Sunchung Park

Plant Body Weight-Induced Secondary Growth in Arabidopsis and Its Transcription Phenotype Revealed by Whole-Transcriptome Profiling

Wood is an important raw material and environmentally cost-effective renewable source of energy. However, the molecular biology of wood formation (i.e. secondary growth) is surprisingly understudied. A novel experimental system was employed to study the molecular regulation of secondary xylem formation in Arabidopsis. First, we demonstrate that the weight carried by the stem is a primary signal for the induction of cambium differentiation and the plant hormone, auxin, is a downstream carrier of the signal for this process. We used Arabidopsis whole-transcriptome (23 K) GeneChip analysis to examine gene expression profile changes in the inflorescent stems treated for wood formation by cultural manipulation or artificial weight application. Many of the genes up-regulated in wood-forming stems had auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth. We identified 700 genes that were differentially expressed during the transition from primary growth to secondary growth. More than 40% of the genes that were up-regulated (>5×) were associated with signal transduction and transcriptional regulation. Biological significance of these regulatory genes is discussed in light of the induction and development of secondary xylem.

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.

Identification of Genes with Potential Roles in Apple Fruit Development and Biochemistry through Large-Scale Statistical Analysis of Expressed Sequence Tags

Advanced studies of apple (Malus domestica Borkh) development, physiology, and biochemistry have been hampered by the lack of appropriate genomics tools. One exception is the recent acquisition of extensive expressed sequence tag (EST) data. The entire available EST dataset for apple resulted from the efforts of at least 20 contributors and was derived from more than 70 cDNA libraries representing diverse transcriptional profiles from a variety of organs, fruit parts, developmental stages, biotic and abiotic stresses, and from at least nine cultivars. We analyzed apple EST sequences available in public databanks using statistical algorithms to identify those apple genes that are likely to be highly expressed in fruit, expressed uniquely or preferentially in fruit, and/or temporally or spatially regulated during fruit growth and development. We applied these results to the analysis of biochemical pathways involved in biosynthesis of precursors for volatile esters and identified a subset of apple genes that may participate in generating flavor and aroma components found in mature fruit.

Novel gene expression profiles define the metabolic and physiological processes characteristic of wood and its extractive formation in a hardwood tree species, Robinia pseudoacacia.

Wood is of critical importance to humans as a primary feedstock for biofuel, fiber, solid wood products, and various natural compounds including pharmaceuticals. The trunk wood of most tree species has two distinctly different regions: sapwood and heartwood. In addition to the major constituents, wood contains extraneous chemicals that can be removed by extraction with various solvents. The composition and the content of the extractives vary depending on such factors as, species, growth conditions, and time of year when the tree is cut. Despite the great commercial and keen scientific interest, little is known about the tree-specific biology of the formation of heartwood and its extractives. In order to gain insight on the molecular regulations of heartwood and its extractive formation, we carried out global examination of gene expression profiles across the trunk wood of black locust (Robinia pseudoacacia L.) trees. Of the 2,915 expressed sequenced tags (ESTs) that were generated and analyzed in the current study, 55.3% showed no match to known sequences. Cluster analysis of the ESTs identified a total of 2278 unigene sets, which were used to construct cDNA microarrays. Microarray hybridization analyses were then performed to survey the changes in gene expression profiles of trunk wood. The gene expression profiles of wood formation differ according to the region of trunk wood sampled, with highly expressed genes defining the metabolic and physiological processes characteristic of each region. For example, the gene encoding sugar transport had the highest expression in the sapwood, while the structural genes for flavonoid biosynthesis were up-regulated in the sapwood-heartwood transition zone. This analysis also established the expression patterns of 341 previously unknown genes.

Natural variation in the C‐repeat binding factor cold response pathway correlates with local adaptation of Arabidopsis ecotypes

The natural range of Arabidopsis thaliana (Arabidopsis) encompasses geographical regions that have greatly differing local climates, including harshness of winter temperatures. A question thus raised is whether differences in freezing tolerance might contribute to local adaptation in Arabidopsis. Consistent with this possibility is that Arabidopsis accessions differ in freezing tolerance and that those collected from colder northern latitudes are generally more tolerant to freezing than those collected from warmer southern latitudes. Moreover, recent studies with Arabidopsis genotypes collected from sites in Sweden (SW) and Italy (IT) have established that the two accessions are locally adapted, that the SW ecotype is more tolerant of freezing than the IT ecotype, and that genetic differences between the two ecotypes that condition local adaptation and freezing tolerance map to a region that includes the C-repeat binding factor (CBF) locus. The CBF locus includes three genes - CBF1, CBF2 and CBF3 - that are induced by low temperature and encode transcription factors that regulate a group of more than 100 genes, the CBF regulon, which impart freezing tolerance. Here we show that cold induction of most CBF regulon genes is lower in IT plants compared with SW plants, and that this is due to the IT CBF2 gene encoding a non-functional CBF2 protein. The non-functional IT CBF2 protein also contributes to the lower freezing tolerance of the IT plants compared with the SW plants. Taken together, studies on the SW and IT ecotypes provide evidence that natural variation in the CBF pathway has contributed to adaptive evolution in these Arabidopsis populations.

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.

Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana

Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.

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.

CAMTA-mediated regulation of salicylic acid immunity pathway genes in arabidopsis exposed to low temperature and pathogen infection

CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves action of a repression module that acts independently of calmodulin binding, a finding that challenges current models. Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

Genomic and Gene-Level Distribution of Histone H3 Dimethyl Lysine-27 (H3K27me2) in Arabidopsis

Histone lysine methylation patterns underlie much of the functional diversity of nucleosomes in eukaryotes, and an interesting aspect of histone methylation is the potential functional specificity for different methylation states on a given lysine. Trimethylation of histone H3 (H3K27me3) is intimately related to developmental gene silencing through the so-called Polycomb Group (PcG) mechanism. How this modification becomes established at PcG-repressed loci is generally not known, but it has been suggested that it may be facilitated by prior occupancy by H3K27me2. In this study we mapped the genomic and gene-level distribution of H3K27me2 in Arabidopsis thaliana using ChIP and a high-density tiling microarray, and integrated this with previous maps of other chromatin features and gene expression data. At the genome level, H3K27me2 enrichment sites were sparsely distributed across chromosomes, within an average size expected for a single nucleosome, and contrasted with the longer domains seen for H3K27me3. In both heterochromatic and euchromatic segments of the genome, H3K27me2 enrichment was often localized within transposon-related genes, with the longest genomic stretches of this modification corresponding to retroelements. However, H3K27me2 was more frequently found within protein-coding genes. These genes generally also showed moderate enrichment for H3K27me3, but H3K27me2 was strongly depleted within those genes most enriched in H3K27me3. H3K27me2 within highly transcribed genes was at highest levels at transcriptional starts and was strongly depleted throughout the transcribed regions, and reached higher levels at active than at silent promoters.