Haloxyfop mode of action in liquid cultures of proso millet: An analysis of haloxyfop sensitivity changes during growth

Gerard Paul Irzyk, Purdue University

Abstract

Haloxyfop is a grass-selective herbicide that inhibits acetyl-CoA carboxylase in species that are not tolerant to the herbicide. Liquid cultures of proso millet (Panicum miliaceum) cells treated with haloxyfop at different phases of growth exhibited different levels of sensitivity to the herbicide. Treatment of 1-d cultures with 1 $\mu$M haloxyfop completely inhibited growth within 48 h. In contrast, 1 mM haloxyfop was required to elicit a similar response in 4-, 7-, or 10-d cultures. Calculated IC$\sb{50}$ values indicated a 300-fold decrease in haloxyfop sensitivity during the period from 1 to 4 d. This period of growth coincided with the greatest increase in cell number during culture growth and suggested that dividing cells are most sensitive to haloxyfop. Uptake and metabolism of $\sp{14}$C-haloxyfop in 1-d and 4-d cultures were compared. In both cultures, amounts of radiolabel uptake were similar. Almost all radioactivity extracted from 1- and 4-d cells was present as the parent compound. These results suggested that the sensitivity change was related to other factors. Acetyl-CoA carboxylase activity of proso millet cells, measured in vitro by the acetyl-CoA-dependent incorporation of $\sp{14}$C-bicarbonate into an acid stable product, was essentially constant during culture growth. Micromolar concentrations of haloxyfop significantly inhibited acetyl-CoA carboxylase activity from both sensitive and insensitive cultures. Thus, the change in the sensitivity of cultures to haloxyfop was not correlated with changes in acetyl-CoA carboxylase abundance, activity, or sensitivity to haloxyfop during culture growth. In vivo incorporation of $\sp{14}$C-acetate into lipids was decreased by 1 $\mu$M haloxyfop in both 1-d and 4-d cultures at the earliest sampling times but the amount of inhibition was significantly greater in the sensitive cultures. In contrast, 1 $\mu$M haloxyfop had relatively little effect on the incorporation of $\sp{14}$C-arabinose into cell walls or $\sp3$H-leucine into proteins. These results established an apparent correlation between sensitivity changes during culture growth and the inhibition of acetyl-CoA carboxylase activity, as reflected in the inhibition of lipid synthesis. Further, these results suggested that dividing cells are more sensitive to the inhibition of lipid synthesis. However, treatment of 1-d and 4-d cultures with 100 $\mu$M cerulenin, which inhibits the growth of both 1-d and 4-d cultures, resulted in similar decreases in $\sp{14}$C-acetate incorporation into lipids. These results do not support a correlation between haloxyfop activity and culture growth or the sensitivity of dividing cells to an inhibition of lipid synthesis. The basis for the haloxyfop-sensitivity changes during the growth of proso millet cells was not determined but may be related to the sequesteration of haloxyfop away from the active site by unknown mechanisms dependent on culture growth.

Degree

Ph.D.

Advisors

Bauman, Purdue University.

Subject Area

Botany|Agronomy

COinS