Open AccessCalcium- and Calmodulin-Mediated Regulation of Plant Responses to Stress
Author(s) -
Hillel Fromm,
Joe Poovaiah
Publication year - 1993
Language(s) - English
Resource type - Reports
DOI - 10.32747/1993.7568096.bard
Subject(s) - calmodulin , glutamate decarboxylase , biochemistry , calcium , mutant , enzyme , biology , glutamate receptor , recombinant dna , chemistry , microbiology and biotechnology , receptor , gene , organic chemistry
We have taken a molecular approach to clone cellular targets of calcium/calmodulin (Ca2+/CaM). A 35S-labeled recombinant CaM was used as a probe to screen various cDNA expression libraries. One of the isolated clones from petunia codes for the enzyme glutamate decarboxylase (GAD) which catalyzes the conversion of glutamate to g-aminobutyric acid (GABA). The activity of plant GAD has been shown to be dramatically enhanced in response to cold and heat shock, anoxia, drought, mechanical manipulations and by exogenous application of the stress phytohormone ABA in wheat roots. We have purified the recombinant GAD by CaM-affinity chromatography and studied its regulation by Ca2+/CaM. At a physiological pH range (7.0-7.5), the purified enzyme was inactive in the absence of Ca2+ and CaM but could be stimulated to high levels of activity by the addition of exogenous CaM (K0.5 = 15 nM) in the presence of Ca2+ (K 0.5 = 0.8 mM). Neither Ca2+ nor CaM alone had any effect on GAD activity. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain, or transgenic plants expressing the intact GAD were prepared and studied in detail. We have shown that the CaM-binding domain is necessary for the regulation of glutamate and GABA metabolism and for normal plant development. Moreover, we found that CaM is tightly associated with a 500 kDa GAD complex. The tight association of CaM with its target may be important for the rapid modulation of GAD activity by Ca2+ signaling in response to stresses.