Abstract

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.

Highlights

  • Tivity, these proteins have been purified from a range of major crops including maize [3], soybean [4], and wheat [5, 6]

  • Proteomic Analysis—To identify GST family members that preferentially bound S-alkyl glutathione derivatives, crude protein samples from Arabidopsis root cultures were subjected to affinity chromatography using GS-hexyl as the ligand

  • Our results suggest that the binding of plant GSTUs to the S-alkylated glutathione derivatives utilized in affinity chromatography relates to their interactions with analogous conjugated products in vivo

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Summary

EXPERIMENTAL PROCEDURES

Chemicals—12-Oxo-10,15(Z)-phytodienoic acid (OPDA) was obtained from Larodan Fine Chemicals AB, Malmo, Sweden. For Arabidopsis studies, plants were stably transformed using floral dipping [16] In both cases the Strep-tagged GSTs were purified by affinity chromatography [12], with bound ligands recovered from the concentrated protein in 2 volumes of methanol. Additional analyses using negative mode ESI were performed To confirm their integrity, precipitated proteins were redissolved in 50% CH3CN, 0.5% formic acid, and their intact masses were determined by ESITOF MS [15]. For OPDA binding/conjugation, 137 ␮M OPDA in HBS was injected into HBS containing 11 ␮M GSTU19 and 1 mM GSH. In both experiments a one-bindingsite model was fitted to the background-subtracted data to calculate binding parameters. Each entire sample was analyzed by two-dimensional-PAGE on a 12% acrylamide gel [18], and polypeptides of interest were excised and identified by peptide mass fingerprinting [19]

RESULTS
Peak area in each GST preparation
DISCUSSION
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