The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22 000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples.
STE 1 was characterized by the accumulation of three Δ7-sterols concomitantly with the decrease of the three corresponding Δ5-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these Δ7-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (1H-NMR). The accumulation of Δ7-sterols suggested that the mutant is deficient in the activity of the Δ7-sterol-C-5-desaturase.
Genetic analysis showed that the accumulation of Δ7-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the Δ7-sterols in STE 1 are good surrogates of physiologically active Δ5-sterols to sustain normal development.
STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Δ7-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in Δ5-sterols and a concomitant decrease in Δ7-sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Δ7-sterols in the STE 1 mutant is due to a deficiency of the Δ7-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.