JXB Advance Access originally published online on July 8, 2009
Journal of Experimental Botany 2009 60(11):2953-2954; doi:10.1093/jxb/erp224
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ARTICLE-COMMENTARY |
The way the dioecious plant Actinidia deliciosa attracts bees: critical role of volatile terpenes released from kiwifruit flowers of both genotypes
Institut de Biologie Moléculaire des Plantes (Centre National de la Recherche Scientifique,UPR 2357), Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France
* E-mail: ma.hartmann{at}ibmp-ulp.u-strasbg.fr
Plants are sessile organisms which have evolved a wide range of strategies to adjust to their environment. As an example, many species of flowering plants need to be pollinated by an insect. To attract pollinators and seed dispersers and thus to ensure their reproductive and evolutionary success, they release diverse blends of volatile compounds from their flowers. The bewildering array of structures identified in floral scents, which may contain up to 100 different molecules, is dominated by terpenoids, with monoterpenes representing the most abundant components, followed by sesquiterpenes (Knudsen and Gershenzon, 2006).
During the last decade, substantial progress has been made in plant volatile research as a result of the development of sensitive methods for dynamic headspace sampling and improvements in the analysis of volatiles by gas chromatography-mass spectrometry. In the context of current research dealing with the terpenoid composition of floral scents, including, for instance, Arabidopsis (Chen et al., 2003), Clarkia (Dudareva et al., 1996), Antirrhinum majus (Dudareva et al., 2003) or Vitis vinifera (Lücker et al., 2004), Nieuwenhuizen et al. (2009) report in this issue a comparative analysis of volatile terpenes released from male and female flowers of Actinidia deliciosa, a dioecious plant for which efficient insect pollination is particularly critical. The kiwifruit flowers of both male and female genotypes are shown to display a similar terpene profile in which two sesquiterpenes, (E,E)-
-farnesene and germacrene D, and one monoterpene, (E)-β-ocimene, are the main constituents. Interestingly, the monoterpene has been found only in petals, suggesting that it plays a specific role. The emission of volatile compounds was observed predominantly during the day when pollinators were at work, but the molecular mechanisms responsible for co-ordinated expression of the corresponding genes and the potential effect of light intensity remain to be investigated.
In the next step, Nieuwenhuizen et al. (2009) used a functional genomics approach to identify the terpene synthases (TPS), which could account for the production of these volatile terpenes in both male and female flowers. To date, more than 100 TPS that synthesize mono-, sesqui-, and diterpenes have been isolated and characterized from different plant species. They belong to the terpene synthase gene family, which comprises seven subfamilies designated as Tps-a to Tps-g (Bohlmann et al., 1998; Trapp and Croteau, 2001). One of the most outstanding properties of these enzymes is their ability to make multiple products from a single prenyl diphosphate substrate. As an example, only two sesquiterpene synthases were found to be responsible for the synthesis of the 20 sesquiterpenes identified in the Arabidopsis floral scent (Tholl et al., 2005). However, some TPS also produce single products. Two specialized single-product monoterpene synthases are responsible for the biosynthesis of myrcene and (E)-β-ocimene, the two major terpenoids of snapdragon floral scent (Dudareva et al., 2003). In A. deliciosa, the authors identified two genes, referred as to AdGDS1 and AdAFS1, which encode two distinct sesquiterpene synthases belonging to subfamilies Tps-a and Tps-f, respectively. The substrates of these enzymes are prenyl diphosphates. Heterologous expression of these cloned Tps indicates that AdGDS1 uses only farnesyl diphosphate (FPP) as a substrate to form (+)-germacrene D as the sole compound while AdFS1 is able to accept both FPP and geranyl diphosphate (GPP) and catalyses the synthesis of -farnesene and (E)β-ocimene, respectively. Interestingly, AdAFS1 belongs to the Tps-f subfamiliy, which comprises only monoterpene synthases. When transiently expressed in Nicotiana benthamiana plants, AdGDS1 and AdAFS induced the formation of the same volatile terpenes as those found for the recombinant proteins in vitro.
As the synthesis of terpene compounds is dependent on the availability of substrates for the TPS, the authors addressed the question of their cellular site of synthesis. In plants, all terpenoids arise from the five-carbon precursors isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are derived from the two alternative pathways. Sesquiterpene synthesis is expected to occur in the cytosol from FPP synthesized by the mevalonate pathway whereas monoterpene synthesis should take place in the plastids, from GPP arising from the methyl erythritol phosphate (MEP) pathway. Expression of GFP fusion proteins in Arabidopsis protoplasts and analysis by confocal scanning microscopy indicate that both A. deliciosa TPS are located exclusively in the cytosol, in agreement with the lack of plastid N-terminus targeting sequence predicted for these TPS. Consequently, both enzymes compete for the same substrate FPP and the final amounts of each sesquiterpene will depend on the concentration, the Km value, and the lifetime of the respective enzymes. The formation of (E)-β-ocimene by AdAFS1 implies that a number of GPP molecules might be available in the cytosol.
In summary, the paper by Nieuwenhuizen et al. (2009) brings new information about the volatile terpenes from kiwifruit flowers of both genotypes. This detailed study goes from an analysis of the compounds released by the different parts of the male and female flowers, the identification of the two Tps genes involved in their synthesis, their functional characterization in vitro and in planta, and the determination of their tissue-specific expression patterns to the subcellular localization of the sesquiterpene synthases.
However, many questions remain unsolved, such as the function of individual compounds and their respective perception by insects as well as the potential involvement of non-terpenoid compounds. The production of the floral scent was shown to be highest during the day, but mechanisms underlying the regulation of the co-ordinated expression of the responsible genes are still unknown. However, there is no doubt that the knowledge derived from this work is of importance in improving kiwifruit pollination.
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