David W Galbraith

PMID: 9808718;PMCID: PMC34784;Abstract:

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

PMID: 10930466;PMCID: PMC14952;Abstract:

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

Abstract:

Spinach (Spinacia oleracea) foliage is known to synthesize and accumulate insect molting hormones, predominantly in the form of 20- hydroxyecdysone (20E). We previously demonstrated that root 20E accumulation is increased following root damage. We designed two further experiments to address root responses to both mechanical and insect damage. In plants grown hydroponically, removal of 35% or less of the root mass did not result in changes in root 20E levels. However, removal of 70% of the root mass stimulated 6.0- and 1.5-fold increases in the root and shoot 20E concentrations, respectively. The effects of insect damage on soil-grown plants were investigated by infesting plant roots with black vine weevil (BVW: Otiorhynchus sulcatus) larvae and allowing them to feed for seven days. Decreases in root mass occurred in young plants; however, no changes were detected in mature plants. In all cases, root herbivory resulted in at least a 3.0-fold increase in root 20E concentrations. Our previous experiments implicated jasmonic acid and the analog methyl jasmonate (MJ) in signaling the damage-induced accumulation of root 20E levels. We investigated the activity of other phytohormones and growth regulators (GRs) on the 20E accumulation patterns of young plants as a means of examining the significance of jasmonates in the induction response. Hydroponic additions of MJ (0.5 μM) and the synthetic auxin, 1-naphthaleneacetic acid (NAA; 0.5 μM), resulted in significant increases in root 20E levels. At the concentrations tested, indole-3-acetic acid (IAA), gibberellic acid (GA3), abscisic acid (ABA), and trans-zeatin (Z) had no effects on root 20E concentrations. However, both NAA (0.5-5.0 μM) and Z (5.0 μM) treatments caused increases in the root/shoot dry mass ratios, indicating shifts in resource allocation to the roots. Treatments involving ABA (5.0 μM) and Z (0.5-5.0 μM) caused significant increases in shoot 20E concentrations. No other hormone treatments altered shoot accumulation patterns. The mechanisms underlying the root 20E induction phenomena were investigated through the incorporation of [2-¹⁴C]mevalonic acid ([¹⁴C]MVA). Within one day, excised roots readily incorporated radioactivity into 20E from [¹⁴C]MVA. In intact plants, [¹⁴C]MVA absorbed by the roots was rapidly incorporated into root 20E pools following damage and MJ treatments. This implies that the wound-induced root 20E accumulation is the result of increased de novo 20E synthesis in the root.

Abstract:

Recent advances in technology and in the ability to acquire, store, and analyze complex biological data sets have provided an unprecedented understanding of the mechanisms regulating living organisms' development and responses to the environment. We are gaining the insights required for rational modification of these organisms toward specific purposes. Microarrays provide an early example of the productive integration of high-throughput technologies with biological inquiry. In this article we discuss the development of this popular platform and its application in crop science and agriculture. © 2010 by American Institute of Biological Sciences. All rights reserved.