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 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 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 (1H-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.
Gachotte
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Département d'Enzymologie Moléculaire et Cellulaire, UPR 406 du CNRS, Strasbourg, France.
Meens
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Benveniste
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D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003433:Crosses, Genetic; D008401:Gas Chromatography-Mass Spectrometry; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013261:Sterols / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D013261:Sterols; D010088:Oxidoreductases; C039306:sterol delta-5 desaturase