Dzidziso dzakasiyana dzekudzivirira zvirimwa dzinopa gwara rakakosha redzidziso yekutsanangura maitiro emetabolism yezvirimwa, asi fungidziro dzadzo huru dzichiri kuyedzwa. Pano, takashandisa ongororo ye unbiased tandem mass spectrometry (MS/MS) kuti tiongorore zvakarongeka metabolome yefodya attenuated strains kubva kumiti yega yega kusvika kumarudzi akasiyana uye marudzi ane hukama, uye takagadzirisa nhamba yakawanda ye mass spectrometric feature theories zvichibva pa compound spectra muruzivo. Framework yekuedza fungidziro huru dze optimal defense (OD) uye moving target (MT) theories. Chikamu cheruzivo che plant metabolomics chinoenderana ne OD theory, asi chinopesana nefungidziro huru ye MT theory pamusoro pe metabolomics dynamics inokonzerwa ne herbivores. Kubva pa micro kusvika pa macro evolutionary scale, jasmonate signal yakaonekwa se main determinant ye OD, nepo ethylene signal yakapa fine-tuning ye herbivore-specific response annotated by MS/MS molecular network.
Mametabolites akakosha ane maumbirwo akasiyana-siyana ndiwo anonyanya kubatanidzwa mukugadzirisa nharaunda yemiti, kunyanya mukudzivirira vavengi (1). Kusiyana-siyana kunoshamisa kwemetabolism chaiyo inowanikwa mumiti kwakakurudzira makumi emakore ekutsvagisa kwakadzama pamusoro pemabasa ayo mazhinji ezvakatipoteredza, uye kwakagadzira runyorwa rurefu rwedzidziso dzekudzivirira zvirimwa, idzo dziri shanduko uye kukura kwezvakatipoteredza kwekudyidzana kwezvirimwa nezvipembenene. Tsvagiridzo yeEmpirical inopa nhungamiro yakakosha (2). Zvisinei, idzi dzidziso dzekudzivirira zvirimwa hadzina kutevera nzira yekufungidzira kwekufungidzira, umo kufanotaura kukuru kwaive padanho rimwe chete rekuongorora (3) uye kwakaedzwa nekuyedza kufambisa kutenderera kunotevera kwekuvandudzwa kwedzidziso (4). Miganhu yehunyanzvi inoganhurira kuunganidzwa kwedata kune chaiwo metabolic categories uye inobvisa kuongororwa kwakazara kwemametabolites akasarudzika, nokudaro ichidzivirira kuenzanisa pakati pezvikamu zvakakosha pakukura kwedzidziso (5). Kushaikwa kwedata rakazara remetabolomics uye mari yakafanana yekuenzanisa mashandiro ekugadzirisa nzvimbo yemetabolism pakati pemapoka akasiyana ezvirimwa kunokanganisa kukura kwesainzi kwemunda.
Zvichangobva kuitika mumunda we tandem mass spectrometry (MS/MS) metabolomics zvinogona kutsanangura zvizere shanduko dze metabolic mukati uye pakati pemhando ye system clade yakapihwa, uye zvinogona kusanganiswa nenzira dzekuverenga kuti zviverenge kufanana kwechimiro pakati pemisanganiswa iyi yakaoma. Ruzivo rwekare rwe chemistry (5). Kusanganiswa kwetekinoroji yepamusoro mukuongorora nekushandisa komputa kunopa hurongwa hunodiwa hwekuyedza kwenguva refu fungidziro dzakawanda dzakaitwa nedzidziso dze ecological ne evolutionary dzekusiyana kwe metabolic. Shannon (6) akatanga dzidziso yeruzivo kekutanga muchinyorwa chake chikuru muna 1948, achiisa hwaro hwekuongorora kwemasvomhu kweruzivo, iyo yakashandiswa muminda yakawanda kunze kwekushandiswa kwayo kwekutanga. Mu genomics, dzidziso yeruzivo yakashandiswa zvinobudirira kuyerwa ruzivo rwekuchengetedza sequence (7). Mukutsvaga kwe transcriptomics, dzidziso yeruzivo inoongorora shanduko dzese mu transcriptome (8). Mukutsvaga kwakapfuura, takashandisa chimiro che statistical chedzidziso yeruzivo ku metabolomics kutsanangura hunyanzvi hwe metabolic hwe tissue level mumiti (9). Pano, tinobatanidza mafambiro ebasa akavakirwa paMS/MS nehurongwa hweruzivo hwehuwandu hwemashoko, hunoratidzwa nekusiyana kwemetabolism mumari yakajairika, kuti tienzanise kufanotaura kukuru kwedzidziso yekudzivirira zvirimwa yemetabolome inokonzerwa nezvinodya miriwo.
Mafungiro edzidziso ekudzivirira zvirimwa anowanzo sanganisira zvese uye anogona kukamurwa kuita mapoka maviri: ayo anoedza kutsanangura kugoverwa kwemetabolites chaiyo yezvirimwa zvichibva pamabasa ekudzivirira, akadai sekudzivirira kwakanaka (OD) (10), chinangwa chinofamba (MT) (11)) Uye chitarisiko (12) dzidziso, nepo vamwe vachitsvaga tsananguro dzemakanika ekuti shanduko mukuwanikwa kwezviwanikwa zvinokanganisa sei kukura kwezvirimwa uye kuunganidzwa kwemetabolites dzakagadzirwa, dzakadai secarbon: nutrient balance hypothesis (13), growth rate hypothesis (14), uye growth and differentiation equilibrium hypothesis (15). Matimu maviri edzidziso ari pamatanho akasiyana ekuongorora (4). Zvisinei, dzidziso mbiri dzinosanganisira mabasa ekudzivirira padanho rekushanda dzinotonga hurukuro pamusoro pekudzivirira kwezvirimwa uye kunokonzerwa: dzidziso yeOD, iyo inofunga kuti zvirimwa zvinoisa mari mukudzivirira kwavo kwemakemikari kunodhura chete kana zvichidikanwa, semuenzaniso, pazvinomedzwa Kana mhuka yeuswa ikarwisa, saka, zvichienderana nekugona kwekurwiswa mune ramangwana, chinhu chine basa rekudzivirira chinopihwa (10); Pfungwa yeMT inoratidza kuti hapana shanduko yekuchinja kwemetabolite inoenderana negwara, asi metabolite inoshanduka zvisina tsarukano, nokudaro ichigadzira mukana wekuti "chinangwa chekufamba" kwemetabolite chekurwisa zvinodya zvinomera. Nemamwe mashoko, dzidziso mbiri idzi dzinopa fungidziro dzakasiyana nezvekugadzirisa metabolic kunoitika mushure mekurwiswa kwezvinodya zvinomera: hukama huripo pakati pekuunganidzwa kwemetabolite nekudzivirira basa (OD) uye shanduko dzemetabolite dzisina kutungamirirwa (MT) (11).
Fungidziro dzeOD neMT hadzingosanganisiri chete shanduko dzinokonzereswa mu metabolome, asiwo mhedzisiro yezvakatipoteredza uye yekushanduka-shanduka kwekuunganidzwa kwemetabolites idzi, dzakadai semitengo inochinjika uye mabhenefiti ekuchinja uku kwemetabolism munzvimbo chaiyo yezvakatipoteredza (16). Kunyangwe fungidziro dzese dziri mbiri dzichiziva basa rekudzivirira remetabolites dzakanongedzerwa, dzingave dzakadhura kana dzisingadhuri, kufanotaura kukuru kunosiyanisa fungidziro dzeOD neMT kuri mugwara rekuchinja kwemetabolism kunokonzerwa. Kufanotaura kwedzidziso yeOD kwave kugamuchira kutariswa kwakanyanya kusvika pari zvino. Miedzo iyi inosanganisira kudzidza kwemabasa ekudzivirira zvakananga kana zvisina kunanga ematishu akasiyana emakomponi chaiwo mugreenhouse nemamiriro echisikigo, pamwe nekuchinja muchikamu chekukura kwezvirimwa (17-19). Zvisinei, kusvika pari zvino, nekuda kwekushaikwa kwekushanda uye hurongwa hwehuwandu hwekuongorora kwakazara kwemetabolism yechero chipenyu, musiyano mukuru wekufanotaura pakati pedzidziso mbiri idzi (ndiko kuti, gwara rekuchinja kwemetabolism) uchiri kuyedzwa. Pano, tinopa ongororo yakadaro.
Chimwe chezvinhu zvinonyanya kukosha zve metabolites dzemiti ndekusiyana kwazvo kwemaumbirwo adzo pamatanho ese kubva pamiti imwe chete, huwandu hwevanhu kusvika kune mhando dzakafanana (20). Shanduko dzakawanda muhuwandu hwe metabolites dzakasarudzwa dzinogona kuonekwa pahuwandu hwevanhu, nepo musiyano wakasimba wemhando unowanzo chengetwa padanho remhando (20). Nokudaro, kusiyana kwemetabolism yemiti ndicho chinhu chikuru chekusiyana kwebasa, zvichiratidza kuchinjika kune nzvimbo dzakasiyana, kunyanya nzvimbo idzodzo dzine mukana wakasiyana wekupinda nezvipembenene zvakasiyana uye zvinodya zvinomera (21). Kubva pane chinyorwa chaFraenkel (22) pamusoro pezvikonzero zvekuvapo kwe metabolites dzemiti, kusangana nezvipembenene zvakasiyana-siyana kwave kuchionekwa sekumanikidzwa kwakakosha kwekusarudza, uye kusangana uku kunofungidzirwa kuti kwakaumba zvirimwa panguva yekushanduka-shanduka. Nzira yemetabolism (23). Kusiyana kwemarudzi akasiyana-siyana mukusiyana kwe metabolites dzakasarudzwa kunogonawo kuratidza kuenzana kwemuviri kunobatanidzwa nekudzivirira kwezvirimwa zvinoumba uye zvinokonzeresa nzira dzekudyara, sezvo mhando mbiri idzi dzinowanzobatanidzwa zvisina kunaka (24). Kunyangwe zvingave zvakanaka kuchengetedza dziviriro yakanaka nguva dzese, shanduko dzemetabolism dzinoenderana nedziviriro dzinopa mabhenefiti akajeka mukubvumira zvirimwa kugovera zviwanikwa zvakakosha kune dzimwe mari dzemuviri (19, 24), uye kudzivirira kudiwa kwekubatana. Kukuvadzwa kwemari (25). Pamusoro pezvo, kurongeka uku kwemetabolism dzakasarudzika dzinokonzerwa nezvipembenene zvinodya zvinomera kunogona kutungamira mukupararira kunoparadza muhuwandu hwevanhu (26), uye kunogona kuratidza kuverenga kwakananga kweshanduko huru dzechisikigo muchiratidzo chejasmonic acid (JA), icho chinogona kuchengetwa muhuwandu hwevanhu. Zviratidzo zveJA zvakakwirira nepasi zvinotsinhana pakati pekudzivirira zvinodya zvinomera uye kukwikwidzana nemarudzi chaiwo (27). Pamusoro pezvo, nzira dzebiosynthetic dzemetabolism dzakasarudzika dzicharasikirwa nekukurumidza uye kushandurwa panguva yekushanduka, zvichikonzera kupararira kwemetabolism pakati pemarudzi ane hukama hwakanyanya (28). Aya mapolymorphisms anogona kukurumidza kusimbiswa mukupindura kuchinja kwemapatani eherbivore (29), zvinoreva kuti kuchinja kwenharaunda dzeherbivore chinhu chikuru chinotyaira kusawirirana kwemetabolism.
Pano, takagadzirisa matambudziko anotevera zvakananga. (I) Chipembenene chinodya miriwo chinogadzirisa sei metabolome yemuti? (Ii) Ndezvipi zvikamu zvikuru zveruzivo rwe metabolic plasticity zvinogona kuverengwa kuti zviedze fungidziro yedzidziso yekudzivirira kwenguva refu? (Iii) Kuti togadzirisa metabolome yemuti nenzira yakasiyana neyeanorwisa, kana zvakadaro, hormone yemuti inoita basa rei mukugadzirisa mhinduro chaiyo ye metabolic, uye ndeapi metabolites anobatsira mukudzivirira kwemhando dzakasiyana dzemhuka? (Iv) Sezvo fungidziro dzakaitwa nedzidziso dzakawanda dzekudzivirira dzichigona kuwedzerwa pamatanho ese ezvipenyu zvemuviri, takabvunza kuti mhinduro ye metabolic inokonzerwa yakabatana sei kubva pakuenzanisa kwemukati kusvika pakuenzanisa kwemarudzi akasiyana? Kuti tiite izvi, takadzidza zvakarongeka metabolome yemashizha efodya nicotine, inova chirimwa chemuenzaniso wezvakatipoteredza chine metabolism yakapfuma, uye chinoshanda pakurwisa honye dzemhuka mbiri dzemuno, Lepidoptera Datura (Ms) (Inorwisa zvikuru, inonyanya kudyiwa). PaSolanaceae neSpodoptera littoralis (Sl), honye dzemashizha edonje rudzi rwe "genus", nezvirimwa zveSolanaceae nezvimwe zvizvarwa nemhuri. Takaongorora MS/MS metabolomics spectrum uye takatora tsananguro dzezviverengero zveruzivo kuti tienzanise dzidziso dzeOD neMT. Gadzira mamepu chaiwo kuti aratidze hunhu hwemametabolites akakosha. Ongororo iyi yakatambanudzirwa kuvanhu vemo veN. nasi uye mhando dzefodya dzine hukama hwakasimba kuti tiongorore zvakanyanya covariance pakati pe plant hormone signaling uye OD induction.
Kuti titore mepu yakazara nezve plasticity uye chimiro che metabolome yemashizha efodya inodya zvinomera, takashandisa ongororo yakaitwa kare uye workflow yekuverenga kuti tiunganidze zvakakwana uye tibvise data re high-resolution rakazvimiririra re MS/MS spectra kubva muzvirimwa (9). Iyi nzira isina musiyano (inonzi MS/MS) inogona kugadzira non-redundant compound spectra, iyo inogona kushandiswa kune ese ma compound-level analysis akatsanangurwa pano. Aya ma deconvoluted plant metabolites ndeemhando dzakasiyana, ane mazana kusvika kuzviuru zvema metabolites (anenge 500-1000-s/MS/MS pano). Pano, tinofunga nezve metabolic plasticity muchimiro cheruzivo rwedzidziso, uye tinotsanangura kusiyana uye hunyanzvi hwe metabolome zvichibva paShannon entropy yekugoverwa kwe metabolic frequency. Tichishandisa fomura yakashandiswa kare (8), takaverenga seti yezviratidzo zvinogona kushandiswa kuyera metabolome diversity (Hj indicator), metabolic profile specialization (δj indicator) uye metabolic specificity ye single metabolite (Si indicator). Pamusoro pezvo, takashandisa Relative Distance Plasticity Index (RDPI) kuti tiverenge metabolome inducibility yezvinomera zvinodya herbivores (Mufananidzo 1A) (30). Mukati mehurongwa uhwu hwezviverengero, tinotora MS/MS spectrum seyuniti yeruzivo rwekutanga, uye tinogadzirisa huwandu hweMS/MS mumepu yekugovera frequency, uye tobva tashandisa Shannon entropy kufungidzira kusiyana kwemetabolome kubva mairi. Kunyanya kushandiswa kwemetabolome kunoyerwa neavhareji chaiyo yeMS/MS spectrum imwe chete. Nokudaro, kuwedzera kwehuwandu hwemamwe makirasi eMS/MS mushure mekutanga kushandiswa kweherbivore kunoshandurwa kuita spectral inducibility, RDPI uye specialization, kureva kuti, kuwedzera kwe δj index, nekuti metabolites dzakawanda dzinogadzirwa uye high Si index inogadzirwa. Kudzikiswa kweHj diversity index kunoratidza kuti huwandu hweMS/MS inogadzirwa hunoderedzwa, kana kuti kugoverwa kweprofile frequency kunochinja munzira isina kufanana, ukuwo kuchideredza kusava nechokwadi kwayo. Kuburikidza nekuverenga kweSi index, zvinokwanisika kuratidza kuti ndeapi MS/MS anokonzerwa nemamwe marudzi emhuka dzinodya miriwo, asi, ndeapi MS/MS asingapindure kuinduction, inova chiratidzo chikuru chekusiyanisa kufanotaura kweMT neOD.
(A) Tsanangudzo dzezviverengero dzinoshandiswa pakushandisa zvinodya mhuka (H1 kusvika Hx) MS/MS data-inducibility (RDPI), kusiyana-siyana (Hj index), specialization (δj index) uye metabolite specificity (Si index). Kuwedzera kwedanho respecialization (δj) kunoratidza kuti, paavhareji, metabolites dzakawanda dzinodya mhuka dzichagadzirwa, nepo kudzikira kwekusiyana-siyana (Hj) kunoratidza kudzikira kwekugadzirwa kwemetabolites kana kusagovaniswa kwemetabolites mumepu yekugovera. Kukosha kweSi kunoongorora kana metabolite yacho yakanangana nemamiriro ezvinhu akapihwa (pano, zvinodya mhuka) kana kuti yakachengetedzwa padanho rimwe chete. (B) Dhayagiramu yepfungwa yekufanotaura dzidziso yekudzivirira uchishandisa information theory axis. OD theory inofanotaura kuti kurwiswa kwezvinodya mhuka kuchawedzera metabolites dzekudzivirira, nokudaro zvichiwedzera δj. Panguva imwe chete, Hj inoderera nekuti profile inorongwazve kusvika pakuderera kwekusaziva kweruzivo rwemetabolism. MT theory inofanotaura kuti kurwiswa kwezvinodya mhuka kuchakonzera shanduko dzisiri dzenzira mumetabolome, nokudaro zvichiwedzera Hj sechiratidzo chekuwedzera kusaziva kweruzivo rwemetabolism uye zvichikonzera kugoverwa kweSi zvisina kurongeka. Takakurudzirawo modhi yakasanganiswa, iyo MT yakanakisa, umo mamwe ma metabolites ane dziviriro yakakwirira achawedzerwa zvakanyanya (high Si value), nepo mamwe achiratidza mhinduro dzisina kurongeka (low Si value).
Tichishandisa tsananguro dzedzidziso yeruzivo, tinodudzira dzidziso yeOD kuti tifembere kuti shanduko dzemetabolite dzakakonzereswa nezvinomera muchimiro chisina kukonzerwa chichatungamira ku (i) kuwedzera kwe metabolic specificity (Si index) driving metabonomic specificity (δj index) Kuwedzera kwe) mamwe mapoka emetabolite akakosha ane dziviriro yakakwira, uye (ii) kudzikira kwe metabolome diversity (Hj index) nekuda kwekuchinja kwekugoverwa kwe metabolic frequency kune leptin body distribution yakawanda. Padanho remetabolite imwe chete, kugoverwa kweSi kwakarongwa kunotarisirwa, uko metabolite ichawedzera kukosha kweSi zvichienderana nekudzivirira kwayo (Mufananidzo 1B). Nenzira iyi, tinotsanangura dzidziso yeMT kuti tifembere kuti kufadza kuchatungamira ku (i) shanduko dzisiri dzenzira mumetabolites zvichikonzera kudzikira kwe δj index, uye (ii) kuwedzera kweHj index nekuda kwekuwedzera kwekusagadzikana kwemetabolite. Kana kusarongeka, izvo zvinogona kuverengerwa neShannon entropy muchimiro chekusiyana-siyana kwakajairika. Nezve metabolic composition, dzidziso yeMT ichafanotaura kugoverwa kweSi kusingarongeki. Tichifunga kuti mamwe ma metabolites ari mumamiriro ezvinhu chaiwo, uye mamwe mamiriro haasi mumamiriro ezvinhu chaiwo, uye kukosha kwawo kwekudzivirira kunoenderana nenzvimbo, takakurudzirawo modhi yekudzivirira yakasanganiswa, umo δj neHj zvinogoverwa muzvikamu zviviri pamwe chete neSi Kuwedzera kumativi ese, mamwe mapoka e metabolites chete, ane dziviriro yakakwira, ndiwo achanyanya kuwedzera Si, nepo mamwe achava nekugoverwa kusingatarisirwi (Mufananidzo 1B).
Kuti tiedze kufanotaura kwedzidziso yekudzivirira patsva padivi petsananguro yedzidziso yeruzivo, takatsvaga nyanzvi (Ms) kana generalist (Sl) herbivore larvae pamashizha eNepenthes pallens (Mufananidzo 2A). Tichishandisa ongororo yeMS/MS, takatora 599 non-redundant MS/MS spectra (data file S1) kubva mu methanol extracts yemashizha akaunganidzwa mushure mekudyiswa nehonye. Tichishandisa RDPI, Hj, uye δj indexes kuti tione kugadziridzwa kweruzivo mumafaira eMS/MS configuration kunoratidza mapatani anonakidza (Mufananidzo 2B). Mafambiro ese ndeekuti, sezvakatsanangurwa netsananguro yeruzivo, sezvo honye dzichiramba dzichidya mashizha, mwero wekugadziriswa kwese kwemuviri unowedzera nekufamba kwenguva: maawa makumi manomwe nemaviri mushure mekudya kwehonye, ​​RDPI inowedzera zvakanyanya. Zvichienzaniswa nekudzora kusina kukuvara, Hj yakaderedzwa zvakanyanya, izvo zvakakonzerwa nekuwedzera kwehunyanzvi hwe metabolic profile, iyo yakayerwa ne δj index. Maitiro aya anoenderana nekufanotaura kwedzidziso yeOD, asi haaenderane nekufanotaura kukuru kwedzidziso yeMT, iyo inotenda kuti shanduko dzisina kurongeka (dzisiri dzenzira) mumazinga emetabolite dzinoshandiswa senzira yekudzivirira (Mufananidzo 1B). Kunyangwe huwandu hwese hwesero rinobuda mumuromo (OS) uye maitiro ekudya kwemhuka idzi mbiri dzinodya miriwo hwakasiyana, kudya kwadzo zvakananga kwakaguma nekuchinja kwakafanana munzira dzeHj neδj panguva yekukohwa kwemaawa makumi maviri nemana uye maawa makumi manomwe nemaviri. Musiyano chete wakaitika pamaawa makumi manomwe nemaviri eRDPI. Zvichienzaniswa neizvo zvinokonzerwa nekudya kweMs, metabolism yese inokonzerwa nekudya kweSl yaive yakakwira.
(A) Dhizaini yekuyedza: nguruve yakajairika (S1) kana nyanzvi (Ms) inodya zvinomera zvinodya zvinomera zvinobviswa mumunyu, nepo kune zvinodya zvinomera zvinotevedzera, OS yeMs (W + OSM) inoshandiswa kubata kuboorwa kwenzvimbo dzakajairwa dzemashizha. Makonye eS1 (W + OSSl) kana mvura (W + W). Kudzora (C) ishizha risina kukuvara. (B) Kusagadzikana (RDPI kana tichienzanisa nechati yekudzora), kusiyana-siyana (Hj index) uye specialization (δj index) index yakaverengerwa pamepu chaiyo yemetabolite (599 MS/MS; data file S1). Nyeredzi dzinoratidza mutsauko mukuru pakati pekudyisa zvinomera zvakananga neboka rekudzora (T-test yemudzidzi ine paired t-test, *P<0.05 uye ***P<0.001). ns, hazvina kukosha. (C) Nguva yekugadziriswa kwechinhu chikuru (bhokisi rebhuruu, amino acid, organic acid neshuga; faira redata S2) uye special metabolite spectrum (bhokisi dzvuku 443 MS/MS; faira redata S1) mushure mekuyedzwa kwekurapwa kwemishonga yemiriwo. Ruvara rwebhandi runoreva 95% confidence interval. Asterisk inoratidza musiyano mukuru pakati pekurapwa uye control [quadratic analysis of variance (ANOVA), ichiteverwa neTukey's honestly significant difference (HSD) yekuenzanisa kwakawanda kwe post hoc, *P<0.05, **P<0.01 Uye *** P <0.001]. (D) Kunyatsoongorora scatter plots uye special metabolite profiles (masampuru akadzokororwa ane kurapwa kwakasiyana).
Kuti tiongorore kana kugadziriswa kwezvinomera padanho remetabolome kunoratidzwa nekuchinja kwehuwandu hwemetabolites, takatanga tatarisa pane metabolites dzakambodzidzwa mumashizha eNepenthes pallens dzine resistance yezvinomera. Phenolic amides ihydroxycinnamamide-polyamine conjugates dzinoungana panguva yekushandiswa kwezvipembenene zvinomera uye dzinozivikanwa kuderedza mashandiro ezvipembenene (32). Takatsvaga precursors yeMS/MS inoenderana uye takaronga cumulative kinetic curves dzavo (Mufananidzo S1). Hazvishamisi kuti, phenol derivatives dzisingabatanidzwi zvakananga mukudzivirira zvinomera, zvakaita sechlorogenic acid (CGA) uye rutin, dzinoderedzwa mushure mezvinomera. Kusiyana neizvi, zvinomera zvinogona kuita kuti phenol amides ive nesimba guru. Kudyiswa kuri kuitwa kwezvinomera zviviri izvi kwakaita kuti phenolamides ive ne excitation spectrum yakafanana, uye maitiro aya aive pachena zvakanyanya pakuumbwa kwe de novo kwe phenolamides. Chiitiko chimwe chete ichi chichaonekwa pakutsvaga nzira ye17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), iyo inogadzira huwandu hwakawanda hweacyclic diterpenes ine mabasa anoshanda ekurwisa herbivore (33), ayo Ms Feeding with Sl yakakonzera chimiro chakafanana chekutaura (Mufananidzo S1)).
Dambudziko ringangoitika rekuyedza kudya miriwo zvakananga ndiro musiyano wehuwandu hwekushandiswa kwemashizha nenguva yekudyisa miriwo zvinodya zvinomera, izvo zvinoita kuti zviome kubvisa mhedzisiro chaiyo yemiriwo inokonzerwa nemaronda nemiriwo zvinodya zvinomera. Kuti tigadzirise zviri nani hunyanzvi hwemarudzi emiriwo hunokonzereswa nekushanduka kwemashizha, takatevedzera kudyisa Ms neSl larvae nekushandisa OS itsva (OSM neOSS1) nekukurumidza kupunhwa W kwenzvimbo dzakafanana dzemashizha. Maitiro aya anonzi W + OS treatment, uye anoenzanisa induction nekunyatso tarisa kutanga kwemhinduro inokonzereswa nemiriwo pasina kukonzera mhedzisiro inovhiringidza yemusiyano muhuwandu kana huwandu hwekurasikirwa kwenyama (Mufananidzo 2A) (34). Tichishandisa MS/MS analysis and calculation pipeline, takatora 443 MS/MS spectra (data file S1), iyo yakabatana nespectra yakambounganidzwa kubva kuongororo dzakananga. Kuongororwa kweruzivo rwedata reMS/MS kwakaratidza kuti kurongwazve kwemametabolome akasarudzika mashizha nekutevedzera zvinodya zvinomera kwakaratidza zvinokurudzira zveOS (Mufananidzo 2C). Zvikuru, zvichienzaniswa nekurapwa kweOSS1, OSM yakakonzera kuwedzera kwehunyanzvi hwemetabolome mumaawa mana. Zvakakosha kuziva kuti zvichienzaniswa nedata rekuyedza rekudyisa zvinomera zvakananga, kinetics yemetabolome yakatariswa munzvimbo mbiri-dimensional uchishandisa Hj na δj sema coordinates uye kutungamira kwehunyanzvi hwemetabolome mukupindura kurapwa kwezvinomera kwakaenzaniswa nekufamba kwenguva Kuwedzera kwakasimba (Mufananidzo 2D). Panguva imwe chete, takayera huwandu hweamino acids, organic acids uye shuga (data file S2) kuti tiongorore kana kuwedzera uku kwakanangana muhunyanzvi hwemetabolome kuri nekuda kwekugadzirisazve metabolism yekabhoni pakati mukupindura kune zvinomera zvakaenzaniswa (Mufananidzo S2). Kuti titsanangure zviri nani pateni iyi, takaongorora zvakare kinetics yekuunganidza metabolism yenzira dzakambokurukurwa dzephenolamide uye 17-HGL-DTG. Kupinza kwezvinomera zvinodya herbivores muOS kunoshandurwa kuita patani yekuronga-ronga yakasiyana mukati me phenolamide metabolism (Mufananidzo S3). Phenolic amides ine coumarin uye caffeoyl moieties inonyanya kukonzerwa neOSS1, nepo maOSM achikonzera kupinza kwakananga kwe ferulyl conjugates. Panzira ye17-HGL-DTG, kupinza kwe differential OS ne downstream malonylation uye dimalonylation products kwakaonekwa (Mufananidzo S3).
Tevere, takadzidza nezve plasticity yeOS-induced transcriptome tichishandisa data renguva-course microarray, iro rinotevedzera kushandiswa kweOSMs kurapa mashizha emashizha emuti werosette mumhuka dzinodya zvinomera. Sampling kinetics inonyanya kupindirana nekinetics inoshandiswa muchidzidzo ichi chemetabolomics (35). Zvichienzaniswa ne metabolome reconfiguration umo metabolic plasticity inowedzera zvakanyanya nekufamba kwenguva, tinoona transient transcription bursts mumashizha anokonzerwa naMs, uko transcriptome inducibility (RDPI) uye specialization (δj) zviri pa1 Pakava nekuwedzera kukuru kwemaawa, uye kusiyana-siyana (Hj) panguva ino, kutaura kweBMP1 kwakaderedzwa zvakanyanya, zvichiteverwa nekuzorora kwe transcriptome specialization (Mufananidzo S4). Mhuri dzemajini emetabolic (dzakadai seP450, glycosyltransferase, uye BAHD acyltransferase) dzinotora chikamu mukuita kwekuunganidza metabolites dzakakosha kubva kumayuniti ekuvaka anobva kumetabolism yekutanga, zvichitevera muenzaniso wepamusoro-soro wataurwa pamusoro apa. Sechidzidzo chemuenzaniso, nzira yephenylalanine yakaongororwa. Ongororo iyi yakasimbisa kuti majini makuru mu metabolism ye phenolamide anokonzerwa zvakanyanya neOS-mumhuka dzinodya zvinomera zvichienzaniswa nemiti isina kukwezvwa, uye anowirirana zvakanyanya mumaitiro awo ekutaura. Transcription factor MYB8 uye structural genes PAL1, PAL2, C4H uye 4CL dziri kumusoro kwenzira iyi dzakaratidza kutanga kwe transcription nekukurumidza. Acyltransferases dzinotamba basa mukuungana kwekupedzisira kwe phenolamide, senge AT1, DH29, uye CV86, dzinoratidza pateni yekukwira kwenguva refu (Mufananidzo S4). Zvataurwa pamusoro apa zvinoratidza kuti kutanga kwekutanga kwe transcriptome specialization uye kuwedzerwa kwe metabolomics specialization inzira yakabatana, iyo inogona kunge iri nekuda kwe synchronous regulatory system inotanga simba rekudzivirira.
Kugadziriswazve kwezviratidzo zvehomoni yezvirimwa kunoshanda sechikamu chekutonga chinobatanidza ruzivo rwezvinomera kuti chigadzirise mashandiro emuviri wezvirimwa. Mushure mekutevedzera zvinomera, takayera huwandu hwemahormone ezvirimwa akakosha uye takaona kubatana kwenguva pfupi pakati pawo [Pearson correlation coefficient (PCC)> 0.4] (Mufananidzo 3A). Sezvaitarisirwa, mahormone ezvirimwa ane chekuita ne biosynthesis akabatana mukati me network ye co-expression yehomoni yezvirimwa. Pamusoro pezvo, metabolic specificity (Si index) yakarongwa kune network iyi kuratidza mahormone ezvirimwa anokonzerwa nekurapa kwakasiyana. Nzvimbo mbiri huru dzemhinduro chaiyo yezvinomera dzinotorwa: imwe iri muboka reJA, uko JA (chimiro chayo che biology JA-Ile) nedzimwe JA derivatives dzinoratidza Si score yakakwira; imwe i ethylene (ET). Gibberellin yakaratidza kuwedzera kwakaringana kwe herbivore specificity, nepo mamwe mahormone ezvirimwa, akadai se cytokinin, auxin, uye abscisic acid, aive ne induction specificity yakaderera yezvinomera zvinomera. Zvichienzaniswa nekushandisa W + W chete, kuwedzera kwehuwandu hwepamusoro hweJA derivatives kuburikidza neOS application (W + OS) kunogona kushandurwa kuita chiratidzo chakasimba cheJAs. Zvisingatarisirwi, OSM neOSS1 zvine exicator content dzakasiyana zvinozivikanwa kuti zvinokonzera kuunganidzwa kwakafanana kweJA neJA-Ile. Kusiyana neOSS1, OSM inonyanya kukonzerwa neOSMs, nepo OSS1 isingawedzere mhinduro yemaronda epazasi (Mufananidzo 3B).
(A) Kuongororwa kweCo-expression network zvichibva pakuverenga kwePCC kwekuyerwa kwe herbivore-induced plant hormone accumulation kinetics simulation. Node inomiririra hormone yemuti mumwe chete, uye saizi yenode inomiririra Si index chaiyo ye plant hormone pakati pekurapa. (B) Kuunganidzwa kweJA, JA-Ile neET mumashizha kunokonzerwa nekurapa kwakasiyana kunoratidzwa nemavara akasiyana: apricot, W + OSM; bhuruu, W + OSSl; dema, W + W; grey, C (control). Nyeredzi dzinoratidza mutsauko mukuru pakati pekurapa nekudzora (two-way ANOVA inoteverwa neTukey HSD post hoc multiple comparison, *** P <0.001). Kuongororwa kweruzivo rwe (C)697 MS/MS (data file S1) muJA biosynthesis uye impairment perception spectrum (irAOC ne irCOI1) uye (D)585 MS/MS (data file S1) muETR1 ine impairment ET signal. Kurapa kwe herbivore kwakatevedzera kwakakonzera mitsara yemiti uye empty vehicle control plants (EV). Nyeredzi dzinoratidza mutsauko mukuru pakati pekurapa kweW+OS nekutonga kusina kukuvara (ANOVA yenzira mbiri ichiteverwa neTukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 uye ***P<0.001). (E) Magirafu akapararira ekurwisana kwakapararira nehunyanzvi. Mavara anomiririra mhando dzakasiyana dzakagadziriswa majini; zviratidzo zvinomiririra nzira dzakasiyana dzekurapa: triangle, W + OSS1; rectangle, W + OSM; denderedzwa C
Tevere, tinoshandisa rudzi rweNepenthes dzakagadziriswa majini (irCOI1 uye sETR1) mumatanho akakosha eJA neET biosynthesis (irAOC uye irACO) uye perception (irCOI1 uye sETR1) kuongorora metabolism yemahormone maviri aya ezvirimwa pazvinodya zvinomera. Zvichienderana nekuyedza kwakapfuura, takasimbisa kupinza kweherbivore-OS muzvirimwa zvisina chinhu (EV) (Mufananidzo 3, C kusvika D) uye kudzikira kwese kweHj index kunokonzerwa neOSM, nepo δj index yakawedzera. Mhinduro yacho inonyanya kuoneka kupfuura mhinduro inokonzerwa neOSS1. Girafu yemitsara miviri inoshandisa Hj naδj semacoordinates inoratidza kudzikiswa kwemutemo chaiwo (Mufananidzo 3E). Chinonyanya kuoneka ndechekuti mumhando dzisina chiratidzo cheJA, kusiyana kwemetabolome uye shanduko dzehunyanzvi dzinokonzerwa nezvinodya zvinomera zvinenge zvabviswa zvachose (Mufananidzo 3C). Kusiyana neizvi, maonero eET akanyarara mumiti yeSETR1, kunyange hazvo mhedzisiro yese pakuchinja kwemetabolism yezvinomera iri pasi zvakanyanya pane yeJA signaling, inoderedza mutsauko muHj neδj indices pakati peOSM neOSS1 excitations (Mufananidzo 3D neMufananidzo S5). . Izvi zvinoratidza kuti pamusoro pebasa guru reJA signal transduction, ET signal transduction inoshandawo se fine-tuning yemhinduro yemetabolism yemarudzi chaiwo ezvinomera zvinodya zvinomera. Zvichienderana nebasa iri re fine-tuning, pakanga pasina shanduko mu metabolome inducibility yese mumiti yeSETR1. Kune rumwe rutivi, zvichienzaniswa nemiti yeSETR1, miti ye irACO yakakonzera amplitudes yakafanana yekuchinja kwemetabolism kunokonzerwa nezvinomera zvinodya zvinomera, asi yakaratidza ma Hj neδj akasiyana zvakanyanya pakati peOSM neOSS1 challenges (Mufananidzo S5).
Kuti tizive metabolites dzakasarudzika dzine mipiro yakakosha mukupindura kwemhuka dzinodya zvinomera uye kugadzirisa kugadzirwa kwadzo kuburikidza nezviratidzo zveET, takashandisa nzira yakagadzirwa kare yeMS/MS. Nzira iyi inoshandisa nzira yekubatanidza zvinhu zviviri kuti tiongororezve mhuri yemetabolic kubva kuMS/MS fragments [normalized dot product (NDP)] uye similar score zvichibva pakurasikirwa kwe neutral (NL). Data reMS/MS rakagadzirwa kuburikidza nekuongorora ET transgenic lines rakagadzira 585 MS/MS (data file S1), iyo yakagadziriswa nekuvabatanidza muzvikamu zvinomwe zvikuru zveMS/MS modules (M) (Mufananidzo 4A). Mamwe emamodule aya akazara ne metabolites dzakasarudzika dzakamboonekwa: semuenzaniso, M1, M2, M3, M4 neM7 dzakapfuma mune akasiyana phenol derivatives (M1), flavonoid glycosides (M2), acyl shuga (M3 Uye M4), uye 17-HGL-DTG (M7). Pamusoro pezvo, ruzivo rwe metabolic specific information (Si index) yemetabolite imwe chete mumodule yega yega inoverengwa, uye kugoverwa kwayo kweSi kunogona kuonekwa nenzira isinganzwisisike. Muchidimbu, MS/MS spectra inoratidza herbivory yakakwira uye genotype specificity inoratidzwa ne high Si values, uye kurtosis statistics inoratidza kugoverwa kwemvere pakona yekurudyi yemuswe. Imwe lean colloid distribution yakadaro yakaonekwa muM1, umo phenol amide yakaratidza highest Si fraction (Mufananidzo 4B). Herbivorous inducible 17-HGL-DTG yambotaurwa muM7 yakaratidza moderate Si score, ichiratidza mwero wepakati wekusiyana kwemaitiro pakati pemhando mbiri dzeOS. Kusiyana neizvi, metabolites dzakawanda dzakagadzirwa, dzakadai serutin, CGA, uye acyl sugar, dziri pakati peSi scores dzakaderera. Kuti tiongorore zviri nani kuomarara kwechimiro uye kugoverwa kweSi pakati pemametabolites akakosha, molecular network yakagadzirwa kune yega yega module (Mufananidzo 4B). Kufanotaura kwakakosha kwedzidziso yeOD (yakapfupikiswa muMufananidzo 1B) ndekwekuti kurongekazve kwemametabolites akakosha mushure mekudyara kwemiti kunofanira kutungamira kushanduko dzenzira imwe chete mumametabolites ane dziviriro yakakwira, kunyanya nekuwedzera hunhu hwawo (kusiyana nekugoverwa kwakasarudzika) Mode) Defensive metabolite yakafanotaurwa nedzidziso yeMT. Mazhinji e phenol derivatives akaunganidzwa muM1 ane chekuita nekushanda kwekuderera kwekushanda kwezvipembenene (32). Pakuenzanisa mavalues ​​eSi mumetabolites eM1 pakati pemashizha akakonzerwa nemashizha ari mumiti yeEV control mumaawa makumi maviri nemana, takaona kuti metabolic specificity yemametabolites akawanda mushure mezvipembenene zvemiti ine trend yakakura inowedzera (Mufananidzo 4C). Kuwedzera kwakananga muSi value kwakaonekwa chete mumaphenolamides ekudzivirira, asi hapana kuwedzera muSi value kwakaonekwa mune mamwe maphenol uye metabolites asingazivikanwe aripo mumodule iyi. Uyu muenzaniso wakasarudzika, une chekuita nedzidziso yeOD. Kufanotaura kukuru kwekuchinja kwemetabolic kunokonzerwa nezvinodya zvinodya zvinomera kunoenderana. Kuti tiongorore kana hunhu uhwu hwe phenolamide spectrum hwakakonzereswa ne OS-specific ET, takaronga metabolite Si index ndokukonzera differential expression value pakati peOSM neOSS1 mu EV ne sETR1 genotypes (Mufananidzo 4D). Mu sETR1, musiyano wakakonzerwa ne phenamide pakati peOSM neOSS1 wakaderedzwa zvakanyanya. Nzira ye bi-clustering yakashandiswawo kune data re MS/MS rakaunganidzwa mumhando dzisina JA yakakwana yekufungidzira ma main MS/MS modules ane chekuita ne JA-regulated metabolic specialization (Mufananidzo S6).
(A) Mhedzisiro ye585 MS/MS zvichibva pachikamu chakagovaniswa (NDP similarity) uye shared neutral loss (NL similarity) zvinoita kuti module (M) ienderane nemhuri inozivikanwa yecompound, kana nekuumbwa kweMetabolite kusingazivikanwe kana kusanyatsogadzirwa zvakanaka. Pedyo nemodule yega yega, kugoverwa kwemetabolite (MS/MS) specific (Si) kunoratidzwa. (B) Modular molecular network: MaNodes anomiririra MS/MS nemipendero, NDP (tsvuku) uye NL (bhuruu) MS/MS scores (cut-off,> 0.6). Classified metabolite specificity index (Si) yakapendwa zvichibva pamodule (kuruboshwe) uye yakarongwa kune molecular network (kurudyi). (C) Module M1 yeEV plant iri mu constitutive (control) uye induced state (simulated herbivore) mumaawa makumi maviri nemana: molecular network diagram (Si value ndiyo node size, defensive phenolamide inoonekwa mubhuruu). (D) Dhayagiramu yeM1 molecular network yemutsetse we spectrum sETR1 ine kusanzwisisika kweEV ne ET: phenolic compound inomiririrwa ne green circle node, uye musiyano mukuru (P value) pakati pekurapa kweW + OSM ne W + OSS1 seNode size. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, N-ferulic acid ester-uric acid; FS, N-ferulyl-spermidine; CoP, N', N “-Coumarolyl-tyrosine; DCS, N', N”-dicaffeoyl-spermidine; CFS, N', N”-caffeoyl, feruloyl-spermidine; Lycium barbarum mu wolfberry Son; Nick. O-AS, O-acyl shuga.
Takawedzera ongororo kubva pamhando imwe chete yeNepenthes yakaderedzwa kusvika pahuwandu hwezvisikwa, uko shanduko dzakasimba dzemukati memazinga eJA anodya zvinomera uye huwandu hwemetabolite hwakatsanangurwa kare muhuwandu hwezvisikwa (26). Shandisa iyi data set kufukidza 43 germplasms. Aya germplasms ane marudzi zana nemakumi maviri nematatu emiti kubva kuN. pallens. Miti iyi yakatorwa kubva kumbeu dzakaunganidzwa munzvimbo dzakasiyana dzemuno muUtah, Nevada, Arizona, neCalifornia (Mufananidzo S7), takaverenga metabolome diversity (pano inonzi population level) β diversity) uye specialization inokonzerwa neOSM. Zvichienderana nezvidzidzo zvekare, takaona shanduko dzakasiyana-siyana dzemetabolism pamwe chete neHj neδj axes, zvichiratidza kuti germplasms dzine mutsauko mukuru mukupfava kwemhinduro dzadzo dzemetabolism kumhuka dzinodya zvinomera (Mufananidzo S7). Iyi sangano inoyeuchidza zvakamboonekwa nezve dynamic range yeJAs diversity inokonzerwa nemhuka dzinodya zvinomera, uye yakaramba ichikosha zvikuru muhuwandu humwe chete (26, 36). Nekushandisa JA neJA-Ile kuyedza hukama hwese pakati peHj neδj, takaona kuti pane hukama hwakakosha pakati peJA ne metabolome β diversity and specialization index (Mufananidzo S7). Izvi zvinoratidza kuti kusiyana kwezvinomera muJAs kwakaonekwa pahuwandu hwevanhu kunogona kunge kuri nekuda kwekuchinja kukuru kwemetabolism kunokonzerwa nekusarudzwa kubva kuzvipembenene zvinodya zvinomera.
Ongororo dzakapfuura dzakaratidza kuti mhando dzefodya dzakasiyana zvikuru mumhando uye zvichienderana nekudzivirira kunokonzerwa uye kunokonzerwa nekuchinja kwesimba remuviri. Zvinotendwa kuti shanduko idzi mukuchinja kwechiratidzo chekurwisa herbivore uye kugona kwekudzivirira zvinodzorwa nekumanikidzwa kwehuwandu hwezvipembenene, hupenyu hwezvirimwa, uye mari yekugadzira dziviriro munzvimbo inokura mhando yakati. Takadzidza kuenderana kwekuchinja kwemashizha metabolome kunokonzerwa nezvinodya miriwo yemarudzi matanhatu eNicotiana anobva kuNorth America neSouth America. Mhando idzi dzakabatana zvakanyanya neNepenthes North America, dzinoti Nicolas Bociflo. La, N. nicotinis, Nicotiana n. attenuated grass, Nicotiana tabacum, linear tobacco, tobacco (Nicotiana spegazzinii) uye tobacco leaf tobacco (Nicotiana obtusifolia) (Mufananidzo 5A) (37). Mhando nhanhatu dzemhando idzi, kusanganisira mhando inozivikanwa zvakanaka N. ndapota, zvirimwa zvegore negore zvepetunia clade, uye obtusifolia N. zvirimwa zvenguva refu zvesister clade Trigonophyllae (38). Zvadaro, W + W, W + OSM uye W + OSS1 zvakaiswa pamhando idzi nomwe kuti dzidzidze kurongwa kwemhando dzemhuka pakudya zvipembenene.
(A) Muti we phylogenetic we bootstrap wakavakirwa pahuwandu hukuru [hwekugadzira nuclear glutamine (38)] uye kugoverwa kwenzvimbo kwemarudzi manomwe eNicotiana ane hukama hwakasimba (mavara akasiyana) (37). (B) Plot yekupararira kwemhando dzakasiyana dze metabolic profiles dzemhando nomwe dzeNicotiana (939 MS/MS; data file S1). Padanho remhando, metabolome diversity haina hukama hwakakwana nedanho re specialization. Kuongororwa kwehukama hwemhando pakati pekusiyana kwe metabolic uye specialization uye JA accumulation kunoratidzwa muMufananidzo 2. S9. Ruvara, mhando dzakasiyana; triangle, W + OSS1; rectangle, W + OSM; (C) Nicotiana JA uye JA-Ile dynamics dzakarongwa zvichienderana neOS excitation amplitude (two-way ANOVA uye Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 uye * ** Pakuenzanisa W + OS na W + W, P <0.001). Chinyorwa chebhokisi che (D) kusiyana-siyana uye (E) kusarudzwa kwemhando yega yega mushure mekutevedzera herbivorous uye methyl JA (MeJA). Chiedza chinoratidza musiyano mukuru pakati peW + OS neW + W kana lanolin pamwe neW (Lan + W) kana Lan pamwe neMeJA (Lan + MeJa) uye Lan control (kuongorora kusiyana kwemarudzi maviri, zvichiteverwa neTukey's HSD post hoc multiple comparison, *P<0.05, **P<0.01 uye ***P<0.001).
Tichishandisa nzira ye dual cluster, takaona ma module mapfumbamwe e939 MS/MS (data file S1). Kuumbwa kweMS/MS kwakagadziriswazve nekurapa kwakasiyana kunosiyana zvikuru pakati pema module akasiyana pakati pemarudzi (Mufananidzo S8). Kuona Hj (inonzi pano se species-level γ-diversity) uye δj kunoratidza kuti marudzi akasiyana anoungana mumapoka akasiyana zvikuru munzvimbo ye metabolic, uko kupatsanurwa kwemarudzi kunowanzoonekwa kupfuura excitation. Kunze kweN. linear uye N. obliquus, anoratidza dynamic range ye induction effects (Mufananidzo 5B). Kusiyana neizvi, marudzi akadai seN. purpurea naN. obtusifolia ane metabolic response isingaonekwe zvakanyanya pakurapa, asi metabolome yakasiyana-siyana. Kugoverwa kwemarudzi chaiwo kwe induced metabolic response kwakakonzera hukama hwakaipa pakati pe specialization uye gamma diversity (PCC = -0.46, P = 4.9×10-8). Shanduko dzinokonzereswa neOS mumazinga eJA dzakabatana zvakanaka ne metabolome specialization, uye dzakabatana zvisina kunaka nekusiyana kwe metabolic gamma kunoratidzwa nerudzi rumwe nerumwe (Mufananidzo 5B neMufananidzo S9). Zvakakosha kuziva kuti mhando dzinonzi "signal response" muMufananidzo 5C, dzakadai seNepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, uye Nepenthes attenuated, dzakakonzera zviratidzo zvakakosha pamaminitsi makumi matatu. Kupararira kweOS-specific JA neJA-Ile munguva pfupi yapfuura, nepo mamwe mabhakitiriya anonzi "signal responsive", akadai seNepenthes mills, Nepenthes powdery neN. obtusifolia anongoratidza JA-Ile Edge induction pasina chero OS specificity (Mufananidzo 5C). Padanho re metabolic, sezvataurwa pamusoro apa, kune Nepenthes attenuated, zvinhu zvinopindura zviratidzo zvakaratidza OS specificity uye zvakawedzera zvakanyanya δj, uku zvichideredza Hj. Iyi OS-specific priming effect haina kuonekwa mumhando dzakarongwa se signal non-reactive species (Mufananidzo 5, D naE). Mametabolites chaiwo eOS anogovaniswa kakawanda pakati pemarudzi anopindura zviratidzo, uye mapoka aya ezviratidzo anoungana nemarudzi ane mhinduro dzisina simba dzezviratidzo, nepo marudzi ane mhinduro dzisina simba dzezviratidzo achiratidza kusawirirana kushoma (Mufananidzo S8). Mhedzisiro iyi inoratidza kuti kupinza kweJAs zvakananga neOS uye kugadziriswazve kweOS zvakananga kwemetabolome iri pasi pemvura zvakabatana padanho remhando.
Tevere, takashandisa lanolin paste ine methyl JA (MeJA) kurapa zvirimwa kuti tiongorore kana nzira idzi dzekubatanidza dzichidziviswa nekuwanikwa kweJA inoshandiswa neJA yekunze, iyo ichave iri mu cytoplasm yezvirimwa. Kukurumidza kuomeswa kwemvura iJA. Takawana maitiro akafanana ekuchinja zvishoma nezvishoma kubva kumarudzi anopindura zviratidzo kuenda kumarudzi asingapinduri zviratidzo anokonzerwa nekuwanikwa kweJA nguva dzose (Mufananidzo 5, D naE). Muchidimbu, kurapwa kweMeJA kwakagadzirisa zvakanyanya metabolomes ye linear nematodes, N. obliquus, N. aquaticus, N. pallens, uye N. mikimotoi, zvichikonzera kuwedzera kukuru kwe δj uye kudzikira kweHj. N. purpurea kwakaratidza kuwedzera kwe δj, asi kwete Hj. N. obtusifolia, iyo yakamboratidzwa kuti inounganidza huwandu hwakaderera hweJAs, inopindurawo zvisina kunaka kurapwa kweMeJA maererano ne metabolome reconfiguration. Mhedzisiro iyi inoratidza kuti kugadzirwa kweJA kana transduction yezviratidzo kwakadziviswa mumuviri mumarudzi asingapinduri zviratidzo. Kuti tiedze fungidziro iyi, takadzidza mhando ina (N. pallens, N. mills, N. pink uye N. microphylla) dzakakonzereswa neW + W, W + OSMs uye W + OSS1 Transcriptome (39). Zvichienderana nemaitiro ekugadzirisa metabolome, mhando idzi dzakaparadzaniswa zvakanaka munzvimbo yetranscriptome, pakati padzo N. attenuated yakaratidza RDPI yepamusoro-soro inokonzerwa neOS, nepo N. gracilis yaive yakaderera (Mufananidzo 6A). Zvisinei, zvakaonekwa kuti kusiyana kwetranscriptome kunokonzerwa neN. oblonga kwaive kwakaderera pakati pemhando ina, zvichipesana nekusiyana kwakanyanya kwemetabonomic kweN. oblonga kwakamboratidzwa mumhando nomwe. Zvidzidzo zvekare zvakaratidza kuti seti yemajini ane chekuita nezviratidzo zvekudzivirira kwekutanga, kusanganisira zviratidzo zveJA, inotsanangura kujeka kwemhinduro dzekudzivirira kwekutanga dzinokonzereswa nezvinodya herbivore mumhando dzeNicotiana (39). Kuenzanisa nzira dzeJA signaling pakati pemhando idzi ina kwakaratidza patani inonakidza (Mufananidzo 6B). Majini mazhinji ari munzira iyi, akadai seAOC, OPR3, ACX neCOI1, akaratidza huwandu hwakanyanya hwekupinza mumarudzi mana aya. Zvisinei, jini guru, JAR4, rinoshandura JA kuita fomu rayo reJA-Ile rakaunganidzwa, uye huwandu hwaro hwekupinza hwakaderera zvikuru, kunyanya muN. mills, Nepenthes pieris neN. microphylla. Pamusoro pezvo, chete gwaro reimwe jini AOS harina kuwanikwa muN. bifidum. Shanduko idzi mukutaura kwemajini dzinogona kunge dziri idzo dzinokonzera ma phenotypes akanyanya anokonzerwa nekuburitswa kweJA kwakaderera mumarudzi ane zviratidzo uye kuburitswa kweN. gracilis.
(A) Kuongororwa kwedzidziso yeruzivo rwekugadziriswazve kwemhinduro dzekutanga dzefodya ina dzine hukama hwakafanana dzakatorwa maminetsi makumi matatu mushure mekuiswa kwemishonga yekudya. RDPI inoverengerwa nekuenzanisa mashizha anokonzerwa nesystem yekudya ...
Muchikamu chekupedzisira, takadzidza kuti zvipembenene zvinogona sei kugadzirisa metabolome yemhando dzakasiyana dzemiti kuti dzisapararire nezvinodya zvinomera. Ongororo yapfuura yakasimbisa rudzi rweNicotiana. Kuramba kwavo kuna Ms nehonye kunosiyana zvikuru (40). Pano, takadzidza hukama huripo pakati pemuenzaniso uyu nekushanduka kwavo kwemetabolism. Tichishandisa mhando ina dzefodya dziri pamusoro apa, uye kuyedza hukama huripo pakati pekusiyana uye kusarudzwa kwe metabolome kunokonzerwa nezvinodya zvinomera uye kuramba kwemiti kuna Ms na Sl, takaona kuti kuramba, kusiyana uye kusarudzwa kune generalist Sl All kwakabatana zvakanaka, nepo hukama huripo pakati pekuramba kune nyanzvi dzezvemiti uye kusarudzwa kwacho husina kusimba, uye hukama nekusiyana hakuna kukosha (Mufananidzo S10). Nezvekuramba kweS1, N. chinensis neN. gracilis dzakaderera, idzo dzakamboratidzwa kuratidza JA signal transduction levels uye metabolome plasticity, dzaive nemhinduro dzakasiyana kwazvo kune herbivore induction, uye dzakaratidzawo kuramba kwakafanana. Bonde.
Mumakore makumi matanhatu apfuura, dzidziso yekudzivirira zvirimwa yakapa hurongwa hwedzidziso, hwakavakirwa pahunotsvagurudza hwakafanotaura huwandu hwakakura hwekushanduka-shanduka uye mabasa emetabolites dzakagadzirwa nemiti. Dzizhinji dzedzidziso idzi hadziteveri maitiro akajairwa ekufungidzira kwakasimba (41). Dzinopa fungidziro huru (3) padanho rimwe chete rekuongorora. Kana kuyedzwa kwefungidziro huru kuchibvumira dzidziso dzakananga kuongororwa, izvi zvichaita kuti munda ufambire mberi. Tsigiro, asi ramba dzimwe (42). Pane kudaro, dzidziso itsva inoita fungidziro pamatanho akasiyana ekuongorora uye inowedzera nhanho itsva yekufunga kunotsanangura (42). Zvisinei, dzidziso mbiri dzakataurwa padanho rekushanda, MT neOD, dzinogona kutsanangurwa zviri nyore sefungidziro dzakakosha dzekuchinja kwakasarudzika kwemetabolites kunokonzerwa nezvinodya zvinodya miriwo: dzidziso yeOD inotenda kuti shanduko mu "nzvimbo" chaiyo yemetabolites dzine divi rakananga. Dzidziso yeMT inotenda kuti shanduko idzi hadzizove dziri munzira uye dziri munzvimbo yemetabolites, uye dzinowanzova nemetabolites dzine dziviriro yakakwira. Ongororo dzekare dzefungidziro dzeOD neMT dzakaedzwa uchishandisa seti diki yemakomputa e "dziviriro" ekutanga. Miedzo iyi yemetabolite-centric inodzivisa kugona kuongorora huwandu uye nzira yekuchinja kwemetabolome panguva yekudyara kwezvirimwa, uye haibvumiri kuyedza mukati mehurongwa hwehuwandu hwezviverengero kuti kuda kufanotaura kwakakosha kunogona kutariswa sese. Kuyera shanduko mumetabolome yezvirimwa. Pano, takashandisa tekinoroji itsva mumetabolomics yakavakirwa pa computational MS uye takaita deconvolution MS analysis mumari yakajairika yezvitsanangudzo zvedzidziso yeruzivo kuti tiedze mutsauko pakati pezviviri zvakatsanangurwa padanho repasi rose remetabolomics. Kufanotaura kukuru kwedzidziso iyi. Dzidziso yeruzivo yakashandiswa munzvimbo dzakawanda, kunyanya mumamiriro ezvinhu ebiologies uye nutrient flow research (43). Zvisinei, sekuziva kwedu, iyi ndiyo yekutanga application inoshandiswa kutsanangura nzvimbo yeruzivo rwemetabolite yezvirimwa uye kugadzirisa matambudziko ezvakatipoteredza ane chekuita nekuchinja kwenguva pfupi kwemetabolite mukupindura kune zviratidzo zvezvakatipoteredza. Kunyanya, kugona kwenzira iyi kuri mukukwanisa kwayo kuenzanisa mapatani mukati uye pakati pemarudzi ezvirimwa kuti iongorore kuti mhuka dzinodya mhuka dzakashanduka sei kubva kumarudzi akasiyana kuenda kune macroevolutionary patterns akasiyana pamatanho akasiyana ekushanduka. Metabolism.
Kuongororwa kwezvikamu zvikuru (PCA) kunoshandura data rakawanda kuita nzvimbo yekuderedza miganhu kuitira kuti mafambiro makuru edata atsanangurwe, saka rinowanzoshandiswa senzira yekuongorora kuti riongorore data, senge deconvolution metabolome. Zvisinei, kuderedza miganhu kucharasikirwa nechikamu cheruzivo rwuri mudata, uye PCA haigone kupa ruzivo rwehuwandu nezvehunhu hwakakosha kune dzidziso yezvakatipoteredza, senge: ma herbivores anogadzirisa sei kusiyana-siyana muminda yakasarudzika (semuenzaniso, kupfuma, kugoverwa) Uye kuwanda) metabolites? Ndeapi metabolites ari kufanotaura mamiriro akakonzerwa ne herbivore yakapihwa? Kubva pamaonero ehunhu, kusiyana-siyana uye kusanzwisisika, ruzivo rwe profile ye metabolite yemashizha inoora, uye zvinowanikwa kuti kudya herbivores kunogona kumutsa metabolism chaiyo. Tisingatarisiri, takaona kuti, sezvakatsanangurwa muzviratidzo zveruzivo zvakashandiswa, mamiriro ekugadzirisa muviri anokonzerwa nekudya kwemhuka mbiri dzinodya mhuka (Sl inodyiswa husiku) uye nyanzvi yeSolanaceae Ms. Kunyangwe maitiro avo ekudya uye huwandu hwavo hwakasiyana zvakanyanya. Fatty acid-amino acid conjugate (FAC) initiator muOS (31). Nekushandisa herbivore OS kurapa maronda akajairwa ekuboorwa, kurapwa kweherbivore kwakatevedzera kwakaratidzawo maitiro akafanana. Maitiro aya akajairwa ekutevedzera mhinduro yemiti kukurwisa herbivore anobvisa zvinhu zvinovhiringidza zvinokonzerwa nekuchinja kwemaitiro ekudya kweherbivore, izvo zvinotungamira kumazinga akasiyana ekukuvara panguva dzakasiyana (34). FAC, inozivikanwa sechikonzero chikuru cheOSM, inoderedza JAS nedzimwe mhinduro dzemahormone ezvirimwa muOSS1, nepo OSS1 ichideredza mazana emazana (31). Zvisinei, OSS1 yakakonzera huwandu hweJA hwakafanana zvichienzaniswa neOSM. Zvakamboonekwa kare kuti mhinduro yeJA muNepenthes dzakadzikira inonzwa zvakanyanya kuOSM, uko FAC inogona kuramba ichiita basa rayo kunyangwe ikaderedzwa 1:1000 nemvura (44). Saka, kana ichienzaniswa neOSM, kunyangwe FAC muOSS1 iri shoma kwazvo, yakakwana kukonzera kubuda kweJA kwakakwana. Zvidzidzo zvekare zvakaratidza kuti mapuroteni akafanana neporin (45) uye oligosaccharides (46) anogona kushandiswa semamorekuru ekukurudzira mhinduro dzekudzivirira zvirimwa muOSS1. Zvisinei, hazvisati zvajeka kana izvi zvinokurudzira muOSS1 zviri izvo zvinokonzera kuunganidzwa kweJA kwakaonekwa muchidzidzo chazvino.
Kunyangwe paine zvidzidzo zvishoma zvinotsanangura kusiyana kweminwe yemuviri inokonzerwa nekushandiswa kwezvinomera zvakasiyana-siyana kana exogenous JA kana SA (salicylic acid) (47), hapana akambokanganisa kukanganiswa kwemarudzi ezvinomera mu network yehuswa hwezvirimwa uye mhedzisiro yazvo pane chaiyo. Mhedzisiro yese yemetabolism inodzidzwa zvakarongeka. Ongororo iyi yakasimbisa zvakare kuti kubatana kwemukati mehomoni nemamwe mahormone ezvirimwa kunze kweJA kunoumba hunhu hwekugadziriswazve kwemetabolism kunokonzerwa nezvinomera zvinodya mhuka. Kunyanya, takaona kuti ET inokonzerwa neOSM yaive yakakura zvakanyanya kupfuura iyo inokonzerwa neOSS1. Iyi nzira inoenderana nehuwandu hwakawanda hweFAC muOSM, inova mamiriro anodiwa uye akakwana ekukonzera kuputika kweET (48). Mumamiriro ekudyidzana pakati pezvirimwa nezvinodya mhuka, basa rekutumira chiratidzo reET pametabolite dynamics yezvirimwa richiri kungoitika uye rinongonanga boka rimwe chete remakemikari. Pamusoro pezvo, zvidzidzo zvakawanda zvakashandisa kushandiswa kweET kana zvinhu zvinoitangira kana zvakasiyana-siyana zvinodzivisa ET kuti zvidzidze kudzorwa kweET, pakati pazvo kushandiswa kwemakemikari aya ekunze kuchaburitsa migumisiro yakawanda isiri chaiyo. Sekuziva kwedu, chidzidzo ichi chinomiririra kuongororwa kukuru kwekutanga kwebasa reET mukushandiswa kweET kugadzira nekuona zvirimwa zvakakanganiswa zve transgenic kuti zvienderane ne metabolome dynamics yezvirimwa. Herbivore-specific ET induction inogona pakupedzisira kugadzirisa mhinduro ye metabolome. Chinonyanya kukosha ndiko kushandiswa kwe transgenic kwemajini eET biosynthesis (ACO) uye perception (ETR1) ayo akaratidza herbivore-specific de novo accumulation ye phenolamides. Zvakamboratidzwa kuti ET inogona kugadzirisa JA-induced nicotine accumulation nekudzora putrescine N-methyltransferase (49). Zvisinei, kubva pakuona kwe mechanical, hazvisi pachena kuti ET inogadzirisa sei induction ye phenamide. Pamusoro pebasa reET rekutumira masaini, kushanduka kwemetabolism kunogona zvakare kuchinjirwa kuS-adenosyl-1-methionine kuti itonge mari inowanikwa mu polyaminophenol amides. S-adenosyl-1-methionine ndiyo ET uye ndiyo nzira yepakati pe polyamine biosynthetic. Nzira iyo ET signal inotonga huwandu hwe phenolamide inoda kuongororwa kwakawanda.
Kwenguva yakareba, nekuda kwehuwandu hwakawanda hwemametabolites chaiwo asina chimiro chisingazivikanwi, kutarisisa kwakanyanya kune mamwe mapoka emetabolites kwave kutadza kunyatsoongorora shanduko dzenguva dzekusiyana kwemetabolites mushure mekudyidzana kwezvipenyu. Parizvino, zvichibva pakuongorora dzidziso yeruzivo, mhedzisiro huru yekuwana MS/MS spectrum zvichibva pamametabolites asina rusaruro ndeyekuti zvinodya kana kutevedzera zvinodya zvinodya zvinoramba zvichideredza kusiyana kwemetabolites kwemashizha metabolome uku zvichiwedzera mwero wayo wehunyanzvi. Kuwedzera kwechinguvana kwemetabolome specificity kunokonzerwa nezvinodya zvinodya zvinosanganiswa nekuwedzera kwekubatana mu transcriptome specificity. Chinhu chinonyanya kubatsira kune iyi metabolome specialization yakakura (ine kukosha kwakakwirira kweSi) imetabolite yakakosha ine basa rekare reherbivorous. Iyi modhi inoenderana nekufanotaura kwedzidziso yeOD, asi kufanotaura kweMT kune chekuita nekusarongeka kwemetabolome reprogramming hakuenderane. Zvisinei, iyi data inoenderanawo nekufanotaura kwemusanganiswa wemodhi (yakanakisisa MT; Mufananidzo 1B), nekuti mamwe mametabolites asina hunhu ane mabasa ekudzivirira asingazivikanwe anogona kuramba achitevera kugoverwa kweSi random.
Nzira inokosha yakawanikwazve nekutsvaga uku ndeyekuti kubva padanho rekushanduka-shanduka kwemakemikari (chirimwa chimwe chete nehuwandu hwefodya) kusvika padanho guru rekushanduka-shanduka (mhando dzefodya dzakabatana), mazinga akasiyana ekurongeka kwekushanduka-shanduka ari mu "dziviriro yakanakisisa". Kune misiyano yakakura mukugona kwemhuka dzinodya miriwo. Moore et al. (20) naKessler naKalske (1) vakazvitsaurira vega kuti vashandure mazinga matatu ekushanda kwemarudzi akasiyana-siyana ezvakasiyana-siyana pakutanga aisiyaniswa naWhittaker (50) kuita shanduko dzenguva dzakagadzirwa uye dzakakonzereswa dzekusiyana-siyana kwemakemikari; vanyori ava havana kupfupisa. Maitiro ekuunganidza data re metabolome rakakura haatsananguriwo maitiro ekuverenga kusiyana-siyana kwemetabolism kubva padata iri. Muchidzidzo ichi, kugadziriswa kudiki kuchikamu chebasa chaWhittaker kuchafunga nezvekusiyana-siyana kweα-metabolic sekusiyana kweMS/MS spectra mumuti wakapihwa, uye kusiyana-siyana kweβ-metabolic senzira yekutanga yekuchinja-shanduka kweboka revanhu Space, uye kusiyana-siyana kweγ-metabolic kuchava kuwedzera kwekuongorora kwemarudzi akafanana.
Chiratidzo cheJA chakakosha pamhinduro dzakasiyana-siyana dzemetabolism yezvinomera. Zvisinei, hapana kuongororwa kwakasimba kwehuwandu hwemupiro we intraspecific regulation yeJA biosynthesis ku metabolome diversity, uye kana JA signal iri nzvimbo yakajairika yekusiyana-siyana kwemetabolism kunokonzerwa nekushushikana pahuwandu hwemacroevolutionary scale ichiri kuomera. Takaona kuti hunhu hweNepenthes herbivorous hwezvinomera hunokonzera metabolome specialization uye kusiyana kwe metabolome specialization mukati mehuwandu hweNicotiana species uye pakati pemhando dzeNicotiana dzine hukama hwapedyo kwakabatana zvakanaka neJA signaling. Pamusoro pezvo, kana JA signal ikakanganiswa, metabolic specificity inokonzerwa negenotype imwe chete herbivore ichamiswa (Mufananidzo 3, C na E). Sezvo shanduko dzemetabolism dzeNepenthes dzakaderera zvakasikwa dziri quantitative, shanduko mu metabolic β diversity uye specificity muongororo iyi inogona kukonzerwa zvakanyanya nekukatyamadzwa kwakasimba kwemetabolite-rich compound categories. Aya ma compound classes anotonga chikamu che metabolome profile uye anotungamira kune hukama hwakanaka neJA signals.
Nekuti mashandiro ebiochemical emhando yefodya ane hukama hwakasimba nayo akasiyana zvikuru, metabolites anonyatsoonekwa muchikamu chequalitative, saka inonyanya kuongorora. Kugadziriswa kweruzivo rwe metabolic profile yeakatorwa kunoratidza kuti herbivorous induction kunowedzera kutengeserana pakati pekusiyana kwe metabolic gamma uye specialization. Chiratidzo cheJA chinoita basa guru mukutengeserana uku. Kuwedzera kwe metabolome specialization kunoenderana nekufanotaura kukuru kweOD uye kwakabatana zvakanaka nechiratidzo cheJA, nepo chiratidzo cheJA chichiwirirana zvisina kunaka nekusiyana kwe metabolic gamma. Mamodheru aya anoratidza kuti kugona kweOD kwezvirimwa kunonyanya kutsanangurwa ne plasticity yeJA, kungave pa microevolutionary scale kana pakukura kwekushanduka-shanduka. Kuedza kwe exogenous JA application kunodzivisa JA biosynthesis defects kunoratidza zvakare kuti mhando dzefodya dzine hukama hwapedyo dzinogona kusiyaniswa mumhando dzinopindura zviratidzo uye dzisingapinduri zviratidzo, kungofanana nemaitiro avo eJA uye metabolome plasticity inokonzerwa ne herbivores. Mhando dzisingapinduri zviratidzo hadzigone kupindura nekuda kwekusakwanisa kwavo kugadzira endogenous JA uye nekudaro dzinosangana nemiganhu ye physiological. Izvi zvinogona kukonzerwa nekuchinja kwemajini akakosha munzira yeJA signaling (AOS neJAR4 muN. crescens). Mhedzisiro iyi inoratidza kuti maitiro aya ekushanduka-shanduka kwemarudzi akasiyana-siyana anogona kunyanya kukonzerwa nekuchinja kwekuona kwemahormone emukati uye kupindura.
Pamusoro pekudyidzana kuripo pakati pemiti nemhuka dzinodya zvinomera, kuongorora kusiyana kwemetabolism kwakabatana nekufambira mberi kwedzidziso kwakakosha mukutsvagisa kwekuchinjana kwezvipenyu kune nharaunda uye kushanduka kwehunhu hwakaoma hwe phenotypic. Nekuwedzera kwehuwandu hwedata rakawanikwa nemidziyo yemazuva ano yeMS, kuyedzwa kwefungidziro pamusoro pekusiyana kwemetabolism ikozvino kunogona kupfuura musiyano wemetabolism yemunhu mumwe nemumwe/boka uye kuita ongororo yepasi rose kuratidza mapatani asingatarisirwi. Mukuita ongororo yakakura, fananidzo yakakosha ipfungwa yekufunga mamepu ane revo anogona kushandiswa kuongorora data. Naizvozvo, mhedzisiro yakakosha yekusanganiswa kwazvino kwemetabolomics yeMS/MS isina rusaruro uye dzidziso yeruzivo ndeyekuti inopa metric iri nyore inogona kushandiswa kugadzira mamepu ekutarisa kusiyana kwemetabolism pamatanho akasiyana etaxonomic. Ndicho chinodiwa chikuru cheiyi nzira. Kudzidza kwekushanduka-shanduka kwemicro/macro uye nharaunda yenharaunda.
Padanho rekushanduka-shanduka kukuru, chinangwa chikuru chedzidziso yekushanduka-shanduka kwezvirimwa nezvipembenene yaEhrlich naRaven (51) ndechekufanotaura kuti kusiyana kwekushanduka-shanduka kwezvirimwa nezvipembenene ndiko kunokonzera kusiyana-siyana kwemarudzi emiti. Zvisinei, mumakore makumi mashanu kubva pakaburitswa basa iri guru, pfungwa iyi haina kumboedzwa (52). Izvi zvinonyanya kukonzerwa nehunhu hwephylogenetic hwehunhu hwakafanana hwekugadzirisa-shanduka mumarudzi emiti ari kure. Kusawanikwa kunogona kushandiswa kusimbisa nzira dzekuongorora chinangwa. Kufambiswa kwemabasa kweMS/MS kwazvino kunogadziriswa nedzidziso yeruzivo kunoyera kufanana kweMS/MS kwemametabolites asingazivikanwe (asina kusarudzwa kwemetabolite yapfuura) uye kunoshandura aya maMS/MS kuita seti yeMS/MS, saka mu metabolism yehunyanzvi Aya mamodheru ekushanduka-shanduka anoenzaniswa muchikamu chekupatsanura. Zviratidzo zvehuwandu zviri nyore. Maitiro acho akafanana nekuongorora kwephylogenetic, iyo inogona kushandisa kurongeka kwematanho kuyera mwero wekusiyana-siyana kana kushanduka kwehunhu pasina kufanotaura kwekutanga.
Padanho rekuongorora biochemical, fungidziro yekuongorora yaFirn naJones (53) inoratidza kuti kusiyana kwemetabolism kunochengetwa pamatanho akasiyana kuti zvipe zvinhu zvekugadzira kuti zviite mabasa ezvehupenyu emetabolism asina hukama kana kuti akatsiviwa. Nzira dzedzidziso yeruzivo dzinopa hurongwa umo shanduko idzi dzemetabolism dzinoitika panguva yekunyanya kushandiswa kwemetabolism dzinogona kuverengerwa sechikamu chemaitiro ekuongorora ekushanduka-shanduka: kugadziriswa kwehupenyu kubva pakuva neruzivo rwakaderera kusvika pakuva neruzivo rwakanyanya. Metabolism dzakadziviswa dzenzvimbo yakapihwa.
Zvose pamwe chete, mumazuva ekutanga ebiology yemamorekuru, dzidziso dzakakosha dzekudzivirira zvirimwa dzakagadzirwa, uye nzira dzinobva mukufungidzira dzinoonekwa sedziri nzira chete yekufambira mberi kwesainzi. Izvi zvinonyanya kukonzerwa nekusakwana kwehunyanzvi hwekuyera metabolome yese. Kunyangwe nzira dzinobva mukufungidzira dzichinyanya kubatsira pakusarudza dzimwe nzira dzinokonzera, kugona kwadzo kusimudzira kunzwisisa kwedu ma network e biochemical kwakaganhurirwa kupfuura nzira dzekuverenga dziripo parizvino musainzi yemazuva ano inoshandisa data rakawanda. Nokudaro, dzidziso dzisingagoni kufanotaurwa dziri kure neruzivo rwuripo, saka fomura/bvunzo yekufambira mberi mumunda wekutsvagisa haigone kubviswa (4). Tinofanoona kuti mashandiro ekuverenga e metabolomics akaunzwa pano anogona kudzorera kufarira munhau dzichangoburwa (sei) uye dzekupedzisira (nei) dzekusiyana kwemetabolism, uye kubatsira kunguva itsva yesainzi yedata inotungamirirwa nedzidziso. Nguva iyi yakaongororazve dzidziso dzakakosha dzakafuridzira zvizvarwa zvakapfuura.
Kudyisa mhuka dzinodya miriwo zvakananga kunoitwa nekusimudza gonye rechipiri kana kuti gonye reSl pashizha rimwe chete remuti rine ruvara rwakachenuruka remuti mumwe chete une maruva, nemiti gumi inofanana nemiti pamuti mumwe chete. Makonye ezvipembenene akasungirirwa nezvidimbu zvemachira, uye nyama yasara yeshizha yakaunganidzwa maawa makumi maviri nemana nemana nemakumi manomwe nemaviri mushure mehutachiona uye yakaomeswa nekukurumidza, uye metabolites dzakabviswa.
Tevedzera kurapwa kwemishonga inodya miriwo nenzira yakabatana zvikuru. Nzira yacho ndeyekushandisa mavhiri ejira kuti uboore mitsara mitatu yeminzwa kumativi ese epakati pemashizha matatu akakura zvakakwana emuti panguva yekukura kweruvara rwejira, uye pakarepo isa 1:5 Diluted Ms. Kana kushandisa minwe ine magirovhosi kuisa S1 OS muronda rekuboorwa. Cheka uye gadzira shizha sezvakatsanangurwa pamusoro apa. Shandisa nzira yakambotsanangurwa kuti ubvise metabolites dzekutanga nemahormone emiti (54).
Pakushandisa JA yekunze, mashizha matatu epetiole emuti mumwe nemumwe wemiti mitanhatu inotumbuka yerudzi rumwe nerumwe anorapwa ne20μl yelanolin paste ine 150μg MeJA (Lan + MeJA), uye 20μl yelanolin pamwe nekurapa maronda (Lan + W), kana kushandisa 20μl pure lanolin senzira yekudzora. Mashizha acho akakohwewa maawa makumi manomwe nemaviri mushure mekurapwa, akaomeswa nechando mu liquid nitrogen, uye akachengetwa pa -80°C kusvika ashandiswa.
Mitsetse mina yeJA neET transgenic, inoti irAOC (36), irCOI1 (55), irACO nesETR1 (48), yakawanikwa muboka redu rekutsvagisa. IrAOC yakaratidza kudzikira kwakanyanya mumazinga eJA neJA-Ile, nepo irCOI1 isina kunzwa maJA. Kana ichienzaniswa neEV, kuunganidzwa kweJA-Ile kwakawedzera. Saizvozvowo, irACO ichaderedza kugadzirwa kweET, uye kana ichienzaniswa neEV, sETR1, iyo isinganzwisisi ET, ichawedzera kugadzirwa kweET.
Chinoongorora chiedza chezuva (sensor Sense ET-300 real-time ET sensor) chinoshandiswa kuyera ET pasina kukuvadza. Mushure mekurapwa, hafu yemashizha yakachekwa ndokuendeswa kuchirongo chegirazi che4-ml chakavharwa, uye nzvimbo yemusoro yakabvumirwa kuungana mukati memaawa mashanu. Munguva yekuyera, chirongo chimwe nechimwe chakasukwa nerukova rwemarita maviri paawa yemhepo yakachena kwemaminitsi masere, iyo yakambopfuura nemuchina unobva kuSensor Sense kubvisa CO2 nemvura.
Data re microarray rakaburitswa pakutanga muna (35) uye rakachengetwa muNational Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (nhamba yekupinda GSE30287). Data rinoenderana nemashizha akakonzerwa nekurapwa kweW + OSMs uye kutonga kusina kukuvara rakatorwa muchidzidzo ichi. Kusimba kwaro kuri log2. Pamberi pekuongorora kwenhamba, baseline yakachinjwa uye yakagadziridzwa kuita 75th percentile yayo uchishandisa R software package.
Data rekutanga reRNA sequencing (RNA-seq) remhando dzeNicotiana rakatorwa kubva kuNCBI Short Reading Archives (SRA), nhamba yepurojekiti iyi iPRJNA301787, iyo yakashumwa naZhou et al. (39) uye inoenderera mberi sezvakatsanangurwa mu (56). Data raw rakagadziriswa neW + W, W + OSM uye W + OSS1 rinoenderana nemhando dzeNicotiana rakasarudzwa kuti riongororwe muchidzidzo ichi, uye rakagadziriswa nenzira inotevera: Kutanga, kuverenga kweRNA-seq kwakashandurwa kuita FASTQ format. HISAT2 inoshandura FASTQ kuita SAM, uye SAMtools inoshandura mafaira eSAM kuita mafaira eBAM akarongeka. StringTie inoshandiswa kuverenga gene expression, uye nzira yayo yekutaura ndeyekuti kune zvidimbu pa 1000 base fragments pa 1000 million sequenced transcription fragments.
Koramu yeAcclaim chromatographic (150 mm x 2.1 mm; saizi yezvikamu 2.2μm) inoshandiswa mukuongorora uye koramu ye4 mm x 4 mm ine zvinhu zvakafanana. Iyo inotevera binary gradient inoshandiswa muDionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system: 0 kusvika 0.5 maminitsi, isocratic 90% A [deionized water, 0.1% (v/v) acetonitrile uye 0.05% formic acid], 10% B (Acetonitrile uye 0.05% formic acid); 0.5 kusvika 23.5 maminitsi, gradient phase i10% A uye 90% B, zvichiteerana; 23.5 kusvika 25 maminitsi, isocratic 10% A uye 90% B. Mwero wekuyerera i400μl/min. Kune ese maMS analysis, isa column eluent mu quadrupole uye time-of-flight (qTOF) analyzer ine electrospray source inoshanda mu positive ionization mode (capillary voltage, 4500 V; capillary outlet 130 V; Kupisa kwekuomesa 200°C; kuomesa mweya 10 litres/min).
Ita ongororo yeMS / MS fragment (inozonzi MS / MS) isingakoshi kana kuti isingasiyaniswi nedata kuti uwane ruzivo rwechimiro nezve metabolic profile yose inogona kuonekwa. Pfungwa yenzira yeMS/MS isingasarudzwe inotsamira pakuti quadrupole ine hwindo guru kwazvo rekupatsanura mass [saka, funga nezve masaini ese e mass-to-charge ratio (m/z) sezvidimbu]. Nekuda kweizvi, nekuti Impact II instrument haina kukwanisa kugadzira CE tilt, ongororo dzakazvimiririra dzakati wandei dzakaitwa uchishandisa increased collision-induced dissociation collision energy (CE) values. Muchidimbu, tanga waongorora sampuro neUHPLC-electrospray ionization/qTOF-MS uchishandisa single mass spectrometry mode (low fragmentation conditions inogadzirwa ne in-source fragmentation), scanning kubva m/z 50 kusvika 1500 pa repetition frequency ye5 Hz. Shandisa nitrogen segasi rekusangana kwemasero (collision gas) pakuongorora MS/MS, uye ita zviyero zvakazvimiririra pamatanho mana akasiyana ekupatsanurana: 20, 30, 40, uye 50 eV. Mukati memaitiro ekuyera, quadrupole ine hwindo guru rekupatsanurana kwemasero, kubva pa m/z 50 kusvika 1500. Kana kuyedza kwemuviri wepamberi m/z nehupamhi hwekupatsanurana kwaiswa pa 200, huwandu hwemasero hunobva hwatanga kushanda otomatiki nesoftware yekushanda yemudziyo uye 0 Da. Skena zvidimbu zvemasero sezviri mu single mass mode. Shandisa sodium formate (50 ml isopropanol, 200 μl formic acid uye 1 ml 1M NaOH aqueous solution) kuti ukwanise kugadzirisa masero. Uchishandisa algorithm yeBruker's high-precision calibration, faira redata rinogadziriswa mushure mekushandisa avhareji yespectrum munguva yakatarwa. Shandisa basa rekutumira data reData Analysis v4.0 software (Brook Dalton, Bremen, Germany) kushandura mafaira edata asina kusimba kuita fomati yeNetCDF. Seti yedata reMS/MS yakachengetwa mudhatabhesi remetabolomics rakavhurika reMetaboLights (www.ebi.ac.uk) nenhamba yekupinda. MTBLS1471.
Kuungana kweMS/MS kunogona kuitwa kuburikidza nekuongorora hukama pakati peMS1 ​​neMS/MS quality signals kuti pave nesimba rakaderera uye rakakwirira rekukonana uye mitemo mitsva yakashandiswa. R script inoshandiswa kuona kuenzanisa kwekugoverwa kwechinhu chinotangira chigadzirwa, uye C# script (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) inoshandiswa kuita mitemo.
Kuti tideredze zvikanganiso zvenhema zvinokonzerwa neruzha rwekumashure uye kubatana kwenhema kunokonzerwa nekuona mamwe maficha e-m/z mumasampuli mashoma chete, tinoshandisa basa rekuti "filled peak" reR package XCMS (rekugadzirisa ruzha rwekumashure). Rinofanira kushandiswa kutsiva "NA" (undetected peak). Kana basa rekuti fill peak richishandiswa, kuchine ma "0" intensity values ​​​​akawanda mu data set ayo achakanganisa kuverenga kwekubatana. Zvadaro, tinoenzanisa mhedzisiro yekugadzirisa data inowanikwa kana basa rekuti filled peak richishandiswa uye kana basa rekuti filled peak risingashandiswe, uye tinoverenga kukosha kweruzha rwekumashure zvichibva paavhareji yakagadziriswa kukosha, uye tobva tatsiva aya ma 0 intensity values ​​​​nekuverengerwa kwekumashure. Isu takangofunga nezvemaficha ane simba rakapfuura katatu kukosha kwekumashure uye takaaona se "true peaks." Pakuverenga kwePCC, zviratidzo zve m/z zve sample precursor (MS1) uye fragment data sets dzine kanenge masere true peaks ndizvo zvinotariswa.
Kana simba rechinhu chemhando yepamusoro che precursor mu sample yese richienderana zvakanyanya nekudzikira kwesimba rechinhu chimwe chete chemhando yepamusoro icho chinosangana nesimba rakaderera kana rakakwirira rekukonana, uye chinhu chacho chisina kunyorwa se isotope peak ne CAMERA, chinogona kutsanangurwa zvakanyanya. Zvadaro, nekuverenga zvese zvinogoneka zve precursor-product mukati memasekondi matatu (nguva inofungidzirwa yekuchengetedza kuti irambe yakakura), ongororo yekubatana inoitwa. Kana m/z value yakaderera pane precursor value uye MS/MS fragmentation ikaitika munzvimbo imwechete ye sample mu data set se precursor yakabva, ndipo painoonekwa se fragment.
Zvichibva pamitemo miviri iyi iri nyore, tinobvisa zvidimbu zvakatarwa zvine m/z values ​​​​yakakura kupfuura m/z ye precursor yakawanikwa, uye zvichibva pane sample position apo precursor inoonekwa uye chidimbu chakatarwa. Zvinokwanisikawo kusarudza hunhu hwemhando hunogadzirwa nezvidimbu zvakawanda zviri mukati mesource zvakagadzirwa muMS1 ​​mode se candidate precursors, nokudaro zvichigadzira refund MS/MS compounds. Kuti tideredze data redundancy iyi, kana NDP kufanana kwe spectra kukadarika 0.6, uye zviri zve chromatogram "pcgroup" yakatsanangurwa ne CAMERA, tichazvibatanidza. Chekupedzisira, tinosanganisa zvese zvina CE results zvine chekuita ne precursor nezvidimbu mu final deconvoluted composite spectrum nekusarudza highest intensity peak pakati pema candidate peaks ese ane m/z value yakafanana pa collision energies dzakasiyana. Matanho ekugadzirisa anotevera akavakirwa papfungwa ye composite spectrum uye anofunga nezvemamiriro akasiyana e CE anodiwa kuti awedzere mukana wekupatsanuka, nekuti zvimwe zvidimbu zvinogona kuonekwa chete pasi pe specific collision energy.
RDPI (30) yakashandiswa kuverenga inducibility ye metabolic profile. Kusiyana kwe metabolic spectrum (Hj index) kunobva muhuwandu hwe MS/MS precursors vachishandisa Shannon entropy yekugoverwa kwe MS/MS frequency vachishandisa equation inotevera yakatsanangurwa naMartínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) apo Pij inoenderana ne relative frequency ye i-th MS/MS mu j-th sample (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Kunyatsotsanangurwa kweMetabolic (Si index) kunotsanangurwa sechiratidzo cheMS/MS yakapihwa maererano nehuwandu huripo pakati pemasampuli ari kutariswa. Kunyatsotsanangurwa kweMS/MS kunoverengerwa seSi = 1t (∑j = 1tPijPilog2PijPi)
Shandisa fomura inotevera kuyera metabolome-specific δj index yemuenzaniso wega wega we j, uye avhareji yeMS/MS specificity δj = ∑i = 1mPijSi
MS/MS spectra dzakarongwa muzvikamu zviviri, uye kufanana kunoverengerwa zvichibva pazvibodzwa zviviri. Kutanga, uchishandisa standard NDP (inozivikanwawo se cosine correlation method), shandisa equation inotevera kuti uwane kufanana kwechikamu pakati pe spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 apo S1 naS2 Saizvozvowo, kune spectrum 1 ne spectrum 2, pamwe neWS1, i naWS2, i inomiririra huremu zvichibva pahukuru hwepamusoro hwekuti musiyano we i-th common peak pakati pe spectra mbiri uri pasi pe 0.01 Da. Huremu hunoverengerwa seizvi: W = [peak intensity] m [quality] n, m = 0.5, n = 2, sezvakakurudzirwa neMassBank.
Nzira yechipiri yekukora yakaitwa, yaisanganisira kuongorora NL yakagovaniswa pakati peMS/MS. Kuti tiite izvi, takashandisa runyorwa rwe52 NL rwaiwanzoonekwa panguva yekupatsanurwa kweMS tandem, uye NL chaiyo (data file S1) yakambonyorwa kare yeMS/MS spectrum yesecondary metabolites yeNepenthes species isina simba (9, 26). Gadzira binary vector ye1 ne0 yeMS/MS yega yega, inoenderana ne current uye isipo yeimwe NL zvakateerana. Zvichibva pakufanana kweEuclidean distance, NL similarity score inoverengerwa papeya yega yega ye binary NL vectors.
Kuti tiite dual clustering, takashandisa R package DiffCoEx, iyo yakavakirwa pakuwedzera kweWeighted Gene Co-expression Analysis (WGCNA). Tichishandisa NDP neNL scoring matrices eMS/MS spectra, takashandisa DiffCoEx kuverenga comparative correlation matrix. Binary clustering inoitwa nekuisa "cutreeDynamic" parameter ku method = "hybrid", cutHeight = 0.9999, deepSplit = T, uye minClusterSize = 10. R source code yeDiffCoEx yakatorwa kubva kufaira rekuwedzera 1 naTesson et al. (57); R WGCNA software package inodiwa inogona kuwanikwa mu https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Kuti tiite ongororo yeMS/MS molecular network, takaverenga kubatana kwe spectral kwakabatana zvichibva pamhando dzeNDP neNL similarity, uye takashandisa Cytoscape software kuti tiratidze network topology tichishandisa organic layout muCyFilescape yFiles layout algorithm extension application.
Shandisa R version 3.0.1 kuti uite ongororo yehuwandu hwe data. Kukosha kwehuwandu hwe data kwakaongororwa uchishandisa two-way analysis of variance (ANOVA), zvichiteverwa neongororo yaTukey ye "honestly significant difference (HSD) post-hoc". Kuti tiongorore musiyano uripo pakati pekurapa herbivorous ne control, kugoverwa kwetwo-tailed kwemapoka maviri esampuli ane variance yakafanana kwakaongororwa uchishandisa bvunzo ye Student's t.
Kuti uwane zvimwe zvinyorwa zvechinyorwa chino, ndapota tarisai http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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