Mizeezo yiindene-indene yakukwabilila zisyango ilapa busolozi buyandika kapati bwakupandulula nzila zyakusanduka kwazyakulya zyazisyango, pele nzyoziyeeyela zipati zyeelede kusunkwa. Aawa, twakabelesya nzila ya tandem mass spectrometry iitakwe kampenda (MS/MS) kulanga-langa munzila iilondokede metabolome ya tombwe uuzwa kuzisyango zyomwe-zyomwe kuya kubantu alimwi amisyobo iili afwaafwi, alimwi twakabamba mweelwe mupati wa mass spectrometric feature theories kweendelana a compound spectra mumakani. Nzila yakusunka mizeezo iiyandika kapati yakukwabilila (OD) antoomwe akweenda (MT). Bbazu lya makani aajatikizya zyakulya zyazisyango lileendelana a OD theory, pesi lilakazyanya a muzeezo mupati wa MT theory aatala a metabolomics dynamics zyeetelezegwa abanyama balya zisyango. Kuzwa kuchiimo chiche kusikila kuchiimo chipati, citondezyo cha jasmonate chakajanwa kuti nchicho chipati chipa kuti kube OD, mpawo citondezyo cha ethylene chakagwasya kubamba kabotu nsandulo ya herbiver-specific yakaambwa a MS/MS molecular network.
Ma metabolites aalibedelede ajisi bupange bwiindene-indene ngaatola lubazu kapati mukuzyibila zisyango kubusena, ikapati mukukwabilila basinkondo (1). Kusiyana kugambya kwa metabolism iilibedelede iijanika muzisyango kwakulwaizya makumi aamyaka yakuvwuntauzya kwamukati kujatikizya mbazu zinji zyamilimo yazisyango, alimwi kwapanga mulongo mulamfu wa njiisyo zyakukwabilila zisyango, eezyo zili mukusanduka alimwi akuyaambele kwazyintu zipona akaambo kakuswaangana kwazisyango atuzunda. Buvwuntauzi bwakuvwuntauzya bupa busolozi buyandika (2). Nokuba boobo, eezyi njiisyo zyakukwabilila zisyango tiizyakali kutobela nzila ya hypothetical deductive reasoning, oomo zyintu zipati zyakali kulangilwa zyakali abulemu bomwe bwakuvwuntauzya (3) alimwi zyakali kusunkwa munzila yakusola kutegwa ziyungizye nzila iitobela yakusumpula njiisyo (4). Kulezya kwabusongo kulesya kuyobolola data kuzibeela zimwi zya metabolic mpawo takugwisyi kuvwuntauzya koonse kwa metabolites zilibedelede, aboobo kulesya kweezyanisya akati kazibeela eezyo ziyandikana mukusumpula njiisyo (5). Kubula data iizwide ya metabolomics antoomwe amali aabelesyegwa kweezyanisya nzila yakubamba zyakulya akati kankamu zyazisyango zisiyene kulesya kukomena kwasayaansi kwamulimo ooyu.
Zyakachitika lino-lino mumulimo wa tandem mass spectrometry (MS/MS) metabolomics zilakonzya kupandulula kuchincha kwa metabolic mukati aakati kamisyobo ya system clade imwi, mpawo zilakonzya kuswanizigwa anzila zyakubala kuti zikonzye kubala kukozyanya kwabupange aakati ka misanganisyo eeyi iikatazya kumvwisisisya. Luzibo lwakusaanguna lwa chemistry (5). Kusanganya minchina iisumpukide mukulanga-langa akubelesya kkompiyuta kupa nzila iiyandika yakusola kwachiindi chilamfu kwazyintu zyiingi zyaambilizyigwa zyakachitika akaambo kazyintu zipona azyakusanduka kwazyintu zipona. Shannon (6) wakatalisya njiisyo yamakani mchiindi chakusanguna muchibalo chakwe chipati mu 1948, mpawo wakabikka ntalisyo yakuvwuntauzya makani kwiinda mukubelesya nsamu, eeyo yakabelesegwa mumbazu zyiingi kunze lyakubelesya kwayo. Mu genomics, njiisyo yamakani yakabelesegwa kabotu mukupima makani aatobelana (7). Mukuvwuntauzya kwa transcriptomics, njiisyo yamakani ilalanga-langa kuchincha koonse kwa transcriptome (8). Mubuvwuntauzi bwakainda, twakabelesya nzila zyakubala zya makani ku metabolomics kupandulula luzibo lwa metabolic lwa tissue muzisyango (9). Aawa, twaswaanganya MS/MS-based workflow antoomwe anzila zyakubala zya makani, zizyibidwe akusiyana kwa metabolic mumali aabelesyegwa abantu boonse, kweezyanisya zyintu zipati zyaambilizyigwa zya njiisyo yakukwabilila misamu ya metabolome iiletwa abanyama balya zisyango.
Nzila zyakukwabilila zisyango kanjikanji zilabikkilizya antoomwe mpawo zilakonzya kwaandaanizyigwa muzibeela zyobile: eezyo zisola kupandulula kwaabwa kwazintu zipangwa azisyango kweendelanya amilimo yakukwabilila, mbuli kukwabilila kubotu (OD) (10), chiyandisyo chikweenda (MT) (11) ) akulangika (12) njiisyo, mpawo zimwi ziyandaula bupanduluzi bwakucinca kwakujanika kwazibelesyo kukomena kwachisyango akuyoboloka kwazintu zilibedelede, zilimbuli kaboni: muzeezo wazyakulya zigwasya mubili (13), muzeezo wakukomena (14), antoomwe akukomena akusiyana kwamizeezo (15). Mizeezo yobile eeyi ili mumasena aasiyene aakulanga-langa (4). Nokuba boobo, mizeezo yobile iijatikizya milimo yakulikwabilila aawo mpoibeleka njiyeendelezya mubandi aatala alukwabililo lwazisyango: njiisyo ya OD, eeyo iiyeeyela kuti zisyango zibikka mali muzikwabilizyo zyabo zyamakemikalu zidula biyo kuti kaziyandika, mbuli, aawo noziligwa Chiindi munyama wamusyokwe nalwana, aboobo, kweendelanya akukonzeka kwakumbele, musamu wakulikwabilila ulabikkwa mbuli nzila yakukwabilila (10); muzeezo wa MT utondeezya kuti taakwe nzila yakusanduka kwa metabolite, pesi metabolite ilachincha biyo, aboobo kupa kuti kube nzila yakukwabilila "kweenda" kwa metabolic kwakulwana banyama balya zisyango. Mumajwi amwi, eezyi mizeezo yobile ilaamba zintu ziimpene kujatikizya kusanduka kwa metabolic ooko kuchitika nikwakamana kulwanwa abanyama balya zisyango: kujatana aakati kakuyoboloka kwa metabolites munzila yomwe antoomwe amulimo wakukwabilila (OD) akusanduka kwa metabolic kutali kweendelezyegwa (MT) (11 ).
Mizeezo ya OD a MT tiijatikizyi buyo kucinca kwa metabolome, pele alimwi azintu zicitika akaambo kakuyoboloka kwa metabolites eezyi, mbuli mali aabelesyegwa alimwi ampindu zyakucinca ooku kwa metabolome mubusena bumwi (16). Nikuba kuti mizeezo yoonse yobile ilaziba mulimo wakukwabilila wa metabolites zilibedelede, eezyo zinga zyadula naa pe, kusinsima kupati kwaandaanya mizeezo ya OD a MT ili mukusololela kwakusanduka kwa metabolic. Kusinsima kwa OD theory kwatambula kubikkilwa maanu kapati kusikila lino. Masunko aaya abikkilizya kwiiya milimo yakulikwabilila iigaminide naa iitaluleme yazibeela zyanyama ziindene-indene zyamisamu imwi mumaanda aajisi busani alimwi abukkale bwacilengwaleza, alimwi akucinca kwaciimo cakukomena kwazisyango (17-19). Nokuba boobo, kusikila lino, akaambo kakubula nzila yakubeleka anzila yakubala yakulanga-langa nyika yoonse mbwiizulwa kujatikizya kusiyana kwazyakulya zili zyoonse, kusiyana kupati aakati kamizeezo yobile (nkokuti, nzila yakusanduka kwazyakulya) kuchili mukusunkwa. Aawa, twapa buvwuntauzi buliboobu.
Chimwi chazintu zipati zitondezya zintu zipangwa azisyango nkusiyana kwazyo muzibeela zyoonse kuzwa kuzisyango zyomwe, kubantu kusikila kumisyobo iikozyenye (20). Kuchincha kunji kwamweelwe muzintu zilibedelede kulakonzya kubonwa aawo mweelwe wabantu, mpawo kwiimpana kupati kwabube kanjikanji kulazumanana aawo mweelwe wamisyobo (20). Aboobo, kusiyana kwazyakulya muzisyango nchintu chipati mukusiyana kwakubeleka, kutondezya kukonzya kuzyibila mumasena aasiyene, ikapati mumasena aayo ajisi nzila ziindene zyakunjililwa atuzunda tulibedelede alimwi abanyama balya zisyango (21). Kuzwa muchibalo cha Fraenkel (22) chakusaanguna aatala atwaambo tupa kuti kube zintu zipangwa muzisyango, kuswaangana atuzunda tuindene-indene kwabonwa kuti nchintu chipati chakusala, mpawo kuswaangana ooku kusyomwa kuti kwakabumba misamu muchiindi chakusanduka. Nzila yakusanduka kwamubili (23). Kusiyana aakati kamisyobo yazisyango mukusiyana kwama metabolites aalibedelede kulakonzya kutondezya kweendelana kwamubili kweendelana akukwabililwa kwazisyango kuzwa kunzila zyakulya zisyango, nkaambo misyobo yobile kanjikanji ilaswaangene munzila iitali kabotu (24). Nikuba kuti chilakonzya kugwasya kubamba lukwabililo lubotu chiindi choonse, kuchincha kwachiindi cheelede kweendelana alukwabililo kupa mpindu zilibonya mukuzumizya misamu kuti ibelesye zintu ziyandikana kuli zimwi zintu (19, 24), akutantamuka kuyandikana kwakubelekela antoomwe. Kunyonyooka kwakukwabilila (25). Mukuyungizya, ooku kubamba zintu zilibedelede ziletwa atuzunda tulya zisyango kulakonzya kuleta kumwaigwa kwakunyonyoona mubantu (26), alimwi kulakonzya kutondezya kubala kwakugaminina kwakucinca kupati kwacilengwaleza mucitondezyo ca jasmonic acid (JA), ooko kukonzya kuzumanana mubantu. Zitondeezyo zya JA zisumpukide aziniini nzintu zipa kuti kube kukwabililwa kuzwa kuzinyama zilya zisyango akuzundana amisyobo imwi (27). Mukuyungizya, nzila zyakupanga zintu zipona ziyoofwambaana kusweeka akusanduka muchiindi chakusanduka, eezi ziyoopa kuti kube kwaandaana kutali kabotu akati kamisyobo iikozyenye (28). Eezyi zintu ziindene-indene zilakonzya kufwambaana kuzyibwa akaambo kakucinca kwabukkale bwabanyama balya zisyango (29), calo caamba kuti kucinca-cinca kwamisyobo yabanyama balya zisyango ncecintu cipati cipa kuti kube kusiyana kwa metabolic.
Aawa, twakamanizya mapenzi aaya. (I) Ino tuzunda tulya zisyango tulabamba biyeni metabolome yazisyango? (Ii) Ino nzibeela nzi zipati zya makani aajatikizya metabolic plasticity zikonzya kubalwa kutegwa zisunkwe zyintu zikonzya kuchitwa muchiindi chilamfu? (Iii) Kuti naa tweelede kubambulula metabolome yamusamu munzila iilibonya kuli yooyo uulwana, ikuti kakuli boobo, ino mahomoni aamusamu ajisi mulimo nzi mukubamba nsandulo imwi ya metabolic, alimwi ino nzintu nzi zigwasya mukukwabilila misyobo yazisyango? (Iv) Mbwaanga zyeezyo zyaambilizyigwa amizeezo minjaanji yakukwabilila zilakonzya kuyungizigwa kuzibeela zyoonse zyanyama, twakabuzya kuti ino nsandulo ya metabolic ilakonzya buti kuzwa mukweezyanisya kwamukati kusikila kukweezyanisya akati kamisyobo? Kusikila kumamanino, twakavwuntauzya kabotu-kabotu nzila zyamafwaafwi aa nicotine ya tombwe, ooyo uuli mbuli musamu uukonzya kugwasya buumi bwamuntu uulaa nzila zyakupanga zintu zilibedelede, alimwi uubeleka kabotu kulwana bana banyama bobile balya zisyango, Lepidoptera Datura (Ms) ( Uulaanguzu kapati, uuligwa kapati) Kuli Solanaceae a Spodoptera (Storal lit), musyobo wamafwaafwi aamafwaafwi aamusamu wa tombwe "musyobo", antoomwe azisyango zya Solanaceae azimwi zisyango zyamisyobo imwi amikwasyi Chakulya chazisyango. Twakavwuntauzya MS/MS metabolomics spectrum mpawo twakagwisya twaambo twakubala twamakani kuti tweezyanisye njiisyo zya OD a MT. Panga maapu aasalala kuti uyubunune zyintu zipati zipangwa mumubili. Buvwuntauzi bwakayungizigwa kubantu bazwa ku N. nasi alimwi amisyobo ya tombwe iikozyenye kapati kutegwa kuyungizye kuvwuntauzya kusiyana aakati kakutondezya mahomoni aazisyango akutalisya OD.
Kuti tujane maapu yoonse aatala aabupange bwamafwaafwi aamafwabi aatombwe uulya zisyango, twakabelesya nzila yakuvwuntauzya akubalila zyakabambilwa kale kuti tuyobolole akugwisya data iilibonya kabotu iitakwe MS/MS spectra kuzwa kuzisyango ( 9). Eeyi nzila iitakwe lwiindano (iitwa kuti MS/MS) ilakonzya kupanga zintu zitabelesyegwi pe, eezyo zikonzya kubelesegwa mukuvwuntauzya koonse kwapandululwa aawa. Eezyi zintu zipangwa muzisyango ziliindene-indene, zijisi myaanda kusikila kuzyuulu zyazintu zipangwa (aali 500-1000-s/MS/MS aawa). Aawa, tulalanga-langa kusanduka kwa metabolic munzila ya njiisyo ya makani, mpawo twapima kusiyana akubeleka kwa metabolome kweendelanya a Shannon entropy ya metabolic frequency distribution. Kubelesya nzila yakabelesegwa musule (8), twakabala zitondezyo zikonzya kubelesegwa kupima kusiyana kwa metabolome (Hj indicator), metabolic profile (δj indicator) antoomwe a metabolic specificity ya metabolite yomwe (Si indicator). Mukuyungizya, twakabelesya Relative Distance Plasticity Index (RDPI) kuti tuzyibe mweelwe wa metabolome inducibility ya banyama balya zisyango (Chitondeezyo 1A) (30). Mukati kachibalo eechi, tulabamba MS/MS spectrum mbuli ntalisyo yamakani, mpawo twabamba bunji bwa MS/MS kuti bube maapu yakumwaika kwa frequency, mpawo tubelesya Shannon entropy kweezyeezya kusiyana kwa metabolome kuzwa kulinjiyo. Busongo bwa metabolome bulapimwa amweelwe waakati-kati wa MS/MS spectrum yomwe. Aboobo, kuyungizigwa kwabunji bwamisyobo imwi ya MS/MS musule lyakunjizigwa kwa herbiver kusanduka kuba spectral inducibility, RDPI a specialization, nkokuti, kuyungizigwa kwa δj index, nkaambo zintu zyiingi zilibedelede zilapangwa mpawo Si index mpati ilapangwa. Ikuselemuka kwa Hj diversity index kutondeezya kuti mweelwe wa MS/MS wakacheya, naakuti kwaabwa kwachiindi chilamfu kwasanduka munzila iiteendelani, kumwi kakucheya kutaziba koonse. Kwiinda mukubalwa kwa Si index, zilakonzeka kutondezya kuti MS/MS nzi ziletwa azinyama zimwi zilya zisyango, mukwiimpana, MS/MS nzi zitavwiilili kuchitwa, eecho nchitondezyo chipati chakwaandaanya MT a OD.
(A) Zitondeezyo zyakubala zyakabelesegwa kuli banyama balya zisyango (H1 kusikila ku Hx) MS/MS data-inducibility (RDPI), kusiyana (Hj index), kuba aluzibo (δj index) antoomwe a metabolite specificity (Si index). Kuyungizya kwakuzyiba (δj) kutondeezya kuti, mweelwe waakati aakati, zintu zyiingi zilya zisyango ziyoopangwa, mpawo ikucheya kwakusiyana (Hj) kutondeezya kucheya kwakupanga zintu naakuti kwaabwa kutali kabotu kwama metabolite mumaapu yakumwaika. Mpindu ya Si ilalanga-langa naa metabolite ili buyo mubukkale bumwi (aawa, iilya zisyango) naa kuti ilazumanana kuba aciimo nciconya. (B) Mufanikisyo wa muzeezo wakusinsima kwa njiisyo yakukwabilila kubelesya njiisyo ya makani. Njiisyo ya OD ilasinsima kuti kulwanwa kwabanyama balya zisyango kuyooyungizya zintu zikwabilila, aboobo kuyooyungizya δj. Aawo awo, Hj ilaselemuka nkaambo chiimo chakabambululwa kuya kukucheya kwakutaziba kwamakani aajatikizya zyakulya. Njiisyo ya MT ilasinsima kuti kulwanwa kwabanyama balya zisyango kuyoopa kuti kube kucinca kutakwe nzila mu metabolome, aboobo kuyooyungizya Hj mbuli citondezyo cakuyungizya kutasinizya kwamakani aajatikizya metabolic akupa kuti Si amwaigwe cakutayeeyelwa. Twakakankaizya lubo nzila iiswaangenyi, MT mbotu, oomo zimwi zintu zili aanguzu zyakulikwabilila ziyooyungizyigwa (Si mpati), mpawo zimwi zitondezya nsandulo zitayeeyelwi (Si nsyoonto).
Kubelesya bupanduluzi bwa njiisyo ya makani, twasandulula njiisyo ya OD kuti tuyeeye kuti kucinca kwa metabolite kwaalubazu kwakachitwa abanyama balya zisyango muchiimo chitakwe kuchincha kuyoopa kuti (i) kuyungizigwa kwa metabolic specificity (Si index) kweendelezya metabolic specificity (δj index) Kuyungizya kwa) tubunga twa metabolite tumwi twaalubazu tujisi mpindu mpati yakukwabilila, antoomwe a (ii) kucheya kwa metabolite (Hj) index kuchincha kwakwaabila kwakuvwula kwa metabolic kuya kukumwaigwa kwa leptin mumubili. Aawo achiimo cha metabolite yomwe, kwaabwa kwa Si kwalondokede kulalangilwa, ooko metabolite nkwayooyungizya mweelwe wa Si kweendelanya abulemu bwayo bwakulikwabilila (Chitondeezyo 1B). Munzila eeyi, twapandulula njiisyo ya MT kuti tuyeezye kuti kukankaminwa kuyoopa kuti (i) kuchincha kutakwe nzila muma metabolites kuyoopa kuti δj index iche, antoomwe a (ii) kuyungizigwa kwa Hj index akaambo kakuyungizya kutazibisya kwa metabolic. Naa kutayeeyelwa, ooko kukonzya kubalwa a Shannon entropy munzila yakusiyana koonse. Kujatikizya bupange bwa metabolic, njiisyo ya MT ilakonzya kusinsima kwaabwa kwa Si. Twabikkila maanu kuti zimwi zintu zipangwa mumubili zili mubukkale bumwi mubukkale bumwi, mpawo zimwi zintu tazili mubukkale bumwi, mpawo mpindu yazyo yakulikwabilila iyeeme abusena, twakakankaizya nzila yakulikwabilila iisanganizidwe, oomo δj a Hj mozimwaigwa muzibeela zyobile kweendelana a Si. 1B).
Mukusola kulingaula njiisyo yakukwabilila yakasandululwa aawo mpokugolela njiisyo yamakani, twakakomezya basyaazibwene (Ms) naakuti basyaazibwene (Sl) banyama balya zisyango atala aamafwabi aa Nepenthes pallens (Chitondeezyo 2A). Kubelesya MS/MS analysis, twakajana 599 non-redundant MS/MS spectra (data file S1) kuzwa ku methanol extracts zyamafwabi zyakayobolwa nikwakamana kulya nkumba. Kubelesya RDPI, Hj, antoomwe a δj indexes kubona kusanduka kwamakani aali muma fayila aa MS/MS kutondezya nzila zibotu (Chitondeezyo 2B). Chiimo choonse nchakuti, mbuli mbokwaambwa amuntu uupandulula makani, mbuli nkumba mbozizumanana kulya matu, mweelwe wakubamba zintu zyoonse mumubili ulatanta muchiindi: mawoola aali makumi musanu atutatu aabili kuzwa aawo munyama uulya zisyango nalya, RDPI ilatanta loko. Mukweezyanisya akweendelezya kutakwe ntenda, Hj yakacheya loko, eecho chakachitika akaambo kakuyungizigwa kwabusongo bwa metabolic profile, eeyo yakapimwa a δj index. Eechi chiimo chilibonya cheendelana azintu zyaambwa a OD theory, pesi tazyeendelani azintu zipati zyaambwa a MT theory, eezyo zisyoma kuti kucinca kwakutayeeyelwa (kutali kweendelezegwa) kwama metabolite kulabelesyegwa mbuli nzila yakukwabilila (Chitondeezyo 1B). Nikuba kuti zyintu zili mumulomo (OS) zipa kuti kube kulya kwabanyama aaba bobile balya zisyango ziliindene, kulya kwabo kwakapa kuti kube kucinca kukozyenye mumabazu aa Hj a δj muchiindi chakutebula chamawoola aali makumi obilwe atune (24) antoomwe amahola aali makumi musanu aabili (72). Kusiyana kwakachitika mumawoola aali 72 aa RDPI. Kweezyanisya aawo chakachitwa akulya kwa Ms, kusanduka koonse kwakachitwa akulya kwa Sl kwakalisumpukide.
(A) Muzeezo wakusola: ngulube (S1) naakuti basyaazibwene (Ms) banyama balya zisyango balapegwa matu aatakwe munyo aamisamu ya pitcher, mpawo kuli banyama balya zisyango, OS ya Ms (W + OSMs) ilabelesyegwa kubamba kugonkwa kwamafwaafwi aazilonda zyamafwaafwi. S1 (W + OSSl) nziba naa maanzi (W + W). Kweendelezya (C) nditu litanyonyooki pe. (B) Inducibility (RDPI kweelanisigwa a control chart), diversity (Hj index) antoomwe a specialization (δj index) index yakabalilwa ku mapu yaalubazu ya metabolite (599 MS/MS; data file S1). Nyenyeezi zitondezya kwiimpana kupati aakati kakulya kwabanyama balya zisyango antoomwe akabunga kakweendelezya (Student's t-test with paired t-test, *P<0.05 and ***P<0.001). ns, taziyandiki pe. (C) Chiindi chakusalazya cha (bbokesi lyamubala wa bbuluu, amino acid, organic acid a syuga; fayela lya data S2) antoomwe a metabolite spectrum (bbokesi lisalazya 443 MS/MS; fayela lya data S1) nikwakamana busilisi bwakulya zisyango. Ibbanti lyamibala lyaamba 95% yakusyoma. Inyenyeezi itondezya kwiimpana kupati aakati kakusilikwa akweendelezya [kuvwuntauzya kwakusiyana (ANOVA), kutobelwa akwiimpana kupati kwa Tukey (HSD) mukweezyanisya kwakumbele, *P<0.05, **P<0.01 And *** P <0.001]. (D) Kusalazya zibeela zyakumwaya antoomwe azibeela zya metabolite (zitondezyo ziindulukwa amisamu yiindene).
Kulanga-langa naa kusanduka kwakachitwa abanyama balya zisyango kuchiimo cha metabolome kulatondezyegwa mukucinca kwachiimo cha metabolites, twakasaanguna kubikkila maanu ku metabolites zyakavwuntauzyigwa musule mumafwaafwi aa Nepenthes pallens aajisi nguzu zyakukazya banyama balya zisyango. Ma phenolic amides nga hydroxycinnamamide-polyamine conjugates aayo aabungika muchiindi chakulya tuzunda mpawo alizyibidwe kuti alacesya kubeleka kwa tuzunda (32). Twakayandaula zintu zyakasaanguna zya MS/MS mpawo twakabamba nzila zyabo zyakuzungaana (Chitondeezyo S1). Kakunyina kugambya, zintu zizwa ku phenol zitajatikizyigwi pe mukulwana banyama balya zisyango, mbuli chlorogenic acid (CGA) a rutin, zilacheya musule lyakulya zisyango. Mukwiimpana, banyama balya zisyango balakonzya kuchita kuti ma phenol amides abe anguzu loko. Kuzumanana kulya kwabanyama bobile balya zisyango kwakapa kuti kube kukankaminwa kweelene kwa phenolamides, mpawo eechi chiimo chakalibonya kapati mukupanga ma phenolamides. Chintu chomwe chiyoobonwa aawo notuvwuntauzya nzila ya 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), eeyo iipanga mweelwe mupati wa acyclic diterpenes iijisi milimo iigwasya kulwana nyama iilya zisyango (33), oomo Ms Feeding a Sl mwaakatalisya kutondezya kukozyenye (Figure S1).
Bubi bukonzya kuba bwakusola kulya banyama balya zisyango nkusiyana kwakulya kwamafwaafwi achiindi chakulya chabanyama balya zisyango, eecho chipa kuti kakutazya kugwisya ntenda zyabanyama balya zisyango akaambo kazilonda abanyama balya zisyango. Kuti tukonzye kujana nsandulo yamisyobo yazinyama zilya zisyango zyakusanduka kwamafwaafwi, twakakozyanisya kulya kwa Ms a Sl larvae kwiinda mukufwambaana kubelesya OS (OSM a OSS1) yakabungikwa ku W yamafwaafwi aatali kabotu. Eeyi nzila yiitwa kuti W + OS treatment, mpo ilabamba nzila yakutalisya kwiinda mukubamba chiindi cheenzu chakutalika kwakwiingula kwakachitwa amuntu uulya zisyango kakunyina kuleta ntenda zyakusiyana kwamweelwe naakuti mweelwe wakusweekelwa kwanyama (Chitondeezyo 2A) (34). Kubelesya nzila yakuvwuntauzya a MS/MS, twakajana 443 MS/MS spectra (data file S1), eeyo yakavwumbilila a spectra yakabungikwa musyule kuzwa kumasunko aakulya. Buvwuntauzi bwa makani aa MS/MS data set bwakatondezya kuti kubambulula ma metabolomes aamafwaafwi kwiinda mukukozyanisya banyama balya zisyango kwakatondezya OS-specific inducements (Chitondeezyo 2C). Mubwini, kweezyanisya abusilisi bwa OSS1, OSM yakachita kuti metabolome iyungizye busongo mumawoola one. Nchintu chibotu kuzyiba kuti kweezyanisya a data yakujana zyakulya zyabanyama balya zisyango, kweenda kwazyakulya kwakabonwa mubusena bujisi zibeela zyobile kubelesya Hj a δj mbuli zyeelelo anzila zya metabolome mukwiingula busilisi bwabanyama balya zisyango mukuya kwachiindi Kuyungizya kweendelana (Chitondeezyo 2D). Aawo awo, twakapima bunji bwa amino acids, organic acids a sugars (data file S2) kuti tuvwuntauzye naa ooku kuyungizigwa kwaluzibo lwa metabolome kwakachitika akaambo kakucinca kwa carbon metabolism yamukati mukuvwiila banyama balya zisyango (Chitondeezyo S2). Kuti tupandulule kabotu nzila eeyi, twakayungizya kulanga-langa nzila zyakuyobolola zyakulya zya phenolamide a 17-HGL-DTG zyakabandikwa musyule. Ikunjizya kwa OS kwa banyama balya zisyango kwasandulwa kuba nzila yakubamba zintu ziindene-indene mukati ka metabolism ya phenolamide (Chitondeezyo S3). Ma amide aa Phenolic ajisi zibeela zya coumarin antoomwe a caffeoyl zilasungwaazigwa a OSS1, mpawo OSMs zipa kuti kube kunjizigwa kwa ferulyl conjugates. Kunzila ya 17-HGL-DTG, kusiyana kwa OS kwiinda mukubelesya malonylation antoomwe a dimalonylation kwakajanika (Chitondeezyo S3).
Kuzwa waawo, twakayiya OS-induced transcriptome plasticity kubelesya time-course microarray data set, eeyo yeezyanisya kubelesya OSMs kusilika matu aamusamu wa rosette mubanyama balya zisyango. Ikweenda kwakusampulwa kweendelana akweenda kwakabelesegwa muchiiyo eechi cha metabolomics (35). Mukweezyanisya akusanduka kwa metabolome ooko metabolic plasticity nkoiyungizigwa muchiindi, twabona kupya kwakulemba kwamafwaafwi kwakachitwa a Ms, ooko transcriptome inducibility (RDPI) antoomwe a specialization (δj) zili a 1 Kwakali kuyungizigwa kupati kwamahola, akusiyana (Hj) achiindi eechi, kutondezya kwa BMP1 kwakaceya kapati, kwakatobelwa akulyookezya kwa BMP1 lwiiyo lwa transcriptome (Chitondeezyo S4). Mikwasyi yamajini aagwasya mubili (mbuli P450, glycosyltransferase, antoomwe a BAHD acyltransferase) ilatola lubazu munzila yakubunganya zintu zilibedelede kuzwa kuzintu zipangwa zizwa kumubili wakusaanguna, kutobela nzila yakusaanguna yabusongo bwakusaanguna. Mbuli chitondezyo, nzila ya phenylalanine yakavwuntauzyigwa. Buvwuntauzi bwakasinizya kuti ma genes mapati mu metabolism ya phenolamide alajanika kapati mu OS-induced muzilenge zilya zisyango kweelanisigwa azisyango zitayandisyi, alimwi ali afwaafwi kapati munzila zyabo zyakutondezya. Ibbazu lyakulemba MYB8 antoomwe amajini aa PAL1, PAL2, C4H antoomwe a 4CL aali atala aanzila eeyi zyakatondezya kutalika kwakulemba. Ma acyltransferases ajisi lubazu mukubunganya kwamamanino kwa phenolamide, mbuli AT1, DH29, antoomwe a CV86, atondezya nzila yakutanta kwachiindi chilamfu (Chitondeezyo S4). Zyakabonwa atala aawa zitondezya kuti kutalika kwakusaanguna kwakuvwuntauzya kwa transcriptome akusumpuka kwakumbele kwakuvwuntauzya kwa metabolomics nzintu zijatikizyidwe, eezyo zinga zyaba akaambo kanzila iilanganya zyintu zyoonse antoomwe eeyo iitalisya nzila yakukwabilila.
Kubambulula kwa mahomoni aazisyango kubeleka mbuli nzila yeendelezya iiswaanganya makani aajatikizya zyakulya zisyango kuti ichite bubambe bupya bwabuumi bwazisyango. Musule akweezyanisya kwabanyama balya zisyango, twakapima kuvwula kwamahomoni aamisamu mpawo twakabona chiindi chilamfu akati kabo [Pearson correlation coefficient (PCC)> 0.4] (Figure 3A). Mbuli mbokulangilwa, mahomoni aazisyango aajatikizya kupanga zintu zipona alaswaangana mukati kanzila yakutondezya mahomoni aazisyango. Mukuyungizya, bube bwakusanduka kwazyakulya (Si index) bulabikkwa mumaapu eeyi kutondezya mahomoni aazisyango aaletwa abusilisi bwiindene-indene. Zibeela zyobile zipati zyakwiingula kwabanyama balya zisyango zyaambwa: limwi lili mukabunga ka JA, oomo JA (musyobo wayo uubeleka mubuumi JA-Ile) azimwi zizwa ku JA zitondezya Si score mpati; aimbi ni ethylene (ET). Gibberellin yakatondeezya buyo kuyungizigwa kwaakati-akati mukulya zisyango, kakuli mahomoni aambi aazisyango, mbuli cytokinin, auxin, antoomwe a abscisic acid, akatondezya kuyungizigwa kusyoonto kuli banyama balya zisyango. Kweezyanisya akubelesya W + W alikke, kuyungizya mpindu ya JA kwiinda mukubelesya OS (W + OS) kulakonzya kusandulwa kuba chitondezyo chiyumu cha JAs. Chakutayeeyelwa, OSM antoomwe a OSS1 zili azintu zisiyene-siyene zilizyibidwe kuti zilakonzya kuleta kuyoboloka kwa JA a JA-Ile. Kwiimpana a OSS1, OSM ilasungwaazigwa loko a OSMs, mpawo OSS1 taiyungizyi nsandulo yazilonda zyaansi (Chitondeezyo 3B).
(A) Kuvwuntauzya kwakutondezya antoomwe kweendelanya akubala kwa PCC kwakuyobolola kwamahomoni aazisyango akaambo kakulya zisyango. Node yiiminina homoni yomwe yamusamu, mpawo bupati bwa node yiiminina Si index iijatikizya homoni yamusamu aakati kakusilikwa. (B) Kuyoboloka kwa JA, JA-Ile a ET mumafwaafwi kwakachitwa abusilisi bwiindene-indene butondezyedwe amibala yiindene-indene: apricot, W + OSM; buluu, W + OSSl; chisiya, W + W; mubala wadota, C (kweendelezya) . Nyenyeezi zitondezya kwiimpana kupati aakati kakusilikwa akweendelezya (nzila zyobile zya ANOVA zitobelwa a Tukey HSD kweezyanisya kwakumbele, *** P <0.001). Kuvwuntauzya kwamakani aa (C)697 MS/MS (data file S1) mu JA biosynthesis akunyongana kwakuzyiba (irAOC a irCOI1) antoomwe a (D)585 MS/MS (data file S1) mu ETR1 kakuli ET signal iitali kabotu. Nzila zyobile zyakusilisya banyama balya zisyango zyakapa kuti kube misamu iikola (EV). Nyenyeezi zitondezya kwiimpana kupati aakati kakusilikwa kwa W+OS akweendelezya kutakwe ntenda (nzila zyobile zya ANOVA zitobelwa a Tukey HSD kweezyanisya kwakumbele, *P<0.05, **P<0.01 antoomwe a ***P<0.001). (E) Ma graph aamwaikide aakukazya kwamwaikide kwabusongo. Mibala yiiminina misyobo yiindene-indene yakacincwa; zitondezyo ziiminina nzila zyakusilikwa zisiyene: nkomo yotatwe, W + OSS1; kkeyala, W + OSM; muzyunguluko C
Kuzwa waawo, twabelesya musyobo wa Nepenthes (irCOI1 a sETR1) wakasandulwa munzila iigaminide munzila zipati zya JA a ET biosynthesis (irAOC a irACO) akuzyiba (irCOI1 a sETR1) kulanga-langa mbozibeleka mahomoni aaya obile aazisyango kuli banyama balya zisyango. Kweendelana amasunko aakainda, twakasinizya kunjila kwa herbivore-OS muzisyango zitakwe chintu (EV) (Mufanikisyo 3, C kusikila ku D) akuseluka koonse kwa Hj index kwakaletwa a OSM, mpawo δj index yakatanta. Ikwiingula kulalibonya kwiinda kwiingula kwakachitwa a OSS1. Igilafu yamitwe yobile ibelesya Hj a δj mbuli zyeelelo itondezya kugwisigwa kwamilawu (Chitondeezyo 3E). Chintu chilibonya loko nchakuti mumisyobo iitakwe chitondezyo cha JA, kusiyana kwa metabolome akusanduka kwazyintu zichitwa abanyama balya zisyango zilamana (Chitondeezyo 3C). Mukwiimpana, kumvwisisisya ET kutakwe jwi muzisyango zya sETR1, nikuba kuti zyoonse zipa kuti kube kucinca kwa metabolism yazisyango zili munsi kapati kwiinda zya JA signaling, zilacesya kwiimpana kwa Hj a δj indices akati ka OSM a OSS1 excitations (Chitondeezyo 3D a Cifwanikiso S5). . Eezi zitondezya kuti kunze lyamulimo mupati wa JA signal transduction, ET signal transduction alimwi ilabeleka mbuli kubamba kabotu-kabotu nsandulo yamisyobo yanyama yazisyango. Kweendelana amulimu ooyu wakubamba kabotu, taakwe kucinca kwakachitika mukusanduka kwa metabolome muzisyango zya sETR1. Kulubazu lumwi, kweezyanisya azisyango zya sETR1, zisyango zya irACO zyakapa kuti kube kuchincha kwakusanduka kwazyakulya kwakachitwa abanyama balya zisyango, asi zyakatondezya kwiindana kupati kwa Hj a δj aakati ka OSM a OSS1 (Chitondeezyo S5).
Kutegwa tuzyibe zintu zilibedelede zijisi lugwasyo lupati mukwiingula kwamisyobo yazilenge zilya zisyango akubamba kabotu kupanga kwazyo kwiinda muzitondezyo zya ET, twakabelesya nzila ya MS/MS yakapangwa musyule. Eeyi nzila iyeeme anzila yakubunganya zintu zyobile kuti ibweze mukwasyi wa metabolic kuzwa kuzibeela zya MS/MS [normalized dot product (NDP)] antoomwe amweelwe wakukozyanya kweendelanya a neutral loss (NL). I data ya MS/MS yakapangwa kwiinda mukulanga-langa ET transgenic lines yakapanga 585 MS/MS (data file S1), eeyo yakakosolwa kwiinda mukuzibika muzibeela zili musanu ababili zya MS/MS (M) (Chitondeezyo 4A). Zimwi zyazibeela eezyi zilizwide zintu zyakale zizyibidwe kale: mbuli, M1, M2, M3, M4 antoomwe a M7 zilivwubide muzintu ziindene-indene zizwa ku phenol (M1), flavonoid glycosides (M2), acyl sugars (M3 And M4), antoomwe a 17-HGL-DTG (M7). Mukuyungizya, makani aajatikizya metabolic (Si index) ya metabolite yomwe muchibeela chimwi achimwi alabalilwa, mpawo kwaabwa kwa Si kulakonzya kubonwa. Mubufwiifwi, MS/MS spectra itondezya kulya zisyango zyiingi antoomwe a genotype specificity ilazyibwa a Si values ​​mpati, mpawo kurtosis statistics itondezya kwaabwa kwaboya kukkona lyalulyo. Imwi nzila ya colloid yakajanwa mu M1, oomo phenol amide moyakatondezya Si mpati (Chitondeezyo 4B). I 17-HGL-DTG yaambwa musule iikonzya kulya zisyango mu M7 yakatondezya Si score iili akati-akati, kutondezya kuti kuli mweelwe waakati-kati wakusiyana kwabweendelezi akati kamisyobo yobile ya OS. Mukwiimpana, bunji bwazintu zipangwa munzila iilibedelede, mbuli rutin, CGA, alimwi a acyl sugars, zili akati kazintu zijisi Si zili munsi kapati. Kuti tukonzye kuvwuntauzya kabotu bupange bwazintu antoomwe akwaabila kwa Si aakati kama metabolites, nzila ya molecule yakayakwa kuli chibeela chimwi achimwi (Chitondeezyo 4B). Kusinsima kuyandika kwa OD theory (kwaambwa muchifanikisyo 1B) nkwakuti kubambululwa kwa metabolites zilibedelede musule lyakulya zisyango kweelede kusololela kukuchincha kwa metabolites zili aampindu mpati yakukwabilila, ikapati kwiinda mukuyungizya bube bwazyo (kwiindana akumwaigwa kutayeeyelwi) Mode) Defensive metabolite yakasinsimwa a MT theory. Bunji bwazintu zizwa mu phenol zyakabungikwa mu M1 zilaswaangene akucheya kwakubeleka kwa tuzunda (32). Notwakali kweezyanisya mweelwe wa Si mu M1 metabolites aakati kamatu aayungizyidwe amafwaafwi aa EV control plants mumahola aali makumi obilwe atune, twakabona kuti metabolic specificity ya metabolites zyiingi nikwakainda tuzunda tulya zisyango kuli akuyungizya kupati (Chitondeezyo 4C). Kuyungizya kwa Si kwakajanwa buyo muma phenolamides aakwabilila, pesi taakwe kuyungizigwa kwa Si kwakajanwa muma phenols aambi alimwi azintu zitazyibidwe zyakaliko antoomwe muchibeela eechi. Eeyi ninzila iilibedelede, eeyo iiswaangene anjiisyo ya OD. Zyakuyeeyela zipati zyakusanduka kwazyakulya zichitwa abanyama balya zisyango zilazumanana. Mukusola kusunka naa ooku kusiyana kwa phenolamide kwakachitwa a OS-specific ET, twakabamba metabolite Si index mpawo twakapa kuti kube kwiimpana kwa OSM a OSS1 mu EV a sETR1 genotypes (Chitondeezyo 4D). Mu sETR1, kwiimpana kwakachitwa a phenamide aakati ka OSM a OSS1 kwakacheya loko. Nzila ya bi-clustering yakabelesegwa ku MS/MS data yakabungikwa mumisyobo iitakwe JA iizulide kuti ikonzye kuzyiba MS/MS modules zipati zijatikizyidwe a JA-regulated metabolic specialization (Chitondeezyo S6).
(A) Ikubunganya kwa 585 MS/MS kweendelanya akwaabana cibeela (NDP kukozyanya) akusweekelwa kwaabwa (NL kukozyanya) kwakapa kuti module (M) kiibe kweendelana amukwasyi uuzyibidwe, naakuti kwiinda mukutazibinkana naakuti kutali kabotu kwa Metabolite. Kumbali a module imwi aimwi, metabolite (MS/MS) iigaminide (Si) kwaabwa kutondezyedwe. (B) Modular molecular network: Nodes ziiminina MS/MS antoomwe ambali, NDP (mubala uusalala pyu) antoomwe a NL (mubala wa bbuluu) MS/MS scores (cut-off,> 0.6). Ibbazu lya metabolite specificity index (Si) lyakabikkwa mubala kweendelanya a module (kulumwensyi) mpawo lyakabikkwa muma molecule network (kululyo). (C) Module M1 ya musamu wa EV muchiimo chakubamba (kweendelezya) akuyungizigwa (chikozyano chakulya zisyango) mumahola aali makumi obilwe atune: mufanikisyo wama molecule (mpi ya Si mbupati bwa node, defensive phenolamide ilatondezyegwa mubala wa bbuluu). (D) M1 molecular network diagram ya spectrum line sETR1 iijisi EV a ET zitali kabotu: musamu wa phenolic wiimininwa a green circle node, akusiyana kupati (P ​​value) aakati ka W + OSM antoomwe a W + OSS1 mbuli bupati bwa Node. 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 sugar.
Twakayungizya buvwuntauzi kuzwa ku Nepenthes genotype yomwe yakacheya kusikila kubantu bachilengwaleza, ooko kucinca kupati kwa intraspecific mu JA levels alimwi a metabolite zimwi zyakapandululwa kale mubantu bacilengwaleza (26). Belesya data eeyi kuvwumbilila ma germplasm aali makumi aane atutatu. Aaya ma germplasms ajisi misyobo iili 123 yazisyango kuzwa ku N. pallens. Eezyi zisyango zyakatolwa kuzwa kunseke zyakayobolwa mumasena aasiyene-siyene mu Utah, Nevada, Arizona, a California (Chitondeezyo S7), twakabala kusiyana kwa metabolome (aawa kwiitwa kuti mweelwe wabantu) β kusiyana) akusiyana kwa OSM. Kweendelana aziiyo zyakachitika musule, twakabona kuchincha kupati kwakucinca kwazyakulya ku Hj antoomwe a δj, kutondezya kuti ma germplasms alijisi kwiimpana kupati mukusandula kwazyakulya kuli banyama balya zisyango (Chitondeezyo S7). Eeyi mbunga ilatuyeezya zyakabonwa musule aatala akusanduka kwa JAs kweetelezegwa abanyama balya zisyango, mpo yakabamba mpindu mpati loko mukabunga komwe (26, 36). Kwiinda mukubelesya JA a JA-Ile kulingaula kujatana aakati ka Hj a δj, twakajana kuti kuli kujatana kubotu aakati ka JA a metabolome β diversity and specialization index (Chitondeezyo S7). Eezi zitondezya kuti kusiyana kwakachitwa abanyama balya zisyango mu JAs induction kwakajanika kubantu banji kulakonzya kuba akaambo kakusiyanasiyana kwa metabolic kwakachitwa akusalwa kuzwa kuzinyama zilya zisyango.
ziiyo zyakachitika musule zyakatondezya kuti misyobo ya tombwe iliindene kapati mumusyobo akuyeeme akukwabilila kwamubili. Kusyoma kuti kucinca ooku mukusanduka kwazitondezyo zyakulwana banyama balya zisyango akukonzya kulikwabilila kweendelezyegwa akuvwula kwatuzunda, buumi bwazisyango, antoomwe amali aabelesyegwa mukupanga nzila zyakulikwabilila mubusena ooko musyobo umwi nkoukomena. Twakaiya kweendelana kwakusanduka kwamafwaafwi kwakachitwa abanyama balya zisyango zyamisyobo iili musanu aimwi ya Nicotiana iizwa kuNorth America akuSouth America. Misyobo eeyi ilaswaangene kapati a Nepenthes North America, nkokuti Nicolas Bociflo. La, N. nicotinis, Nicotiana n. bwizu butakwe nguzu, Nicotiana tabacum, tombwe uuli mumulongo, tombwe (Nicotiana spegazzinii) antoomwe a tombwe wamafwaafwi (Nicotiana obtusifolia) (Chitondeezyo 5A) (37). Misyobo iili musanu aimwi, kubikkilizya amusyobo uuzyibidwe kabotu N. please, nzisyango zimena amwaka zya petunia clade, mpawo obtusifolia N. nzisyango zizyala lyoonse zya sister clade Trigonophyllae (38). Kuzwa waawo, W + W, W + OSM antoomwe a W + OSS1 zyakachitilwa kumisyobo eeyi iili musanu ababili kuti twiiye mbozibambululwa zyakulya zyatuzunda.
(A) Musamu wa bootstrap phylogenetic uuyeeme akulangilwa kupati [kwakupanga glutamine ya nyukiliya (38)] akumwaigwa kwamisyobo ya Nicotiana iili musanu ababili (mibala yiindene) (37). (B) Chitondeezyo chakusiyana kwamisyobo ya Nicotiana iili musanu ababili (939 MS/MS; data file S1). Aawo musyobo, kusiyana kwa metabolome takuli kweendelana abulemu bwakuziba. Kuvwuntauzya kwakujatana kwamisyobo yazilenge aakati kakusiyanasiyana kwazyakulya antoomwe akuvwula kwa JA kutondezyedwe muchifanikisyo 2. S9. Mubala, misyobo isiyene; makona otatwe, W + OSS1; kkeyala, W + OSM; (C) Nicotiana JA a JA-Ile zyeendelana a OS excitation amplitude (two-way ANOVA a Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 and * ** Kweezyanisya W + OS a W + W, P <0.001). Chitondeezyo cha (D) kusiyana a (E) kusiyana kwamusyobo umwi aumwi nikwakamana kweezyanisya zyakulya zilya zisyango a methyl JA (MeJA). Nyenyeezi itondezya kwiimpana kupati aakati ka W + OS a W + W naa lanolin antoomwe a W (Lan + W) naa Lan antoomwe a MeJA (Lan + MeJa) a Lan control (kuvwuntauzya kwakusiyana kwanzila zyobile, kutobelwa a Tukey's HSD post hoc multiple comparison , *P<0.05, **P<0.01 a ***P<0.01).
Kubelesya nzila yazibeela zyobile, twakajana zibeela zili 9 zya 939 MS/MS (data file S1). Bupange bwa MS/MS bwakabambululwa kwiinda mukubelesya misamu yiindene-indene buliindene kapati akati kazibeela ziindene-indene akati kamisyobo (Mufanikisyo S8). Kulanga mumizeezo Hj (yaambwa aawa kuti γ-kwiindana kwamusyobo) a δj kutondeezya kuti misyobo isiyene-siyene ilabungana mutubunga tuindene-indene muchibaka cha metabolic, oomo kwaandaana kwamisyobo-misyobo mokulibonya kwiinda kukkomana. Kusiya buyo N. linear a N. obliquus, zitondezya nzila zyiingi zyakuchita zintu (Chitondeezyo 5B). Mukwiimpana, misyobo iili mbuli N. purpurea a N. obtusifolia ilaa nsandulo iitaboneki kabotu yakusilikwa, asi nzila ya metabolome iliindene. Kwaabila kwamisyobo yazilenge kwakapa kuti kube kujatana kutali kabotu aakati kakusiyanasiyana kwa gamma (PCC = -0.46, P = 4.9×10-8). Kuchincha kwa OS mu JAs kulaswaangene kabotu a metabolome, mpawo kuliswaangene munzila mbyaabi a metabolic gamma diversity itondezyedwe amusyobo umwi aumwi (Chitondeezyo 5B aChitondeezyo S9). Nchintu chibotu kuzyiba kuti misyobo yazilenge zyaambwa kuti "ziingula" muchifanikisyo 5C, zilimbuli Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, antoomwe a Nepenthes attenuated, zyakapa kuti kube zitondezyo zipati mumaminiti aali 30. Kuzwa kwa OS-specific JA antoomwe a JA-Ile, kakuli zimwi zilwazi zyaambwa kuti "zitavwiilili", zilimbuli Nepenthes mills, Nepenthes powdery antoomwe a N. obtusifolia zitondezya buyo JA-Ile Edge induction kakunyina OS specificity (Figure 5C). Aawo mpokugolela, mbuli mbukwaambidwe atala aawa, ku Nepenthes yakacheya, zintu zitambula zitondezyo zyakatondezya OS specificity mpawo zyakayungizya δj, mpawo kazicesya Hj. Ooku OS-specific priming effect tiikwakajanwa mumisyobo yazilenge zitabeleki kabotu (Chitondeezyo 5, D a E). OS-specific metabolites zilaabwa chiindi chiingi aakati kazilenge zitambula zitondezyo, mpawo eezyi zitondezyo zilabungana amisyobo iitakwe nguzu zyakuvwiila zitondezyo, kakuli misyobo iijisi zitondezyo zitakwe nguzu zitondezya kujatana kusyoonto (Chitondeezyo S8). Eezyi mpindu zitondezya kuti OS-specific induction ya JAs antoomwe a OS-specific reconfiguration ya downstream metabolome zilaswaanganizyigwa a species level.
Kuzwa waawo, twakabelesya musamu wa lanolin uujisi methyl JA (MeJA) kusilika misamu kutegwa tuvwuntauzye naa nzila eezyi zyakuswaanganya zilalesyegwa akaambo kakujanika kwa JA iibelesyegwa a JA iizwa anze, eeyo iyooba mu cytoplasm yazisyango. Kufwambaana kugwisigwa kwamaanzi nku JA. Twakajana nzila njiyonya yakucinca asyoonto-asyoonto kuzwa kumisyobo iitambula zitondezyo kuya kumisyobo iitavwili zitondezyo akaambo kakuzumanana kwa JA (Mufanikisyo 5, D a E). Mubufwiifwi, busilisi bwa MeJA bwakabambulula loko ma metabolomes aa linear nematodes, N. obliquus, N. aquaticus, N. pallens, antoomwe a N. mikimotoi, zyakapa kuti kube kuyungizigwa kupati kwa δj akucheya kwa Hj. N. purpurea yakatondezya buyo kuyungizigwa kwa δj, pesi kutali Hj. N. obtusifolia, eeyo yakatondeezyegwa kuti ilayobolola ma JAs masyoonto loko, ayalo tiikonzyi kugwasyigwa a MeJA mukubambulula metabolome. Eezyi mpindu zitondezya kuti kupanga JA naakuti kusandula zitondezyo zilalesyegwa mumubili wazilenge zitavwiilili zitondezyo. Kusunka muzeezo ooyu, twakaiya misyobo yone (N. pallens, N. mills, N. pink antoomwe a N. microphylla) yakayungizigwa a W + W, W + OSMs antoomwe a W + OSS1 Transcriptome (39). Kweendelana anzila yakubambulula metabolome, misyobo eeyi iliimpene kabotu mubusena bwakulemba, aakati ka N. attenuated yakatondezya OS-induced RDPI mpati, mpawo N. gracilis yakatondezya nsyoonto (Chitondeezyo 6A). Nokuba boobo, kwakajanwa kuti kusiyana kwama transcriptome kwakachitwa a N. oblonga kwakali munsi kapati akati kamisyobo yone, mukwiimpana akusiyana kwa metabolomic kwa N. oblonga kwakatondezyegwa musyule mumisyobo iili musanu ababili. Ziiyo zyakachitika musule zyakatondezya kuti tubunga twama genes tujatikizya zitondezyo zyakulikwabilila, kubikkilizya azitondezyo zya JA, zipandulula bube bwakulikwabilila kwakusaanguna kuchitwa abasikulya zisyango mumusyobo wa Nicotiana (39). Kweezyanisya nzila zya JA akati kamisyobo yone eeyi kwakatondeezya nzila iikkomanisya (Chitondeezyo 6B). Bunji bwama genes munzila eyi, mbuli AOC, OPR3, ACX antoomwe a COI1, zyakatondezya zyeelelo zisumpukide zyakutalisya mumisyobo yone eeyi. Nokuba boobo, gene iiyandika kapati, JAR4, ilasandula JA kuba nzila yayo iibeleka ya JA-Ile, mpawo mweelwe wayo wakulemba uli munsi loko, ikapati mu N. mills, Nepenthes pieris a N. microphylla. Mukuyungizya, buyo lugwalo lwa gene limbi AOS tiilwakajanwa mu N. bifidum. Kuchincha ooku mukutondezya kwamajini kulakonzya kuba akaambo kakuboneka kupati kwakachitika akaambo kakucheya kwa JA mumisyobo yazilenge zitakwe nguzu zyakutondezya akutalika kwa N. gracilis.
(A) Kuvwuntauzya kwamakani aakusanduka kwakusaanguna kwakulemba kwamisyobo yone ya tombwe yakasampulwa maminiti aali 30 kuzwa nikwakatalisyigwa kulya zisyango. RDPI ilabalilwa kwiinda mukweezyanisya matu aakaletwa a herbivore OS antoomwe akweendelezya zilonda. Mibala itondezya misyobo yiindene, mpawo zitondezyo zitondezya nzila zyakusilikwa ziindene. (B) Kuvwuntauzya kwakutondezya kwamajini munzila zya JA akati kamisyobo yone. Nzila ya JA iitakatazyi itondezyedwe afwaafwi achifanikisyo chakabbokesi. Mibala yiindene itondezya nzila zyakuchita zintu ziindene. Inyenyeezi itondezya kuti kuli kwiindana kupati aakati ka W + OS abusilisi bwa W + W (ku Student's t-test yakwiimpana kwababili, *P<0.05, **P<0.01 antoomwe a ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Muchibeela chamamanino, twakayiya mbuli misyobo yatuzunda iikonzya kuchincha mubili wamisyobo yiindene-indene yazisyango mboikonzya kukazya banyama balya zisyango. Buvwuntauzi bwakainda bwakakankaizya musyobo wa Nicotiana. Kukazya kwabo kuli Ms a larvae kuliindene loko (40). Aawa, twakayiya kujatana aakati kachikozyano eechi akusanduka kwabo. Kubelesya misyobo yone ya tombwe iili atala aawa, akusola kujatana aakati kakusiyana akusiyana kwa metabolome kweetelezegwa abanyama balya zisyango akukazya kwazisyango kuli Ms a Sl, twakajana kuti kukazyigwa, kusiyana akuba aluzibo kuli generalist Sl Zyoonse zilaswaangene kabotu, kakuli kujatana aakati kakukazyigwa kuli bamakaintu basyaazibwene aluzibo takuli kabotu pe, mpawo kusiyana ooku takuyandiki pe (Mufanikisyo 10). Kujatikizya kukazyigwa kwa S1, zyoonse zyobile N. chinensis a N. gracilis, eezyo zyakatondezyegwa kale kuti zitondezya zyoonse zyobile JA signal transduction levels antoomwe a metabolome plasticity, zyakalijisi nsandulo ziindene kapati kukunjizya kwa herbiver induction, mpawo azyalo zyakatondezya kukazyigwa kupati kukozyenye. Koonana.
Muminyaka iili makumi musanu aibili yainda, njiisyo yakukwabilila zisyango yakapa nzila yakuyiisya, oomo basikuvwuntauzya mobakasinsima mweelwe mupati wakusanduka amilimo yazisyango zilibedelede. Bunji bwamizeezo eeyi taitobeli nzila iili kabotu yakujana twaambo tuyumu (41). Bakapa mizeezo iiyandika kapati (3) aawo mpaakali kulanga-langa. Eelyo kulingaula kwazyintu zipati zyakuyeeyela nokwazumizya kuti mizeezo imwi ilangwe-langwe, eezi ziyoopa kuti mulimo uya kunembo. Kogwasigwa, pesi kokaka bamwi (42). Muciindi, njiisyo mpya ilasinsima mumasena aasiyene-siyene aakulanga-langa mpawo iyungizya nzila mpya yakupandulula (42). Nokuba boobo, mizeezo yobile yakaambwa aawo mpobeelede kubeleka, MT a OD, ilakonzya kupandululwa kuti nzintu ziyandika kapati zyakusinsima kucinca kwamubili kwalo kucitika akaambo kazyintu zilya zisyango: njiisyo ya OD isyoma kuti kucinca kwa “busena” bwakucinca kwamubili kulasololela kapati. Njiisyo ya MT isyoma kuti kuchincha ooku takukonzyi kweenda mpawo kuyooba biyo muchibaka cha metabolic, mpawo kulakonzya kuba azintu zigwasya kapati. Kulangisisya kwakaindi kwa OD antoomwe a MT kwakasunkwa kubelesya nzila nsyoonto ya "kwabilila" misamu. Ooku kulingaula kwa metabolite-centric kulesya nguzu zyakulanga-langa bupati anzila yakusanduka kwa metabolome muchiindi chakulya zisyango, mpawo takuzumizyi kulingaula mukati kachibalo chili kabotu kuti kuyandika kuyeeyelwa kupati ooko kukonzya kubonwa mbuli chintu choonse. Aawa, twakabelesya nzila mpya ya metabolomics kweendelanya a computational MS mpawo twakachita deconvolution MS analysis mubweendelezi bwa data theory descriptors kuti tusunke kwaandaana aakati kazeezi zyobile zyaambwa a metabolomics yanyika yoonse. Kusinsima kupati kwa njiisyo eeyi. Njiisyo yamakani yakabelesegwa mumbazu zyiingi, kampatila mubuvwuntauzi bwazintu zipona azyakulya (43). Nokuba boobo, mbuli mbutuzyi, eeyi njenzila yakusaanguna kubelesegwa kupandulula busena bwamakani aazyakulya zyazisyango akumanizya mapenzi aajatikizya kucinca kwazyakulya kwachiindi chifwiifwi mukuvwiila zitondezyo zyabusena. Mubwini, inguzu zyanzila eeyi zili mukukonzya kwayo kweezyanisya nzila zili mukati aakati kamisyobo yazisyango kuti ibone mbuli banyama balya zisyango mbozyakasanduka kuzwa kumisyobo yiindene kuya kunzila zyakusanduka kwamisyobo yiindene-indene mumasena aaindene-indene aakusanduka. Kusanduka kwamubili.
Kuvwuntauzya kwazibeela zipati (PCA) kusandula data iijisi zintu zyiingi kuba busena bwakucesya bupati kuchitila kuti nzila mpati ya data ipandululwe, aboobo kanjikanji ibelesyegwa mbuli nzila yakuvwuntauzya yakuvwuntauzya data, mbuli deconvolution metabolome. Nokuba boobo, kucesya bupati kuyoosweekelwa cibeela chamakani aali mu data, mpawo PCA taikonzyi kupa makani aabunji aatala abukkale bujatikizya njiisyo zyabuumi, mbuli: ino mbuti banyama balya zisyango mbobukonzya kubamba zintu ziindene-indene mumbazu zilibedelede (mbuli buvubi, kwaabila) akuvwula) zintu zipangwa mumubili? Ino nzintu nzi zipangwa mumubili zikonzya kusinsima chiimo chamunyama uulya zisyango? Kuzwa mukulanga kwakusalazya, kusiyana akukonzya kuchitwa, makani aali mukati ka metabolite yamafwaafwi alabola, mpawo kwajanika kuti kulya banyama balya zisyango kulakonzya kuchita kuti metabolism imwi ibeleke. Kakunyina kulangila, twakabona kuti, mbuli mbokwaambidwe muzitondezyo zya makani zyakabelesegwa, bukkale bwakusanduka kwazyakulya bwakazwa musule lyakulwanwa kwabanyama bobile balya zisyango (Sl) a Solanaceae syaazibwene Ms. Nikuba kuti kulya kwabo akubikkila maanu kwabo kuliindene kapati. Mafuta-amino acid conjugate (FAC) initiator mu OS (31). Kwiinda mukubelesya OS ya herbivore kusilika zilonda zyakuumwa, busilisi bwa herbivore bwakatondezya nzila iikozyenye. Eeyi nzila iizumizyidwe yakukozyanisya nsandulo yazisyango kukulwanwa abanyama balya zisyango ilagwisya twaambo tunyonganya tuboola akaambo kakucinca kwabukkale bwakulya bwabanyama balya zisyango, eezyo zipa kuti kube kunyonyooka kwamisyobo yiindene-indene muziindi ziindene-indene (34). FAC, eeyo iizyibidwe kuti njenzila mpati ya OSM, ilacesya JAS azimwi nsandulo zya mahomoni aazisyango mu OSS1, mpawo OSS1 ilacesya ziindi zyiingi (31). Nokuba boobo, OSS1 yakapa kuti kube kuvwula kwa JA kweelanisigwa a OSM. Cakatondezyegwa kale kuti nsandulo ya JA mu Nepenthes iitakwe nguzu ilakonzya kumvwisisisya OSM, ooko FAC nkoikonzya kuzumanana kubeleka nikuba kuti yasanganyigwa 1:1000 amaanzi (44). Aboobo, kweezyanisya a OSM, nikuba kuti FAC mu OSS1 ili munsi loko, ilazula kuchita kuti JA izwe. Ziiyo zyakachitika musule zyakatondezya kuti ma protein aali mbuli porin (45) antoomwe ama oligosaccharides (46) alakonzya kubelesegwa mbuli zitondezyo zya molecule kuti zisyango zikwabilile mu OSS1. Nokuba boobo, tachilibonyi kuti naa eezyi zintu zili mu OSS1 nzizyo zipa kuti JA iyungizigwe muchiiyo chiliwo lino.
Nikuba kuti kuli ziiyo ziche zipandulula kusiyana kwakusanduka kwaminwe kwachitika akaambo kakubelesya banyama balya zisyango zisiyene naakuti JA naakuti SA (salicylic acid) (47), taakwe naba omwe wakanyonganya kunyongana kwamisyobo yabanyama balya zisyango mumulongo wamasokwe aazisyango anzyozichita kuzintu zimwi. makani aajatikizya muntu. Ooku kuvwuntauzya kwakasinizya kuti kujatana kwamahomoni aamukati amahomoni aambi aamisamu kunze lya JAs kubumba bube bwakusanduka kwazyakulya kweetelezegwa abanyama balya zisyango. Mubwini, twakabona kuti ET yakaletwa a OSM yakali mpati kwiinda eeyo yakaletwa a OSS1. Eeyi nzila ileendelana azintu zyiingi zya FAC mu OSM, eezyo ziyandikana alimwi zizulide mukutalisya ET burst (48). Mubukkale bwakuyanzana aakati kazisyango abanyama balya zisyango, mulimo wakutondezya wa ET kuzyintu zipangwa muzisyango uchichili chiindi chifwiifwi mpawo ulanganya buyo kabunga komwe. Mukuyungizya, ziiyo zyiingi zyakabelesya kubelesya ET kunze naakuti zintu zyakasaanguna naakuti zintu zimwi zilesya ET kwiiya kweendelezyegwa kwa ET, aakati kazyeezyo kubelesya misamu eeyi kuyoopa kuti kube ntenda zyiingi zitali zyamasimpe. Kweelana aluzibo ndotujisi, chiiyo eechi nchitondezyo chakusaanguna chipati chakuvwuntauzya mulimo wa ET mukubelesya ET kupanga akuziba misamu iitabeleki kabotu kuchitila kuti misamu ichite kabotu. Kuyungizya kwa ET kwa banyama balya zisyango kulakonzya kuchincha nzila zya metabolome. Chipati loko nkucinca kwa ET biosynthesis (ACO) antoomwe a perception (ETR1) ma genes kwalo kwakayubununa kuyoboloka kwa phenolamides kwalo kuli banyama balya zisyango. Cakatondezyegwa kale kuti ET ilakonzya kubamba kabotu kuyoboloka kwa nicotine akaambo ka JA kwiinda mukubamba putrescine N-methyltransferase (49). Nokuba boobo, kuzwa munzila yakulanga zintu, tachilibonyi kabotu kuti ET ibamba biyeni kunjila kwa phenamide. Mukuyungizya kumulimo wakusandula twaambo twa ET, kweenda kwamubili kulakonzya kusandulwa kuya ku S-adenosyl-1-methionine kuchitila kuti kube kweendelezya kubikka mali mu polyaminophenol amides. S-adenosyl-1-methionine ninzila yakupanga polyamine. Nzila eeyo ET signal njiyeendelezya mweelwe wa phenolamide iyandika kuvwuntauzyigwa.
Kwachiindi chilamfu, akaambo kamweelwe mupati wama metabolites aatazyibidwe bupange, kubikkila maanu kapati kuzibeela zimwi zya metabolic tiikwakali kukonzya kulanga-langa kucinca kwachiindi kwakusiyana kwa metabolic nikwakamana kujatana kwa zintu zipona. Kwalino, kweendelanya akuvwuntauzya kwamakani, mpindu mpati ya MS/MS spectrum acquisition kweendelanya a metabolites iitakwe lusalululo njakuti banyama balya zisyango balya naa kweezyanisya banyama balya zisyango bazumanana kucesya kusiyana kwa metabolic yoonse ya metabolome yamafwaafwi kumwi kabayungizya lwiiyo lwabo. Ooku kuyungizigwa kwakaindi kaniini kwa metabolome kwakachitwa abanyama balya zisyango kulaswaangene akuyungizya kwa synergistic kwa transcriptome. Chintu chigwasya kapati mukupa kuti metabolome ibe mpati (iijisi Si value mpati) ni metabolite iilibedelede iijisi mulimo wakulya zisyango wakazyibidwe kale. Eeyi nzila ileendelana akusinsima kwa OD theory, asi kusinsima kwa MT kweendelana akutayeeyelwa kwa metabolome reprogramming takuli kweendelana pe. Nokuba boobo, data eeyi ilazuminana akuyeeyelwa kwamusyobo uusanganizidwe (MT mubotu; Cifwanikiso 1B), nkaambo zimwi zintu zitazyibidwe zitazyibidwe zyakukwabilila zilakonzya kutobela kwaabwa kwa Si kutalangilwi.
Chimwi chintu chibotu chakajanika mubuvwuntauzi oobu nchakuti kuzwa kukusanduka kusyoonto (musamu omwe atombwe) kusikila kukusanduka kupati (musyobo watombwe uuli afwaafwi), zibeela zisiyene zyakusanduka zili "mukwabilila kubotu" Kuli kwiindana kupati mukukonzya kwabanyama balya zisyango. Moore abamwi. (20) a Kessler a Kalske (1) bakakankaizya lwabo beni kuti basandule zibeela zyotatwe zibeleka zyabuumi bwiindene-indene zyakasaanguna kwaandaanizyigwa a Whittaker (50) kuti zibe zintu zipa kuti kube kucinca kwakaindi kasyoonto kwakusiyana kwamakemikalu; aaba balembi tiibakapandulula mubufwiifwi Nzila zyakuyobolola data ya metabolome mpati azyalo tazitondezyi mbokukonzya kubalilwa kusiyana kwa metabolome kuzwa ku data eeyi. Mukuvwuntauzya ooku, kuchincha kusyoonto kwa Whittaker's functional classification kuyoolanga α-metabolic diversity mbuli kusiyana kwa MS/MS spectra mumusamu umwi, antoomwe a β-metabolic diversity mbuli basic intraspecific metabolism ya nkamu yabantu.
Ichitondezyo cha JA nchintu chipati kulibanyama balya zisyango. Nokuba boobo, kuli kubula kwakusunkwa kwakusinizya kwakugwasizya kwa intraspecific regulation ya JA biosynthesis ku metabolome diversity, alimwi akuti naa JA signal mbusena bupati bwa metabolic diversification akaambo kakukatazyigwa mumizeezo a macroevolutionary scale kucili kukatazya kuzyiba. Twakabona kuti bube bwakulya zisyango bwa Nepenthes herbivorous bupa kuti kube kuchincha kwa metabolome mukati kabantu ba Nicotiana alimwi aakati ka Nicotiana species iili afwaafwi kweendelana a JA signaling. Mukuyungizya, eelyo chitondezyo cha JA nichakachilwa, kusanduka kwamubili kwakachitwa amuntu omwe uulya zisyango kuyoomana (Chitondeezyo 3, C a E). Mbwaanga kucinca kwa metabolic spectrum yabantu ba Nepenthes batakwe nguzu zyachilengwa leza nchintu chijatikizya mweelwe, kucinca kwa metabolic β diversity a specificity mukulanga-langa ooku kulakonzya kuchitwa akukankaminwa kupati kwa metabolite-rich compound categories. Eezyi zibeela zya misamu zyeendelezya chibeela cha metabolome mpawo zisololela kukujatana kubotu azitondezyo zya JA.
Nkaambo kakuti nzila zya biochemical zyamusyobo wa tombwe uujatikizyidwe kapati ziliindene kapati, zintu zipangwa mumubili zilazyibwa kapati munzila yakupima, aboobo ncintu ciyandika kapati mukuvwuntauzya. Njiisyo zya makani aakubamba zyintu zyakajatwa zya metabolic zitondezya kuti kulya zisyango kupa kuti kutabi kusiyana aakati ka metabolic gamma diversity akuba aluzibo. Chitondeezyo cha JA chili amulimu mupati mukuzuminana ooku. Kuyungizya kwabusongo bwa metabolome kweendelana akuyeeyelwa kwa OD mpawo kuli kweendelana a JA signal, mpawo JA signal tiiliswaangene a metabolic gamma diversity. Eeyi mikozyanisyo itondezya kuti nguzu zya OD zyazisyango zyeendelezyegwa a plasticity ya JA, nikuba a microevolutionary scale naakuti a evolutionary scale mpati. Kusola kwakubelesya JA kwaanze kwalo kuzyunguluka tumpenda twa JA biosynthesis kwakayubununa kuti misyobo ya tombwe iiswaangene kapati ilakonzya kwaandaanizyigwa kuba misyobo iitambula zitondezyo alimwi amisyobo iitavwili zitondezyo, mbubwenya mbuli nzila yabo ya JA alimwi a metabolome plasticity iiletwa abanyama balya zisyango. Misyobo iitavwiili zitondezyo taikonzyi kuvwiila akaambo kakutakonzya kupanga JA yamukati aboobo ilakonzya kuba amapenzi aamubili. Eezi zilakonzya kuchitwa akusanduka kwamajini amwi aayandika kapati munzila ya JA (AOS a JAR4 mu N. crescens) ya. Eechi chakazwa chitondezya kuti eezyi nzila zyakusanduka kwamisyobo yiindene-indene zilakonzya kuchitwa akuchincha kwakuzyiba mahomoni aamukati akuvwiila.
Mukuyungizya kukuswaangana aakati kazisyango abanyama balya zisyango, kuvwuntauzya kwakusiyana kwakusanduka kwazyakulya kulaswaangene akusumpuka kwamizeezo yoonse iiyandikana mukuvwuntauzya kwakuzyibilana kwabuumi kubusena akusanduka kwabube bwazintu zikatazya kumvwisisisya. Akuyungizya mweelwe wa data iijanwa azibelesyo zyamazubaano zya MS, kulingaula kwakuyeeyela aatala akusiyana kwa metabolic lino kulakonzya kwiinda kwiimpana kwa metabolite yamuntu omwe-omwe akuchita buvwuntauzi bwanyika yoonse kuchitila kuyubununa nzila zitalangilwi. Munzila yakuvwuntauzya kupati, chikozyano chiyandikana muzeezo wakuyeeya maapu aagwasya aakonzya kubelesegwa kuvwuntauzya data. Aboobo, mpindu mpati yakusanganya kwa MS/MS metabolomics iitakwe kampenda antoomwe a njiisyo yamakani njakuti ipa nzila nguba iikonzya kubelesegwa kupanga maapu kulanga kusiyana kwa metabolic muzibeela zya taxonomic zisiyene. Nchintu chiyandikana loko munzila eyi. Chiiyo chakusanduka kwazintu ziniini/zipati antoomwe abukkale bwabantu.
Aawo mpokugolela kusanduka kupati, njiisyo yakusanduka antoomwe kwa Ehrlich a Raven (51) nkuyeyela kuti kusiyana kwamisyobo yazisyango nchintu chipa kuti misyobo yazisyango iindene. Nokuba boobo, muminyaka iili makumi musanu kuzwa muchiindi eechi chakasimbwa, muzeezo ooyu teensi wakasunkwa pe (52). Eezi zilachitika akaambo kakuti kuli bube bwakuzyalana bwamisyobo yazisyango zikozyania mumisyobo yazisyango zili kulaale. Kubula kujanika kulakonzya kubelesegwa kubamba nzila zyakuvwuntauzya. Inzila ya MS/MS yamazubaano iibelesyegwa a njiisyo ya makani ilapima kukozyanya kwa MS/MS kwama metabolites aatazyibidwe (kakunyina kusala metabolite) mpawo isandula ma MS/MS aaya kuba MS/MS, aboobo mu metabolism yabasyaazibwene Eezyi zikozyanyo zya macro-evolutionary zilakozyanisyigwa muchiimo chakusalazya. Zitondezyo zyakubala ziuba. Nzila eeyi ilikozyene akuvwuntauzya kwa phylogenetic, ooko kukonzya kubelesya nzila zyakutobelana kutegwa kuzyibe mweelwe wakusiyana naa kusanduka kwabuntu kakunyina kusinsima.
Kuchiimo cha biochemical, muzeezo wakulanga-langa wa Firn a Jones (53) utondezya kuti kusiyana kwa metabolic kulajatwa mumasena aasiyene-siyene kuti kube zintu zitabelesyegwi zyakubelesya kuchita milimo ya biochemical ya metabolites zyatakali kweendelana naakuti zyakanjililwa busena. Nzila zyakuyiisya makani zipa nzila oomo kusanduka kwa metabolite-specific kuchitika muchiindi chakusanduka kwa metabolite kulakonzya kubalwa mbuli chibeela cha nzila yakulanga-langa kusanduka: kusanduka kwabuumi kuzwa kukubula kusalazya kuya ku kusalazya kupati.
Zyoonse antoomwe, kumatalikilo aa biology ya molecule, njiisyo ziyandikana zyakukwabilila zisyango zyakabambwa, mpawo nzila zyeendelezyegwa akuyeyela zyaambwa kuti njenzila ilikke yakusumpula sayensi. Eezi zilachitika akaambo kakulezya kwabusongo bwakupima metabolome yoonse. Nikuba kuti nzila zyeendelezyegwa akuyeeyela zilagwasya kapati mukusala nzila zimbi zipa kuti kube ntenda, inguzu zyazyo zyakusumpula kumvwisisisya kwesu kwazyintu zipona zili munsi kwiinda nzila zyakubala ziliwo lino musayensi iibelesya data mazubaano. Aboobo, mizeezo iitakonzyi kweezyeezyegwa ili kule loko kwiinda data iiliwo, aboobo nzila yakuyeeyela/kusunkwa kwakuyaambele mumulimo wakuvwuntauzya takukonzyi kugwisigwa pe (4). Tulabona kuti nzila yakubelesya kkompiyuta ya metabolomics yakatalisyigwa aawa ilakonzya kubusya luyandisisyo mumakani aamazubaano (mbuti) alimwi aamamanino (nkaambo nzi) aajatikizya kusiyana kwa metabolic, alimwi akugwasya kuciindi cipya ca sayensi ya data iisololelwa anjiisyo. Chiindi eecho chakalanga lubo njiisyo ziyandika zyakasungwaazya mazyalani aakainda.
Kusanina banyama balya zisyango kuchitwa kwiinda mukukomezya nziba zyabili naakuti nziba ya Sl aacisamu comwe cijisi mubala uusiya-siya wacisamu comwe cijisi maluba aa rose, antoomwe azisyango zili kkumi zya musamu omwe. Inswi zya tuzunda zyakajatwa aama clamps, mpawo nyama yakasyaala yamafwaafwi yakabwezelwa mawoola aali 24 a 72 kuzwa nikwakachitwa bulwazi mpawo yakafwambaana kuyobolwa, mpawo zintu zyakagwisigwa.
Kozyanisya busilisi bwazisyango munzila iibambidwe kabotu. Nzila njakubelesya mavwili aachikozyano chachisani kugonka milongo yotatwe yamaamvwa kulubazu lomwe-lomwe lwamukati wamafwaafwi otatwe aamusamu muchiindi chakukomena kwachisani, mpawo mpawo ubike 1:5 Diluted Ms. Naa kubelesya minwe iijisi magilovu kubikka S1 OS muchilonda. Tebula akubamba tuzunda mbuli mbokwaambidwe atala aawa. Belesya nzila yakaambwa musule kuti ugwisye zintu zipangwa mumubili alimwi amahomoni aazisyango (54).
Kukubelesya kwa JA, matu otatwe aamaluba aali cisambomwe aamusyobo umwi aumwi alasilikwa a 20μl ya lanolin paste iijisi 150μg MeJA (Lan + MeJA), antoomwe a 20μl ya lanolin antoomwe abusilisi bwazilonda (Lan + W), naa kubelesya 20μl ya lanolin mbuli nzila yakulesya. Matu aka tebulwa mawoola aali makumi musanu aabili aabili kuzwa muchiindi chakusilikwa, mpawo akabikkwa mumuwo wa nitrogen, mpawo akabikkwa a -80°C kusikila akabelesegwe.
Misyobo yone ya JA antoomwe a ET, irAOC (36), irCOI1 (55), irACO antoomwe a sETR1 (48), yakajanwa mukabunga kesu kakuvwuntauzya. irAOC yakatondeezya kucheya kwa JA a JA-Ile, mpawo irCOI1 tiiyakali kukonzya kumvwisisisya JAs. Kweezyanisya a EV, kuyoboloka kwa JA-Ile kwakatanta. Mbubwenya buyo, irACO iyoocheesya kupanga kwa ET, mpawo kweelanisigwa a EV, sETR1, eeyo iitakwe maanu ku ET, iyooyungizya kupanga kwa ET.
I photoacoustic laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) ilabelesyegwa kuchita ET yakupima kakunyina kunjila mumubili. Mpawo biyo nikwakamana kusilikwa, chisela chamafwaafwi chakatemwa mpawo chakabikkwa mumupika wagilazi uujalilidwe wa 4-ml, mpawo busena bwamutwe bwakazumizigwa kuti buvwule mukati kamawoola aali 5. Muchiindi chakupima, chikopo chimwi achimwi chakali kusanzyigwa amuuya uusalala uusika ku 2 litres/hour kwamaminiti aali 8, walo wakainda muchibelesyo chakapegwa a Sensor Sense kuti chigwisye CO2 amaanzi.
Data ya microarray yakasaanguna kumwaigwa mu (35) mpawo yakabambwa mu National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (namba yakunjilila GSE30287). Data iijatikizya matu aakaletwa a W + OSMs abusilisi butanyonyooki yakagwisigwa muchiiyo eechi. Kuyuma kwa raw nku log2. Kakutanaba kuvwuntauzya kwamweelwe, mweelwe wakusaanguna wakasandulwa mpawo wakabambululwa kusika ku 75th percentile kubelesya R software package.
Data yakusaanguna ya RNA sequencing (RNA-seq) yamusyobo wa Nicotiana yakajanwa kuzwa ku NCBI Short Reading Archives (SRA), namba yapulojekiti ngu PRJNA301787, eeyo yakaambwa a Zhou abamwi. (39) mpawo uzumanane mbuli mbokwaambidwe muli (56). Data iitaninga yakabambwa a W + W, W + OSM antoomwe a W + OSS1 yeendelana amusyobo wa Nicotiana yakasalwa kuti ivwuntauzyigwe muchiiyo eechi, mpawo yakabambwa munzila itobela: Chakusanguna, kubala kwa RNA-seq kwakasandulwa kuba FASTQ. HISAT2 isandula FASTQ kuba SAM, mpawo SAMtools isandula ma fayela aa SAM kuba ma fayela aa BAM aasalala. StringTie ibelesyegwa kubala zitondezyo zya gene, anzila yayo yakutondezya njakuti kuli zibeela zili zyuulu zya zibeela zya base zili 1 miliyoni zya zibeela zyakulemba zitobelene.
Ibbazu lya Acclaim chromatographic (150 mm x 2.1 mm; bupati bwazibeela 2.2μm) lyakabelesegwa mukuvwuntauzya antoomwe a 4 mm x 4 mm guard column zilijisi zintu zyomwe. Inzila yabili iitobela ibelesyegwa mu Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system: 0 kusikila ku 0.5 maminiti, isocratic 90% A [maanzi aatakwe ion, 0.1% (v/v) acetonitrile a 0.05% formic acid] B, 10% acetonitrile 0.05% asidi yafoomu); 0.5 kusikila ku 23.5 maminiti, chiindi chakusanduka chili 10% A a 90% B, kweendelana; 23.5 kusikila ku 25 maminiti, isocratic 10% A a 90% B. Mweelwe wakuzuza ngu 400μl/min. Kuzyoonse zyakuvwuntauzya zya MS, bikka musamu uuzwa mumulongo mu quadrupole a time-of-flight (qTOF) analyzer iijisi electrospray source iibeleka munzila ya positive ionization (capillary voltage, 4500 V; capillary outlet 130 V ; Drying temperature 200°C; drying airflow 10 min/liter).
Chita MS / MS fragment analysis (aawa yiitwa kuti MS / MS) eeyo iitakwe makani naa iitakonzyi kwaandaanizyigwa kuzwa ku data kuti ujane makani aajatikizya mbozipangidwe zintu zyoonse. Muzeezo wanzila ya MS/MS iitakwe kusalulula uyeeme akuti quadrupole ilijisi windo pati lyakugwisya bunji bwazintu [aboobo, kolanga zitondezyo zyoonse zya mweelwe-a-nguzu (m/z) mbuli zibeela-beela]. nkaambo kaako, nkaambo kakuti chibelesyo cha Impact II tiichakali kukonzya kupanga CE tilt, buvwuntauzi bwiingi bwakachitilwa kubelesya nguzu zyakudonkolana zyakayungizyigwa (CE). Mubufwiifwi, sanguna kulanga musamu kwiinda mukubelesya UHPLC-electrospray ionization/qTOF-MS kubelesya nzila ya mass spectrometry yomwe (bukkale bwakutyompwa buniini buzwa kukutyoka kwamukati), kulanga kuzwa ku m/z 50 kusikila ku 1500 a frequency yakwiinduluka ya 5 Hz. Belesya nitrogen mbuli muya wakudonkolana mukuvwuntauzya kwa MS/MS, mpawo uchite zipimyo zyakulisalila kuma voltage one aatobela aakudonkolana: 20, 30, 40, antoomwe a 50 eV. Muchiindi choonse chakupima, quadrupole njiijisi windo pati lyakukwabilila, kuzwa ku m/z 50 kusika ku 1500. Eelyo mubili wakumbele m/z abwaanda bwakusunka bwabikkwa a 200, mweelwe wabulemu ulabeleka buya apulogilamu yachibelesyo a 0 Da. Langa zibeela zyabunji mbuli mubunji bomwe. Belesya sodium formate (50 ml isopropanol, 200 μl formic acid antoomwe a 1 ml 1M NaOH aqueous solution) mukupima bunji bwazintu. Kubelesya nzila ya Bruker yakupima kabotu, fayela lya data lilapimwa musule aakubelesya mweelwe waakati aakati muchiindi cheelede. Belesya nzila yakutumina kunze ya Data Analysis v4.0 (Brook Dalton, Bremen, Germany) kusandula ma fayela aa data aatabelesyegwi kuba NetCDF. I data ya MS/MS yakabambwa mubbuku lya metabolomics lya MetaboLights (www.ebi.ac.uk) antoomwe anamba yakunjilila. MTBLS1471.
Kubunganya kwa MS/MS kulakonzya kuchitwa kwiinda mukulanga-langa kweendelana aakati ka MS1 a MS/MS zitondezyo zyabubotu bwanguzu zyakudonkolana zili ansi azyaatala amilawu mipya yakabikkwa. Ilembedwe lya R lilabelesyegwa kuchita kuvwuntauzya kweendelana kwakwaabila kwachintu chitalikila kuchintu, mpawo ilembedwe lya C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) lilabelesyegwa kuchita milawu.
Kutegwa tucesye kulubizya kwakubeja kwakachitwa amuzuzumo wakusyule akujatana kwakubeja kwakaletwa akujana zibeela zimwi zya m/z mumasampu masyoonto, twabelesya mulimo wa "filled peak" wa R package XCMS (kululamika muzuzumo wakusyule) Yeelede kubelesegwa kunjila mubusena bwa "NA" (undetected peak) intensity. Eelyo mulimo wakuzuzya nsingo nuubelesyegwa, kuchili "0′′ mipati myiingi mu data set iikonzya kuchincha kubala kwakujatana. Mpawo, twakozyanisya mpindu zya data zijanwa aawo mulimo wakuzuzya nsingo nuubelesyegwa akuti mulimo wakuzuzya nsumbu nuutabelesyegwi, mpawo twabala mweelwe wamuzuzumo wakusyule kweendelanya amweelwe waakati-kati wakalulamikwa, mpawo twabikka mweelwe ooyu wa 0′′ mweelwe wamusyule wakabalilwa. Twakalanga buyo zibeela eezyo zyakali kwiinda mweelwe wamusyule ziindi zyotatwe mpawo twakazibona kuti "nzintu zyamasimpe." Kubalilwa kwa PCC, buyo zitondezyo zya m/z zya sampulu precursor (MS1) antoomwe amaseti aa data aajisi zibeela zili musanu atutatu zyamasimpe zilalangwa-langwa.
Kuti bupati bwachiimo chakusaanguna muchikozyano choonse kabuswaangene akucheya kwachiimo chabubotu mbubonya oobo buli munguzu zyakudonkolana zisyoonto naa zipati, mpawo chiimo eecho tachilembedwe kuti isotope peak a CAMERA, chilakonzya kupandululwa lubo. Mpawo, kwiinda mukubalila zyoonse zikonzya kuchitwa mukati ka 3 s (chiindi chakujatisya chikonzya kuchitwa), kuvwuntauzya kwakujatana kwachitwa. Lilikke eelyo m/z value noili munsi aamuulo wa precursor alimwi MS/MS fragmentation noicitika mubusena bomwe bwa sampulu mu data set mbuli precursor oomo moyakazwa, nceciindi nocibonwa kuti ncibeela.
Kwiinda mumilawu eeyi yobile ngubauba, tulagwisya zibeela zyaambwa zili a m/z values ​​zipati kwiinda m/z ya precursor yakazyibwa, alimwi kweendelanya abusena bwasampulu ooko precursor nkoilibonya alimwi acibeela caambwa. Alimwi zilakonzeka kusala zibeela zibotu zipangwa azibeela zyiingi zipangwa munzila ya MS1 mbuli zintu zikonzya kuchitwa, aboobo zipa kuti kube zintu zitayandiki zya MS/MS. Mukuchitila kuchesya kuvwula kwa data, ikuti naa kukozyanya kwa NDP kwa spectra kwayinda 0.6, mpawo zili mu chromatogram "pcgroup" yakalembedwe a CAMERA, tulaziswaanganya. Kumamanino, twaswaanganya zyoonse zyone zya CE zijatikizyidwe a precursor a fragments mu deconvoluted composite spectrum yamamanino kwiinda mukusala peak mpati loko akati ka peaks zyoonse zili a m/z value yomwe kuma collision energy aasiyene. Ntaamu zyakuchita zitobela zyeendelana amuzeezo wa composite spectrum mpawo zibikkila maanu kubukkale bwiindene-indene bwa CE buyandika kutegwa kube kutyompokelwa kupati, nkaambo zimwi zibeela zilakonzya kujanwa buyo munguzu zyakudonkolana zimwi.
RDPI (30) yakabelesegwa kubala nzila zyakusanduka kwa metabolic profile. Kusiyana kwa metabolic spectrum (Hj index) kuzwa kukuvwula kwa MS/MS precursors kubelesya Shannon entropy ya MS/MS frequency distribution kubelesya equation itobela yakapandululwa a Martínez abamwi. (8). Hj = −∑i = 1mPijlog2(Pij) aawo Pij nkweendelana a frequency ya i-th MS/MS mu j-th sampulu (j = 1, 2,..., m) (i = 1, 2, …, m) t).
Kusalazya kwa metabolic (Si index) kwapandululwa kuti nkutondezya kwa MS/MS kweendelanya akuvwula aakati kamasampu aali mukulangwa-langwa. MS/MS specificity ilabalilwa kuti Si = 1t (∑j = 1tPijPilog2PijPi)
Belesya fomuyula itobela kupima metabolome-specific δj index ya j sampulu imwi aimwi, mpawo mweelwe waakati-kati wa MS/MS specificity δj = ∑i = 1mPijSi
MS/MS spectra ziliimpene muzibeela zyobile, mpawo kukozyanya kwabalilwa kweendelanya amakki obile. Chakusanguna, kubelesya NDP (alimwi iizyibidwe kuti nzila ya cosine correlation), kobelesya equation itobela kuti ujane kukozyanya kwazibeela aakati ka spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 aawo S1 a S2 Kweendelana, ku spectrum 1 a 2, iWS1, iWS, i2 wiiminina kulema kweendelanya abwaanda bwaatala bwakuti kwiimpana kwa i-th common peak aakati kama spectra obile kuli munsi aa 0.01 Da. Kulema kwabalilwa boobu: W = [kuyuma kwaatala] m [bubotu] n, m = 0.5, n = 2, mbuli mbokwakaambwa a MassBank.
Nzila yabili yakupima yakabelesegwa, eeyo yakabikkilizya kulanga-langa NL yakaabana aakati ka MS/MS. Kusikila kumamanino, twakabelesya milongo ya NL iili 52 iijanika chiindi chiingi muchiindi chakupambuka kwa MS antoomwe, antoomwe a NL iigaminide (data file S1) eeyo yakalembedwe kale ya MS/MS spectrum ya metabolites yabili ya Nepenthes species yakakompama ( 9, 26). Panga vekita yobile ya 1 a 0 kuli MS/MS imwi aimwi, kweendelana a NL imwi iiliwo lino aitako. Kwiinda mukukozyanya kwakulampa kwa Euclidean, mweelwe wakukozyanya wa NL ulabalilwa kuli umwi awumwi wa NL vectors zyobile.
Kuchita kubunganya zintu zyobile, twakabelesya R package DiffCoEx, eeyo iiyeeme aakuyungizya kwa Weighted Gene Co-expression Analysis (WGCNA). Kubelesya NDP antoomwe a NL zyakubala zya MS/MS spectra, twakabelesya DiffCoEx kubala nzila zyakukozyanisya. Kubunganya zintu zyobile kuchitwa akubikka "cutreeDynamic" parameter kuti ibe nzila = "hybrid", cutHeight = 0.9999, deepSplit = T, antoomwe a minClusterSize = 10. R source code ya DiffCoEx yakagwisigwa kuzwa kufailo lyakuyungizya 1 a Tesson abamwi. (57); Ipulogilamu ya R WGCNA iyandika ilakonzya kujanwa a https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Kuti tuchite MS/MS molecular network analysis, twakabala kujatana kwa spectral kweendelanya a NDP a NL kukozyanya, mpawo twakabelesya pulogilamu ya Cytoscape kubona network topology kubelesya organic layout mu CyFilescape yFiles layout algorithm extension application.
Belesya R version 3.0.1 kuchita buvwuntauzi bwa data. Bupati bwakubala bwakavwuntauzyigwa kubelesya nzila zyobile zyakuvwuntauzya (ANOVA), kutobelwa a Tukey's honestly significant difference (HSD) post-hoc test. Kuti tuvwuntauzye kusiyana aakati kakubelesya misamu yakulya zisyango akubelesya misamu iikola, kwaabwa kwamisela yobile kwatubunga tobile twamasampu aajisi kusiyana komwe kwakavwuntauzyigwa kubelesya nzila ya Student's t test.
Kuti ujane zimwi zilembedwe muchibalo eechi, langa http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Eechi nchibalo chabantu boonse chakamwaigwa kweendelana amalailile aa Creative Commons Attribution-Non-Commercial License, aayo aazumizya kubelesya, kwaabila akuzyala munzila iili yoonse, kufwumbwa buyo kuti kubelesya kwamamanino takuli kwakujana mpindu alimwi akuti mulimo wakusaanguna uliluzi. Kulanga kulichimwi.
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Njiisyo zyamakani zipa mali yanyika yoonse yakukozyanisya ma metabolomes aalibedelede akusinsima njiisyo zyakulikwabilila zyakusunkwa.
Njiisyo zyamakani zipa mali yanyika yoonse yakukozyanisya ma metabolomes aalibedelede akusinsima njiisyo zyakulikwabilila zyakusunkwa.
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