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Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificitydetermining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A thaliana to its fully self-incompatible ancestral state.

The self-incompatibility (SI) system of the Brassicaceae is based on allele-specific interactions among haplotypes of the S locus. In all tested self-incompatible Brassicaceae, the S haplotype encompasses two linked genes, one encoding the S-locus receptor kinase (SRK), a transmembrane kinase displayed at the surface of stigma epidermal cells, and the other encoding its ligand, the S-locus cysteine-rich (SCR) protein, which is localized in the pollen coat. Transfer of the two genes to self-fertile Arabidopsis thaliana allowed the establishment of robust SI in several accessions, indicating that the signaling cascade triggered by this receptor–ligand interaction and the resulting inhibition of \"self\" pollen by the stigma have been maintained in extant A. thaliana. Based on studies in Brassica species, the membrane-tethered kinase MLPK, the ARM repeat-containing U-box protein ARC1, and the exocyst subunit Exo70A1 have been proposed to function as components of an SI signaling cascade. Here we tested the role of these molecules in the SI response of A. thaliana SRK-SCR plants. We show that the A. thaliana ARC1 ortholog is a highly decayed pseudogene. We also show that, unlike reports in Brassica, inactivation of the MLPK ortholog AtAPK1b and overexpression of Exo70A1 neither abolish nor weaken SI in A. thaliana SRK-SCR plants. These results do not support a role for these molecules in the SI response of A. thaliana.

A common yet poorly understood evolutionary transition among flowering plants is a switch from outbreeding to an inbreeding mode of mating. The model plant Arabidopsis thaliana evolved to an inbreeding state through the loss of self-incompatibility, a pollen-rejection system in which pollen recognition by the stigma is determined by tightly linked and co-evolving alleles of the S-locus receptor kinase (SRK) and its S-locus cysteine-rich ligand (SCR). Transformation of A. thaliana, with a functional AlSRKb-SCRb gene pair from its outcrossing relative A. lyrata, demonstrated that A. thaliana accessions harbor different sets of cryptic self-fertility-promoting mutations, not only in S-locus genes, but also in other loci required for self-incompatibility. However, it is still not known how many times and in what manner the switch to self-fertility occurred in the A. thaliana lineage. Here, we report on our identification of four accessions that are reverted to full self-incompatibility by transformation with AlSRKb-SCRb, bringing to five the number of accessions in which self-fertility is due to, and was likely caused by, S-locus inactivation. Analysis of S-haplotype organization reveals that inter-haplotypic recombination events, rearrangements, and deletions have restructured the S locus and its genes in these accessions. We also perform a Quantitative Trait Loci (QTL) analysis to identify modifier loci associated with self-fertility in the Col-0 reference accession, which cannot be reverted to full self-incompatibility. Our results indicate that the transition to inbreeding occurred by at least two, and possibly more, independent S-locus mutations, and identify a novel unstable modifier locus that contributes to self-fertility in Col-0.

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.

Plant reproductive development is dependent on successful pollen-pistil interactions. In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. The specialized functions of stigma epidermal cells and transmitting tract cells are likely to depend on the activity of genes expressed specifically in these cells. In order to identify these genes, we used the Arabidopsis (Arabidopsis thaliana) ATH1 microarray to compare the whole-genome transcriptional profiles of stigmas and ovaries isolated from wild-type Arabidopsis and from transgenic plants in which cells of the stigma epidermis and transmitting tract were specifically ablated by expression of a cellular toxin. Among the 23,000 genes represented on the array, we identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis and transmitting tract, respectively. Both gene sets were significantly enriched in predicted secreted proteins, including potential signaling components and proteins that might contribute to reinforcing, modifying, or remodeling the structure of the extracellular matrix during pollination. The possible role of these genes in compatible and incompatible pollen-pistil interactions is discussed.

As a major agent of rapid speciation, interspecific hybridization has played an important role in plant evolution. When hybridization involves species that exhibit self-incompatibility (SI), this prezygotic barrier to self-fertilization must be overcome or lost to allow selfing. How SI, a normally dominant trait, is lost in nascent hybrids is not known, however. Here we demonstrate that hybrid self-fertility can result from epigenetic changes in expression of the S-locus genes that determine specificity in the SI response. We analyzed loss of SI in synthetic hybrids produced by crossing self-fertile and self-incompatible species in each of two crucifer genera. We show that SI is lost in the stigmas of A. thaliana–lyrata hybrids and their neo-allotetraploid derivatives and in the pollen of C. rubella–grandiflora hybrids and their homoploid progenies. Aberrant processing of S-locus receptor kinase gene transcripts as detected in Arabidopsis hybrids and suppression of the S-locus cysteine-rich protein gene as observed in Capsella hybrids are two reversible mechanisms by which SI might break down upon interspecific hybridization to generate self-fertile hybrids in nature.

Detlef Weigel and colleagues report the genome sequence of Arabidopsis lyrata . In comparison with the much smaller genome of A. thaliana , from which A. lyrata diverged about 10 million years ago, they find that the reduction in genome size is attributed to a large number of deletions across the genome. We report the 207-Mb genome sequence of the North American Arabidopsis lyrata strain MN47 based on 8.3× dideoxy sequence coverage. We predict 32,670 genes in this outcrossing species compared to the 27,025 genes in the selfing species Arabidopsis thaliana . The much smaller 125-Mb genome of A. thaliana , which diverged from A. lyrata 10 million years ago, likely constitutes the derived state for the family. We found evidence for DNA loss from large-scale rearrangements, but most of the difference in genome size can be attributed to hundreds of thousands of small deletions, mostly in noncoding DNA and transposons. Analysis of deletions and insertions still segregating in A. thaliana indicates that the process of DNA loss is ongoing, suggesting pervasive selection for a smaller genome. The high-quality reference genome sequence for A. lyrata will be an important resource for functional, evolutionary and ecological studies in the genus Arabidopsis .

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allelespecific 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 coordinate evolution of self-incompatibility (SI) and stigma-anther separation, two mechanisms that promote cross-pollination in plants, has been a long-standing puzzle in evolution and development. Using a transgenic self-incompatible Arabidopsis thaliana model, we performed screens for mutants exhibiting a modified SI response. A mutation in the RNA-dependent RNA polymerase RDR6, which functions in trans-acting short interfering RNA (ta-siRNA) production, was found that simultaneously enhances SI and causes stigma exsertion, without associated increases in SRK transcript levels. While rdr6 mutants had been previously shown to exhibit stochastic stigma exsertion, our results demonstrate that the S-locus receptor kinase (SRK) gene further enhances pistil elongation and stigma exsertion in this mutant background, a process that requires SRK catalytic activity and correlates with SRK transcript levels. These results suggest that positive regulators or effectors of SI and pistil development are regulated by ta-siRNA(s). By establishing complex connections between SI and stigma exsertion through the sharing of a ta-siRNA-mediated regulatory pathway and the dual role of SRK in SI and pistil development, our study provides a molecular explanation for the coordinate evolution of these processes.

Self-incompatibility and recurrent transitions to self-compatibility have shaped the extant mating systems underlying the nonrandom mating critical for speciation in angiosperms. Linkage between self-incompatibility and speciation is illustrated by the shared pollen rejection pathway between self-incompatibility and interspecific unilateral incompatibility (UI) in the Brassicaceae. However, the pollen discrimination system that activates this shared pathway for heterospecific pollen rejection remains unknown. Here we show that Stigma UI3.1 , the genetically identified stigma determinant of UI in Arabidopsis lyrata × Arabidopsis arenosa crosses, encodes the S-locus-related glycoprotein 1 (SLR1). Heterologous expression of A. lyrata or Capsella grandiflora SLR1 confers on some Arabidopsis thaliana accessions the ability to discriminate against heterospecific pollen. Acquisition of this ability also requires a functional S-locus receptor kinase (SRK), whose ligand-induced dimerization activates the self-pollen rejection pathway in the stigma. SLR1 interacts with SRK and interferes with SRK homomer formation. We propose a pollen discrimination system based on competition between basal or ligand-induced SLR1–SRK and SRK–SRK complex formation. The resulting SRK homomer levels would be sensed by the common pollen rejection pathway, allowing discrimination among conspecific self- and cross-pollen as well as heterospecific pollen. Our results establish a mechanistic link at the pollen recognition phase between self-incompatibility and interspecific incompatibility. Through genetic and molecular analyses of interspecific stigma–pollen interactions, the authors show that Brassicaceae plants use an integrated pollen discrimination system and a shared pollen rejection pathway to reject conspecific self-pollen and heterospecific pollen. This establishes a mechanistic link between self-incompatibility and speciation in this clade.