An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Abstract

Clutathione S-transferases (CSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max 1.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic CST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic CST but did not affect the intracellular isoforms. The discovery of apoplastic inducible CST will be discussed in light of the putative function of these enzymes in plants.The GSTs (EC 2.5.1.18) are a family of proteins with severa1 activities (Wilce and Parker, 1994;Marrs, 1996). GSTs catalyze the nucleophilic attack of the thiol of GSH to electrophilic substrates, typically resulting in the formation of GSH conjugates. GSTs are usually dimeric proteins with subunit molecular masses of 24 to 30 kD; they are organized in gene families that produce multiple isoenzymes in eukaryotic organisms. GSTs are mostly soluble cytoplasmic enzymes, but microsomal isoforms are also known in both plants and animals. GSTs conjugate GSH to various xenobiotics, e.g. drugs and pesticides, which is often a key step in their metabolic detoxification and elimination from the cytoplasm (Lamoureux and Rusness, 1989;Kreuz et al., 1996;Reinemer et al., 1996).The multitude of GST isoenzymes in plants and their inducibility by diverse biotic and abiotic factors has been the primary focus of research in recent years. Yet, knowledge about the physiological substrate(s) and function(s) of plant GSTs is only slowly beginning to emerge. Thus, GSTs have been implicated in the conjugation of cinnamic acid and of anthocyanins and in the binding of IAA (Edwards and Dixon, 1991;Marrs, 1996). Some GST isoenzymes display additional activities; for example, the selenium-independent GSH peroxidases catalyze the reduction of fatty acid hydroperoxides with concomitant formation of GSSG (Bartling et al., 1993). Such hydroperoxides are formed by the action of active oxygen species that are generated both as normal by-products of aerobic metabolism and as the result of pathogen infection or exposure to certain abiotic * Corresponding author; e-mail klaus-eugen.kreuz@chbs.mhs. ciba.com; fax 41-61-697-8455.agents. Organic hydroperoxides are potentially cytotoxic, and their removal by GSH peroxidase activity has thus been implicated in the protection of tissues against oxidative stress (Ketterer et al., 1990).In this report we describe the isolation and characterization of GSTs from soybean (Glycine max L.) that display high GSH peroxidase activity toward hydroperoxides of linolenic acid and arachidonic acid. In addition to the soluble intracellular isoenzymes, GST and GSH peroxidase were also found in the intercellular washing fluid of hypocotyls. These apoplastic enzyme ac...

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

“…In contrast, a GST isolated from wheat flour exhibited a preference for 9 c,t-HPO over 13 c,t-HPO (Williamson and Beverley, 1988) similar to what was observed for sorghum GST A1/A1 (Table III). The level of GSH peroxidase activity of GST A1/A1 and GST B1/B2 with linoleic hydroperoxides is similar to that reported for linolenic and arachidonic hydroperoxides (0.4-1.2 mol min Ϫ1 mg Ϫ1 ) in a purified GST fraction from soybean hypocotyls (Flury et al, 1996). GSH peroxidase activity of GST A1/A1 and B1/B2 with linoleic hydroperoxides is within the range of activities reported for mammalian ␣-GSTs, which are distinguished by their activity with products of oxidative stress .…”

“…An auxin-binding, plasma membrane-associated GST from Arabidopsis exhibited activity with cumene hydroperoxide (Zettl et al, 1994). GSH peroxidase activity with lipid hydroperoxides as substrates has been reported for GSTs from pea seeds (Williamson and Beverley, 1987), wheat flour (Williamson and Beverley, 1988), soybean hypocotyls (Flury et al, 1996), and Arabidopsis (Bartling et al, 1993).…”

“…It has been proposed that -GSTs originally evolved in prokaryotes to protect against oxidative stress (Pemble and Taylor, 1992). In plants, oxidative stress may signal the induction of certain GSTs (Flury et al, 1996;Marrs, 1996). Several treatments known to induce GSTs, such as wounding or exposure to ozone, ethylene, heavy metals, or pathogens, cause oxidative stress (Marrs, 1996, and refs.…”

Differential Gene Expression and Subcellular Targeting of Arabidopsis Glutathione S-Transferase F8 Is Achieved through Alternative Transcription Start Sites

“…Current knowledge of plant GSTs indicates that nearly all are soluble cytoplasmic enzymes (2,5,7,10,59,62,65), with the studies presented here representing the first case of a plant GST being targeted both to a subcellular organelle and to the cytoplasm. Although GSTF8 has been identified in plastid pro-teomes by MS (39 -42) and even in the nuclear proteome (66), in another study of proteins purified from Arabidopsis chloroplasts, no GSTF8 was detected (67).…”

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

“…It is known that pathogens are able to induce changes in the antioxidant status in the apoplast (Bolwell et al, 2001;Hu et al, 2005;DiazVivancos et al, 2006) and that dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase are present in the apoplast (Vanacker et al, 1998). An inducible glutathione S-transferase was also localized in the apoplast of soybean hypocotyls (Flury et al, 1996).…”