Roles for Stress-inducible Lambda Glutathione Transferases in Flavonoid Metabolism in Plants as Identified by Ligand Fishing

David P Dixon,Robert Edwards

doi:10.1074/jbc.m110.164806

Abstract

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K(d) = 25 nM), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.

Highlights

These TaGSTLs are of particular interest as some are strongly induced by safeners, which are a group of agrochemicals which enhance herbicide detoxification, and selectivity in this major crop (7–9)
For comparison we have utilized AtGSTLs, the homologous proteins previously identified in Arabidopsis thaliana (6), In each case, the ligands retained by these GSTLs have been identified by high resolution mass spectrometry, with compounds whose binding is dependent on the presence of GSH, being of particular interest
Identification, Cloning, and Expression of GSTLs—Using the sequence of TaGSTL1 ϭ Cla30 (7) for homology searching, over 100 related sequences were identified in the full-length and EST datasets derived from Triticum species

Summary

EXPERIMENTAL PROCEDURES

Enzyme Cloning and Expression—The cDNA sequence of TaGSTL1 was available from previous studies (7). Products were cloned after restriction enzyme digestion into the bacterial expression vector pET-STRP3 (10). The clarified extract was either used directly, or after the addition of either 5 mM DTT, or 1 mM oxidized glutathione (GSSG) and 5 ml loaded onto the GSTL-affinity column. For the in vivo capture of ligands, the Strep-tagged enzymes were transiently expressed in unwounded Nicotiana benthamiana leaves prior to affinity recovery and analysis (5). Extracts containing recovered protein and bound ligands were concentrated to 50 ␮l using a Vivaspin 2 centrifugal concentrator (Sartorius Stedim, Epsom, UK). Ligand mixtures were resolved by reversed phase HPLC, with the eluate analyzed by photodiode array detection and electrospray mass spectrometry in series. Where additives (GSH, DTT, GSSG) were used, these were added to both protein and ligand. Enzyme activities of TaGSTLs as thiol transferases towards HED, as glutathione transferases towards CDNB and as DHAR

28 Ϯ 1 106 Ϯ 2 449 Ϯ 8

RESULTS

78 Ϯ 11 60 Ϯ 3 82 Ϯ 4 111 Ϯ 8

DISCUSSION