Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

An HPLC method for simultaneous determination of residual benomyl and MBC on apple foliage without cleanup

A simple High Performance Liquid Chromatograph (HPLC) method has been developed to identify benamyl (methyl 1- (butylcarbamoyl)-2-benzimidazole carbamate) and MBC (methyl 2-benzimidazole carbamat~ residues on apple leaves without cleanup. Sample leaves are freeze dried in a Mason jar and residues are then extracted by tumbling them in chloroform containing 5,000 microgram per milliliter of n-propyl isocyanate (PIC) at 10 C. To the extract, n-butyl isocyanate (BIC) was added at 5,000 microgram per milliliter and 20 microliter of this mixture injected onto the HPLC system. Separation is accomplished by the use of a Brownlee LiChrosorb silica gel column with a guard column and' operated with a mixed mobile phase consisting of chloroform and hexane (4:1) saturated with water. MBC, a degradation product of benomyl is identified if present as methyl l-(npropyl carbamoyl)-2-benzimidazole carbamate (MBC-n-PIC). Both benomyl and MBC-n-PIC can be detected with aKUltraviolet (UV) detector (280nm) at a concentration as low as 0.2 microgram per milliliter in apple leaves. The fate of benomyl on apple foliage after spray application of benomyl (Ben late 50 per cent wettable powder) was investigated by the method thus described. Benomyl quickly dissipated during the first 3-7 days, but the dissipatio'n sltowed down thereafter. In contrast, the concentration of MBC in leaves gradually increased after repeated applications of Benlate.