Molecular cloning and characterization of a type III glutathione S-transferase from cell suspension cultures of opium poppy treated with a fungal elicitor

Glutathione S-transferases (GSTs) catalyze the conjugation of electrophilic compounds to glutathione (GSH), and are involved in the detoxification of xenobiotics in plants, However, little is known about the endogenous substr ates of plant GSTs, or their role in normal cellular processes, A cDNA libr ary was constructed with poly(A)(+) RNA isolated from cell suspension cultu res of opium poppy (Papaver somniferum L.) treated with a fungal elicitor. A putative type III GST fragment was amplified by PCR from the cDNA library using degenerate primers designed from conserved domains found in several plant GSTs, The cDNA library was screened with the PCR product and 3 homolo gous clones were isolated. Deduced amino acid sequences from these clones d isplayed extensive homology to other plant type III GSTs, especially within the N-terminal domain. One full-length cDNA, designated pGST2, was express ed in Escherichia coli, and the recombinant enzyme exhibited strong GST act ivity in bacterial protein extracts, using 1-chloro-2,4-dinitrobenzene as t he substrate. Recombinant GST2 activity was inhibited by p-coumaroyl- and f eruloyl-CoA esters, and p-coumaric- and ferulic-acid amides of tyramine, bu t not by free hydroxycinnamic acids or aromatic amines. Although type III G STs in opium poppy appear to bind select endogenous phenolics, the in vitro conjugation of GSH to these compounds was not detected. Genomic DNA gel bl ot-hybridization analysis confirmed the existence of a small. family of typ e III GST genes in opium poppy, RNA gel blot-hybridization analysis showed that type III GST transcripts were most abundant in the roots of mature opi um poppy plants, and were induced in developing seedlings and in cell suspe nsion cultures treated with a fungal elicitor. Relative transcript levels w ere generally consistent with the levels of total GST enzyme activity in ea ch tissue.