Mating system is associated with seed phenotypes upon loss of RNA-directed DNA methylation in Brassicaceae.

In plants, de novo DNA methylation is guided by 24-nt short interfering (si)RNAs in a process called RNA-directed DNA methylation (RdDM). Primarily targeted at transposons, RdDM causes transcriptional silencing and can indirectly influence expression of neighboring genes. During reproduction, a small number of siRNA loci are dramatically upregulated in the maternally derived seed coat, suggesting that RdDM might have a special function during reproduction. However, the developmental consequence of RdDM has been difficult to dissect because disruption of RdDM does not result in overt phenotypes in Arabidopsis (Arabidopsis thaliana), where the pathway has been most thoroughly studied. In contrast, Brassica rapa mutants lacking RdDM have a severe seed production defect, which is determined by the maternal sporophytic genotype. To explore the factors that underlie the different phenotypes of these species, we produced RdDM mutations in 3 additional members of the Brassicaceae family: Camelina sativa, Capsella rubella, and Capsella grandiflora. Among these 3 species, only mutations in the obligate outcrosser, C. grandiflora, displayed a seed production defect similar to Brassica rapa mutants, suggesting that mating system is a key determinant for reproductive phenotypes in RdDM mutants.

Mapping a mutation causing pale yellow petals in Brassica rapa.

Petal color is an important trait for both ornamental purposes and also for attracting pollinators. Here, we report a mutation of Brassica rapa R-o-18 with pale yellow petals that we retrieved from an EMS population and named whiter shade of pale ( wsp ). Phenotypic segregation ratio of an F2 mapping population indicates the phenotype is controlled by a single recessive gene. Mapping data from the whole genome sequencing coupled with allele frequency analysis suggests the mutation is located in a ~2 Mbp interval on chromosome 2. The interval contains a putative esterase/lipase/thioesterase protein previously demonstrated to account for floral color in B. rapa . We demonstrate that wsp carries a G to A missense mutation causing an aspartate to asparagine substitution within the putative lysophospholipid acyltransferase domain.

A novel CLAVATA1 mutation causes multilocularity in Brassica rapa.

Locules are the seed-bearing structure of fruits. Multiple locules are associated with increased fruit size and seed set, and therefore, control of locule number is an important agronomic trait. Locule number is controlled in part by the CLAVATA-WUSCHEL pathway. Disruption of either the CLAVATA1 receptor-like kinase or its ligand CLAVATA3 can cause larger floral meristems and an increased number of locules. In an EMS mutagenized population of Brassica rapa, we identified a mutant allele that raises the number of locules from four to a range of from six to eight. Linkage mapping and genetic analysis support that the mutant phenotype is due to a missense mutation in a CLAVATA 1 (CLV1) homolog. In addition to increased locule number, additional internal gynoecia are formed in brclv1 individuals, suggesting a failure to terminate floral meristem development, which results in decreased seed production.

Small RNA-mediated DNA methylation during plant reproduction.

Reproductive tissues are a rich source of small RNAs, including several classes of short interfering (si)RNAs that are restricted to this stage of development. In addition to RNA polymerase IV-dependent 24-nt siRNAs that trigger canonical RNA-directed DNA methylation, abundant reproductive-specific siRNAs are produced from companion cells adjacent to the developing germ line or zygote and may move intercellularly before inducing methylation. In some cases, these siRNAs are produced via non-canonical biosynthesis mechanisms or from sequences with little similarity to transposons. While the precise role of these siRNAs and the methylation they trigger is unclear, they have been implicated in specifying a single megaspore mother cell, silencing transposons in the male germ line, mediating parental dosage conflict to ensure proper endosperm development, hypermethylation of mature embryos, and trans-chromosomal methylation in hybrids. In this review, we summarize the current knowledge of reproductive siRNAs, including their biosynthesis, transport, and function.

Ovule siRNAs methylate protein-coding genes in trans.

Twenty-four-nucleotide (nt) small interfering RNAs (siRNAs) maintain asymmetric DNA methylation at thousands of euchromatic transposable elements in plant genomes in a process called RNA-directed DNA methylation (RdDM). RdDM is dispensable for growth and development in Arabidopsis thaliana, but is required for reproduction in other plants, such as Brassica rapa. The 24-nt siRNAs are abundant in maternal reproductive tissue, due largely to overwhelming expression from a few loci in the ovule and developing seed coat, termed siren loci. A recent study showed that 24-nt siRNAs produced in the anther tapetal tissue can methylate male meiocyte genes in trans. Here we show that in B. rapa, a similar process takes place in female tissue. siRNAs are produced from gene fragments embedded in some siren loci, and these siRNAs can trigger methylation in trans at related protein-coding genes. This trans-methylation is associated with silencing of some target genes and may be responsible for seed abortion in RdDM mutants. Furthermore, we demonstrate that a consensus sequence in at least two families of DNA transposons is associated with abundant siren expression, most likely through recruitment of CLASSY3, a putative chromatin remodeler. This research describes a mechanism whereby RdDM influences gene expression and sheds light on the role of RdDM during plant reproduction.

RNA-directed DNA Methylation and sexual reproduction: expanding beyond the seed.

Two trends are changing our understanding of RNA-directed DNA methylation. In model systems like Arabidopsis, tissue-specific analysis of DNA methylation is uncovering dynamic changes in methylation during sexual reproduction and unraveling the contribution of maternal and paternal epigenomes to the developing embryo. These studies indicate that RNA-directed DNA Methylation might be important for mediating balance between maternal and paternal contributions to the endosperm. At the same time, researchers are moving beyond Arabidopsis to illuminate the ancestral role of RdDM in non-flowering plants that lack an endosperm, suggesting that RdDM might play a broader role in sexual reproduction.

Regulation of miR163 and its targets in defense against Pseudomonas syringae in Arabidopsis thaliana.

Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. miR163 is a non-conserved miRNA and its locus has evolved recently through inverted duplication of its target genes to which they belong to the SABATH family of related small-molecule methyltransferases (MTs). In Arabidopsis thaliana, previous study demonstrated that miR163 accumulation was induced by alamethicin treatment, suggesting its roles in defense response pathways. Enhanced resistance against Pseudomonas syringae pv. tomato (Pst) was observed in the mir163 mutant, whereas transgenic lines overexpressing miR163 showed increase sensitivity to Pst, suggesting that miR163 is a negative regulator of defense response. Elevated level of miR163 and its targets in A. thaliana were observed upon Pst treatment, suggesting a modulating relationship between miR163 and its targets. In addition, miR163 and histone deacetylase were found to act cooperatively in mediating defense against Pst. Transgenic plants overexpressing miR163-resistant targets suggested their different contributions in defense. Results from this study revealed that the stress-inducible miR163 and its targets act in concert to modulate defense responses against bacterial pathogen in A. thaliana.