Functional Evolution Of Avian Taste Genes And Preliminary Study On Bitter Taste Receptors Detecting Non-bitter Substances

The five basic taste modalities(sweet,umami,bitter,sour,salty)are essential for the survival of vertebrates in nature.Among them,the sweet and umami tastes are mediated by GPCRs(G protein-coupled receptors),such as Tas1r1,Tas1r2 and Tas1r3,which detect corresponding ligands in the form of heterologous dimers.The bitter taste is mediated by a group of G protein-coupled receptors encoded by the type 2 taste receptor genes(Tas2rs).In this work,we studied the functional evolution of avian taste receptors by molecular analysis combined with functional assay methods from the perspective of two taste modalities(umami and bitter).We further conducted a preliminary study on bitter taste receptors involving in detecting non-bitter substances,which reveals the molecular mechanism underlying the metallic taste other than the five basic taste modalities.First,we comprehensively investigated the functional evolution of avian taste receptor genes(Tas1r1/Tas1r3).Previous studies have shown that birds have a widespread absence of the sweet taste receptor gene Tas1r2,whereas one specialized nectar-feeding bird,Anna's hummingbird(Calypte anna)is able to detect sweet compounds because of the functional transformation from the umami to sweet taste using the heterologous dimer:Tas1r1+Tas1r3.Passerida is one of the largest clades of passerines,with a great species richness and diet diversity,and some species in Passerida have a diet with a substantial proportion of nectar.We examined 31 species from major lineages of Passerida,covering Passeroidea,Musicicapoidea and Sylvioidea.The third exon of Tas1r3 was obtained by PCR amplification and genomic sequence identification.With the exception of the hill niltava(Niltava banyumas)and the daurian redstart(Phoenicurus auroreus),the Tas1r3 sequences of other species are free from frameshift mutations.We then performed selective pressure analysis on these Tas1r3 sequences.The result indicated that the Tas1r3 gene evolved faster in nectar-eating sunbirds and flowerpeckers than in the non-nectar feeding birds,but no signal of positive selection was detected.We also conducted molecular convergence analysis for passerine Tas1r3 and hummingbird Tas1r3 sequences.The results revealed that the most recent common ancestor of the sunbirds and flowerpeckers,and the ancestor of the white-eyes,as well as the bohemian waxwing(Bombycilla garrulus),have undergone convergent evolution with the Anna's hummingbird.However,the cell-based functional assays indicated that Tas1r1+Tas1r3 of the fork-tailed sunbird and dark green white-eye showed no functional transformation compared with their outgroups,both of which can only detect umami compounds.These results also showed that the loss of Tas1r2 gene in birds can lead to the loss of sweet taste.Secondly,we performed an in-depth study to examine bitter taste receptor genes(Tas2rs)in the Anna's hummingbird.Nectar may contain many secondary metabolites,acting as a form of natural defense.The ecological hypothesis suggests that such bitter-tasting secondary metabolites might keep the nectar exclusive to only a few pollinators and maintain the fidelity of pollinators.To test this hypothesis,we examined the functional changes of a specialized nectar-feeding bird(Anna's hummingbird),and compared it with two outgroups,the chimney swift(Chaetura pelagica)and the chuck-will' s widow(Caprimulgus carolinensis).With the release of avian genomes,our previous work identified all complete and intact Tas2r sequences of 48 bird species.A lineage-specific Tas2r gene duplication(n=5)was found to have occurred in the Anna's hummingbird,who carries a larger Tas2r gene repertoire(n=6)compared with its two close relatives,the chimney swift(n=4)and the chuck-will's widow(n=3).The improved branch-site model and site models were used to test selective pressures,and it showed hummingbird specific duplicated Tas2rl copies are under positive selection.The result of functional assay indicated that the Anna's hummingbird Tas2r1 copies have higher sensitivity and new functions,which may lead to increased sensitivities and specialized abilities of the hummingbird to detect bitter-tasting nectar.Although it is long believed that the bitter taste is a natural defense against the bitter-tasting and toxic foods in the wild,the functional enhancement and expansion of the hummingbird receptor genes indicates that this nectar-feeding bird may prefer some bitter tastes compared to their insect-feeding relatives.Moreover,this study supports the hypothesis that bitter-tasting nectar may have been specialized for some specialized pollinators,thus enforcing plant-pollinator mutualism.In addition to the sweet,umami,bitter,sour and salty taste,the so-called five"basic" taste modalities,there are some other flavors such as spicy,cold,fatty and metallic.The molecular mechanism of metallic taste,which has a complex taste profile,is still largely unknown.To test whether bitter taste receptor is able to detect non-bitter substances,we screened several metal ions with 25 human bitter taste receptors.The result showed that Tas2r7 responds to all the divalent and trivalent salts we tested.In contrast,potassium chloride,which is a monovalent,does not activate Tas2r7.It suggests that Tas2r7 may act as a metal cation receptor mediating bitterness of divalent and trivalent salts.Molecular modeling and initial mutagenesis analysis identified two residues,H943.37 and E26 4 7.32,in Tas2r7 that appear to be responsible for the interaction of Tas2r7 with metallic ions.Our study suggests the molecular mechanism of metallic taste,and demonstrates that the complex profile of metallic taste is perceived by the bitter taste receptor Tas2r7 in human.This finding expands our understanding to the ligand range of bitter taste receptors,and provides the foundation for our future work on bitter taste receptors involved in detecting non-bitter substances in birds.