MOLECULAR CHARACTERIZATION OF TOBACCO SQUALENE SYNTHASE AND REGULATION IN RESPONSE TO FUNGAL ELICITOR

The enzyme squalene synthase (SS) represents the first commitment of c arbon from the general isoprenoid pathway toward sterol biosynthesis a nd is a potential point for regulation of sterol biosynthesis. The iso lation and characterization of tobacco (Nicotiana tabacum) squalene sy nthase (TSS) cDNA and genomic DNA clones, as well as determination of the steady state level of TSS mRNA in response to elicitor treatment, were investigated, cDNA clones for TSS were isolated from poly (A)(+) RNA using a reverse transcription/polymerase chain reaction (RT/PCR) m ethod. A 1233-bp cDNA clone was generated that contained an open readi ng frame of 411 amino acids giving a predicted molecular mass of 46.9 kDa. Comparison of the TSS deduced amino acid sequence with currently described SS from different species showed the highest identity with N icotiana benthamiana (97%), followed by Glycyrrhiza glabra (81%), Arab idopsis thaliana (74%), rat (40%), and yeast (37%), Expression of a so luble form of the TSS enzyme with enzymatic activity in Escherichia co li was achieved by truncating 24 hydrophobic amino acids at the carbox y terminus. Characterization of genomic TSS (gTSS) revealed a gene of 7.086 kb with a complex organization of small exons and large introns not typical of plant genes. Southern blot hybridization indicated only two copies of the SS gene in the tobacco genome. Treatment of tobacco cell suspension cultures with a fungal elicitor dramatically reduced TSS enzymatic activity, lowering it to zero within 24 h, Analysis of T SS mRNA levels, by RNA blot hybridization and primer extension assays, in elicitor-treated cells indicated that the transcript level remaine d largely unchanged over this 24-h period. These results suggest that the suppression of TSS enzyme activity in elicitor-treated cells may r esult from a posttranscriptional modification of TSS. (C) 1998 Academi c Press.