RESUMO
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.
Assuntos
Metilação de DNA , RNA Polimerases Dirigidas por DNA , Evolução Molecular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , Carofíceas/genética , Carofíceas/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Clorófitas/genética , Clorófitas/enzimologia , Subunidades Proteicas/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassicaceae , Metilação de DNA/genética , Brassicaceae/genética , Brassicaceae/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , RNA Interferente Pequeno/genética , RNA de Cadeia Dupla , Fenótipo , Sementes/genética , Sementes/metabolismo , Reprodução , RNA de Plantas/genética , RNA de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cromatina/genética , Heterocromatina/genética , Código das Histonas , Metilação de DNA , Arabidopsis/genética , Arabidopsis/metabolismo , RNA Polimerase II/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de PlantasRESUMO
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.
RESUMO
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.
Assuntos
Metilação de DNA , RNA Polimerases Dirigidas por DNA , Metilação de DNA/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , ReproduçãoRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas/genética , Nucleotídeos/metabolismo , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The revolution in sequencing has created a wealth of plant genomes that can be mined to understand the evolution of biological complexity. Complexity is often driven by gene duplication, which allows paralogs to specialize in an activity of the ancestral gene or acquire novel functions. Angiosperms encode a variety of gene silencing pathways that share related machinery for small RNA biosynthesis and function. Recent phylogenetic analysis of these gene families plots the expansion, specialization, and occasional contraction of this core machinery. This analysis reveals the ancient origin of RNA-directed DNA Methylation in early land plants, or possibly their algal ancestors, as well as ongoing duplications that evolve novel small RNA pathways.
Assuntos
Evolução Molecular , RNA , Metilação de DNA/genética , Duplicação Gênica , Genoma de Planta , Filogenia , Plantas/genética , Plantas/metabolismo , RNA/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Oryza , Alelos , Proteínas de Arabidopsis/metabolismo , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Oryza/genética , Oryza/metabolismo , RNA Polimerase II/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genéticaRESUMO
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.
Assuntos
Epigenômica , Sequências Reguladoras de Ácido Nucleico , Cromatina/genética , Genoma de Planta/genética , Plantas/genética , Controle de QualidadeRESUMO
BACKGROUND: RNA-directed DNA methylation (RdDM) initiates cytosine methylation in all contexts and maintains asymmetric CHH methylation. Mature plant embryos show one of the highest levels of CHH methylation, and it has been suggested that RdDM is responsible for this hypermethylation. Because loss of RdDM in Brassica rapa causes seed abortion, embryo methylation might play a role in seed development. RdDM is required in the maternal sporophyte, suggesting that small RNAs from the maternal sporophyte might translocate to the developing embryo, triggering DNA methylation that prevents seed abortion. This raises the question of whether embryo hypermethylation is autonomously regulated by the embryo itself or influenced by the maternal sporophyte. RESULTS: Here, we demonstrate that B. rapa embryos are hypermethylated in both euchromatin and heterochromatin and that this process requires RdDM. Contrary to the current models, B. rapa embryo hypermethylation is not correlated with demethylation of the endosperm. We also show that maternal somatic RdDM is not sufficient for global embryo hypermethylation, and we find no compelling evidence for maternal somatic influence over embryo methylation at any locus. Decoupling of maternal and zygotic RdDM leads to successful seed development despite the loss of embryo CHH hypermethylation. CONCLUSIONS: We conclude that embryo CHH hypermethylation is conserved, autonomously controlled, and not required for embryo development. Furthermore, maternal somatic RdDM, while required for seed development, does not directly influence embryo methylation patterns.
Assuntos
Brassica rapa/embriologia , Metilação de DNA/genética , RNA de Plantas/metabolismo , Sementes/genética , Brassica rapa/genética , Centrômero/metabolismo , Endosperma/embriologia , Endosperma/genética , GenótipoRESUMO
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.
Assuntos
Brassica rapa/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , RNA de Plantas , Sementes/crescimento & desenvolvimento , Alelos , Arabidopsis/metabolismo , Brassica rapa/genética , Brassica rapa/crescimento & desenvolvimento , Brassica rapa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Interferente Pequeno , Sementes/genética , Sementes/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Metilação de DNA , Endosperma , Regulação da Expressão Gênica de Plantas , RNA , SementesRESUMO
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.
Assuntos
Arabidopsis/embriologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Impressão Genômica , Óvulo Vegetal , RNA de Plantas/fisiologia , RNA Interferente Pequeno/genética , Sementes/crescimento & desenvolvimento , Proteínas de Arabidopsis/genéticaRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Grão Comestível/enzimologia , Duplicação Gênica , Poaceae/enzimologia , Seleção Genética , Sequência de Aminoácidos , Grão Comestível/genética , Proteínas de Grãos , Filogenia , Poaceae/genéticaRESUMO
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.
Assuntos
Brassica rapa/genética , Metilação de DNA , RNA Interferente Pequeno/genética , Sementes/genética , Brassica rapa/embriologia , Brassica rapa/enzimologia , Brassica rapa/fisiologia , Elementos de DNA Transponíveis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Diploide , Genótipo , Mutação , Fenótipo , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , Reprodução , Sementes/embriologia , Sementes/enzimologia , Sementes/fisiologiaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Glicoproteínas de Membrana/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Endosperma/citologia , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Fertilização , Glicoproteínas de Membrana/genética , Mutação , Especificidade de Órgãos , Óvulo Vegetal/citologia , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Pólen/citologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Tubo Polínico/citologia , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Polinização , Plântula/citologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/citologia , Sementes/genética , Sementes/crescimento & desenvolvimento , ZigotoRESUMO
ARGONAUTE (AGO) proteins are eukaryotic RNA silencing effectors that interact with their binding partners via short peptide motifs known as AGO hooks. AGO hooks tend to cluster in one region of the protein to create an AGO-binding platform. In addition to the presence of AGO hooks, AGO-binding platforms are intrinsically disordered, contain tandem repeat arrays, and have weak sequence conservation even between close relatives. These characteristics make it difficult to identify and perform evolutionary analysis of these regions. Because of their weak sequence conservation, only a few AGO-binding platforms are characterized, and the evolution of these regions is only poorly understood. In this chapter we describe modules developed for computational identification and evolutionary analysis of AGO-binding platforms, with particular emphasis on understanding evolution of the tandem repeat arrays.