The Mechanism of Herbicide Resistance in Tobacco Cells with a New Mutation in the Q B Protein

Abstract

A new mutant of the psbA gene conferring resistance to 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine) was obtained by selection of photomixotrophic tobacco (Nko-tiana tabacum cv Samsun NN) cells. The 264th codon AGT (serine) in the wild psbA gene was changed to ACT (threonine) in these mutant tobacco cells. All other higher plants resistant to atrazine exhibit a change to GGT (glycine) in this codon. Measurements of Hill reaction activity and chlorophyll fluorescence showed that the threonine 264-containing plastoquinone serving as secondary stable electron acceptor of PSII (QB protein) had not only strong resistance to triazine-type herbicides but also moderate resistance to substituted urea-type herbicides. Threonine-type QB protein showed especially strong resistance to methoxylamino derivatives of the substituted urea herbicides. The projected secondary structures of the mutant Q5 proteins indicate that the cross-resistance of threonine 264 QB protein to triazine and urea her-bicides is mainly due to a conformational change of the binding site for the herbicides. However, the glycine 264 Os protein is resistant to only triazine herbicides because of the absence of an hydroxyl group and not because of a conformational change. Photosystem II (PSII) complex has the important function of splitting water to form molecular oxygen in photosynthesis. Among the components of the PSII complex, the 32 kD polypeptide (known as the QB3 or Dl protein) has a special importance since it forms part of the reaction center which transports electrons from the primary electron acceptor, QA, to the secondary stable electron acceptor, QB (20). The QB protein is also important as the primary target site of many herbicides which act by inhibiting photosynthesis (triazines, substituted ureas, triazinones, uracils and so on) (9, 24). The amino acid sequence ofthe QB protein was determined from the sequence ofthe psbA gene which encodes this protein (11, 13, 33). The 3-dimensional structure of the QB protein, however, has not been determined since it is extremely unstable in the isolated state. Thus, the mechanisms of electron transport and herbicide-binding in PSII are not fully under-' QB, plastoquinone serving as secondary stable electron acceptor of PSII; QA, primary electron accepting plastoqui-none of PSII; atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine; diuron, 3-(3,4-dichloro-phenyl)-1, l-dimethyl-urea. stood. Recent x-ray analysis of the reaction center complex (L/M subunits) of a purple photosynthetic bacterium, Rho-dopseudomonas viridis (4) and the large degree of amino acid sequence homology between the bacterial L/M subunits and the PSII reaction center complex (QB/QA) in higher plants have allowed speculation on the structure of the QB protein (16, 19, 31, 32). Investigations of herbicide-resistant mutants represent another approach to studying the structure of the herbicide-binding site in the QB protein. Genetic analysis of triazine-resistant weeds (2, 11, 13, 14, 29) collected in fields where a triazine herbicide was used repeatedly, revealed that a point mutation ofthe 264th codon was responsible for the herbicide resistance. Investigations in Chlamydomonas reinhardtii and Anacystis nidulans revealed that the amino acids at positions 219, 251, 255, 264, and 275 also contributed to herbicide binding (5-7, 10, 15). Recently, we selected a new triazine-resistant cell line from cultured cells oftobacco which can grow photoautotrophically in the light. This resistant cell line had a mutation in the psbA gene at the 264th codon as did the triazine-resistant weeds. In constract to weeds, where the mutation causes a substitution of glycine for serine, this mutation resulted in threonine at position 264 (27). This new mutation is interesting because it brought about resistance not only to atrazine but also to diuron, a different type of photosynthesis-inhibiting herbicide (27). We report here the further characterization of our resistant tobacco cells and discuss the possible mechanism of herbicide resistance in these cells. MATERIALS AND METHODS Cell and Plant Materials Green tobacco (Nicotiana tabacum L. cv Samsun NN) cells were cultured photomixotrophically in modified Linsmaier-Skoog medium with 10 gM l-naphthaleneacetic acid, 1 gM kinetin, and 3% sucrose. The atrazine-resistant tobacco cells were selected in a medium containing atrazine as described elsewhere (27). To isolate chloroplasts, both atrazine-resistant and-susceptible tobacco cells were harvested at late log phase, 10 to 14 d after inoculation. The seeds of triazine-resistant and-susceptible biotypes of redroot pigweed (Amaranthus retroflexus L) were kindly provided by Dr. Radosevich of Oregon State University. They were grown in a greenhouse, and their leaves harvested from 6-to 8-week-old seedlings. 986