GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

The identification, distribution and persistence of oxamyl and its degradation products in planted corn seed, seedling root and soil from oxamyl-treated corn seeds

A simple method was developed for treating corn seeds with oxamyl. It involved soaking the seeds to ensure oxamyl uptake, centrifugation to draw off excess solution, and drying under a stream of air to prevent the formation of fungus. The seeds were found to have an even distribution of oxamyl. Seeds remained fungus-free even 12 months after treatment. The highest nonphytotoxic treatment level was obtained by using a 4.00 mg/mL oxamyl solution. Extraction methods for the determination of oxamyl (methyl-N'N'-dimethyl-N-[(methylcarbamoyl)oxy]-l-thiooxamimidate), its oxime (methyl-N',N'-dimethyl-N-hydroxy-1-thiooxamimidate), and DMCF (N,N-dimethyl-1-cyanoformanade) in seed" root, and soil were developed. Seeds were processed by homogenizing, then shaking in methanol. Significantly more oxamyl was extracted from hydrated seeds as opposed to dry seeds. Soils were extracted by tumbling in methanol; recoveries range~ from 86 - 87% for oxamyl. Root was extracted to 93% efficiency for oxamyl by homogenizing the tissue in methanol. NucharAttaclay column cleanup afforded suitable extracts for analysis by RP-HPLC on a C18 column and UV detection at 254 nm. In the degradation study, oxamyl was found to dissipate from the seed down into the soil. It was also detected in the root. Oxime was detected in both the seed and soil, but not in the root. DMCF was detected in small amounts only in the seed.