Wheat cytosolic acetyl-CoA carboxylase complements an ACC1 null mutation in yeast

Abstract

Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3' tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose, the activity of the "wheat gene" driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors. An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM. In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM. The activity of yeast ACCase is less affected. The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM. Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases.

Figure 1

(a) Structure of yeast expression plasmids encoding yeast and wheat cytosolic ACCase. (b) Structure of ACCase inhibitors used in this study.

Figure 2

Biotinylated proteins in yeast strains. Western blot was probed with [³⁵S]streptavidin to reveal biotinylated peptides. Equal amounts of protein from strains 1–10 listed in Table 1 were loaded (lanes 1–10); wge, wheat germ extract; PCase, pyruvate carboxylase (lanes 1–10).

Figure 3

Growth curves (a) and galactose dependence (b) of gene-replacement and wild-type yeast strains. Growth in YPRG medium (a) or in the presence of varying amounts of galactose in YPR medium (b) was monitored at 600 nm. Galactose induction was measured 24 hr after inoculation from cultures grown in YPR medium for about 24 hr.

Figure 4

Growth inhibition of yeast strains expressing wheat cytosolic or yeast ACCase. Growth in YPR medium containing 0.01% galactose and varying amounts of inhibitors was monitored at 600 nm. (a) Relative culture densities 24 hr after inoculation. (b) Growth curves in the presence of cethoxydim.

Figure 5

In vitro inhibition of wheat cytosolic ACCase expressed in the yeast gene-replacement strain, along with yeast and wheat germ ACCases. Relative acetyl-CoA-dependent conversion of radioactive bicarbonate into acid-stable malonyl-CoA was measured in the presence of varying amounts of the inhibitors.