Alan W. Bown

Metabolism and functions of gamma-aminobutyric acid

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.

The Metabolism and Functions of [gamma]-Aminobutyric Acid

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.

The Synthesis of [gamma]-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH

[gamma]-Aminobutyric acid (GABA) synthesis (L-glutamic acid + H+ -> GABA + CO2) is rapidly stimulated by a variety of stress conditions including hypoxia. Recent literature suggests that GABA production and concomitant H+ consumption ameliorates the cytosolic acidification associated with hypoxia or other stresses. This proposal was investigated using isolated asparagus (Asparagus sprengeri Regel) mesophyll cells. Cell acidification was promoted using hypoxia, H+/L-glutamic acid symport, and addition of butyrate or other permeant weak acids. Sixty minutes of all three treatments stimulated the levels of both intracellular and extracellular GABA by values ranging from 100 to 1800%. At an external pH of 5.0, addition of 5 mM butyrate stimulated an increase in overall GABA level from 3.86 (0.56 [plus or minus] SE) to 20.4 (2.16 [plus or minus] SE) nmol of GABA/106 cell. Butyrate stimulated GABA levels by 200 to 300% within 15 s, and extracellular GABA was observed after 10 min. The acid load due to butyrate addition was assayed by measuring [14C]butyrate uptake. After 45 s of butyrate treatment, H+-consuming GABA production accounted for 45% of the imposed acid load. The cytosolic location of a fluorescent pH probe was confirmed using fluorescent microscopy. Spectrofluorimetry indicated that butyrate addition reduced cytosolic pH by 0.60 units with a half-time of approximately 2 s. The proposal that GABA synthesis ameliorates cytosolic acidification is supported by the data. The possible roles of H+ and Ca2+ in stimulating GABA synthesis are discussed.

The rapid determination of γ-aminobutyric acid

A rapid procedure for the extraction and assay of γ-aminobutyric acid (GABA) is described. The extraction procedure prevents rapid GABA accumulation during the sampling of tissue for analysis. It also removes over 95% of pigments absorbing at 340 nm which otherwise reduce the sensitivity of the spectrophotometric coupled enzyme assay. The absorbance increase at 340 nm was linear for GABA levels ranging from 10 to 100 nmol per cuvette.

Extracellular γ-Aminobutyrate Mediates Communication between Plants and Other Organisms

Plants exhibit mutually beneficial and antagonistic interactions with a variety of prokaryotic and eukaryotic organisms. We argue that these interactions are mediated in part by extracellular plant-derived γ -aminobutyrate (GABA). GABA is a ubiquitous four-carbon, nonprotein amino acid that is

Insect Footsteps on Leaves Stimulate the Accumulation of 4-Aminobutyrate and Can Be Visualized through Increased Chlorophyll Fluorescence and Superoxide Production

A substantial literature has demonstrated that within 2 to 3 h of insect herbivory or mechanical damage, plants synthesize wound-induced proteinase inhibitors that inhibit digestion ([Bergey et al., 1996][1]; [Ryan, 2000][2]). In contrast, we demonstrate here that the simple non-wounding crawling of

Rapid [gamma]-Aminobutyric Acid Synthesis and the Inhibition of the Growth and Development of Oblique-Banded Leaf-Roller Larvae.

The hypothesis that rapid [gamma]-aminobutyrate (GABA) accumulation is a plant defense against phytophagous insects was investigated. Increasing GABA levels in a synthetic diet from 1.6 to 2.6 [mu]mol g-1 fresh weight reduced the growth rates, developmental rates, and survival rates of cultured Choristoneura rosaceana cv Harris larvae. Simulation of the mechanical damage resulting from phytophagous activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2.15 ([plus or minus]0.11 SE) [mu]mol GABA g-1 fresh weight.

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.

OVEREXPRESSION OF GLUTAMATE DECARBOXYLASE IN TRANSGENIC TOBACCO PLANTS DETERS FEEDING BY PHYTOPHAGOUS INSECT LARVAE

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid synthesized by glutamate decarboxylase. Previous research suggests that the endogenous synthesis of GABA, a naturally occurring inhibitory neurotransmitter at neuromuscular junctions, serves as a plant resistance mechanism against invertebrate pests. In this study, two homozygous transgenic tobacco lines constitutively overexpressing a single copy of a full-length chimeric glutamate decarboxylase cDNA and possessing enhanced capacity for GABA accumulation (GAD plants), a homozygous transgenic line lacking the gene insert, and wild-type tobacco were employed. Tobacco budworm larvae were presented with plantattached wild type and transgenic leaves for 4 hr in a feeding preference study. Larvae consumed six to twelve times more leaf tissue from wild-type plants than from GAD plants. These results suggest that leaf GABA accumulation, which is known to occur in response to insect larval walking and feeding, represents a rapidly deployed localresistance mechanism.

Footsteps from insect larvae damage leaf surfaces and initiate rapid responses

Plant resistance to insect herbivory involves gene expression in response to wounding and the detection of insect elicitors in oral secretions (Kessler and Baldwin, 2002, Ann. Rev. Plant/ Biol. 53: 299–328). However, crawling insect larvae stimulate the synthesis of 4-aminobutyrate within minutes and imprints of larval footsteps can be visualized within seconds through superoxide production or transient increases in chlorophyll fluorescence (Bown et al., 2002, Plant Physiol. 129: 1430–1434). Here cryo-scanning electron microscopy was used to demonstrate that larval feet, which are equipped with a perimeter row of hook-like crochets, damage leaf tissue and result in larval footprints. Staining for cell death shows that areas of wounding correspond to footsteps detected through increased chlorophyll fluorescence. Superoxide production in response to footsteps was inhibited by diphenyleneiodonium, an inhibitor of the plasma membrane NADPH oxidase enzyme. Inhibition of superoxide production, however, did not eliminate the detection of cell death. The results demonstrate that larval footsteps damage leaf tissue, and initiate rapid local responses which are not dependent on herbivory or oral secretions. It is proposed that superoxide production at the wound site prevents opportunistic pathogen infection.