Glutathione transferases and herbicide detoxification in suspension-cultured cells of giant foxtail (Setaria faberi)

Abstract

Glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene, the chloro-s-triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen have been identified in suspension-cultured cells derived from the grass weed giant foxtail (Setaria faberi Herrm.). In contrast to suspension-cultured cells of maize, where atrazine-conjugating GSTs are lost during de-differentiation, the GSTs active toward this herbicide in S. faberi plants were also expressed in cultures, suggesting that these isoenzymes are subject to different regulation in the crop and weed. As a result, glutathione conjugation was the major route of atrazine metabolism in S. faberi cultures. Activities of these GSTs were maximal three days after sub-culturing when the cells were dividing most actively, when they were determined to be in the order CDNB>alachlor>metolachlor= fluorodifen>atrazine. This indicated that GSTs which are enhanced during cell division can metabolise herbicides. On the basis of activity per mg protein, GST activities in the cultures were between 20 and 60-fold higher than those determined in the foliage of S. faberi seedlings. The GSTs with activity towards CDNB were resolved into three peaks following anion-exchange chromatography at pH 7·8 using Q-Sepharose. Peak 1 GSTs were not retained, while peak 2 and peak 3 were sequentially resolved with an increasing concentration of salt. Peak 1 GSTs showed activity toward metolachlor and atrazine but showed little activity toward fluorodifen. Peak 2 and peak 3 GSTs were active toward atrazine and metolachlor, with peak 3 being particularly associated with activity toward fluorodifen. The GSTs in these peaks were then further purified using S-hexyl-glutathione-agarose affinity chromatography. In each case, the affinity-bound fraction of the GSTs consisted of 28 kDa and 26 kDa polypeptides, suggesting that the GST isoenzymes in S. faberi cultures are composed of related subunits. Our results demonstrate that the GST isoenzymes involved in herbicide metabolism in suspension cultures of a grass weed show a similar level of complexity to that determined in maize cell cultures. © 1998 SCI