David W Galbraith

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.

PMID: 13677474;Abstract:

Using a cDNA microarray containing 7943 ESTs, the behavior of the maize root transcriptome has been monitored in a time course for 72 h after imposition of salinity stress (150 mM NaCl). Under these conditions, root sodium amounts increased faster than in leaves, and root potassium decreased significantly. Although the overall free amino acid concentration was not affected, amino acid composition was changed with proline and asparagine increasing. Microarray analysis identified 916 ESTs representing genes whose steady-state RNA levels were significantly altered at various time points, corresponding to 11% of the ESTs printed. The response of the transcriptome to sub-lethal salt stress was rapid and transient, leading to a burst of changes at the three-hour time point. The salt-regulated ESTs represented 472 tentatively unique genes (TUGs), which, based on functional category analysis, are involved in a broad range of cellular and biochemical activities, prominent amongst which were transport and signal transduction pathways. Clustering of regulated transcripts based on the timing and duration of changes suggests a structured succession of induction and repression for salt responsive genes in multiple signal and response cascades. Within this framework, 16 signaling molecules, including six protein kinases, two protein phosphatases and eight transcription factors, were regulated with distinct expression patterns by high salinity.

PMID: 11289513;Abstract:

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2)most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²-10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) Contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.