Mathiason, K., Dong, H. e., Grimplet, J., Venkateswari, J., Galbraith, D. W., Etti, O. r., & Fennell, A. (2009). Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break. Functional and Integrative Genomics, 9(1), 81-96.
PMID: 18633655;Abstract:
Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. © Springer-Verlag 2008.
Boyce, J. M., Knight, H., Deyholos, M., Openshaw, M. R., Galbraith, D. W., Warren, G., & Knight, M. R. (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal, 34(4), 395-406.
PMID: 12753580;Abstract:
The sfr6 mutant of Arabidopsis displays a deficit in freezing tolerance after cold acclimation. We previously observed that the transcripts of three cold-, ABA- and drought-inducible genes, each having a C-repeat motif or the drought-responsive element (CRT/DRE) in its promoter, failed to normally accumulate in this mutant. We now report that the effects of sfr6 upon transcript levels are reflected in the levels of the encoded proteins, confirming that the cold-inducible protein expression is affected by the sfr6 mutation. Using microarray analysis, we found not only that this effect may be general to cold-inducible genes with CRT/DRE promoter elements, but also that it extends to some other genes whose promoters lack a CRT/DRE element. The role of the CRT/DRE has been empirically tested by use of a synthetic promoter, confirming that the CRT/DRE is sufficient to confer the sfr6 effect upon expression. Tolerance of osmotic stress was also found to be reduced in sfr6, consistent with a role in osmotic stress tolerance for the cold-, ABA-and drought-inducible genes whose expression is affected by the sfr6 mutation.
Galbraith, D. W. (2007). Analysis of Plant Gene Expression Using Flow Cytometry and Sorting. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes, 405-422.
Birnbaum, K., Jung, J. W., Wang, J. Y., Lambert, G. M., Hirst, J. A., Galbraith, D. W., & Benfey, P. N. (2005). Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nature Methods, 2(8), 615-619.
PMID: 16170893;Abstract:
To investigate the relationship between developmental events and gene expression, cell-specific resolution of gene activity is critical. Such high-resolution data have been difficult to obtain at a genomic level because cells first need to be isolated, and then sufficient amounts of mRNA must be collected, or subsequently amplified, for a large-scale profiling analysis. Genomics methods have tremendous potential to infer developmental circuits and, in combination with genetic tools, to discover the unknown downstream targets of known developmental regulators. We have developed a method that can be used to isolate up to hundreds of thousands of plant cells of a specific cell type, with very high purity, which can then be used for microarray analysis. The method makes use of reporter lines expressing green fluorescent protein (GFP) in histologically defined cell types, of which large collections are now available (Table 1). The GFP Line of interest is bulked and the tissue is collected and rapidly converted into protoplasts. GFP-positive cells are then isolated using a fluorescence-activated cell sorter (FACS). Total RNA is isolated, labeled using standard procedures and applied to microarrays (Fig. 1). The technique has been used to generate expression profiles of cell types and tissues in the Arabidopsis thaliana root, although it can be used for any tissue whose cell walls can be readily digested. The protocol presented here has been optimized for roots.
Galbraith, D., Gong, F. C., Giddings, T. H., Meehl, J. B., Staehelin, L. A., & Galbraith, D. W. (1996). Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 93(5).
We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
