic Analysis Reveals the Crucial Biological Pathways Involved in Self-Incompatibility Responses in Camellia oleifera.

Camellia oleifera is a valuable woody oil plant belonging to the Theaceae, Camellia oil extracted from the seed is an excellent edible oil source. Self-incompatibility (SI) in C. oleifera results in low fruit set, and our knowledge about the mechanism remains limited. In the present study, the Tandem mass tag (TMT) based quantitative proteomics was employed to analyze the dynamic change of proteins response to self- and cross-pollinated in C. oleifera. A total of 6,616 quantified proteins were detected, and differentially abundant proteins (DAPs) analysis identified a large number of proteins. Combined analysis of differentially expressed genes (DEGs) and DAPs of self- and cross-pollinated pistils based on transcriptome and proteome data revealed that several candidate genes or proteins involved in SI of C. oleifera, including polygalacturonase inhibitor, UDP-glycosyltransferase 92A1-like, beta-D-galactosidase, S-adenosylmethionine synthetase, xyloglucan endotransglucosylase/hydrolase, ABC transporter G family member 36-like, and flavonol synthase. Venn diagram analysis identified 11 proteins that may participate in pollen tube growth in C. oleifera. Our data also revealed that the abundance of proteins related to peroxisome was altered in responses to SI in C. oleifera. Moreover, the pathway of lipid metabolism-related, flavonoid biosynthesis and splicesome were reduced in self-pollinated pistils by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In summary, the results of the present study lay the foundation for learning the regulatory mechanism underlying SI responses as well as provides valuable protein resources for the construction of self-compatibility C. oleifera through genetic engineering in the future.

The Brassica napus GATA transcription factor BnA5.ZML1 is a stigma compatibility factor.

Self-incompatibility (SI) is a genetic mechanism that rejects self-pollen and thus prevents inbreeding in some hermaphroditic angiosperms. In the Brassicaceae, SI involves a pollen-stigma recognition system controlled by a single locus known as the S locus, which consists of two highly polymorphic genes that encode S-locus cysteine-rich protein (SCR) and S-receptor kinase (SRK). When self-pollen lands on the stigma, the S-haplotype-specific interaction between SCR and SRK triggers SI. Here, we show that the GATA transcription factor BnA5.ZML1 suppresses SI responses in Brassica napus and is induced after compatible pollination. The loss-of-function mutant bna5.zml1 displays reduced self-compatibility. In contrast, overexpression of BnA5.ZML1 in self-incompatible stigmas leads to a partial breakdown of SI responses, suggesting that BnA5.ZML1 is a stigmatic compatibility factor. Furthermore, the expression levels of SRK and ARC1 are up-regulated in bna5.zml1 mutants, and they are down-regulated in BnA5.ZML1 overexpressing lines. SRK affects the cellular localization of BnA5.ZML1 through direct protein-protein interaction. Overall, our findings highlight the fundamental role of BnA5.ZML1 in SI responses in B. napus, establishing a direct interaction between BnA5.ZML1 and SRK in this process.

Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize.

In maize (Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination (G_GG, G_GB, B_BB, B_BG). A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S.

NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance.

Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.

Intercontinental dispersal and whole-genome duplication contribute to loss of self-incompatibility in a polyploid complex.

PREMISE OF THE STUDY: Angiosperm species often shift from self-incompatibility to self-compatibility following population bottlenecks. Across the range of a species, population bottlenecks may result from multiple factors, each of which may affect the geographic distribution and magnitude of mating-system shifts. We describe how intercontinental dispersal and genome duplication facilitate loss of self-incompatibility. METHODS: Self and outcross pollinations were performed on plants from 24 populations of the Campanula rotundifolia polyploid complex. Populations spanned the geographic distribution and three dominant cytotypes of the species (diploid, tetraploid, hexaploid). KEY RESULTS: Loss of self-incompatibility was associated with both intercontinental dispersal and genome duplication. European plants were largely self-incompatible, whereas North American plants were intermediately to fully self-compatible. Within both European and North American populations, loss of self-incompatibility increased as ploidy increased. Ploidy change and intercontinental dispersal both contributed to loss of self-incompatibility in North America, but range expansion did not affect self-incompatibility within Europe or North America. CONCLUSIONS: When species are subject to population bottlenecks arising through multiple factors, each factor can contribute to self-incompatibility loss. In a widespread polyploid complex, the loss of self-incompatibility can be predicted by the cumulative effects of whole-genome duplication and intercontinental dispersal.

Insights into the Prunus-Specific S-RNase-Based Self-Incompatibility System from a Genome-Wide Analysis of the Evolutionary Radiation of S Locus-Related F-box Genes.

Self-incompatibility (SI) is an important plant reproduction mechanism that facilitates the maintenance of genetic diversity within species. Three plant families, the Solanaceae, Rosaceae and Plantaginaceae, share an S-RNase-based gametophytic SI (GSI) system that involves a single S-RNase as the pistil S determinant and several F-box genes as pollen S determinants that act via non-self-recognition. Previous evidence has suggested a specific self-recognition mechanism in Prunus (Rosaceae), raising questions about the generality of the S-RNase-based GSI system. We investigated the evolution of the pollen S determinant by comparing the sequences of the Prunus S haplotype-specific F-box gene (SFB) with those of its orthologs in other angiosperm genomes. Our results indicate that the Prunus SFB does not cluster with the pollen S of other plants and diverged early after the establishment of the Eudicots. Our results further indicate multiple F-box gene duplication events, specifically in the Rosaceae family, and suggest that the Prunus SFB gene originated in a recent Prunus-specific gene duplication event. Transcriptomic and evolutionary analyses of the Prunus S paralogs are consistent with the establishment of a Prunus-specific SI system, and the possibility of subfunctionalization differentiating the newly generated SFB from the original pollen S determinant.

CrWSKP1, an SKP1-like Gene, Is Involved in the Self-Incompatibility Reaction of "Wuzishatangju" (Citrus reticulata Blanco).

Plant S-phase kinase-associated protein 1 (SKP1) genes play crucial roles in plant development and differentiation. However, the role of SKP1 in citrus is unclear. Herein, we described a novel SKP1-like gene, designated as CrWSKP1, from "Wuzishatangju" (Citrus reticulata Blanco). The cDNA sequence of CrWSKP1 is 779 base pairs (bp) and contains an open reading frame (ORF) of 477 bp. The genomic sequence of the CrWSKP1 gene is 1296 bp with two exons and one intron. CrWSKP1 has high identity with SKP1-like genes from other plant species within two conserved regions. Approximately 85% of pollen tubes of self-pollinated CrWSKP1 transgenic tobaccos became twisted at four days after self-pollination. Pollen tube numbers of self-pollinated CrWSKP1 transformants entering into ovules were significantly fewer than that of the control. Seed number of self-pollinated CrWSKP1 transformants was significantly reduced. These results suggested that the CrWSKP1 is involved in the self-incompatibility (SI) reaction of "Wuzishatangju".

Dissection of the style's response to pollination using transcriptome profiling in self-compatible (Solanum pimpinellifolium) and self-incompatible (Solanum chilense) tomato species.

BACKGROUND: Tomato (Solanum lycopersicum) self-compatibility (SC) is defined as self-pollen tubes that can penetrate their own stigma, elongate in the style and fertilize their own ovules. Self-incompatibility (SI) is defined as self-pollen tubes that are prevented from developing in the style. To determine the influence of gene expression on style self-pollination, a transcriptome-wide comparative analysis of SC and SI tomato unpollinated/pollinated styles was performed using RNA-sequencing (RNA-seq) data. RESULTS: Transcriptome profiles of 24-h unpollination (UP) and self-pollination (P) styles from SC and SI tomato species were generated using high-throughput next generation sequencing. From the comparison of SC self-pollinated and unpollinated styles, 1341 differentially expressed genes (DEGs) were identified, of which 753 were downregulated and 588 were upregulated. From the comparison of SI self-pollinated and unpollinated styles, 804 DEGs were identified, of which 215 were downregulated and 589 were upregulated. Nine gene ontology (GO) terms were enriched significantly in SC and 78 GO terms were enriched significantly in SI. A total of 105 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified in SC and 80 enriched KEGG pathways were identified in SI, among which "Cysteine and methionine metabolism pathway" and "Plant hormone signal transduction pathway" were significantly enriched in SI. CONCLUSIONS: This study is the first global transcriptome-wide comparative analysis of SC and SI tomato unpollinated/pollinated styles. Advanced bioinformatic analysis of DEGs uncovered the pathways of "Cysteine and methionine metabolism" and "Plant hormone signal transduction", which are likely to play important roles in the control of pollen tubes growth in SI species.

Site-specific N-glycosylation of the S-locus receptor kinase and its role in the self-incompatibility response of the brassicaceae.

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

The ARC1 E3 ligase promotes a strong and stable self-incompatibility response in Arabidopsis species: response to the Nasrallah and Nasrallah commentary.

Following the identification of the male (S-locus Cysteine Rich/S-locus Protein 11) and female (S Receptor kinase [SRK]) factors controlling self-incompatibility in the Brassicaceae, research in this field has focused on understanding the nature of the cellular responses activated by these regulators. We previously identified the ARM Repeat Containing1 (ARC1) E3 ligase as a component of the SRK signaling pathway and demonstrated ARC1's requirement in the stigma for self-incompatible pollen rejection in Brassica napus, Arabidopsis lyrata, and Arabidopsis thaliana. Here, we discuss our findings on the role of ARC1 in reconstructing a strong and stable A. thaliana self-incompatibility phenotype, in the context of the putative issues outlined in a commentary by Nasrallah and Nasrallah. Additionally, with their proposed standardized strategy for studying self-incompatibility in A. thaliana, we offer our perspective on what constitutes a strong and stable self-incompatibility phenotype in A. thaliana and how this should be investigated and reported to the greater community.

The timetable for allopolyploidy in flowering plants.

BACKGROUND: Our understanding of the processes and dynamics of allopolyploid speciation, the long-term consequences of ploidal change, and the genetic and chromosomal changes in new emerged allopolyploids has substantially increased during the past few decades. Yet we remain uncertain about the time since lineage divergence when two taxa are capable of spawning such entities. Indeed, the matter has seemed intractable. Knowledge of the window of opportunity for allopolyploid production is very important because it provides temporal insight into a key evolutionary process, and a temporal reference against which other modes of speciation may be measured. SCOPE: This Viewpoint paper reviews and integrates published information on the crossability of herbaceous species and the fertility of their hybrids in relation to species' divergence times. Despite limitations in methodology and sampling, the estimated times to hybrid sterility are somewhat congruent across disparate lineages. Whereas the waiting time for hybrid sterility is roughly 4-5 million years, the waiting time for cross-incompatibility is roughly 8-10 million years, sometimes considerably more. Strict allopolyploids may be formed in the intervening time window. The progenitors of several allopolyploids diverged between 4 and 6 million years before allopolyploid synthesis, as expected. This is the first study to propose a general temporal framework for strict allopolyploidy. This Viewpoint paper hopefully will stimulate interest in studying the tempo of speciation and the tempo of reproductive isolation in general.

The Tarenaya hassleriana genome provides insight into reproductive trait and genome evolution of crucifers.

The Brassicaceae, including Arabidopsis thaliana and Brassica crops, is unmatched among plants in its wealth of genomic and functional molecular data and has long served as a model for understanding gene, genome, and trait evolution. However, genome information from a phylogenetic outgroup that is essential for inferring directionality of evolutionary change has been lacking. We therefore sequenced the genome of the spider flower (Tarenaya hassleriana) from the Brassicaceae sister family, the Cleomaceae. By comparative analysis of the two lineages, we show that genome evolution following ancient polyploidy and gene duplication events affect reproductively important traits. We found an ancient genome triplication in Tarenaya (Th-α) that is independent of the Brassicaceae-specific duplication (At-α) and nested Brassica (Br-α) triplication. To showcase the potential of sister lineage genome analysis, we investigated the state of floral developmental genes and show Brassica retains twice as many floral MADS (for minichromosome maintenance1, AGAMOUS, DEFICIENS and serum response factor) genes as Tarenaya that likely contribute to morphological diversity in Brassica. We also performed synteny analysis of gene families that confer self-incompatibility in Brassicaceae and found that the critical serine receptor kinase receptor gene is derived from a lineage-specific tandem duplication. The T. hassleriana genome will facilitate future research toward elucidating the evolutionary history of Brassicaceae genomes.

Molecular characterization and evolution of self-incompatibility genes in Arabidopsis thaliana: the case of the Sc haplotype.

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.