Plant Science Research Unit, U.S. Department of Agriculture, Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55108.
Plant Physiol. 1991 May;96(1):104-9. doi: 10.1104/pp.96.1.104.
We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or "wild-type" velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K(m) values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V(max) for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V(max) for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.
我们之前曾报道，马里兰州发现的一种苘麻（Abutilon theophrasti Medic）生物型对莠去津具有抗药性，这是因为其通过谷胱甘肽结合解毒的能力增强（JW Gronwald，Andersen RN，Yee C [1989] Pestic Biochem Physiol 34: 149-163）。本研究旨在探讨该生物型增强莠去津结合能力的生化基础。我们测定了来自抗莠去津生物型和对莠去津敏感或“野生型”苘麻生物型的提取物中的谷胱甘肽水平和谷胱甘肽 S-转移酶活性。在两种生物型中，叶组织中的谷胱甘肽浓度最高（约 500 纳摩尔/克鲜重）。然而，两种生物型的根、茎或叶中的谷胱甘肽水平没有显著差异。在两种生物型中，用 1-氯-2,4-二硝基苯或莠去津作为底物测定的谷胱甘肽 S-转移酶活性在叶组织中最高。与敏感生物型相比，敏感生物型叶和茎组织中用 1-氯-2,4-二硝基苯作为底物测定的谷胱甘肽 S-转移酶分别高 40%和 25%。相比之下，用莠去津作为底物测定的谷胱甘肽 S-转移酶活性在抗莠去津生物型的叶和茎组织中分别高 4.4 倍和 3.6 倍。用抗莠去津和敏感生物型的叶提取物进行谷胱甘肽 S-转移酶活性的动力学分析，使用的底物为谷胱甘肽、1-氯-2,4-二硝基苯和莠去津。谷胱甘肽、莠去津或 1-氯-2,4-二硝基苯的表观 K(m)值几乎没有或没有变化。然而，在抗莠去津生物型中，谷胱甘肽和莠去津的 V(max)值约为敏感生物型的 3 倍。相比之下，抗莠去津生物型中 1-氯-2,4-二硝基苯的 V(max)值低 30%。用莠去津和 1-氯-2,4-二硝基苯表现出活性的叶谷胱甘肽 S-转移酶同工酶通过快速蛋白液（阴离子交换）色谱法分离。敏感生物型有三个峰表现出对莠去津的活性，而抗莠去津生物型有两个。抗莠去津生物型中与莠去津表现出两个谷胱甘肽 S-转移酶活性的峰与敏感生物型中的两个峰共洗脱，但在抗莠去津生物型中的峰高为三到四倍。在两种生物型中，与莠去津表现出谷胱甘肽 S-转移酶活性的两个峰也与 1-氯-2,4-二硝基苯表现出活性，敏感生物型中的峰高更高。结果表明，马里兰州苘麻生物型对莠去津的抗性是由于叶和茎组织中莠去津的谷胱甘肽 S-转移酶活性增强，从而通过谷胱甘肽结合增强了对除草剂的解毒能力。
