ISHS Acta Horticulturae 682: V International Postharvest Symposium
CLONING AND FUNCTIONAL EXPRESSION OF AN (E,E)-α-FARNESENE SYNTHASE CDNA FROM APPLE FRUIT |
| Authors: | S.W. Pechous, B.D. Whitaker |
| Keywords: | Malus x domestica, apple fruit, (E,E)-α-farnesene, ethylene regulation, sesquiterpene synthesis, storage disorder, superficial scald |
| Abstract:
Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apples (Malus x domestica). The gaseous 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, RT-PCR with RNA isolated from peel tissue of ‘Law Rome’ apples yielded an ~800-bp gene fragment. This was used to screen a cDNA library generated from peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1931 nucleotides long was isolated. The 1728-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. The TS includes an RR(X8)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 TS gene in E. coli gave myc-epitope-tagged and untagged proteins estimated at ~68 and ~66 kDa, respectively. In sesquiterpene synthase assays, with farnesyl diphosphate as substrate, the untagged bacterially-expressed TS gene product synthesized (E,E)-α-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the apple TS produced trace levels of (E)-β-ocimene. This is the first report of an (E,E)-α-farnesene synthase gene (AFS1; GenBank accession number AY182241) from a flowering plant. RNA gel blots showed that AFS1 transcript increased about fourfold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5°C. In contrast, when fruit were treated at harvest with 1 methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined over the initial 4 weeks of cold storage and was nearly undetectable by 8 weeks. | |
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