Sookyung Oh

Genic and global functions for Paf1C in chromatin modification and gene expression in arabidopsis

In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 3′ shift in the distribution of H3K4me3 and a 5′ shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory.

Genome Sequencing of Arabidopsis abp1-5 Reveals Second-Site Mutations That May Affect Phenotypes

Auxin regulates numerous aspects of plant growth and development. For many years, investigating roles for AUXIN BINDING PROTEIN1 (ABP1) in auxin response was impeded by the reported embryo lethality of mutants defective in ABP1. However, identification of a viable Arabidopsis thaliana TILLING mutant defective in the ABP1 auxin binding pocket (abp1-5) allowed inroads into understanding ABP1 function. During our own studies with abp1-5, we observed growth phenotypes segregating independently of the ABP1 lesion, leading us to sequence the genome of the abp1-5 line described previously. We found that the abp1-5 line we sequenced contains over 8000 single nucleotide polymorphisms in addition to the ABP1 mutation and that at least some of these mutations may originate from the Arabidopsis Wassilewskija accession. Furthermore, a phyB null allele in the abp1-5 background is likely causative for the long hypocotyl phenotype previously attributed to disrupted ABP1 function. Our findings complicate the interpretation of abp1-5 phenotypes for which no complementation test was conducted. Our findings on abp1-5 also provide a cautionary tale illustrating the need to use multiple alleles or complementation lines when attributing roles to a gene product.

PLANT HOMOLOGOUS TO PARAFIBROMIN Is a Component of the PAF1 Complex and Assists in Regulating Expression of Genes within H3K27ME3-Enriched Chromatin

The human Paf1 complex (Paf1C) subunit Parafibromin assists in mediating output from the Wingless/Int signaling pathway, and dysfunction of the encoding gene HRPT2 conditions specific cancer-related disease phenotypes. Here, we characterize the organismal and molecular roles of PLANT HOMOLOGOUS TO PARAFIBROMIN (PHP), the Arabidopsis (Arabidopsis thaliana) homolog of Parafibromin. PHP resides in an approximately 670-kD protein complex in nuclear extracts, and physically interacts with other known Paf1C-related proteins in vivo. In striking contrast to the developmental pleiotropy conferred by mutation in other plant Paf1C component genes in Arabidopsis, loss of PHP specifically conditioned accelerated phase transition from vegetative growth to flowering and resulted in misregulation of a very limited subset of genes that included the flowering repressor FLOWERING LOCUS C. Those genes targeted by PHP were distinguished from the bulk of Arabidopsis genes and other plant Paf1C targets by strong enrichment for trimethylation of lysine-27 on histone H3 (H3K27me3) within chromatin. These findings suggest that PHP is a component of a plant Paf1C protein in Arabidopsis, but has a more specialized role in modulating expression of a subset of Paf1C targets.

Cloning, characterization, and heterologous expression of a functional geranylgeranyl pyrophosphate synthase from sunflower (Helianthus annuus L.).

Summary Geranylgeranyl pyrophosphate (GGPP) synthase is a key enzyme for the biosynthesis of terpenoid compounds that play vital roles in plant growth and development, and interactions between organisms. We have cloned and characterized a sunflower cDNA encoding GGPP synthase. Sequence analysis showed that the gene contained a 1 071-bp open reading frame coding for a peptide of 356 amino acid residues with a calculated molecular mass of 38.7 kDa. The predicted amino acid sequence of this sunflower GGPS has high similarity to other plant GGPP synthases (63–79% ). In vitro activity assay using recombinant protein and genetic complementation experiments have shown that the cDNA we cloned encodes for functional GGPP synthase. Gene expression studies using sunflower seedlings showed that the gene was expressed after two days of seed imbibition. Abscisic acid treatment down-regulated the expression of the gene.

Phytochrome-dependent coordinate control of distinct aspects of nuclear and plastid gene expression during anterograde signaling and photomorphogenesis

Light perception by photoreceptors impacts plastid transcription, development, and differentiation. This photoreceptor-dependent activity suggests a mechanism for photoregulation of gene expression in the nucleus and plastid that serves to coordinate expression of critical genes of these two organelles. This coordinate expression is required for proper stoichiometric accumulation of components needed for assembly of plastids, photosynthetic light-harvesting complexes and components such as phytochromes. Chloroplast-targeted sigma factors, which function together with the plastid-encoded RNA polymerase to regulate expression of plastid-encoded genes, and nuclear-encoded plastid development factors, such as GLK1 and GLK2, are targets of phytochrome regulation. Such phytochrome-dependent functions are hypothesized to allow light-dependent regulation, and feasibly tuning, of plastid components and function in response to changes in the external environment, which directly affects photosynthesis and the potential for light-induced damage. When the size and protein composition of the light-harvesting complexes are not tuned to the external environment, imbalances in electron transport can impact the cellular redox state and cause cellular damage. We show that phytochromes specifically regulate the expression of multiple factors that function to modulate plastid transcription and, thus, provide a paradigm for coordinate expression of the nuclear and plastid genomes in response to changes in external light conditions. As phytochromes respond to changes in the prevalent wavelengths of light and light intensity, we propose that specific phytochrome-dependent molecular mechanisms are used during light-dependent signaling between the nucleus and chloroplast during photomorphogenesis to coordinate chloroplast development with plant developmental stage and the external environment.

Detection and Species Identification of Wood-Decaying Fungi by Hybridization of Immobilized Sequence-Specific Oligonucleotide Probes with PCR-Amplified Fungal Ribosomal DNA Internal Transcribed Spacers

Summary We developed an effective detection method for wood-decaying fungi by hybridization of immobilized Sequence-Specific Oligonucleotide Probes with florescent-labeled PCR-amplified fungal rDNA internal transcribed spacer sequences. This method takes advantage of both the sequence specificity of Southern blot hybridization and the sensitivity of the previously reported PCR-based fungal species identification methods. Both in vitro cultured fungal strains and naturally decaying wood samples were used to demonstrate that this method is robust and practical for detection of incipient wood-decaying fungi. It can be a useful tool for microbial ecology, plant pathology, protection of wood products in service, preservation efforts for high-value furniture and wood-based art and DNA fingerprinting for tracking the source of contamination of wood decay fungi.

Phytochrome-induced SIG2 expression contributes to photoregulation of phytochrome signalling and photomorphogenesis in Arabidopsis thaliana

Chloroplast-localized sigma factor (SIG) proteins promote specificity of the plastid-encoded RNA polymerase. SIG2 function appears to be necessary for light-grown Arabidopsis thaliana plants. Specific photoreceptors or light-dependent factors that impact the light-induced accumulation of SIG2 have not been reported. A molecular link between phytochromes and nuclear-encoded SIG2, which impacts photomorphogenesis specifically under red (R) and far-red (FR) light, is described here. Both phyA and phyB promote SIG2 transcript accumulation. Disruption of SIG2 results in R- and FR-specific defects in the inhibition of hypocotyl elongation and cotyledon expansion, although no impairments in these responses are detected for sig2 mutants under blue (B) or white (W) light. SIG2 also impacts root elongation under W and R, and the R-dependent expression of PIF4, encoding a phytochrome-interacting factor, and HY2, which encodes a phytochrome chromophore biosynthetic enzyme. Whereas SIG2 apparently impacts the accumulation of the phytochromobilin (PΦB) phytochrome chromophore, sig2 mutants differ significantly from PΦB mutants, primarily due to wavelength-specific defects in photomorphogenesis and disruption of a distinct subset of phytochrome-dependent responses. The molecular link between phytochromes and SIG2 is likely to be an important part of the co-ordination of gene expression to maintain stoichiometry between the nuclear-encoded phytochrome apoprotein and plastid-derived PΦB, which combine to form photoactive phytochromes, and/or light-dependent SIG2 accumulation is involved in an inductive light signalling pathway co-ordinating components between nucleus and plastids.

Large-scale computational analysis of poplar ESTs reveals the repertoire and unique features of expressed genes in the poplar genome

Perennial woody plants differ from annual herbaceous plants in several ways and are expected to have evolved to adopt a unique repertoire and expression profiles of functional genes. Poplar, a model tree species for which a large number of ESTs are publicly available, was used to carry out a large-scale comparative analysis with the expressed sequences of eight plant species. First, we obtained 105,831 poplar ESTs from public databases and identified a set of 25,282 unigenes (i.e., tentative non-redundant sequences). The majority of the unigenes (56%) had significant matches to Arabidopsis genes. We then estimated poplar multigene families by counting the tBLASTX matches of each unigene against the poplar unigene dataset itself. Forty-seven percent of the 25,282 unigenes were subsequently organized into 3,481 multigene families 89% of which had less than five copy members. In poplar, protein kinases represent the largest family followed by GTP-binding proteins and Myb transcription factors. Several multigene families had a higher copy number in poplar than in Arabidopsis hinting potential lineage-specific proliferation of poplar protein families. Such expansion may be related to the adaptation of perennial poplars for the high degree of environmental stresses that affects growth and survival. Comparison of poplar unigenes with the Arabidopsis transcriptome revealed that genes involved in transcriptional regulation are the most divergent while metabolism-related genes are the most conserved.

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana.

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.

Frequency-based time-series gene expression recomposition using PRIISM

BackgroundCircadian rhythm pathways influence the expression patterns of as much as 31% of the Arabidopsis genome through complicated interaction pathways, and have been found to be significantly disrupted by biotic and abiotic stress treatments, complicating treatment-response gene discovery methods due to clock pattern mismatches in the fold change-based statistics. The PRIISM (Pattern Recomposition for the Isolation of Independent Signals in Microarray data) algorithm outlined in this paper is designed to separate pattern changes induced by different forces, including treatment-response pathways and circadian clock rhythm disruptions.ResultsUsing the Fourier transform, high-resolution time-series microarray data is projected to the frequency domain. By identifying the clock frequency range from the core circadian clock genes, we separate the frequency spectrum to different sections containing treatment-frequency (representing up- or down-regulation by an adaptive treatment response), clock-frequency (representing the circadian clock-disruption response) and noise-frequency components. Then, we project the components’ spectra back to the expression domain to reconstruct isolated, independent gene expression patterns representing the effects of the different influences.By applying PRIISM on a high-resolution time-series Arabidopsis microarray dataset under a cold treatment, we systematically evaluated our method using maximum fold change and principal component analyses. The results of this study showed that the ranked treatment-frequency fold change results produce fewer false positives than the original methodology, and the 26-hour timepoint in our dataset was the best statistic for distinguishing the most known cold-response genes. In addition, six novel cold-response genes were discovered. PRIISM also provides gene expression data which represents only circadian clock influences, and may be useful for circadian clock studies.ConclusionPRIISM is a novel approach for overcoming the problem of circadian disruptions from stress treatments on plants. PRIISM can be integrated with any existing analysis approach on gene expression data to separate circadian-influenced changes in gene expression, and it can be extended to apply to any organism with regular oscillations in gene expression patterns across a large portion of the genome.