The Regulation of GABA Accumulation by Heat Stress in Arabidopsis
GABA (4-Aminobutyric acid) is a non-protein amino acid that is made in response to a number of environmental stresses in plants including high temperature stress. Glutamate decarboxylase (GAD) is the enzyme responsible for catalyzing this increase in GABA, and it has been shown to be a calmodulin binding protein in plant systems. It is our hypothesis that high temperature stresses generate an increase in cytoplasmic calcium ([Ca’+]y,), that combines with calmodulin to activate GAD, producing the high-temperature induced accumulation of GABA in Arabidopsis roots but not in shoot tissues. We have previously shown that the calmodulin inhibitors chlorpromazine and trifluoperazine inhibit heat-induced GABA accumulation. We have further examined the effectiveness of calcium transport inhibitors (EGTA, BAPTA, verapamil, methoxyverapamil, ruthenium red, dantrolene, thapsigargin, nifedipine, and 3,4,5trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMBA) on the heat-induced accumulation of GABA in Arabidopsis seedlings. Only ruthenium red and dantrolene were found to be effective in blocking heat-induced GABA accumulation. Ca’ ionophore stimulated GABA accumulation even in the absence of heat stress. All of the above agents that inhibited heat-induced GABA accumulation were also shown to increase the sensitivity of Arabidopsis seedlings to lethal temperature treatments, while Ca’+ ionophore decreased this sensitivity. We have also utilized transgenic Arabidopsis plants expressing the chemiluminescent calcium sensing protein aequorin in an attempt to directly demonstrate the effects of the calcium transport inhibitors and Ca’ ionophore on calcium accumulation in Arabidopsis roots and shoots. Western blot analysis further suggests that the levels of GAD protein present in tissues does not change in response to heat stress. These data collectively demonstrate that cytoplasmic calcium sensed during heat stress is sufficient to account for the accumulation of GABA during heat stress, and that other mechanisms of regulating GABA accumulation may not be involved in heat-stress regulation.
KeywordsHeat Stress Arabidopsis Seedling Calcium Ionophore High Temperature Stress Arabidopsis Root
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