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Kiwifruit species are vigorously growing dioecious vines that rely on bees and other insects for pollen transfer between spatially separated male and female individuals. Floral volatile terpene cues for insect pollinator attraction were characterized from flowers of the most widely grown and economically important kiwifruit cultivar Actinidia deliciosa ‘Hayward’ and its male pollinator ‘Chieftain’. The sesquiterpenes α-farnesene and germacrene D dominated in all floral tissues and the emission of these compounds was detected throughout the day, with lower levels at night. Two terpene synthase (TPS) genes were isolated from A. deliciosa petals that produced (+)-germacrene D and (E,E)-α-farnesene respectively. Both TPS genes were expressed in the same tissues and at the same times as their corresponding floral volatiles. Here we discuss these results with respect to plant and insect ecology and the evolution and structure of sesquiterpene synthases.
Angiosperms rely on pollination for sexual reproduction. In dioecious species, the male plant is specialized in the production of pollen, while the female plant bears the seed and fruit-producing organs. Dioecy goes hand-in-hand with the spatial separation of the sexes and with a complete dependence on vectors to carry the pollen, such as insects, wind and water. This sexual system has evolved to promote outbreeding, which generates heterozygosity, often accompanied by increased vigor and genetic variation. It may also be influenced by other ecologic factors such as allocation of resources for male and female functions, sexual selection, seed dispersal, pollination and predation.1
Terpenoids are a class of volatile secondary compounds that fulfill many ecological roles in plants interacting with their biotic environment, including pollinator attraction, direct and indirect defense against insects, bacteria, fungi and even mammals,2 as well as in intraand inter-plant signaling.3,4 Terpenes are produced from a small number of simple prenyl diphosphate substrates, but terpene synthase (TPS) enzymes are capable of producing many terpene skeletons.2 Complexity is further increased by the action of several modifying enzymes leading to the production of even larger numbers of volatile secondary metabolites.
TPS enzyme function can evolve rapidly by means of mutations which may lead to changes in spacial and temporal expression patterns,5 changes in intracellular localization by gain or loss of plastid targeting6 or by generating novel TPS functionalities altogether.7 In the presence of large positive selection pressures such as attraction to certain resident pollinators or through the acquisition of direct or indirect resistance against insects or pathogens, these mutations may contribute significantly to the evolution of new traits and even speciation.
In the wild, kiwifruit vines intertwine and grow to the top of trees or creep over the undergrowth forming dense almost impenetrable tangles.8 The flowers are typically large, visually attractive and fragrant. In A. deliciosa, female flowers are about twice the size of the male flowers but the latter are produced in larger numbers. Dioecy is maintained in the female flowers by the production of stamens with inviable pollen, while in male flowers a central pistil is lacking.9 This system of reproduction is linked to an active Y sex chromosome system (XX/XY female/male) proposed to be in the early stages of evolution.10
In their natural habitats in south-western China, A. deliciosa flowers are pollinated by a large array of insects, mainly bees, including honey bees, carpenter bees, smaller native bees and bumble bees, but other insects such as hoverflies have also been implicated.11 In A. polygama it was shown that the major flower visitors were bumble bees, small solitary bees and hoverflies.12 No consistent sex-based preference was shown by the visiting insects. However, when the stamens were removed from the female flowers these flowers were then rarely visited by the insects and fruit set in these flowers decreased significantly. This is not surprising, since the flowers of both sexes are devoid of any nectar,13 making pollen the only immediate reward for the visiting insects in kiwifruit. The infertile pollen from the female flowers are a particularly poor reward as they have no protoplast which results in poor nutritional value to collecting insects.9 In the orchard, where male and female plants are planted in close vicinity, honey bee pollination is most commonly employed to promote high levels of pollination to obtain economically viable fruit yields and sizes. However, wind pollination can also contribute, albeit at low efficiency.14 Male pollinator plants occupy only 10% of an orchard whereas the reverse is true in the wild where male plants predominate.
Despite the importance of insect pollination for fruit formation in kiwifruit, studies on floral volatiles produced by the Actinidia genus are limited. A. deliciosa flowers produce around 30% of their total scent output as terpenes, mainly the sesquiterpene α-farnesene,15 but also small amounts of (+)-germacrene D and the monoterpene (E)-β-ocimene. In contrast, only a minute amount of α-farnesene was detected in the floral volatiles emitted from flowers of several A. arguta accessions, which emit a scent dominated by the monoterpene β-linalool and its derivatives, including lilac alcohols and lilac aldehydes.16 Analysis of volatiles emitted by flowers of four Actinidia species revealed that terpenoid fractions vary in their composition and constitute from 13% to 46% of total scent output.17
In kiwifruit flowers, the presence of high levels of (E,E)-α-farnesene is likely to be an important part of the volatile blend that helps in the attraction and conditioning of pollinators as has been shown for honey bees.18 (E,E)-α-farnesene is also produced by ripe apples so may also be important for attraction of seed dispersal agents in ripe fruits. (+)-Germacrene D may have a role in pollinator attraction as it is found in flowers of many plants such as rose19 but it can also be emitted from vegetative tissues20,21 where it may contribute to insect defense. Germacrene D has been shown to act as an insect pheromone mimic22 but also to stimulate oviposition.
In Nieuwenhuizen et al.23 we identified two kiwifruit TPS genes from a petal-specific A. deliciosa EST library. The AdAFS1 and AdGDS1 enzymes were shown to produce (E,E)-α-farnesene and (+)-germacrene D in vitro respectively and the genes were expressed spatially and temporally in the same pattern as their corresponding floral volatiles. Both gene products were shown to be localized in the cytoplasm, the main site of sesquiterpene production. We also observed that the monoterpene (E)-β-ocimene was released from female flowers, but only during the daytime and only from petal tissue. Despite the fact that the α-farnesene synthase gene was able to produce (E)-β-ocimene in vitro and to a lesser extent in planta, this compound is likely to be produced by an as yet unidentified petal expressed monoterpene synthase gene. The monoterpene (E)-β-ocimene is a very commonly released plant compound involved in indirect defense to insects.24 Its release from wounded tissues after attack from herbivores may lead to the attraction of predatory insects.25 In A. deliciosa female flowers, expression of a TPS enzyme producing (E)-β-ocimene appears to be induced by wounding in petals and we suspect it has evolved to play a role in protecting the female reproductive structures from herbivory during flowering.
TPS enzymes are thought to have evolved via the duplication of an ancestral conifer di-TPS gene that existed prior to the divergence of TPS genes in angiosperms and gymnosperms. One copy of the duplicated ancestral gene has remained highly conserved in both structure and function and is the parent to TPS enzymes involved in gibberellin biosynthesis.26 The structural and functional diversity of TPS enzymes involved in secondary metabolism is believed to result from divergence of the second ancestral gene as a consequence of adaptive evolutionary processes. Despite its in vivo sesquiterpene synthase activity, the kiwifruit AdAFS1 enzyme is more structurally akin to a di-TPS, having retained a conifer diterpene internal sequence (CDIS) domain, an approximate 200-residue region of unknown function commonly found in di-TPS enzymes. The kiwifruit AdGDS1 enzyme appears to have evolved from a more progressive loss of exons resulting in a much smaller sized protein. The AdGDS1 gene has also acquired an N-terminal RRX8W motif which is more commonly associated with cyclic mono-TPS enzymes and is thought to assist in the unique diphosphate migration step accompanying formation of the linalyl diphosphate intermediate preceding the final cyclisation reaction.27 Mutation of the RRX8W into an AAX8W domain resulted in complete loss of AdGDS1 activity (unpublished data) when both farnesyl diphosphate and an alternative sesquiterpene substrate nerolidyl diphosphate were used. These results suggest that the RRX8W motif may also play an important, but as yet undefined, role in cyclic sesquiterpene synthesis.
A unique aspect of terpene synthases is that most of these enzymes show promiscuous function28 despite having a highly conserved active site scaffold composed largely of inert residues.29 Numerous structural and biochemical analyses have pointed to the ability of a protein to evolve novel activities or altered functions via a small number of amino acid alterations (plasticity).30 It is also generally accepted that proteins that exhibit promiscuous functions acquire higher specificity and activity through divergent evolution31 and that this process is highly dependent upon plasticity.30 AdGDS1 is likely to have evolved as a consequence of divergence from a di-TPS while the evolution of AdAFS1 could have simply been initiated by loss of the plastid targeting signal containing exon 1 in the di-TPS ancestor. This would have resulted in access to a new substrate (i.e., FDP) pool which it could then utilize without significant active site alterations. The presence of multiple α-farnesene producing enzymes in a number of phylogenetically distinct TPS subgroups indicates that this activity is easily acquired. Future studies looking at the ability of AdAFS1 to utilize the di-TPS precursor geranylgeranyl diphosphate could provide additional insight into its evolution.
Although floral scent is often dominated by monoterpene and sesquiterpene compounds, individual terpene volatiles are nearly always present in the context of a mixture, either with other terpenoids or non-terpene volatiles such as benzenoids, phenylpropanoids and aliphatics.32 This makes it very difficult to determine the relative contributions of individual terpene compounds to their perception by various insects, as well as the potential involvement of non-terpenoid compounds. In dioecious kiwifruit, the challenge now is to understand the importance of each of these compounds in insect and plant ecology. In a wider context, the challenge is to better understand how evolutionary pressure drives change in the structure and function of terpene synthase enzymes. To do this, future analyses will need to be carried out in the context of relevant structural information. Understanding how the three dimensional structures of these enzymes govern their chemistry will also lead us to understand how their chemistry has evolved over time.
Previously published online: www.landesbioscience.com/journals/psb/article/10161