ABSTRACT
Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared with Arabidopsis Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Chromatin/genetics , Heterochromatin/genetics , Histone Code , DNA Methylation , Arabidopsis/genetics , Arabidopsis/metabolism , RNA Polymerase II/genetics , Arabidopsis Proteins/genetics , Gene Expression Regulation, PlantABSTRACT
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.
Subject(s)
DNA Methylation , DNA-Directed RNA Polymerases , DNA Methylation/genetics , RNA, Plant/genetics , RNA, Plant/metabolism , DNA-Directed RNA Polymerases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , ReproductionABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Brassicaceae , DNA Methylation/genetics , Brassicaceae/genetics , Brassicaceae/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , RNA, Small Interfering/genetics , RNA, Double-Stranded , Phenotype , Seeds/genetics , Seeds/metabolism , Reproduction , RNA, Plant/genetics , RNA, Plant/metabolism , Gene Expression Regulation, PlantABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Chromatin/metabolism , DNA Methylation/genetics , DNA Transposable Elements/genetics , Gene Expression Regulation, Plant/genetics , Nucleotides/metabolism , Ovule/genetics , Ovule/metabolism , RNA, Plant/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.
Subject(s)
Epigenomics , Regulatory Sequences, Nucleic Acid , Chromatin/genetics , Genome, Plant/genetics , Plants/genetics , Quality ControlABSTRACT
All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt short interfering RNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects nor embryo lethality, although adult plants are dwarf and sterile.
Subject(s)
Arabidopsis Proteins , Oryza , Alleles , Arabidopsis Proteins/metabolism , DNA Methylation/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Plant/genetics , Oryza/genetics , Oryza/metabolism , RNA Polymerase II/metabolism , RNA, Plant/genetics , RNA, Small Interfering/geneticsABSTRACT
Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.
Subject(s)
Brassica rapa/embryology , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Plant/physiology , RNA, Plant , Seeds/growth & development , Alleles , Arabidopsis/metabolism , Brassica rapa/genetics , Brassica rapa/growth & development , Brassica rapa/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , RNA, Small Interfering , Seeds/genetics , Seeds/metabolismABSTRACT
Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.
Subject(s)
Arabidopsis/embryology , Arabidopsis/genetics , Gene Expression Regulation, Plant/physiology , Genomic Imprinting , Ovule , RNA, Plant/physiology , RNA, Small Interfering/genetics , Seeds/growth & development , Arabidopsis Proteins/geneticsABSTRACT
RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. RdDM requires a specialized transcriptional machinery that comprises two plant-specific RNA polymerases - Pol IV and Pol V - and a growing number of accessory proteins, the functions of which in the RdDM mechanism are only partially understood. Recent work has revealed variations in the canonical RdDM pathway and identified factors that recruit Pol IV and Pol V to specific target sequences. RdDM, which transcriptionally represses a subset of transposons and genes, is implicated in pathogen defence, stress responses and reproduction, as well as in interallelic and intercellular communication.
Subject(s)
DNA Methylation/physiology , DNA-Directed RNA Polymerases/metabolism , Epigenesis, Genetic/physiology , Eukaryota/physiology , RNA, Small Untranslated/metabolism , DNA-Directed RNA Polymerases/genetics , RNA, Small Untranslated/geneticsABSTRACT
Small RNAs trigger repressive DNA methylation at thousands of transposable elements in a process called RNA-directed DNA methylation (RdDM). The molecular mechanism of RdDM is well characterized in Arabidopsis, yet the biological function remains unclear, as loss of RdDM in Arabidopsis causes no overt defects, even after generations of inbreeding. It is known that 24 nucleotide Pol IV-dependent siRNAs, the hallmark of RdDM, are abundant in flowers and developing seeds, indicating that RdDM might be important during reproduction. Here we show that, unlike Arabidopsis, mutations in the Pol IV-dependent small RNA pathway cause severe and specific reproductive defects in Brassica rapa. High rates of abortion occur when seeds have RdDM mutant mothers, but not when they have mutant fathers. Although abortion occurs after fertilization, RdDM function is required in maternal somatic tissue, not in the female gametophyte or the developing zygote, suggesting that siRNAs from the maternal soma might function in filial tissues. We propose that recently outbreeding species such as B. rapa are key to understanding the role of RdDM during plant reproduction.
Subject(s)
Brassica rapa/genetics , DNA Methylation , RNA, Small Interfering/genetics , Seeds/genetics , Brassica rapa/embryology , Brassica rapa/enzymology , Brassica rapa/physiology , DNA Transposable Elements/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Diploidy , Genotype , Mutation , Phenotype , Plant Breeding , Plant Proteins/genetics , Plant Proteins/metabolism , RNA, Plant/genetics , Reproduction , Seeds/embryology , Seeds/enzymology , Seeds/physiologyABSTRACT
Gene duplication is an important driver for the evolution of new genes and protein functions. Duplication of DNA-dependent RNA polymerase (Pol) II subunits within plants led to the emergence of RNA Pol IV and V complexes, each of which possess unique functions necessary for RNA-directed DNA Methylation. Comprehensive identification of Pol V subunit orthologs across the monocot radiation revealed a duplication of the largest two subunits within the grasses (Poaceae), including critical cereal crops. These paralogous Pol subunits display sequence conservation within catalytic domains, but their carboxy terminal domains differ in length and character of the Ago-binding platform, suggesting unique functional interactions. Phylogenetic analysis of the catalytic region indicates positive selection on one paralog following duplication, consistent with retention via neofunctionalization. Positive selection on residue pairs that are predicted to interact between subunits suggests that paralogous subunits have evolved specific assembly partners. Additional Pol subunits as well as Pol-interacting proteins also possess grass-specific paralogs, supporting the hypothesis that a novel Pol complex with distinct function has evolved in the grass family, Poaceae.
Subject(s)
DNA-Directed RNA Polymerases/genetics , Edible Grain/enzymology , Gene Duplication , Poaceae/enzymology , Selection, Genetic , Amino Acid Sequence , Edible Grain/genetics , Grain Proteins , Phylogeny , Poaceae/geneticsABSTRACT
In flowering plants, the female gametophyte controls pollen tube reception immediately before fertilization and regulates seed development immediately after fertilization, although the controlling mechanisms remain poorly understood. Previously, we showed that LORELEI (LRE), which encodes a putative glycosylphosphatidylinositol-anchored membrane protein, is critical for pollen tube reception by the female gametophyte before fertilization and the initiation of seed development after fertilization. Here, we show that LRE is expressed in the synergid, egg, and central cells of the female gametophyte and in the zygote and proliferating endosperm of the Arabidopsis (Arabidopsis thaliana) seed. Interestingly, LRE expression in the developing seeds was primarily from the matrigenic LRE allele, indicating that LRE expression is imprinted. However, LRE was biallelically expressed in 8-d-old seedlings, indicating that the patrigenic allele does not remain silenced throughout the sporophytic generation. Regulation of imprinted LRE expression is likely novel, as LRE was not expressed in pollen or pollen tubes of mutants defective for MET1, DDM1, RNA-dependent DNA methylation, or MSI-dependent histone methylation. Additionally, the patrigenic LRE allele inherited from these mutants was not expressed in seeds. Surprisingly, and contrary to the predictions of the parental conflict hypothesis, LRE promotes growth in seeds, as loss of the matrigenic but not the patrigenic LRE allele caused delayed initiation of seed development. Our results showed that LRE is a rare imprinted gene that functions immediately after double fertilization and supported the model that a passage through the female gametophyte establishes monoalleleic expression of LRE in seeds and controls early seed development.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Membrane Glycoproteins/metabolism , Arabidopsis/cytology , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Endosperm/cytology , Endosperm/genetics , Endosperm/growth & development , Fertilization , Membrane Glycoproteins/genetics , Mutation , Organ Specificity , Ovule/cytology , Ovule/genetics , Ovule/growth & development , Pollen/cytology , Pollen/genetics , Pollen/growth & development , Pollen Tube/cytology , Pollen Tube/genetics , Pollen Tube/growth & development , Pollination , Seedlings/cytology , Seedlings/genetics , Seedlings/growth & development , Seeds/cytology , Seeds/genetics , Seeds/growth & development , ZygoteABSTRACT
Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.
Subject(s)
DNA-Directed RNA Polymerases/genetics , Phylogeny , Plant Proteins/genetics , Plants/enzymology , Plants/genetics , Amino Acid Sequence , DNA-Directed RNA Polymerases/chemistry , Evolution, Molecular , Flowers/genetics , Gene Duplication , Gene Silencing , Genes, Plant , Magnoliopsida/enzymology , Molecular Sequence Data , Plant Proteins/chemistry , Protein Structure, Tertiary , Protein Subunits/genetics , Species SpecificityABSTRACT
Argonaute (Ago) proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Ago proteins, but little is known about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. Here we investigated the evolutionary dynamics of an Ago-binding platform in NRPE1, the largest subunit of RNA polymerase V. We compared NRPE1 sequences from > 50 species, including dense sampling of two plant lineages. This study demonstrates that the Ago-binding platform of NRPE1 retains Ago hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is the result of relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million yr. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.
Subject(s)
Argonaute Proteins/metabolism , Evolution, Molecular , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Repetitive Sequences, Amino Acid , Amino Acid Sequence , Conserved Sequence , Gene Duplication , Models, Biological , Phylogeny , Protein Binding , Structure-Activity RelationshipABSTRACT
Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.
Subject(s)
Arabidopsis/embryology , Arabidopsis/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Plant , RNA, Plant/genetics , RNA, Small Interfering/genetics , Seeds/genetics , Arabidopsis/enzymology , DNA-Directed RNA Polymerases/genetics , Genome, Plant/genetics , Genomic Imprinting , RNA Interference , RNA, Plant/biosynthesis , RNA, Small Interfering/biosynthesis , Seeds/embryology , Seeds/enzymologyABSTRACT
In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae.
Subject(s)
DNA Methylation , DNA-Directed RNA Polymerases , Evolution, Molecular , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Phylogeny , Charophyceae/genetics , Charophyceae/enzymology , Plant Proteins/genetics , Plant Proteins/metabolism , Chlorophyta/genetics , Chlorophyta/enzymology , Protein Subunits/geneticsABSTRACT
The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5'-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1Delta cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.
Subject(s)
Centromere/ultrastructure , Gene Expression Regulation, Fungal , RNA, Small Interfering/metabolism , Schizosaccharomyces/physiology , Base Sequence , Centromere/metabolism , Heterochromatin/chemistry , Molecular Sequence Data , Multigene Family , Mutation , Nucleic Acid Conformation , RNA/chemistry , RNA/metabolism , RNA Interference , RNA, Double-Stranded/chemistry , RNA, Small Interfering/chemistry , Schizosaccharomyces/metabolism , Sequence Homology, Nucleic AcidABSTRACT
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.
ABSTRACT
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.