Characterization of atrazine resistance in photoautotrophic cell cultures and weed biotypes

Carla Nanette Duncan Yerkes, Purdue University

Abstract

An atrazine resistant photoautotrophic potato cell line (PO-VAR), resistant due to a Ser-264 to Thr substitution in D1 (chloroplast psbA-encoded herbicide/Q$\rm\sb{B}$-binding protein), has similar growth and electron transport characteristics to the susceptible cell line (PO-WT) from which it was selected. For comparison, triazine resistant (VAR) and susceptible (WT) biotypes (whole plants) of common groundsel (CG) and redroot pigweed (PW) were established in photoautotrophic cell culture. As cells developed to photoautotrophic growth, differences between WT and VAR cells in response to atrazine were enhanced. Sequencing of psbA encoding the herbicide binding niche showed CG-VAR and PW-VAR cells were resistant due to Ser-264 to Gly substitutions in D1, the commonly observed mutation at the intact plant level. CG-WT and CG-VAR cells and intact plants had common growth and resistance characteristics. Cross resistance of PO and CG cells to various photosystem II inhibitors was evaluated. All VAR cells were resistant to triazines, triazinones, and uracils. Only PO-VAR cells were resistant to ureas, suggesting that the Ser-264 to Thr mutation alters the Q$\rm\sb{B}$-binding niche resulting in an expanded spectrum of resistance. PO-WT and PO-VAR cells were inhibited similarly by short and long-term exposures to high light intensities; however, only PO-VAR cells were able to recover from photoinhibitory conditions. The ability to recover was not due to gross changes in photosynthetic pigments nor to differences in non-photochemical energy quenching. When grown under intraspecific competition, PO-WT cells were more competitive than PO-VAR cells; however, the fitness penalty of PO-VAR cells was not as great as observed in Ser-264 to Gly mutants. Determining if the unique characteristics of PO-VAR cells would be maintained at the intact plant level required a protocol to isolate, fuse, and regenerate somatic hybrids from PO-VAR cell protoplasts and protoplasts from a nuclear donor plant line. Techniques to alter protoplast organelle composition were employed so that somatic cybrids could also be produced. An atrazine resistant biotype of jimsonweed (the first report of triazine resistance in jimsonweed) was characterized. Responses to atrazine, the effect of atrazine on photosynthesis, basis of resistance, and the effect of resistance on vegetative growth were evaluated for susceptible (S) and resistant (R) jimsonweed (JW) biotypes. S and R biotypes of redroot pigweed (PW) were used for comparison in several studies. Diallel analysis of JW-S and JW-R plants showed the resistance trait to be maternal. Resistance in JW-R and PW-R biotypes was due to a Ser-264 to Gly substitution in D1. JW inherently possesses a Lys-238 where other dicots possess an Arg. This difference, coupled with the Ser-264 to Gly mutation, was predicted through protein modeling to alter D1 topology. Topological changes in the binding niche may account for the reduced level of JW-R resistance relative to other triazine resistant species and the inherent JW-S hypersensitivity to atrazine. Photosynthetic analysis showed atrazine transiently altered photosynthesis in JW-R plants. Plants recovered within 5 d after treatment. JW-S and JW-R plants were similar in vegetative fitness when grown under non-competitive conditions.

Degree

Ph.D.

Advisors

Weller, Purdue University.

Subject Area

Botany|Molecular biology

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