Regulation of Arabidopsis Glutamate Decarboxylase in Response to Heat Stress: Modulation of Enzyme Activity and Gene Expression

Abstract

Most plants accumulate the nonprotein amino acid, g-aminobutyric acid (GABA), in response to heat stress. GABA is made from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), an enzyme that has been shown by the Israeli PI to be a calmodulin (CaM) binding protein whose activity is regulated in vitro by calcium and CaM. In Arabidopsis there are at least 5 GAD genes, two isoforms of GAD, GAD1 and GAD2, are known to be expressed, both of which appear to be calmodulin-binding proteins. The role of GABA accumulation in stress tolerance remains unclear, and thus the objectives of the proposed work are intended to clarify the possible roles of GABA in stress tolerance by studying the factors which regulate the activity of GAD in vivo. Our intent was to demonstrate the factors that mediate the expression of GAD activity by analyzing the promoters of the GAD1 and GAD2 genes, to determine the role of stress induced calcium signaling in the regulation of GAD activity, to investigate the role of phosphorylation of the CaM-binding domain in the regulation of GAD activity, and to investigate whether ABA signaling could be involved in GAD regulation via the following set of original Project Objectives: 1. Construction of chimeric GAD1 and GAD2 promoter/reporter gene fusions and their utilization for determining cell-specific expression of GAD genes in Arabidopsis. 2. Utilizing transgenic plants harboring chimeric GAD1 promoter-luciferase constructs for isolating mutants in genes controlling GAD1 gene activation in response to heat shock. 3. Assess the role of Ca2+/CaM in the regulation of GAD activity in vivo in Arabidopsis. 4. Study the possible phosphorylation of GAD as a means of regulation of GAD activity. 5. Utilize ABA mutants of Arabidopsis to assess the involvement of this phytohormone in GAD activation by stress stimuli. The major conclusions of Objective 1 was that GAD1 was strongly expressed in the elongating region of the root, while GAD2 was mainly expressed along the phloem in both roots and shoots. In addition, GAD activity was found not to be transcriptionally regulated in response to heat stress. Subsequently, The Israeli side obtained a GAD1 knockout mutation, and in light of the objective 1 results it was determined that characterization of this knockout mutation would contribute more to the project than the proposed Objective 2. The major conclusion of Objective 3 is that heat-stress-induced changes in GAD activity can be explained by heat-stress-induced changes in cytosolic calcium levels. No evidence that GAD activity was transcriptionally or translationally regulated or that protein phosphorylation was involved in GAD regulation (objective 4) was obtained. Previously published data by others showing that in wheat roots ABA regulated GABA accumulation proved not to be the case in Arabidopsis (Objective 5). Consequently, we put the remaining effort in the project into the selection of mutants related to temperature adaptation and GABA utilization and attempting to characterize events resulting from GABA accumulation. A set of 3 heat sensitive mutants that appear to have GABA related mutations have been isolated and partially characterized, and a study linking GABA accumulation to growth stimulation and altered nitrate assimilation were conducted. By providing a better understanding of how GAD activity was and was not regulated in vivo, we have ruled out the use of certain genes for genetically engineering thermotolerance, and suggested other areas of endeavor related to the thrust of the project that may be more likely approaches to genetically engineering thermotolerance.