Abstract
Floral scents, forming a complex mixture of volatile organic compounds (VOCs), play a major role in attracting pollinators, and thus in plant reproduction, but also in defense against biotic and abiotic stresses. As different factors can influence the production of these VOCs, their emission is very variable, at the inter- and intra-specific levels but also with time. In this interdisciplinary study, we have integrated metabolomics, bioinformatics, biochemistry and molecular biology methodologies to study the evolution, spatio-temporal variation and biosynthesis of the emission of attractive signals for pollinators using fig trees and in particular the Mediterranean fig tree Ficus carica as a model. Each species of the genus Ficus (Moraceae) is pollinated by a specific pollinator of the family Agaonidae (Hymenoptera). They are tiny wasps that develop exclusively in the figs, i.e. closed inflorescences with a single entrance (the ostiole), of their host species and use the VOCs emitted by the receptive figs (i.e. at the stage of development when they are ready to be pollinated) to locate their host. In the case of Ficus carica, it has been shown that its specific pollinator (Blastophaga psenes) is attracted to receptive figs by a combination of four VOCs in a particular proportion. At the level of the genus Ficus, we compared phylogenetic and chemical data of 32 fig species and found a strong phylogenetic signal in the VOCs emitted by receptive figs. This signal could be due to either constraints in VOC biosynthetic pathways or selection to attract pollinators that evolved along with the host fig. However, using the same analysis, we found no effect of pollinator phylogeny. By comparing chemical profiles of receptive figs of eight fig varieties of F. carica in Morocco, we found that human domestication resulted in inter-varietal differences, although the chemical signal responsible for pollinator attraction was highly conserved between varieties. Also, in F. carica, we studied the emission patterns of VOCs responsible for pollinator attraction, by performing real-time measurements using a PTR-TOF-MS. We found that VOCs emitted by receptive figs followed a diurnal emission pattern, with a peak at midday corresponding to the period when the pollinator is most active. The VOC emission pattern in F. carica may be optimized to attract its exclusive pollinators. We also highlighted that temperature and humidity can also influence the emission of these VOCs but in a non-linear manner. Moreover, still with these real-time measurements, we have highlighted a very fast change in the emission of floral VOCs after pollination. Chemical profile analyses and comparative transcriptomics between receptive and pollinated figs allowed us to highlight some genes whose expression is down-regulated after pollination. The heterologous expression in Escherichia coli of two genes coding for terpene synthases (TPS) allowed us to confirm their function. Finally, three complementary experiments (VOC analysis, transcript analysis and histology of different tissues) showed that the ostiole is the source of production of volatile compounds that attract fig wasps. This study examined some key factors affecting the emission of pollinator attractive signals in fig trees and elucidated some molecular mechanisms of the biosynthesis of these VOCs. Our work thus provides a solid basis for future studies on the evolution of VOC biosynthesis in Ficus.