75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

439 A Comparison of Full Versus Partial 15 N Incorporation for Metabolic Labeling in Arabidopsis
Thaliana
Edward Huttlin 2, 1 , Adrian Hegeman 2, 1 , Amy Harms 1 , Michael Sussman 2, 1
1
University of Wisconsin Biotechnology Center, 2 University of Wisconsin Biochemistry Department
Over the past several years a variety of isotope-assisted quantitative proteomics techniques have been developed, allowing
use of tandem mass spectrometry for the simultaneous comparison of the identities and abundances of hundreds or thousands
of proteins within pairs of biological samples. These approaches include in vitro labeling techniques such as ICAT and 18 O
labeling which introduce an isotopically-labeled tag onto each sample during sample preparation, as well as in vivo metabolic
labeling techniques in which an isotopic label is incorporated into the organism from its media during normal growth and
development. For all of these approaches, after incorporation of either a light or a heavy isotopic tag, the control and experimental
samples are combined and processed together. Because the tagged peptides are essentially chemically identical, they provide
an excellent internal control for all subsequent steps in sample preparation and analysis. Yet using mass spectrometry we can
differentiate among peptides from each sample due to the mass difference between the heavy and light isotopic tags. Because
metabolic labeling allows combination of control and experimental samples through all steps of sample processing including
tissue homogenization and protein extraction, it provides perhaps the ideal internal control.
While metabolic labeling provides an elegant control for all steps in sample preparation, presently some technical challenges
limit its widespread application, especially in intact organisms such as plants. First, achieving full incorporation of an isotopic
label such as 15 N into plants and other higher organisms is challenging and may require unnatural growth conditions that limit
the biological questions to which it may be applied. Additionally, when applied in a high throughput manner these approaches
require informatics tools for automated data analysis. We have compared traditional ubiquitous 15 N metabolic labeling in
Arabidopsis with a new approach using partial incorporation of 15 N (based on Whitelegge et al. Phytochemistry 65 (2004)
1507-1515) to produce changes in the isotopic envelopes of each peptide that can be used for quantitative comparison. While
both approaches require significant informatics tools for analysis, the latter is perhaps more amenable for labeling under a
wider variety of conditions. We will present a comparison of traditional metabolic labeling with partial metabolic labeling in
Arabidopsis whole tissue with respect to numbers of peptide and protein identifications as well as quantitative accuracy and
dynamic range. We will also discuss our current strategies for automated data analysis using both approaches.
440 Understanding the TGA transcriptional network using ChIP-chip and expression arrays
Francoise Thibaud-Nissen 1 , Christopher Johnson 2 , Hank Wu 1 , Julia Redman 1 , Yuelin Zhang 3 , Xin Li 3 , Jonathan Arias 2 ,
Christopher Town 1
1
The Institute for Genomic Research, Rockville MD, USA., 2 University of Maryland, Baltimore County, MD,
USA., 3 University of British Columbia, Vancouver BC, Canada
In order to understand the transcriptional network of TGA factors in Arabidopsis and its role in systemic acquired
resistance, we are conducting ChIP-chip studies using TGA antibodies and analysis of SA-induced changes in expression
in wild-type and tga mutants. The TGA transcription factors TGA2, TGA5 and TGA6 have been found to play a redundant
and essential role in the onset of systemic acquired resistance. In a preliminary analysis we have identified over 50 putative
binding sites for TGA2 through a ChIP-chip approach using Arabidopsis whole-genome arrays, 55% of which contain a
single palindromic octamer TGACGTCA. Additional ChIP-chip data using TGA5 show substantial overlap in the binding
sites of TGA2 and TGA5. In parallel, the effect of the SA treatment on gene expression in wild-type plants and tga2tga5,
tga6 and tga2tga5tga6 mutants was determined using Affymetrix ATH1 arrays. We observed substantial and very similar
changes in expression upon SA application in the wild-type, the single and the double mutant. In particular, genes coding
for kinases and proteins involved in the response to biotic stimuli are overrepresented among the genes differentially
regulated by SA. However, SA induced very few changes in gene expression in the triple mutant. Hierarchical clustering
of experimental samples based on gene expression revealed that the 18-hour mock-treated samples of the triple mutant
cluster with SA-treated samples of the wild-type, single and double mutants, indicative of high basal levels of genes
usually induced by SA in the untreated tga2tga5tga6 plants.
Research supported by the NSF 2010 Program