An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast
Article first published online: 5 MAR 2002
The Plant Journal
Volume 8, Issue 3, pages 407–416, September 1995
How to Cite
Gachotte, D., Meens, R. and Benveniste, P. (1995), An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast. The Plant Journal, 8: 407–416. doi: 10.1046/j.1365-313X.1995.08030407.x
- Issue published online: 5 MAR 2002
- Article first published online: 5 MAR 2002
- Received 10 March 1995; revised 24 May 1995; accepted 26 June 1995.
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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.