Two terpene synthases are responsible for the major sesquiterpenes emitted from the flowers of kiwifruit (Actinidia deliciosa)

Abstract

Kiwifruit vines rely on bees for pollen transfer between spatially separated male and female individuals and require synchronized flowering to ensure pollination. Volatile terpene compounds, which are important cues for insect pollinator attraction, were studied by dynamic headspace sampling in the major green-fleshed kiwifruit (Actinidia deliciosa) cultivar ‘Hayward’ and its male pollinator ‘Chieftain’. Terpene volatile levels showed a profile dominated by the sesquiterpenes α-farnesene and germacrene D. These two compounds were emitted by all floral tissues and could be observed throughout the day, with lower levels at night. The monoterpene (E)-β-ocimene was also detected in flowers but was emitted predominantly during the day and only from petal tissue. Using a functional genomics approach, two terpene synthase (TPS) genes were isolated from a ‘Hayward’ petal EST library. Bacterial expression and transient in planta data combined with analysis by enantioselective gas chromatography revealed that one TPS produced primarily (E,E)-α-farnesene and small amounts of (E)-β-ocimene, whereas the second TPS produced primarily (+)-germacrene D. Subcellular localization using GFP fusions showed that both enzymes were localized in the cytoplasm, the site for sesquiterpene production. Real-time PCR analysis revealed that both TPS genes were expressed in the same tissues and at the same times as the corresponding floral volatiles. The results indicate that two genes can account for the major floral sesquiterpene volatiles observed in both male and female A. deliciosa flowers.

Variation of Herbivore-Induced Volatile Terpenes among Arabidopsis Ecotypes Depends on Allelic Differences and Subcellular Targeting of Two Terpene Synthases, TPS02 and TPS03

“…3C), which is consistent with the formation of (E)-b-ocimene and (E,E)-afarnesene as common constituents of floral volatile blends in plants (Dudareva et al, 2003;Nieuwenhuizen et al, 2009). However, for the TPS03 gene, the strong promoter-GUS activity and transcript abundance in Col-0 flowers (Fig.…”

“…S5). Several other TPSs producing (E)-b-ocimene or (E,E)-a-farnesene or both of these terpenes have been identified previously from gymnosperms (TPS-d; Martin et al, 2004) and in the TPS-a, -b, -f, and -g subfamilies of angiosperms (Dudareva et al, 2003;Arimura et al, 2004;Mercke et al, 2004;Nieuwenhuizen et al, 2009; Supplemental Fig. S5).…”

“…Formation of only trace amounts of (E)-b-ocimene in the Col-0 ecotype indicates the absence of a high GPP level in the cytosol and rules out an efficient export of GPP from plastids to the cytosol. Experimental evidence for small cytosolic levels of GPP has recently been provided from tobacco (Nicotiana tabacum) and kiwifruit (Actinidia deliciosa; Wu et al, 2006;Nieuwenhuizen et al, 2009).…”

“…In contrast to the bifunctional (E,E)-a-farnesene/ (E)-b-ocimene synthases from apple and kiwifruit (AdAFS1, TPS-f subfamily), which preferably produce (E,E)-a-farnesene from FPP Nieuwenhuizen et al, 2009), the recombinant TPS02 and TPS03 enzymes exhibit 19-and 3-fold higher catalytic efficiency for GPP than FPP, respectively (Table II), suggesting their original function as monoterpene synthases. The cytosolic Col-0 TPS03 enzyme is slightly more efficient in converting FPP to (E,E)-afarnesene than the plastidic Ws TPS02 protein and less efficient in producing (E)-b-ocimene from GPP (Table II).…”

“…We further presumed that the actively transcribed TPS03 gene in Col-0 might encode a protein that produced (E,E)-a-farnesene, the C 15 analog of (E)-b-ocimene from FPP, instead of (E)-bocimene itself derived from GPP. Several enzymes with (E)-b-ocimene synthase activity from GPP have been shown to also catalyze the formation of (E,E)-afarnesene in vitro when supplied with FPP (Pechous and Whitaker, 2004;Nieuwenhuizen et al, 2009;see below). As an intermediate of terpene biosynthesis, GPP is thought to be largely restricted to the plastids, while FPP is restricted mainly to the cytosol (Aharoni et al, 2005).…”