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.
PMID: 3409786;Abstract:
We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.
PMID: 18510771;PMCID: PMC2435114;Abstract:
Background. We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results. We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion. We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals. © 2008 Edwards et al; licensee BioMed Central Ltd.
PMID: 9668984;Abstract:
The Biomek® 2000 Laboratory Automation Workstation is used for liquid handling and other repetitive operations in many laboratories. Since it has very good spatial positioning capabilities, we have modified this workstation to deliver samples at high densities onto microscope slides to produce DNA microarrays. The workstation tool, originally designed for bacterial colony replication, was adapted to carry special printing pins and was further modified to improve its positional accuracy. Software written in the Tool Command Language was concurrently developed to control the movements of the workstation arm during the process of printing. With these modifications, the workstation can reliably deliver individual samples at a spacing of 0.5 mm, corresponding to a total of more than 3000 samples on a single slide. Arrays prepared in this way were successfully tested in hybridization experiments.
Abstract:
Some plant defenses are known to be rapidly induced following attack by phytophagous insects. Plant-produced insect molting hormones, trained phytoecdysteroids, are believed to aid plant resistance; however, their dynamics are poorly understood. Using spinach (Spinacia oleracea) as a model system, we examined the inducibility of phytoecdysteroids, primarily 20-hydroxyecdysone (20E), in an effort to characterize potential interactions with herbivorous insects. Rapid phytochemical induction was investigated using damage treatments and applications of defense-related plant-signal analogs, specifically methyl jasmonate (MJ) and methyl salicylate (MSA). Within two days, mechanically damaged roots exhibited two to three fold increases in phytoecdysteroid concentrations. Four days after root damage, small increases in shoot levels were also detectable. Unlike roots, foliar 20E concentrations were unaltered over a range of shoot treatments including insect herbivory (Spodoptera exigua), mechanical damage, and MJ applications. Additions of MJ (12.5-50 μg/liter) to the root systems of hydroponically grown plants stimulated accumulations of root phytoecdysteroids in a dose-dependent manner, similar in magnitude to the response induced by root damage. Under identical conditions, MSA did not affect the accumulation of 20E when added to the hydroponic solutions of undamaged plants. Moreover, MSA inhibited the induction of 20E in wounded roots, but did not interfere with the action of applied MJ. In contrast to mechanical damage, roots did not induce 20E levels when challenged with two different fungal pathogens (Pythium aphanidermatum and Phytophthora capsici). We propose that wound- induced accumulations of 20E are generated in the roots, signaled via endogenous jasmonates, and may confer enhanced resistance against subterranean herbivorous insects.