Research Description - Arabidopsis functional genomics at TIGR

Research Description

Genomics has produced a revolution in plant biology and genome and EST sequencing projects of various plants are progressing rapidly. The completion of the Arabidopsis genome sequence at the end of the year 2000 was a noteworthy achievement, and the rapid progress that has been made in the sequencing of Rice promises to provide an important additional plant resource for comparative analysis of genes and gene function. A significant challenge in all genome projects is the identification and annotation of the genes and the roles they play in development, disease, and response to environmental stresses.

In Arabidopsis, genome sequencing has allowed the identification of approximately 25,500 predicted coding sequences, but significant work remains to provide functional annotation for these. Our goal is to provide experimental validation for these gene predictions and to begin to assign functional roles to genes using DNA microarray technology as a key tool.

Current research

I. Validation of gene predictions using chromosome 2 array.
With the completion of sequencing of the Arabidopsis thaliana genome, wealth of information about the genetic make-up of this model plant has become available. However, much of the annotation of the genome still needs tremendous amount of validations and refinements, and furthermore our ultimate challenge lies in linking these identified genes to specific biological processes. To initiate the expression analysis of the Arabidopsis chromosome 2 in a high-throughput manner, we constructed a microarray representing nearly the entire predicted genes in the chromosome 2. We surveyed diverse Arabidopsis samples using this array, and obtained evidence suggesting the validation of the presence of as much as 84% of the chromosome 2 gene-predictions. We also observed distinct expression patterns in genes that reflect the physiological and structural characteristics of the samples, and these provided us with additional clues to functions encoded by the genes. Additionally, we found a region exhibiting significantly less gene expression activities than anywhere else on the chromosome in all of the samples except seedlings and salt-stressed plants. This may be an example of a chromosomal-level gene expression regulation.
See the PCR primer designing strategy used to construct chromosome 2 microarray.

Heenam Kim, Erik C. Snesrud, Baoping Zhao, Christopher D. Town and John Quackenbush 2002. Expression analysis of the Arabidopsis chromosome 2 using a genomic DNA amplicon microarray. Manuscript in preparation.

II. Evaluation of common references for their relative reliability.
In most microarray assays, labeled cDNA molecules derived from reference and query RNA samples are co-hybridized to probes arrayed on glass surfaces. Gene expression profiles are then calculated for each gene based on the relative hybridization intensities measured between the two samples. The most commonly used reference samples are typically isolates from a single representative RNA source (RNA-0) or pooled mixtures of RNA derived from a plurality of sources (RNA-p). Genomic DNA (gDNA) offers an alternative reference nucleic acid with a number of potential advantages, including stability, reproducibility, and a potentially uniform representation of all genes. Using hydrogen peroxide treated Arabidopsis thaliana plants as a model, we evaluated gDNA and RNA-p as reference samples, and compared expression levels to those measured using an RNA-0 reference.

Kim, H., Zhao, B., Snesrud, E.C., Haas, B.J., Town, C.D., and J. Quackenbush. 2002. Use of RNA and genomic DNA references for inferred comparisons in DNA microarray analyses. BioTechniques. 33: 924-930.

III. Plant response to oxidative stress.
Immediate defense response in plants to various biotic and abiotic stresses often involves a process called oxidative burst that accumulates reactive oxygen species (ROS), mainly superoxide (O2-) and hydrogen peroxide (H2O2), in the challenged plant tissue. H2O2 is an active signaling molecule and its accumulation leads to concerted gene expression and ultimately to enhanced stress and pathogen tolerance. However, the detailed mechanism by which H2O2 triggers defense responses in plants is still largely unknown. In an attempt to identify elements of the signaling pathways triggered by H2O2, we exposed Arabidopsis Col-0 plants to H2O2 and profiled gene expression at 0, 1, 3, 6, and 12 hrs using a DNA microarray representing the nearly 4400 genes on Arabidopsis chromosome 2. We have found a total of 541 genes significantly up- or down-regulated (at 95% confidence) at least one time point. These were then grouped into clusters based on expression pattern. One cluster showing high level expression peaking at 1 hr point includes genes coding for PAL1 and GST6, two marker enzymes for oxidative stress. Our data showed up-regulation of a number of genes that may code for proteins involved in protection and defense. In addition to these, we observed various patterns of regulation with the genes that may encode components of the MAPK signaling pathway, plant hormone synthesis, Ca2+/calmodulin signaling systems, programmed cell death, guanine nucleotide-pool control, cell-cycle control, a ubiquitin/proteasome pathway, and transcription factors. The diversity of the expression patterns and the functional roles represented suggests the presence of an integrated network of signaling pathways linked to the H2O2 signal transduction pathway.

Heenam Kim, Erik C. Snesrud, Baoping Zhao, Brian J. Haas, Christopher D. Town, and John Quackenbush. Activation of a plant defense network in response to H2O2 examined with Arabidopsis chromosome 2 microarrays. Manuscript in preparation.

IV. Construction of the genomic DNA amplicon array for the whole Arabidopsis genome. We recently have expanded our research scope from chromosome 2 to the whole genome, and constructed an amplicon array that represents all the genes and pseudogenes identified in nuclear, mitochondria, and chloroplast genomes. All our future experiments will be conducted using this array. See the information about primers used for the whole genome array construction and PCR scores. All of our primers were synthesized by Invitrogen based on our design.

Future research

Future projects will deal with various aspects of plant biology. Some of the subjects may include,