Helen Reynolds

Evidence for a Direct Link between Glutathione Biosynthesis and Stress Defense Gene Expression in Arabidopsis

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had ≥50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic γ-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

Elevated Glutathione Biosynthetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased Oxidative Stress

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted γ-glutamylcysteine synthetase (γ-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity–dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and γ-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of γ-ECS. Given the results of these experiments, we suggest that γ-ECS–transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

Agrobacterium - and microprojectile - mediated viral DNA delivery into barley microspore-derived cultures.

SummaryAnther cultures of barley (Hordeum vulgare L. var. “Igri”) were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.

Manipulation of glutathione reductase in transgenic plants: implications for plants' responses to environmental stress

The activities of a number of enzymes of the ascorbate–glutathione pathway have been shown to rise under conditions of increased oxidative stress. The potential to alter the expression of specific enzymes of this pathway by genetic manipulation has provided the opportunity to attempt to develop transgenic plants with altered levels of oxidative stress defense enzymes which should have improved stress tolerances. We have cloned a cDNA for glutathione reductase from a higher plant ( Pisum sativum L.) and have used this to construct chimeric genes for the expression of the pea enzyme in the chloroplast, mitochondrion or cytosol of transgenic tobacco plants. Some of the transformed lines with elevated levels of expression of glutathione reductase accumulate higher concentrations of glutathione and show increased tolerance to paraquat, however, no evidence was found for elevated tolerance to ozone fumigation.

Systemic Sensing of Light in Plants; A Key Regulatory Role of Photosynthetic Electron Transport

H2O2 is the most stable of the active oxygen species (AOS) and can behave both as an oxidant and as a reductant (1). The electron transfer chain of the chloroplasts is the best documented source of H2O2 (2). Mitochondria and peroxisomes are also major sources of H2O2 (3). In the light, the key enzyme involved in H2O2 scavenging is ascorbate peroxidase (APX; EC 1.11.1.11), which catalyzes the reaction; 2 ascorbate + H2O2 → 2 monodehydroascorbate + 2H2O. In Arabidopsis, there are at least five different APX isoforms and the induction of APX1 and APX2 is controlled by the redox status of the plastoquinone pool (PQ; 4, data not shown).