AN ARABIDOPSIS MUTANT DEFICIENT IN STEROL BIOSYNTHESIS - HETEROLOGOUS COMPLEMENTATION BY ERG-3 ENCODING A DELTA(7)-STEROL-C-5-DESATURASE FROM YEAST

The mutant STE 1 was isolated by screening an ethyl-methane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22 000 M(2) 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 Delta(7)-sterols concomitantly with the decrease of the three corresponding Delta(5)-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these Delta(7)-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 (H-1-NMR). The accumulation of Delta(7)-sterols suggested that the mutant is deficient in the activity of the Delta(7)-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of Delta(7)-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 Delta(7)-sterols in STE 1 are good surrogates of physiologically active Delta(5)-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Delta(7)-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in Delta(5)-sterols and a concomitant decrease in Delta(7)-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 Delta(7)-sterols in the STE 1 mutant is due to a deficiency of the Delta(7)-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.