Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

ISOPRENOID BIOSYNTHESIS IN FRUITS AND VEGETABLES 295
1998; Rupasinghe et al., 2000b, c). Recently, as a first step toward understanding the regulation
of α-farnesene accumulation by HMGR, total in vitro HMGR enzyme activity and
expression of two novel cDNA clones, hmg1 and hmg2, encoding HMGR were studied in
the skin tissue of apple in relation to low-temperature storage and C 2 H 4 action.
Accumulation of the stress metabolite α-farnesene in the skin of apples is triggered
by low-temperature storage and reaches a peak during 4–12 weeks in storage (Rupasinghe
et al., 2000a, b). In contrast, in vitro HMGR activity was the highest at the time of harvest and
gradually decreased during the first 8 weeks of storage, and then remained constant during
the remainder of the storage period. Incorporation of radiolabeled or unlabeled mevalonic
acid into α-farnesene is favored over a mixture of GAP and pyruvic acid (precursors of
Rohmer pathway) in isolated apple skin tissues. Therefore, it is evident that the biosynthesis
of α-farnesene occurs predominantly through the classical mevalonate (MVA) pathway in
apple fruit. This conclusion is supported also by the observation that lovastatin, a competitive
inhibitor of HMGR, inhibits α-farnesene accumulation significantly (by 25–54%) in apple
skin during storage. Recently, Ju and Curry (2000) also found that when lovastatin is
applied to apple fruit tissue at high concentrations, α-farnesene biosynthesis is suppressed
to undetectable levels in “Delicious” and “Granny Smith” apples. Together these results
imply that in apple fruit the biosynthesis of α-farnesene occurs predominantly through the
classical MVA pathway.
Cloning and expression of an α-farnesene synthase cDNA from apple (“Law Rome”)
peel tissue was achieved by Pechous and Whitaker (2004). Using degenerate primers, an 800-
bp cDNA fragment of α-farnesene synthase was isolated. A cDNA library generated from
peel tissue mRNA was screened to isolate a 1,931-bp long full length terpene synthase cDNA
(AFS1; GenBank accession number AY182241), which contained an open reading frame of
1,728 bp encoding a protein containing 576 amino acids and having a relative molecular mass
of 66 kDa. The sequence had characteristics similar to monoterpene synthases. The protein
was functionally expressed in E. coli and converted farnesyl pyrophosphate into (E,E)-αfarnesene.
Northern blots showed that the AFS1 transcript levels increased fourfold in apple
peel tissue during storage for a period of 4 weeks at 0.5 ◦ C. There appears to be a better
correlation between an increase in α-farnesene synthase activity and α-farnesene synthase
transcript levels than HMGR activity. The expression of AFS1 is also ethylene-dependent
since treatment with 1-methylcyclopropene (1-MCP), an ethylene receptor blocker, reduced
the AFS1 transcript levels during the initial 4-week period of storage, with further inhibition
to undetectable levels during extended storage up to 8 weeks. α-Farnesene levels were
also reduced in apples by treatment with aminoethoxyvinylglycine and 1-MCP, further
supporting the role of ethylene in farnesene biosynthesis (Rupasinghe et al., 2000c).
13.13 Isolation of hmg1 and hmg2 cDNA from apple
and their expression during storage
To further study the regulation of HMGR activity in relation to the accumulation of α-
farnesene in apple fruit, a molecular approach was employed. With these objectives, a full
length (hmg1) and a fragment (hmg2) of cDNAs of two HMGR genes from the skin of apple
fruit were cloned using a strategy, utilizing sequence similarities among previously cloned
plant HMGR genes (Rupasinghe et al., 2001). All plant HMGR genes identified to date
share some common structural features (Maldonado-Mendoza et al., 1997). They are highly