David W Galbraith
Work Summary
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
Abstract:
Analysis of gene expression on a genome scale can provide useful insights into plant growth and development, and an understanding of the mechanisms used by plants to cope with biotic and abiotic stress. To facilitate analysis of genome-wide gene expression in maize, we have assembled a large collection of maize EST and genomic sequences, designed a set of 57,442 maize 70-mer oligonucleotides to represent these sequences, and printed a two-slide microarray set (MOA and MOB) which is available to the maize research community at minimal cost. To monitor array quality, we have developed a series of printing controls and procedures that when coupled with a 9-mer hybridization assay, allow tracking of spot morphology and printing pin carryover. An optimized hybridization protocol has been developed by testing a series of hybridization temperatures and performing detailed statistical analyses. To facilitate management of all long-oligonucleotide associated array data, Zeamage, a Sybase relational database has been developed and is available at www.maizearray.org. Zeamage contains the appropriate tables and fields for tracking the oligonucleotide sequences and associated annotation, array design, and biological information associated with the microarray hybridizations. The www.maizearray.org web-site provides additional information on the project, array content, and data analysis tools.
PMID: 20833729;PMCID: PMC2971586;Abstract:
The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species. © American Society of Plant Biologists.
Flow cytometry, and the accompanying technology of cell sorting, represents an established and valuable experimental platform for the analysis of cellular populations. Applications involving higher plants, which started to emerge around 30 years ago, are now widely employed both to provide unique information regarding fundamental questions in basic and applied bioscience and to advance agricultural productivity in practical ways. Further developments of this platform are being actively pursued, promising additional advances in our understanding of the interactions of cells within the complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types and second that their individual cells are generally larger than those of mammals.This chapter focuses on the use of flow cytometry and cell sorting with the model species Arabidopsis thaliana, in particular addressing (1) fluorescence in vivo labeling of specific cell types, (2) fluorescence-activated sorting of protoplasts and nuclei, and (3) transcriptome analyses using sorted protoplasts and nuclei.