| Plant-pollinator interactions fuel the diversification of living organisms and form an essential foundation for the maintenance of ecosystem functioning.Chemical communications are critical in mediating plant-pollinator interactions.However,the underlying molecular mechanisms still remain unknow.Volatile organic compounds(VOCs)emited by plants are the key traits in response to the selection from pollinators,but the VOCs that truly participate in pollinator attraction are seldomly known.Therefore,it is necessary to identify the attractive VOCs and uncover the molecular processes regulating the generation of these attractants.Moreover,the VOC profiles of plants are also likely to be shaped by antagonists(e.g.herbivores and florivores which can undermine fitness of plants).Yet,it is unclear whether such selection can exert influences on chemical communications in plant-pollinator mutualisms.Therefore,obtaining comprehensive answers for these two questions will substantially improve our understanding of coadaptation and coevolution between these two mutualistic partners.Species from genus Ficus and their pollinating wasps constitute obligate mutualisms(fig-pollinator mutualisms),which have a surprisingly long coevolutionary history initializing at late Cretaceous(c.75 Ma).Host identification by pollinating wasps is mediated by a few attractive VOCs,and such specialized chemical communications provided an excellent chance to unravel the underlying molecular mechanisms.In this study,we focused on Ficus pumila var.pumila which attracts its specific pollinating wasps by decanal.We used a series of experiments and analyzes including genomics,transcriptomics and proteomics and in vitro functional validation experiments to find out the pathways and genes contributing crucially to regulating the biosynthesis of decanal.Furthermore,we developed a simulation model based on the information theory with three evolutionary scenarios to test the roles of selection from pollinators and that from antagonists in shaping the chemical communication mode between Ficus species and their pollinators.The main results and conclutions are as following:1)The draft de novo assembly of F.pumila var.pumila genome was achieved using the data from Illumina and Pac Bio sequencing,with the genome size of 315.7Mb.To further improve the quality of genome assembly,we used the Hi-C technique to scaffold contigs into 13 pseudo-chromosomes,and the contig N50 reached to 2.3 Mb.BUSCO assessment detected complete structure for 94.3% of 1440 conserved plant genes in our assembled genome,suggesting high-quality of genome assembly.A total of 28,187 protein-coding genes were functionally annotated,and the gene annotation rate was 91.9%.Therefore,a high-quality reference genome was generated for the subsequent omics analysis.Analysis of comparative genomics using the genomes of 13 angiosperm species showed that totally 1,473 contracted and 888 expanded gene families were found in the common ancestor of the selected Ficus species,implying some cues for adaptive evolution of Ficus species.We did not detect recent whole-genome duplication event,suggesting that polyploidization may not be associated with the adaptation of F.pumila var.pumila.2)After measuring the concentration of decanal using solid-phase microextraction(SPME)and GC-MS,we detected that ostiolar tissues were the main part releasing decanal.We then identified the biosynthesis pathways of decanal and nonanal(the repellent for pollinating wasps of F.pumila var.pumila),which is involved in the pathways of fatty acid biosynthesis(ko00061),elongation(ko00062)and metabolism(ko00071 and ko00592).To identify key genes involved in the biosynthesis of decanal,we conducted transcriptomic and proteomic analyses on ostiolar tissues collected at the pre-receptive and the receptive stages.Four proteins with significant differences in quantity(PSDs)were identified,with three Down-regulated PSDs including two longchain acyl-Co A synthetases Fpum ACSL8 and Fpum ACSL10 and an alcohol dehydrogenase Fpum ADH4 and one Up-regulated PSD(an acetaldehyde dehydrogenase Fpum ALDH1).To validate the function of these key genes in decanal biosynthesis,we produced the recombinant proteins of these genes by prokaryotic expression system of Escherichia coli,and conducted in vitro enzyme activity assay.All recombinant proteins could catalyze substrates and generate target products,showing that they are functional.All above results confirmed that we have uncovered the key regulated genes influencing the biosynthesis of decanal in the pathways of fatty acid biosynthesis,elongation and metabolism.3)We scanned the binding motifs present in the upstream of the above key genes using cis-element prediction.With the help of comparative transcriptomics and WGCNA,we screened the differentially expressed transcriptional factors that were predicted to bind to the key genes and were allocated in the same modules with the key genes.Four transcriptional factors were therefore identified,which were Fpum HDZIP1,Fpum HD-ZIP2,Fpumb HLH1 and Fpumb ZIP1.Among them,Fpumb HLH1 and Fpumb ZIP1,which can bind G-box binding motif,were expected to suppress the expression of the key gene Fpum ACSL10 but facilitate the expression of Fpum ALDH1.In addition,Fpum HD-ZIP1 and Fpum HD-ZIP2,which can bind HD-Zip binding motifs,were likely to facilitate and suppress the expression for Fpum ALDH1,respectively.To validate the function of four transcriptional factors,we conducted Ch IP-q PCR experiments.The percentage input(0.267?0.652)and fold enrichment values(3.422?18.332)showed that all these transcriptional factors can bind with target genes.These results revealed that the four transcriptional factors contributed importantly to the regulation of decanal biosynthesis.4)To explore the evolutionary trend of chemical communications in fig-pollinator mutualisms and the roles of different selection forces,we collected data of chromatograms from 45 Ficus species(244 VOCs)and the information of pollinating wasps.Integrating information theory,we modeled three coevolutionary scenarios(I?III),where the pollinator fitness is always optimized by the highest certainty of chemical information provided by plants,but plant fitness is determined by(I)the certainty of chemical information attracting pollinators,(II)the uncertainty of chemical information confusing antagonists,or(III)both aspects.We found that the empirical plant volatiles from 45 pairs of fig-pollinator mutualisms were best explained by the selection from both pollinators and antagonists(scenario III).Under this scenario,plant-pollinator mutualisms evolve to be specialized,and cumulative mutual information curve showed that as few as two VOCs could supply sufficient information for pollinators' host identification.These results proved that the chemical communications in fig-pollinator mutualisms are evolving towards specialization under the selection from both pollinators and antagonists.In summary,we assembled the high-quality genome of F.pumila var.pumila,with further identification of biosynthesis pathways of decanal and the relevant key genes.After that,we detected some key transcriptional factors regulating the biosynthesis of decanal and validated their functions.Moreover,we provided evidence supporting that chemical communications between fig and fig wasps are under the selection from both pollinators and antagonists.Such a conflicting selection has driven the specialization of chemical communications,and as few as two VOCs can enable the host identification by pollinators,consistent with existing empirical studies.Our study anchored the genes determining plant-pollinator mutualisms and developed novel approaches to characterize the plant-pollinator coevolution by means of chemical communications,offering a paradigm for studies on more diffuse interactions. |
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