Cloning and functional expression of an ( E , E )-a-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)-a-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only (E)-b-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)-a-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°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.

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

“…The apparent K m value of snapdragon nerolidol/linalool synthases for GPP is three times lower than that for FPP ($7.6 versus $24 lM for GPP and FPP, respectively; Table 1), in contrast to FaNES1, which has an apparent K m for GPP that is 3.6 times higher than that for FPP (29.0 versus 8.1 lM for GPP and FPP, respectively; Aharoni et al, 2004). Although the apparent K m values of the AmNES/LIS enzymes for GPP and FPP are relatively high, when compared with those of monoterpene synthases for GPP (the low lM range) and sesquiterpene synthases for FPP (0.1-10 lM), the turnover rates (k cat ) with both substrates are within the range reported for monoterpene (0.01-1.0 sec )1 ) and sesquiterpene (0.03-0.5 sec )1 ) synthases (Cane, 1999;Wise and Croteau, 1999 (Green et al, 2007;Pechous and Whitaker, 2004), Abies grandis (Bohlmann et al, 1998a;Steele et al, 1998), L. esculentum (Colby et al, 1998), A. annua L (Mercke et al, 1999), A. thaliana (Tholl et al, 2005), O. basilicum (Iijima et al, 2004) and Zea mays (Kollner et al, 2004). However, their sesquiterpene synthase activities significantly exceeded their monoterpene synthase activities.…”

The Tomato Terpene Synthase Gene Family

“…However, in Arabidopsis ecotype Wassilewskija, an ortholog of a TPS-b protein that is lacking a transit peptide is found in the cytosol and it uses e,e-FPP to make farnesene, although it can use GPP in vitro to make ocimene (Huang et al, 2010). Similar TPS-b cytosolic sesquiterpene synthases have been reported in kiwifruit and apple (Malus domestica; Pechous and Whitaker, 2004;Nieuwenhuizen et al, 2009), among others. Tomato has three TPS-b genes, TPS25, TPS27, and TPS38, encoding proteins that appear to lack a transit peptide.…”

“…Insect herbivory as well as jasmonic acid (JA) treatment increase the production of some monoterpenes such as linalool as well as of (E,8,3,7,, a C16 homoterpene derived from oxidative cleavage of the diterpene geranyllinalool (Ament et al, 2004;Herde et al, 2008). However, in contrast to the fruits of many other species, where volatile monoterpenes and sesquiterpenes are a major determinant of flavor (Sharon-Asa et al, 2003;Aharoni et al, 2004;Pechous and Whitaker, 2004), the tomato fruit is mostly devoid of such volatile terpenes, although some aroma compounds are derived from the degradation of carotenoids (Lewinsohn et al, 2005;Goff and Klee, 2006;Davidovich-Rikanati et al, 2007).…”

Analyses of Expressed Sequence Tags from Apple

“…The sesquiterpenes (E,E)-and (Z,E)-a-farnesene are produced from farnesyl diphosphate by the enzyme a-farnesene synthase. The gene encoding a-farnesene synthase has been isolated and shown to be up-regulated in fruit during ripening (Pechous and Whitaker, 2004). The a-farnesene synthase gene is represented by three NR sequences in the apple dataset.…”