Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. inx Hu Xiangxu pandas giant Qinling in high-quercitrin leaves to bamboo adaptation dietary confer variants TAS2R20 nmltsus niaigta itr oisaeamjrslciefresaigteeouino TAS2Rs of evolution than the compounds shaping toxic more force contain selective tissues major plant al., a because et are diet, (Li toxins its evolution of dietary in animal tens plants that of to of indicating course fraction few tissues, the the a 2010). animal in and from frequently al., species species gained et a among and in (Meyerhof greatly lost compounds al et vary been et (Bufe bitter Hayakawa TAS2R have compounds 2013; several of they environment bitter to and numbers the to responsive receptors, the in responsive these commonly that exists are is demonstrated that that may TAS2R have bitterness receptors which each ubiquitous Studies of of the family and most Given alkaloids, a 2002), plant harbor 2016). al., as animals such al., occupy, substances et aversive species bitter (Lossow trigger of that and harmful variety bitter wide and taste as a usually toxic 2015). viewed toxins are be as is Fujikura, there (TAS2Rs), food, nature, 2013; receptors in 2 thus, In compounds Breslin, stimulate taste toxic and, reactions. against bitter 2006; compounds animals by sensations Food-derived of al., mediated unpleasant system 2010; taste, 2018). et defense or bitter al., a al., (Chandrashekar sensations, pleasant et et taste animals causing possible Zhao Shan of buds, the 2016; 2005; Among preferences taste al., al., dietary within et et the cells Liu (Li influencing 2012; specialized diets al., on species-specific et receptors to Sato taste 2012; response al., in et evolve Jiang genes receptor taste Animals fine a provides and panda. environments INTRODUCTION in giant their the quercitrin to in to pandas selection their sensitivity Qinling directional influences decreased of of thus, the adaptation and effects them that genetic of functional to the that suggest the bitter of than illustrates results less example higher study Our tasting significantly This be be areas. to leaves preference. to other bamboo found commonly dietary in quercitrin higher-quercitrin-containing was most habitats in the which is results agonist. quantified panda Mountains, pandas which subsequently Qinling the from Qinling We Q296H, the bamboo to in and quercitrin. of response distributed A52V to leaves bamboo their sensitivity with the of variant decreased of specifically leaves significantly receptor is sensitivity the a pTAS2R20 the of the confers that content areas, in show pandas, functional other results Qinling differences used from Our in we exhibit pandas found agonists. variants differences Here, in the pTAS2R20 the pTAS2R20 to interrogated to pandas. that variants and pTAS2R20 Compared other and TAS2R20) of panda’s with quercitrin responses giant comparison by vitro (i.e. in changes in activated pTAS2R20 base-pair leaves the of causative in bamboo agonists the perception identify for be in to preference to cells are speculated higher gene engineered TAS2R20 are in the the which expression in for in Mountains, variation Q296H, Qinling pandas and to the Qinling A52V contribute from sites, of pandas types nonsynonymous giant two receptor in that thus, distinct showed selected and functionally study environment directionally previous their encoding with Our variants interacting sensitivity. Genetic of taste means bitter important preferences. an dietary with their animals provides influences tastes bitter to Sensitivity Abstract: Abstract 2020 5, May 3 2 1 ajn omlUiest olg fLf Sciences Life of College University Normal Nanjing Sciences available of not Academy Affiliation Chinese Zoology of Institute 1 unWang Guan , . 04 i ta. 06.Acreainhsbe eeldbtenteTSRnumber TAS2R the between revealed been has correlation A 2016). al., et Liu 2014; , 2 e Shan Lei , 3 hynSun Shuyan , 1 1 ioHu Yibo , 1 n ue Wei Fuwen and , 1 Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. xmnn h ffc fec ftetonnyoyosstso h ucino TSR0i epnet its to in response found in pTAS2R20 are of AQ function and the VH on giving for sites haplotypes used encoded, variants nonsynonymous two is mutated two are only histidine the AH of nature, a and each VQ In or of whereas glutamine respectively, effect variants. areas, a the receptor other examining either from VH where pandas two and and 296, The AH, pandas position Qinling VQ, and quercitrin. AQ, encoded, with to is challenged rise valine and a generated in or were alanine sites variants nonsynonymous of pTAS2R20 selected leaves four significantly the used Finally, of we contents prefer. Then, quercitrin pandas the bamboos. with of results including leaves the plants the of various contents specifically quercitrin in was the pTAS2R20 fargesii quantify found to compounds, and monomer (HPLC) bitter chromatography amygdalin flavonoid liquid these coumarin, high-performance a Among aloin, quercitrin, system. salicin, by and expression activated tannin, nicotine), heterologous (quercitrin, a and (caffeine, chemicals in substances the cycloheximide, galangin) bitter bitter picrotoxinin, to bamboo-derived common chloroquine, bitter several known benzoate, with less some pTAS2R20 denatonium taste challenged lactone, first to we sesquiterpene leaves hypothesis, bamboo this address causing To compounds, areas. other bitter from question to pandas the the sensitivity pandas. raise than taste leaves collectively in pandas’ bamboo findings sites more nonsynonymous Qinling These adaptation two of the consumption genetic that 2014). the showed hypothesized for al., We and genetic in pandas et ago Qinling population sites Wei of 2001); nonsynonymous years consume 2013; preference two al., million pandas al., et the ˜0.3 Qinling Pan et whether pandas that 1985; of (Zhao al., showed other environments sites et observations from their nonsynonymous (Schaller field 2013; divergence to two areas al., finding, their other at et in this indicated (Zhao selected pandas pandas) with data directionally than (Qinling Consistent leaves Mountains been Qinling bamboo 2018). the has more from al., gene population et this panda the Shan and in selection (http://www.genenames.org/, 2016), Q296H Organization positive and 30, Genome of A52V Human April for signatures the important Additionally, accessed of more Committee last diet. functionally Nomenclature panda Gene are the last that the in sites compounds some specifically for bitter are of detected sequences were certain presence gene of the receptor three detection ( of taste these genes the because bitter that TAS2R suggesting probably for three TAS2Rs species, on conserved, bitter the pressure functional abundant highly in selection more the functional strengthened purifying for markedly because putatively The requirement is expected more 2018). ) a tolerating be al., are to of might et there challenge lead which (Shan could carnivores, the that perception other pandas showed poses by in work diet encountered than of previous panda substances type addition, Our been giant this complex have In the plants and more compounds. 2010). in that years, had TAS2Rs indicate bitter al., have evidences million et of of may several Zhao amounts lines pandas for 2007; multiple large ancient These al., diet that et pandas’ 2019). (Jin suggests al., of et ago analyses components However, (Han years isotope pandas million 2019). stable modern 7.0 al., than on et diets based least Nie report at 2015a; that recent diet and al., omnivorous plant a et or a carnivorous Nie were to 2015; pandas switched ancient al., they that et suggests (Wei et evidence molecular bamboo Nei and fibrous 2006; paleontological highly al., of on et exclusively Wooding transformation ( almost 2005; the panda al., in giant et population-level variants Soranzo The and/or these 2005; al., species- of et to roles (Bufe could 2008). leading potential adaptation variants al., compounds, dietary the receptor and that bitter reflecting feeding suggested same preferences, animal reports with the dietary These correlated to in 2010). and are sensitivities differences al., chimpanzees, gene different et humans, different TAS2R38 Suzuki in in five the (PTC) 2006; result and bitter phenylthiocarbamide al., in human arbutin, compound et variants the bitter salicin, (Wooding sequence in the macaques to site 2005); to sensitivity K172N sensitivity al., nonsynonymous increased taste the et variable an that (Soranzo with reported glycosides associated been cyanogenic has was it gene addition, TAS2R16 In receptor 2013). al., et (Li and agsaqinlingensis Fargesia TAS2R42 iuooamelanoleuca Ailuropoda and TAS2R49 o hc iln adsso h togs rfrne n compared and preference, strongest the show pandas Qinling which for npandas. in saseilzdhrioei h re anvr htfeeds that Carnivora order the in herbivore specialized a is ) TAS2R20 TAS2R20 2 TAS2R49 TAS2R20 r h astv aepi hne eutn nthe in resulting changes base-pair causative the are agsadenudata Fargesia cu taioai oiin5,weeete an either where 52, position acid amino at occur snwdsgae as designated now is noercpo ainsta a decrease may that variants receptor encode and sai faberi Bshania TAS2R20 TAS2R1 hc other which , codn to according , 9 Bashania and , 38 Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. tec iepitwr esrdwt mgJsfwr.W one tlat2 el neeygopand group every in cells cells 20 the least of values at fluorescence counted The We compounds. software. bitter ImageJ of addition with the measured within after were s s point 3 120 every and time recorded before each were the s images to at 30 Fluorescence led G-CaMP. of receptors indicator bitter period calcium the a the of medium with activation detected The serum-free was (v/v). in which 1% dissolved of Ca were concentration compounds and of cells final mG15 bitter the release a receptor, h, of exceeding bitter 36 concentration not After the DMSO, activation 2017). of and al., optimal vectors et the the (Behrens culture, with cells adherent HEK-293T stimulated into of cotransfected h 3 transiently 24 were of After G-CaMP density USA). a (Corning, at plates crotiter seeded were cells HEK-293T by prepared was assay background observed. curve functional for response fitting was pTAS2R20 corrected maximal the were method the curves and standard to at dose–response sample, external normalized The each performed The and samples. of fluorescence 2010). was standard repetitions of al., extraction concentrations three et flavonoids, different and with (Wu of using analysis methanol, previously properties quantitative the described ml for to as 1 According used were system. to conditions chromatographic sample added reaction the standard the was in the used sample as was mg system used chromatography USA) 50 (Sigma-Aldrich, A quercitrin 98%. 65 the than of greater purity was The crushed. and freeze-dried TCS denudata Leica a fargesii with bamboo B. taken in were contents Photomicrographs quercitrin 2011). a of al., in Determination et incubated usually (Singh microscope. were Abcam), temperature Inverted coverslips ab150116, first Scan (1:500, the room IgG Laser The times, at mouse SP2 five hours anti receptors, washing goat few h. the After antibody a 1 secondary of membrane. for fluorescent for cell expression the the of albumin the solution label detect serum dilute to min. to bovine used 15 added 4% was for 4 was (mGFP) in at temperature Signaling) overnight incubated room incubation Cell at were by transfection 8146S, followed paraformaldehyde cells after (1:1000, 4% h the with antibody 24 binding, fixed anti-FLAG coverslips then nonspecific times, poly-L-lysine-treated reduce three onto To PBS seeded with were washed cells and HEK-293T observed were transfected fluorescence The Campus, in Research changes Farm The Janelia 2006). ImageJ. Institute, Immunocytochemistry al., with generously Medical et measured was and Hughes (Marella G-CaMP microscopy (Howard indicator used transduction. fluorescence fluorescent was Looger signal by School a GPCR L. Medical for as four Harvard Loren VA) necessary of The is Ashburn, Libeles which reconstituted 1B). Prof. Stephen 2002) directionally We by al., Dr. two 2007). (Fig. et from provided The al., (Bufe gift red a effector et indicated mG15, an (Pronin 2013). and as were cells al., system, pTAS2R20 HEK-293T reaction et 2018) in of GPCR al., expressed (Coin the Q296H, et functionally heptahelical analysis and (Masataka of were immunocytochemistry variants signaling signal A52V pTAS2R20 the for targeting sites, with surface used interfere nonsynonymous cell not be selected a dose which can as 1A), SSTR3 and (Fig. rat receptors (DYKDDDDK) of tag acids FLAG by amino followed 45 with p tagged of minally region coding The receptors pTAS2R20 chimeric of Generation METHODS AND adaptation. MATERIALS population polymorphisms how local reveal in and selection hypothesis, directional the of verify to expected we p strategies, in these combining By agonist. ° o .TeL-S-Saayi ehdwsue nti xeiet lr ihpromneliquid high-performance ultra A experiment. this in used was method analysis LC-ESI-MS The h. 4 for C TAS2R20 ape eecletdfo h inliadMnhnMutis eena abolae were leaves bamboo Perennial Mountains. Minshan and Qionglai the from collected were samples and 2+ nuneqecti ecpini in ads rvdn neapeo h ucinleffects functional the of example an providing pandas, giant in perception quercitrin influence rmteedpamcrtclmada nraei h ocnrto fctpamcCa cytoplasmic of concentration the in increase an and reticulum endoplasmic the from .qinlingensis F. TAS2R20 ape eecletdfo h iln onan,and Mountains, Qinling the from collected were samples a lndit h cN31vco Ivtoe,UA n mn ter- amino and USA) (Invitrogen, vector pcDNA3.1 the into cloned was ° .Tepam ebaelclzto akrpoenfsdwt GFP with fused protein marker localization membrane plasma The C. × 10 3 el e elit 6wl lc-al la-otmmi- clear-bottom black-wall, 96-well into well per cells 3 .faberi B. and 2+ F. Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. oml o acltn h ai ftecl ursec nraecmae oteiiilvlewsa follows: as was value initial the to The were compared observed. increase curves response (F fluorescence response maximal = cell The the F the stage. to of ratio initial normalized the and the calculating fluorescence to for background compared formula the values to fluorescence according their corrected in changes the calculated ra.Teqecti otn of content quercitrin of leaves The the areas. in of four and leaves pandas, the all Qinling in that of quercitrin showed of with contents results the stimulated quantified The quercitrin. We were chemicals. to cells bitter responsive pTAS2R20-expressing cell specifically bamboo-derived Henceforth, the were and variants markers 2C). to pTAS2R20 compounds two (Fig. localized the bitter immunocyto- membrane that pTAS2R20 common The indicated cell that several 2A). which yellow, showed the Fig. appeared 2B) at fluorescence, fluorescence Fig. green colocalized (green and membrane. fluorescence, mGFP red cell (red using Superimposed the pTAS2R20 membrane. by at of visualized localized successfully detection was was membrane chemical pTAS2R20 cell whether determine the to Then, (mGFP) GFP with fused p before cells the RESULTS of F intensity points, fluorescence time average the three is first Fo the value, F fluorescence at the agonists of the change adding fold the is F where itdwt erae estvt oqecti nQnigpna,wihcnesuo h adsadietary a pandas the upon asso- confers are which Q296H, pandas, and Qinling A52V in sites, nonsynonymous quercitrin selected to sensitivity directionally two decreased that with bamboo showed ciated we in study, quercitrin this to In response the 3.34, in AH receptor follows: sites, the as DISCUSSION nonsynonymous were of two groups sensitivity the other the that the 5). Ca reduced suggested in of consistently (Fig. greatly fold those results Q296H, of and These value and 2.80, maximum 4.10. A52V the was pTAS2R20-AQ that group and showed mutation results 42 2.50, VQ statistical at VH The the group each in 5). in change cells (Fig. signal single ImageJ representative using of by intensity points fluorescence time the quantified we molecules, order pTAS2R20-AQ quercitrin the was Overall, groups experimental reaction. the weakest strongest > of the pTAS2R20 showed the activities variant reaction expressing exhibited pTAS2R20-VH which variant the the cells pTAS2R20-AQ of to with mG15), after the responsive the group pTAS2R20, was harboring the and cells of (pcDNA, whereas group HEK-293T before reaction, intensity groups experimental in expressed the control points fluorescence pTAS2R20 of Therefore, that the time The activa- confirmed quercitrin. in function This five optimal that change. 4. at the significant the with no obtained showed on comparison with Fig. images variant in sites in pTAS2R20 increased fluorescence shown nonsynonymous significantly each Cell are activated selected treatment we quercitrin. directionally quercitrin quercitrin, of two to concentration these response tion of the 3C). 285 in effect (Fig. was pTAS2R20 micromolar (EC50) the effect thousand maximal characterize several demonstrated of of To was 50% concentration pTAS2R20 in a dose-response of resulting at a concentration activation occurred the the in the activation which concentration, measured increased, maximum in molar was we curve low quercitrin sigmoidal pTAS2R20, a a of activate by at concentration to pTAS2R20 the (P activated required Mountains When which quercitrin Qinling curve. quercitrin, the of of in concentration potency sampled optimum highest species the bamboo two determine the To of those than lower of much that are by followed in quantified ng/mg, contents 222.3 reaching species, TAS2R20 x TSR0V.T ute uniytedffrne ntersosso ieetpA22 ainsto variants pTAS2R20 different of responses the in differences the quantify further To pTAS2R20-VH. stefloecneitniyo h el eoddeey3s. 3 every recorded cells the of intensity fluorescence the is x F - a ornfce noHK23 el ihtepam ebaelclzto akrprotein marker localization membrane plasma the with cells HEK-293T into cotransfected was cx )/(F 0 F - .denudata F. c0 ) .qinlingensis F. and .denudate F. .faberi B. evswr 82n/gad6. gm,rsetvl,which respectively, ng/mg, 66.4 and ng/mg 98.2 were leaves evswstehgetaogtefu xmndbamboo examined four the among highest the was leaves and C0 .fargesii B. 4 n F and .fargesii B. .faberi B. CX t137n/g ncnrs,tequercitrin the contrast, In ng/mg. 143.7 at r h akrudfloecnevle,and values, fluorescence background the are osmdi h ito adsfo other from pandas of diet the in consumed , and .qinlingensis F. > pTAS2R20-AH osmdi h diet the in consumed , > pTAS2R20-VQ < 0.05). μ ,and M, 2+ Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. td sas yia xml o ikn eoesa addt eet clgclaatto tfunctional adaptation at diet adaptation of ecological evolution to This the gene animals. candidate explaining non-model genome-scan associated in in linking gap is especially for receptors, example which a bitterness typical TAS1R1, fills of a research gene function also This receptor is molecular study the taste 2017). of exception umami of al., perspective the behavioral, the basis the et with emphasized from of molecular (Hu function, have pseudogenization receptors the meat bitterness reveal studies the eating of to with on These perspective rare reports the is similar 2015a). from it pandas 2017). of al., giant However, al., et in et adaptations. adaptation (Nie Hu certain feeding genetic 2007; diet evolved and al., bamboo has et morphological, fibrous low-energy (Zhang physiological, it a bamboo a for nevertheless, handling to suitable for 2014); are adaptation used al., that specifically dentition et is in and that (Wei Mutations musculature pandas pseudothumb Carnivora jaw giant a skull, order and since a diet the surprising, acid. including not tannic herbivores to recognize of is belongs correct characteristics specifically result the panda may this is them giant tannin 2013), of The Although some al., an and preference. et is receptors, feeding (Zhao quercitrin bitter in conjectured numerous that important role previously possess as confirmed significant tannin we experiments a and as play expression quercitrin may listed functional agonist, bamboos we that Here, ingest 2010), pTAS2R20 bamboos to al., of receptor. in inclined et agonist the are Hansen found 2001; of pandas chemicals giant al., substances. agonists common et that bitter potential of (Zhao indicating of tannin number studies reports less previous limited systematic contain to a few that and According are from nutrition there plants. pTAS2R20 on but other for focused 2015b), and have agonists al., bamboo screened et food we Nie staple 2015; Thus, panda al., giant et the (Christian in minerals substances chemical do. prefer on pandas pandas studies other Qinling Recent why than explain leaves from may leaves bamboo sites bamboo more nonsynonymous consume the selected was directionally to taste quercitrin two pandas the higher other Therefore, although than areas. Consequently, better quercitrin, leaves pandas. to from the sensitivity leaves Qinling taste decreased selected bamboo in compounds, directionally significantly the quercitrin flavone two a in to produces the with contained However, aversion TAS2R20 bamboo reduce greater. conferred that may be indeed and may which Q296H, TAS2R20 here that and for assayed Given agonists A52V were dietary 2010). sites, of al., compounds number et bitter groups Meyerhof actual chemical of 2005; the the common al., number of various et possessing receptor limited (Bufe compounds in bitterness interactions a numerous receptor-agonist a found recognize mediating of to monomer for tuned agonist responsible flavonoid broadly the usually a as are identified quercitrin, receptors direct been by providing previously activated insect has preferences, herbivorous specifically that dietary ecology. bamboos feeding be panda including pandas’ giant to plants the on shown and provided function selection has was taste Although study directional pTAS2R20 between present effects of The correlation 2018). functional close effects examined. al., the a been the et revealed, of not of been (Shan have evidence has perception example diets taste pandas in fine bitter the compounds a on in pressure bitter genes selective pressure, TAS2R more selective the several many experienced of panda re- on have with taste to giant signatures from challenged bitter seem selective the Our pandas functional genes are natural TAS2R of putative of species consumption. their more genes shift of these harbor bamboo TAS2R several dietary pandas as The for and red the carnivores, specialization and other 2018). of giant their than al., course the ceptors finally, et that et the (Shi and, demonstrated Shan during sensitivity herbivorous study 2013; taste pressure previous to bitter al., selective omnivorous in et experienced to differences Li have carnivorous pres- individual to 2006; selective to al., expected imposes leading which et are animals genes, As bitterness, Wooding receptor occupy. distinct an 2003; (p taste they encounter provides al., gene bitter that may perception environments receptor their they the taste bitter on diets, in Bitter sure a and compounds in environments bitter recognition. variations detect new (quercitrin) to genetic explore 2005; bitterness animals al., that in for et evidence differences means (Bufe new important with types add correlated receptor sen- distinct we are in functionally 2006), ) polymorphisms encoding that al., by hypothesis et perception the alter Wooding with may agre DUOX2 aii hospiton Papilio e oadces ntyoielvl,wihhle ordc nrycnupinfor consumption energy reduce to helped which levels, thyroxine in decrease a to led Sla ta. 05.Suisi uashv ugse htbte taste bitter that suggested have humans in Studies 2015). al., et (Sollai .fargesii B. and 5 .qinlingensis F. nQnigMutis iln pandas Qinling Mountains, Qinling in .denudate F. and .faberi B. TAS2R20 nother in Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. i . n hn,J 21) itsae h vlto ftevrert itrtsercpo eereper- gene receptor taste bitter vertebrate of the skull of first evolution The the (2007). shapes Q. Diet Zhu, and (2013). M., J. Jaeger, toire. Zhang, J., and Liu, D., M., loss Li, taste R. Major Hunt, panda. W., (2012). giant Dong, K. earliest G. L., the Beauchamp, R. and Ciochon, G., C., J. Jin, Brand, reveals W., genomics Li, Comparative D., mammals. Glaser, (2017). carnivorous X., in F. Li, Wei, J., Josue, and P., X., Jiang, Wang, L., Shan, pandas. red Sciences T., and of giant Ma, bamboo-eating S., the between Ma, evolution convergent Q., taste Wu, bitter Y., the clade. Hu, of Euarchontoglires expansions the Frequent in mammals (2014). of Y. evolution Evolution Go, consumption. during and habitat part al. repertoire A., et and gene plant J., Matsui, receptor bamboo evolution A. N., to Kouba, Diet Suzuki-Hashido, L., relationships T., B. Hayakawa, (2019). behavior: Gocinski, A., feeding W. H. panda Bissell, Chen, giant P., Biology and Jiang, in Zoo J., L., shifts C. Seasonal Yan, Apanavicius, M., X., (2010). M. Carr, Ji, L., analysis. Y., R. isotope Hansen, Nie, stable via humans. Y., pandas modern Hu, giant of in W., contraction receptors Wei, taste H., of Han, variants binding loss-of-function the Multiple reveal cells Reports in (2015). probes GPCR. chemical K. encoded B Fujikura, Genetically class CRF (2013). to al. urocortin-I et of et T., path J., Sun, V., A. Katritch, Kouba, I., C., of Coin, G. species Jr, seven Fahey in L., growth L. Bauer, shoot 1172-1183. F., during J. composition Falcone, mineral K., seudosasa and C. Nutrient Vance, (2015). K., mammalian K. for al. cells Knott, and L., receptors TAS2R16 The A. human Christian, (2006). S. The C. Zuker, (2002). and J., W. N. Ryba, taste. Meyerhof, A., and M. Hoon, D., to J., Chandrashekar, J. response The in Raguse, (2005). taste D., W. bitter Krautwurst, Meyerhof, mediates and T., receptor P., Hofmann, J. B., Slack, perception. Bufe, D., bitterness C. propylthiouracil and Tharp, phenylthiocarbamide R., in D. Biology differences Reed, Current taste. individual C., human of Kuhn, basis and A., molecular P. food Breslin, on B., perspective Bufe, evolutionary An (2013). R409-R418. re- A. taste P. human The Breslin, (2004). W. compounds. Meyerhof, bitter and different of M., variety Communications Winnig, a B., to Bufe, responds C., hTAS2R14 ceptor Kuhn, cyclamate A., inhibition. and Brockhoff, saccharin receptor M., sweeteners bitter Behrens, non-caloric TAS2R of to Blends due (2017). off-taste W. reduced Meyerhof, show and K., Blank, M., Behrens, REFERENCES level. verification oeua ilg n Evolution and Biology Molecular Nature ,12349. 5, , 18,2018-2031. 31(8), , abocnue ygatpanda. giant by consumed bamboo 1() 1081-1086. 114(5), , 94,470-483. 29(4), , 4(17,288-294. 444(7117), , 54,322-327. 15(4), , 1() 479-485. 319(2), , rceig fteNtoa cdm fSciences of Academy National the of Proceedings rceig fteNtoa cdm fSciences of Academy National the of Proceedings 12,303-309. 31(2), , Cell β- ora fAia hsooyadAia Nutrition Animal and Physiology Animal of Journal glucopyranosides. 5() 1258-1269. 155(6), , 6 urn Biology Current elCeia Biology Chemical Cell aueGenetics Nature iceia n ipyia Research Biophysical and Biochemical rceig fteNtoa Academy National the of Proceedings 94,664-669. 29(4), , 0(6,10932-10937. 104(26), , 0(3,4956-4961. 109(13), , urn Biology Current 23,397-401. 32(3), , oeua ilg and Biology Molecular hyllostachys P 41) 1199-1204. 24(10), , Scientific 23(9), , 99(6), , and P Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. oli . abrsa .T,Slr,P,adCna,R 21) at iciiaigcpblt odifferent to capability herbivore discriminating insect Taste the (2015). in R. sensilla Physiology Crnjar, styloconic Insect larval and receptor of P., the taste by Solari, bitter compounds T., of bitter I. activation Barbarossa, of G., basis Sollai, Structural receptors. (2011). G-protein-coupled al. A et class 36032-36041. J., with genes Upadhyaya, comparison receptor P., and taste S. T2R1 bitter of Pydi, diversification N., Adaptive Singh, (2003). P. Y. Zhang, evolution. and receptor mammalian H., taste in Yang, adaptation. bitter Chicago J., dietary of Zhang, implies of evolution P., pandas Lineage-specific University Shi, (2018). red Wolong, F. and Wei, of giant and Panda the L., Giant in Zhang, L., genes The Wang, Q., (1985). Wu, J. L., Shan, Zhu, pinnipeds. and in W., sensation Pan, taste J., Press. umami Hu, of reduction G.B., major genes Schaller, receptor or bitter Loss of (2012). alleles M. Specific Wolsan, wissenschaften (2007). saccharin. and X. and J., Li, aloin J. and of Sato, Q., bitterness Li, the L., to survival. Zhang, lasting sensitivity H., for human Tang, chance H., influence A Xu, (2001). al. N., et A. X., Pronin, Zhou, in Y., herbivory Long, H., Obligate Wang, University. geome- D., (2015b). Beijing Wang, nutritional X., Beijing: F. of Zhu, Wei, Z., perspective Lu, and the W., from Pan, W., bamboo Wei, pandas and J., Giant panda J. (2019). giant al. Elser, the try. et lineage: D., carnivorous D., Raubenheimer, ancestrally Raubenheimer, Z., an Q., Zhang, Wu, low M., Y., Exceptionally A. Nie, (2015a). Senior, al. Y., et Hu, carnivores. J., W., 171-174. macronutritional Zhang, Zhou, 349(6244), are M., F., Science, Xia, Wei, panda. Y., Y., giant Hu, Nie, bamboo-eating C., the reper- Zhang, in gene Q., expenditure Wu, receptor energy R., chemosensory daily J. animal Speakman, of Y., evolution Nie, The The necessity. (2010). (2008). and M. chance M. of Behrens, Nozawa, roles and and toires: B., Y., Niimura, receptors. Bufe, taste M., E., bitter Nei, Chudoba, TAS2R A., human Brockhoff, of C., ranges receptive Kuhn, molecular of C., bitterness Batram, detecting W., for Meyerhof, mice. receptor of main cavity the oral as the 2301-2305. functions behavior. Tas2r125 in and (2018). catechins category responses M. tea taste taste Takumi, of Imaging and (2006). map N., K. functional Masataka, Scott, a and reveals E., brain Rueckert, S., or- fly Asgarian, the for P., in Kong, ranges W., receptive Fischler, molecular S., different Marella, humans. reveals and receptors mice taste in receptors bitter thologous mouse gains of and analysis losses Comprehensive independent Mammals. multiple H drives Laurasiatherian of K., specialization of Pseudogenization Lossow, Dietary repertoire (2005). (2016). gene G. taste al. J. bitter et Brand, the F., and Hailer, in G., L., sugar. Liu, Huang, toward Z., K., indifference Liu, cats’ Maehashi, for J., accounts Cao, gene H., sweet-receptor Wang, a W., Li, X., Li, ucinlEcology Functional be,S,Runtk,N,Sak .P,Plato . ern,M,adMyro,W (2016). W. Meyerhof, and M., Behrens, F., Pollastro, P., J. Slack, N., Roudnitzky, S., ubner, ¨ 98,655-659. 99(8), , 4 45-55. 74, , 91,26-34. 29(1), , oeua ilg n Evolution and Biology Molecular urn Biology Current aueRves Genetics Reviews. Nature iceia n ipyia eerhCommunications Research Biophysical and Biochemical ora fBooia Chemistry Biological of Journal 91) 1677-1682. 29(10), , 7 05,805-814. 20(5), , nertv Zoology Integrative (2,951-963. 9(12), , rnir nZoology in Frontiers ora fBooia Chemistry Biological of Journal LSGenetics PLoS hmclSenses Chemical urn Biology Current Neuron aii hospiton Papilio 9(9,15358-15377. 291(29), , 92,285-295. 49(2), , 32,152-159. 13(2), , () 27-35. 1(1), , 52,157-170. 35(2), , 71) 1403-1408. 17(16), , 31,28. 13(1), , (G´en´e) . 286(41), , 503(4), , Journal Natur- Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. ra.()Ds–epnecre fqecti nteatvto fpA22.EC non-QIN points. pTAS2R20. Ca and of time QIN activation 4. of the leaves in bamboo Fig. quercitrin in of content curves from quercitrin Dose–response leaves of (C) analysis bamboo areas. difference quercitrin. of Significant of food (B) staple curves Mountains). panda dose–response (non-QIN). (QIN). giant areas and Mountain the other leaves of Qinling and bamboo samples (QIN) representative in 10 Mountains in quercitrin = Qinling quercitrin bar of of Scale contents Contents antibody (B). The (A) primary and is anti-FLAG pTAS2R20 (A) 3. an of of by terminus Overlap Fig. detected amino (C) is antibody. The epitope secondary (B) FLAG Alexa561-conjugated (mGFP). the an surface. GFP and and with cell (DYKDDDDK), fused sequence. FLAG the protein acid with at membrane amino tagged pTAS2R20 plasma pTAS2R20 red-labeled signal, a pandas’ The of surface-targeting pTAS2R20. by Qinling detection of cell the labeled sites Immunocytochemical a of mutation 296 as 2. acid and which used amino Fig. 52 immunohistochemistry, two SSTR3, positions by The rat mutated (B) expression of are line. protein acids wavy acids green amino detect amino Forty-five a to box. with used indicated red was are the tag by epitopic surrounded and variations. FLAG is experiments SNP The functional for dots. of pTAS2R20 red of sequence the structure on acid the effect of amino its representation and Schematic bamboo 1. in Fig. tannin the of Determination LEGENDS (2001). FIGURE F. Z. Ma, and sequencing P., G. Whole-genome panda. Liu, (2013). adaptation. H., al. local X. et Zhao, L., and Zhu, history X., demographic Guo, into Q., insights gene Wu, 67-71. provides X., receptor pandas Zhan, taste S., giant umami Dong, of P., the bamboo. Zheng, of to S., Pseudogenization switch Zhao, ( dietary (2010). its J. panda with Zhang, giant coincided and 2669-2673. the panda 27(12), H., giant of Xu, the Mandible R., in Tas1r1 J. (2007). Yang, F. chemical H., Wei, of Zhao, and Analysis (2010). C., R. Oxnard, Society Y. M., Linnean Liang, Li, and R., L., melanoleuca Pan, J. Lu, S., H., Zhang, J. Ye, (2006). L., HPLC. 421-426. J. and X. 4(5), GC/MS M. Wang, , by flowers Bamshad, Z., indicum and chrysanthemum H. of M., composition Vazquez, Gao, ecology C., chimpanzees. Y., the A. and L. in humans Stone, Wu, Progress in T., (2015). sensitivity M. al. bitter-taste Howard, evolutionary et of C., evolution L., an Grassi, Independent Zhu, not B., Z., Bufe, are Zhang, S., pandas L., Wooding, Yan, Giant Y., pandas. (2014). giant Nie, Z. of Y., conservation Zhang, Hu, non-taster and and R., of Q., Swaisgood, Identification Wu, F., Wei, (2010). Y., research. H. Nie, multidisciplinary L., Imai, from Yan, evidence & Y., cul-de-sac: H., Hu, Hirai, F., taste. Y., Wei, bitter Go, specific A., a for Matsui, macaques T., Japanese (2005). Sugawara, B. TAS2R16. D. N., Goldstein, receptor Suzuki, and bitter-taste R., human Marguerie, the E., of M. Weale, allele 1257-1265. F., high-sensitivity 15(14), J. a Wilson, on C., selection P. Positive Sabeti, B., Bufe, N., Soranzo, ora fNrhatFrsr University Forestry Northeast of Journal 2+ oprdwt te hns anvrs ucinladaptation. functional carnivores: chinese other with compared ) urirnwsadda h iepit h oto rus(cN,m1,pTAS2R20) mG15, (pcDNA, groups control The point. time s 0 the at added was Quercitrin ursec mgso el eoeadatrtetetwt urirna five at quercitrin with treatment after and before cells of images fluorescence 23,449-456. 92(3), , .denudata F. osrainBiology Conservation and .faberi B. Primates A mn cd uae nQnigpna r hw as shown are pandas Qinling in mutated acids Amino (A) 92,67-71. 29(2), , oeua ilg n Evolution and Biology Molecular 8 eecletdi te ra Qoga n Minshan and (Qionglai areas other in collected were .fargesii B. 96,1497-1507. 29(6), , 14,285-289. 51(4), , and ora fMdcnlPat Research Plants Medicinal of Journal .qinlingensis F. oeua ilg n Evolution and Biology Molecular Nature 50 ilgclJunlo the of Journal Biological aueGenetics Nature =285 A h elsraeis surface cell The (A) 4(06,930. 440(7086), , 21,4-12. 32(1), , eecletdi the in collected were μ urn Biology Current M μ m Ailuropoda 45(1), , , , Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. tA2 n 26,wihaemsl on nQnigpna;pDAi h etrcnrl n G5is mG15 and control; vector the is pcDNA pandas; sites The Qinling mutant Mountains; in two mutant found carries control. Qinling variant the mostly molecule the pTAS2R20-VH are signaling carries The of which the variant types; Q296H, pTAS2R20-VQ outside lab-produced and the pandas are A52V and variants in at two Q296H, found These site mostly A52V. mutant site is the pTAS2R20 carries common variant Ca pTAS2R20-AH The the course. of time Ca change the Fold taste in bitter change the 5. of 285 activation (quercitrin, the Fig. agonist for the necessary to are (mG15) respond molecule not receptors. signaling did and they (pTAS2R20) receptor and the mG15, receive not did 2+ ursec inli oeta 0rpeettv igeclsi ahgopdrn the during group each in cells single representative 20 than more in signal fluorescence 2+ hneatrqecti treatment. quercitrin after change 9 μ ) niaigta both that indicating M), ahln ersnsthe represents line Each Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. 10 Posted on Authorea 18 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158204133.30523716 — This a preprint and has not been peer reviewed. Data may be preliminary. 11