Cloning and Characterization of a Glutathione S-Transferase That Can Be Photolabeled with 5-Azido-indole-3-acetic Acid

Abstract

Previously, we identified a soluble protein from Hyoscyamus muticus that was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (CST; J. Bilang, H. Macdonald, P.J. King, and A. Sturm i19931 Plant Physiol 102: 29-34). We have examined the effect of auxin on the activity of this H. muticus CST. Auxins reduced enzyme activity only at high concentrations, with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid being more effective than indole-3-acetic acid (IAA) and naphthylacetic acid. IAA was a noncompetitive inhibitor, whereas inhibition by 2,4-D was competitive with respect to 1 -chloro-2,4-dinitro-benzene. We also present the sequence of a full-length cDNA clone that codes for a CST and contains all partia1 amino acid sequences of the purified protein.The auxin-binding CST was found in high amounts in roots and stems and low amounts in leaves and flower buds. The steady-state mRNA level was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a model in which 2,4-D i s a substrate for CST, whereas IAA binds at a second site, known as a ligandinbinding site for the purpose of intracellular transport.

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

“…In addition to porphyrins (14), in vitro studies have shown that specific plant GSTs can bind auxins (39), cytokinins (40), and flavonoid and anthocyanin pigments (41). In the case of the flavonoid metabolites, this ligandin activity also operates in planta, being essential for anthocyanin accumulation in maize and petunia (42).…”

Glutathione-Indole-3-Acetonitrile Is Required for Camalexin Biosynthesis in Arabidopsis thaliana

“…Asterisks indicate statistically significant differences between MKK9 DD and MKK9 DD / GSTFs seedlings, between MKK9 DD and MKK9 DD /gstf6, and between Col wild-type WT and GSTF6 or gstf6 seedlings. *P < 0.05; **P < 0.01 (paired sample t test) binding proteins (Bilang and Sturm, 1995;Mueller et al, 2000;Gonneau et al, 2001;Smith et al, 2003). However, GSH-dependent catalytic activities are the most important characteristics of GSTs.…”

Early Genes and Auxin Action

“…In addition to the catalytic site, animal GSTs possess a noncatalytic site that is thought to be involved in intracellular transport of a wide range of hydrophobic and amphipathic molecules. Based on the enzymatic characterization of a GST from Hyoscyamus muticus, which was identified by photolabeling with 5-azido-IAA, Bilang and Sturm (1995) proposed that IAA binds to such a noncatalytic site. This site, as proposed in analogy to cytoplasmic retinoic acid binding proteins, may control intracellular concentrations and gradients of auxin or may regulate GST enzyme activity, which then modulates the cellular redox potential (Bilang and Sturm, 1995).…”

“…Based on the enzymatic characterization of a GST from Hyoscyamus muticus, which was identified by photolabeling with 5-azido-IAA, Bilang and Sturm (1995) proposed that IAA binds to such a noncatalytic site. This site, as proposed in analogy to cytoplasmic retinoic acid binding proteins, may control intracellular concentrations and gradients of auxin or may regulate GST enzyme activity, which then modulates the cellular redox potential (Bilang and Sturm, 1995). It is interesting that in a similar scenario, noncompetitive binding of salicylic acid to catalase inhibits enzyme activity and increases hydrogen peroxide concentrations, which finally leads to modulation of specific gene expression associated with systemic acquired resistance (Chen et al, 1993).…”