Charophytic Green Algae Encode Ancestral Polymerase IV/Polymerase V Subunits and a CLSY/DRD1 Homolog.

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.

A null allele of the pol IV second subunit impacts stature and reproductive development in Oryza sativa.

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.

CHD Chromatin Remodeling Protein Diversification Yields Novel Clades and Domains Absent in Classic Model Organisms.

Chromatin remodelers play a fundamental role in the assembly of chromatin, regulation of transcription, and DNA repair. Biochemical and functional characterizations of the CHD family of chromatin remodelers from a variety of model organisms have shown that these remodelers participate in a wide range of activities. However, because the evolutionary history of CHD homologs is unclear, it is difficult to predict which of these activities are broadly conserved and which have evolved more recently in individual eukaryotic lineages. Here, we performed a comprehensive phylogenetic analysis of 8,042 CHD homologs from 1,894 species to create a model for the evolution of this family across eukaryotes with a particular focus on the timing of duplications that gave rise to the diverse copies observed in plants, animals, and fungi. Our analysis confirms that the three major subfamilies of CHD remodelers originated in the eukaryotic last common ancestor, and subsequent losses occurred independently in different lineages. Improved taxon sampling identified several subfamilies of CHD remodelers in plants that were absent or highly divergent in the model plant Arabidopsis thaliana. Whereas the timing of CHD subfamily expansions in vertebrates corresponds to whole genome duplication events, the mechanisms underlying CHD diversification in land plants appear more complicated. Analysis of protein domains reveals that CHD remodeler diversification has been accompanied by distinct transitions in domain architecture, contributing to the functional differences observed between these remodelers. This study demonstrates the importance of proper taxon sampling when studying ancient evolutionary events to prevent misinterpretation of subsequent lineage-specific changes and provides an evolutionary framework for functional and comparative analysis of this critical chromatin remodeler family across eukaryotes.

Evidence for a Unique DNA-Dependent RNA Polymerase in Cereal Crops.

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.

Identification and Evolutionary Characterization of ARGONAUTE-Binding Platforms.

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.

Detoxification of polycyclic aromatic hydrocarbons (PAHs) in Arabidopsis thaliana involves a putative flavonol synthase.

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with cytotoxic, teratogenic and carcinogenic properties. Bioremediation studies with bacteria have led to the identification of dioxygenases (DOXs) in the first step to degrade these recalcitrant compounds. In this study, we characterized the role of the Arabidopsis thaliana AT5G05600, a putative DOX of the flavonol synthase family, in the transformation of PAHs. Phenotypic analysis of loss-of-function mutant lines showed that these plant lines were less sensitive to the toxic effects of phenanthrene, suggesting possible roles of this gene in PAH degradation in vivo. Interestingly, these mutant lines showed less accumulation of H2O2 after PAH exposure. Transgenic lines over-expressing At5g05600 showed a hypersensitive response and more oxidative stress after phenanthrene treatments. Moreover, fluorescence spectra results of biochemical assays with the recombinant His-tagged protein AT5G05600 detected chemical modifications of phenanthrene. Taken together, these results support the hypothesis that AT5G05600 is involved in the catabolism of PAHs and the accumulation of toxic intermediates during PAH biotransformation in plants. This research represents the first step in the design of transgenic plants with the potential to degrade PAHs, leading to the development of vigorous plant varieties that can reduce the levels of these pollutants in the environment.

The Argonaute-binding platform of NRPE1 evolves through modulation of intrinsically disordered repeats.

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.

Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP.

The Leguminosae has emerged as a model for studying angiosperm plastome evolution because of its striking diversity of structural rearrangements and sequence variation. However, most of what is known about legume plastomes comes from few genera representing a subset of lineages in subfamily Papilionoideae. We investigate plastome evolution in subfamily Mimosoideae based on two newly sequenced plastomes (Inga and Leucaena) and two recently published plastomes (Acacia and Prosopis), and discuss the results in the context of other legume and rosid plastid genomes. Mimosoid plastomes have a typical angiosperm gene content and general organization as well as a generally slow rate of protein coding gene evolution, but they are the largest known among legumes. The increased length results from tandem repeat expansions and an unusual 13 kb IR-SSC boundary shift in Acacia and Inga. Mimosoid plastomes harbor additional interesting features, including loss of clpP intron1 in Inga, accelerated rates of evolution in clpP for Acacia and Inga, and dN/dS ratios consistent with neutral and positive selection for several genes. These new plastomes and results provide important resources for legume comparative genomics, plant breeding, and plastid genetic engineering, while shedding further light on the complexity of plastome evolution in legumes and angiosperms.

Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues.

PREMISE OF THE STUDY: Variation in the distribution of methylated CpG (methyl-CpG) in genomic DNA (gDNA) across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2) to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich) vs. organellar and prokaryotic (methyl-CpG-poor) elements for genomic and metagenomic sequencing projects. • METHODS: MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1) untreated gDNA, (2) a methyl-CpG-depleted fraction, and (3) a methyl-CpG-enriched fraction. • RESULTS: Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2-11.2-fold and 3.4-11.3-fold increase in chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), respectively. Methyl-enriched fractions showed a 1.8-31.3-fold and 1.3-29.0-fold decrease in cpDNA and mtDNA, respectively. • DISCUSSION: The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae). When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues.