The physiological and molecular characterization of atrazine resistance in photoautotrophic potato cells

Reid John Smeda, Purdue University

Abstract

Atrazine (an s-triazine herbicide) inhibits photosynthetic electron transport by reversibly binding to the thylakoid Q$\sb{\rm B}$ protein. Mutations to the chloroplast psbA gene, which encodes the Q$\sb{\rm B}$ protein, have been determined for various weed biotypes repeatedly treated with s-triazines, and these mutations alter the binding domain of atrazine and strongly reduce the affinity of atrazine for its active site. Resistance is controlled by a single gene and is therefore a desirable trait to incorporate into crop species presently sensitive to atrazine. However, concomitant with resistance, photosynthetic and growth productivity are reduced up to 30%, making a crop resistant to atrazine as a result of this mutation, undesirable. The specific objective of this research was to in vitro select photoautotrophic potato cells resistant to atrazine, but not exhibiting any negative affects on productivity. Cell cultures of photoautotrophic potato provided a desirable system to optimize conditions for selecting resistant cells because they were acutely sensitive to atrazine. An observation by the author resulted in the isolation of a line of resistant cells with 50% enhanced resistance compared to unselected susceptible (wild type) cells. Photosynthetic electron transport and growth of these resistant cells was initially reduced in 1.0 $\mu$M atrazine (minimum lethal concentration for wild type cells), but recovered after a lag period. Continued maintenance of resistant cells in atrazine resulted in isolation of a cell line (variant) exhibiting growth and photosynthesis in atrazine concentrations 100-fold higher than for wild type cells. Evidence is presented that this shift in resistance may be explained by a slow elimination of wild type cells from a mixed cell population of wild type and variant cells which initially comprised the resistant cell line. Unlike other higher plants, variant cell growth and photosynthesis, measured by oxygen evolution and fluorescence, were not affected by atrazine in the absence of herbicide. Differential uptake and metabolism were not the basis for variant cell resistance. Photoaffinity labeling of thylakoid membranes from variant and wild type cells revealed an altered binding site on the Q$\sb{\rm B}$ protein for variant cells. Subsequently, sequencing a portion of the psbA identified that the amino acid serine at position 264 of the Q$\sb{\rm B}$ protein was altered to threonine, a unique mutation among higher plants. Variant cells displayed a 30-fold increase in cross-resistance to diuron compared to wild type cells. This research supports the concept that atrazine resistant higher plants, without reduced fitness, can be selected and potentially utilized.

Degree

Ph.D.

Advisors

Weller, Purdue University.

Subject Area

Botany

COinS