Cloning and functional expression of an ( E, E)-alpha-farnesene synthase cDNA from peel tissue of apple fruit.

Abstract

Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple ( Malus domestica Borkh.) fruit. The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood. Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, reverse transcription-polymerase chain reaction with RNA isolated from peel tissue of 'Law Rome' apples yielded an approx. 800-bp gene fragment. This was used to screen a cDNA library generated from the peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1,931 nucleotides long was isolated. The 1,728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa. Sequence analysis of the apple TS showed it to be most similar to several monoterpene synthases. Oddly, the TS includes an RR(X(8))W motif near the N-terminus that is common among monoterpene synthases but it lacks the plastid transit peptide sequence typically associated with genes of that group. Expression of the apple TS gene in Escherichia coli gave myc-epitope-tagged and untagged proteins estimated at approx. 68 and approx. 66 kDa, respectively. In assays of sesquiterpene synthase activity, with farnesyl diphosphate as substrate, the untagged bacterially expressed TS gene product synthesized ( E, E)-alpha-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only ( E)-beta-ocimene, albeit at much reduced levels. Addition of a C-terminal myc tag appeared to completely prevent production of soluble protein under all of the expression conditions tested. This is the first report of an ( E, E)-alpha-farnesene synthase gene ( AFS1; GenBank accession number AY182241) from a flowering plant. RNA gel blots showed that AFS1 transcript increased about 4-fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5 degrees C. In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and fell to nearly undetectable levels by 8 weeks.