Iingcamango ezahlukeneyo zokukhusela izityalo zibonelela ngesikhokelo esibalulekileyo sethiyori sokuchaza iipateni zemetabolism ekhethekileyo yezityalo, kodwa izibikezelo zazo eziphambili zisaza kuvavanywa. Apha, sisebenzise uhlalutyo lwe-tandem mass spectrometry (MS/MS) olungenamkhethe ukuhlola ngokucwangcisiweyo i-metabolome yeentlobo zecuba ezincitshisiweyo ukusuka kwizityalo ngazinye ukuya kubemi kunye neentlobo ezinxulumene ngokusondeleyo, kwaye sicubungule inani elikhulu leethiyori zeempawu ze-mass spectrometric ezisekelwe kwi-compound spectra kulwazi. Isakhelo sokuvavanya izibikezelo eziphambili ze-optimal defense (OD) kunye ne-moving target (MT) theories. Icandelo lolwazi lwe-plant metabolomics lihambelana nethiyori ye-OD, kodwa liphikisana noqikelelo oluphambili lwethiyori ye-MT kwi-metabolomics dynamics ebangelwa zizityalo ezitya utyani. Ukusuka kwi-micro ukuya kwi-macro evolution scale, isignali ye-jasmonate ichongiwe njengeyona nto iphambili echaza i-OD, ngelixa isignali ye-ethylene ibonelela ngokulungiswa kakuhle kwempendulo ethile ye-herbivore echazwe yinethiwekhi ye-MS/MS molecular.
Iimetabolites ezikhethekileyo ezinezakhiwo ezahlukeneyo zezona zithatha inxaxheba eziphambili ekuziqhelaniseni nezityalo nokusingqongileyo, ngakumbi ekukhuseleni iintshaba (1). Ukwahlukahluka okumangalisayo kwemetabolism ekhethekileyo efumaneka kwizityalo kuye kwavuselela amashumi eminyaka ophando olunzulu kwiinkalo zayo ezininzi zemisebenzi yendalo, kwaye kwenze uluhlu olude lweethiyori zokhuselo lwezityalo, eziluphuhliso lwendalo kunye nolwendalo lokunxibelelana kwezityalo nezinambuzane. Uphando olusebenzayo lubonelela ngesikhokelo esibalulekileyo (2). Nangona kunjalo, ezi thiyori zokhuselo lwezityalo azilandelanga indlela eqhelekileyo yokuqiqa okucingelwayo, apho uqikelelo oluphambili lwalukwinqanaba elifanayo lohlalutyo (3) kwaye lwavavanywa ngovavanyo ukuze kuqhutyelwe phambili umjikelo olandelayo wophuhliso lwethiyori (4). Imida yobuchwephesha ithintela ukuqokelelwa kwedatha kwiindidi ezithile zemetabolism kwaye ikhuphe uhlalutyo olupheleleyo lweemetabolites ezikhethekileyo, ngaloo ndlela ithintela uthelekiso phakathi kwezigaba ezibalulekileyo kuphuhliso lwethiyori (5). Ukungabikho kwedatha epheleleyo yemetabolomics kunye nemali efanayo yokuthelekisa indlela yokusebenza yokucubungula indawo yemetabolism phakathi kwamaqela ezityalo ahlukeneyo kuthintela ukukhula kwesayensi kwentsimi.
Uphuhliso lwamva nje kwicandelo le-tandem mass spectrometry (MS/MS) metabolomics lunokubonisa ngokupheleleyo utshintsho lwe-metabolic ngaphakathi naphakathi kweentlobo ze-system clade ethile, kwaye lunokudityaniswa neendlela zokubala ukubala ukufana kwesakhiwo phakathi kwezi ngxubevange zintsonkothileyo. Ulwazi lwangaphambili lwekhemistri (5). Ukudibana kwetekhnoloji eziphambili kuhlalutyo nakwikhompyutha kubonelela ngesakhelo esifunekayo sovavanyo lwexesha elide lwezibikezelo ezininzi ezenziwe ziingcamango zendalo kunye ne-evolutionary zokwahluka kwe-metabolic. UShannon (6) wazisa ithiyori yolwazi okokuqala kwinqaku lakhe eliphambili ngo-1948, ebeka isiseko sohlalutyo lwezibalo lolwazi, oluye lwasetyenziswa kwiindawo ezininzi ngaphandle kwesicelo salo sokuqala. Kwi-genomics, ithiyori yolwazi isetyenziswe ngempumelelo ukulinganisa ulwazi olugcina ulandelelwano (7). Kuphando lwe-transcriptomics, ithiyori yolwazi ihlalutya utshintsho olupheleleyo kwi-transcriptome (8). Kuphando lwangaphambili, sisebenzise isakhelo sezibalo sethiyori yolwazi kwi-metabolomics ukuchaza ubuchule be-metabolic yenqanaba lezicubu kwizityalo (9). Apha, sidibanisa indlela yokusebenza esekelwe kwi-MS/MS kunye nesakhelo sezibalo sethiyori yolwazi, ebonakaliswa kukwahluka kwemetabolism kwimali eqhelekileyo, ukuze sithelekise izibikezelo eziphambili zethiyori yokukhusela izityalo ye-metabolome ebangelwa zizilwanyana ezitya utyani.
Izikhokelo zethiyori zokuzikhusela kwezityalo zihlala zibandakanya kwaye zinokwahlulwa zibe ngamacandelo amabini: ezo zizama ukuchaza ukusasazwa kweemetabolites ezithile zezityalo ngokusekelwe kwimisebenzi yokuzikhusela, njengokhuselo olufanelekileyo (OD) (10), ithagethi ehambayo (MT) (11)) kunye nethiyori yokubonakala (12), ngelixa ezinye zifuna iinkcazo zoomatshini malunga nendlela utshintsho ekufumanekeni kwezixhobo oluchaphazela ngayo ukukhula kwezityalo kunye nokuqokelelwa kweemetabolites ezikhethekileyo, ezifana nekhabhoni: i-nutrient balance hypothesis (13), i-growth rate hypothesis (14), kunye ne-growth and differentiation equilibrium hypothesis (15). Ezi seti zimbini zethiyori zikwinqanaba ezahlukeneyo lohlalutyo (4). Nangona kunjalo, iingcamango ezimbini ezibandakanya imisebenzi yokuzikhusela kwinqanaba lokusebenza zilawula incoko malunga nokhuselo oluquka izityalo nolungenasiphumo: i-OD theory, ecinga ukuba izityalo zityala imali kwiikhemikhali zazo ezibizayo kuphela xa zifuneka, umzekelo, xa ziginywa. Xa isilwanyana sengca sihlasela, ke ngoko, ngokwethuba lokuhlaselwa kwixesha elizayo, i-compound enomsebenzi wokuzikhusela iyabelwa (10); Ingcamango ye-MT icebisa ukuba akukho axis yotshintsho lwe-metabolite kwicala elithile, kodwa i-metabolite itshintsha ngokungacwangciswanga, ngaloo ndlela idala ithuba lokuthintela "ithagethi yentshukumo" ye-metabolic yokuhlasela i-herbivores. Ngamanye amazwi, ezi ngcamango zimbini zenza uqikelelo oluchaseneyo malunga nokuguqulwa kwe-metabolic okwenzeka emva kokuhlaselwa kwe-herbivores: ubudlelwane phakathi kokuqokelelwa kwe-metabolites kwicala elinye nomsebenzi wokuzikhusela (OD) kunye notshintsho lwe-metabolic olungalawulwanga (MT) (11).
Iingcamango ze-OD kunye ne-MT azibandakanyi nje kuphela utshintsho olubangelwa yi-metabolome, kodwa kunye nemiphumo yendalo kunye nokuguquka kokuqokelelwa kwezi metabolites, njengeendleko zokuguquguquka kunye neenzuzo zolu tshintsho lwe-metabolic kwindawo ethile yendalo (16). Nangona zombini ezi ngcamango ziqaphela umsebenzi wokuzikhusela wee-metabolites ezikhethekileyo, ezinokuba zibiza kakhulu okanye zingabi zibiza kakhulu, uqikelelo oluphambili oluhlula iingcamango ze-OD kunye ne-MT lukwicala lotshintsho lwe-metabolic olubangelwayo. Uqikelelo lwethiyori ye-OD lufumene ingqwalaselo yovavanyo kakhulu ukuza kuthi ga ngoku. Ezi vavanyo ziquka uphononongo lwemisebenzi yokuzikhusela ethe ngqo okanye engathanga ngqo yezicubu ezahlukeneyo zeekhompawundi ezithile kwiindawo zokugcina izityalo kunye neemeko zendalo, kunye notshintsho kwinqanaba lophuhliso lwezityalo (17-19). Nangona kunjalo, ukuza kuthi ga ngoku, ngenxa yokungabikho komsebenzi kunye nesakhelo sezibalo sohlalutyo olupheleleyo lwehlabathi lokwahluka kwe-metabolic yayo nayiphi na into ephilayo, umahluko ophambili phakathi kwezi ngcamango zimbini (oko kukuthi, indlela yotshintsho lwe-metabolic) usaza kuvavanywa. Apha, sinikezela ngohlalutyo olunjalo.
Enye yezona mpawu zibalulekileyo ze-metabolites ezithile zezityalo kukwahluka kwazo okugqithisileyo kwesakhiwo kuzo zonke iinqanaba ukusuka kwizityalo ezizodwa, inani ukuya kwiintlobo ezifanayo (20). Utshintsho oluninzi lobuninzi kwi-metabolites ezikhethekileyo lunokubonwa kwinqanaba labemi, ngelixa umahluko onamandla womgangatho uhlala ugcinwa kwinqanaba leentlobo (20). Ke ngoko, ukwahluka kwe-metabolism yezityalo yeyona nto iphambili yokwahluka kokusebenza, okubonisa ukulungelelaniswa kwiindawo ezahlukeneyo, ngakumbi ezo ndawo zinamathuba ahlukeneyo okuhlasela yizinambuzane ezikhethekileyo kunye nezilwanyana ezitya utyani (21). Ukususela kwinqaku likaFraenkel (22) eliphambili malunga nezizathu zokubakho kwe-metabolites ezithile zezityalo, ukusebenzisana nezinambuzane ezahlukeneyo kuthathwe njengoxinzelelo lokukhetha olubalulekileyo, kwaye olu nxibelelwano lukholelwa ukuba luye lwabumba izityalo ngexesha lokuvela. Indlela ye-Metabolic (23). Umahluko phakathi kweentlobo ezahlukeneyo kwi-metabolites ezikhethekileyo unokubonakalisa ibhalansi ye-physiological enxulumene nokukhuselwa kwezityalo ezidityanisiweyo nezingena kwi-inducible kwizicwangciso ze-herbivorous, njengoko ezi ntlobo zimbini zihlala zinxulumene kakubi (24). Nangona kunokuba luncedo ukugcina ukhuselo oluhle ngamaxesha onke, utshintsho lwe-metabolic oluhambelana nokhuselo lubonelela ngeenzuzo ezicacileyo ekuvumeleni izityalo ukuba zabelane ngezixhobo ezixabisekileyo kwezinye utyalo-mali lwe-physiological (19, 24), kwaye ziphephe imfuneko ye-symbiosis. Umonakalo we-colateral (25). Ukongeza, olu hlengahlengiso lwe-metabolites ezikhethekileyo ezibangelwa zizinambuzane ezitya utyani zinokukhokelela ekusasazweni okutshabalalisayo kuluntu (26), kwaye zinokubonisa ukufundwa ngokuthe ngqo kotshintsho olukhulu lwendalo kwisignali ye-jasmonic acid (JA), enokugcinwa kuluntu. Izibonakaliso ze-JA eziphezulu neziphantsi ziindlela zokurhweba phakathi kokuzikhusela kwizilwanyana ezitya utyani kunye nokhuphiswano neentlobo ezithile (27). Ukongeza, iindlela ze-biosynthetic ze-metabolite ezikhethekileyo ziya kulahlekelwa ngokukhawuleza kunye notshintsho ngexesha lokuvela, okubangela ukusasazwa kwe-metabolic okuneziphene phakathi kweentlobo ezinxulumene ngokusondeleyo (28). Ezi polymorphisms zinokusekwa ngokukhawuleza ukuphendula kwiipateni ze-herbivore ezitshintshayo (29), oko kuthetha ukuba ukuguquguquka kweendawo ze-herbivore yinto ephambili eqhuba ukungafani kwe-metabolic.
Apha, sizisombulule ngokukodwa ezi ngxaki zilandelayo. (I) Izinambuzane ezitya utyani ziwuphinda njani umzobo we-metabolome yesityalo? (Ii) Zeziphi izinto eziphambili zolwazi lwe-metabolic plasticity ezinokulinganiswa ukuvavanya uqikelelo lwethiyori yokuzikhusela yexesha elide? (Iii) Nokuba kufuneka kuhlelwe ngokutsha i-metabolome yesityalo ngendlela eyahlukileyo kumhlaseli, ukuba kunjalo, idlala yiphi indima i-hormone yesityalo ekulungiseni impendulo ethile ye-metabolic, kwaye zeziphi ii-metabolites ezinegalelo ekukhethekeni kweentlobo zokuzikhusela? (Iv) Ekubeni uqikelelo olwenziwe ziithiyori ezininzi zokuzikhusela lunokwandiswa kuwo onke amanqanaba ezicubu zebhayoloji, sibuze ukuba impendulo ye-metabolic ebangelwayo ihambelana kangakanani ukusuka kuthelekiso lwangaphakathi ukuya kuthelekiso lwe-interspecies? Ngenxa yoku, sifunde ngokucwangcisiweyo i-metabolome yamagqabi e-nicotine yecuba, esisityalo esingumzekelo wendalo esine-metabolism ekhethekileyo, kwaye sisebenza kakuhle ngokuchasene nezibungu zezilwanyana ezimbini ezitya utyani, iLepidoptera Datura (Nks) (Inolaka kakhulu, idla kakhulu) KwiSolanaceae kunye neSpodoptera littoralis (Sl), iimpethu zamagqabi ekotoni luhlobo "lwe-genus", kunye nezityalo ezihlala kwiSolanaceae kunye nezinye izizukulwana zezinye izizukulwana kunye neentsapho Ukutya kwezityalo. Sihlolisise i-MS/MS metabolomics spectrum kwaye sakhupha iinkcazo zezibalo zethiyori yolwazi ukuze sithelekise iingcamango ze-OD kunye ne-MT. Yenza iimephu ezithile ukuze kutyhile ubuwena bee-metabolites eziphambili. Uhlalutyo lwandisiwe kubemi bomthonyama be-N. nasi kunye neentlobo zecuba ezinxulumene ngokusondeleyo ukuze kuhlalutywe ngakumbi i-covariance phakathi kokubonisana kwehomoni yezityalo kunye nokungeniswa kwe-OD.
Ukuze sibambe imephu iyonke malunga nokwakheka kwe-metabolome yamagqabi ecuba elitya utyani, sisebenzise uhlalutyo oluphuhliswe ngaphambili kunye nomsebenzi wokubala ukuqokelela ngokupheleleyo kunye nokususa idatha ye-MS/MS spectra ezimeleyo kwi-high-resolution data kwi-extracts zezityalo (9). Le ndlela ingahlukanisiweyo (ebizwa ngokuba yi-MS/MS) inokwakha i-non-redundant compound spectra, enokusetyenziswa kuzo zonke ii-compound-level analysis ezichazwe apha. Ezi metabolites zezityalo ezisusiweyo ziindidi ezahlukeneyo, eziquka amakhulu ukuya kumawaka ee-metabolites (malunga ne-500-1000-s/MS/MS apha). Apha, sicinga nge-metabolic plasticity kwisakhelo sethiyori yolwazi, kwaye silinganise ukwahluka kunye nobungcali be-metabolome ngokusekelwe kwi-Shannon entropy yokusasazwa kwe-metabolic frequency. Sisebenzisa ifomyula esetyenziswe ngaphambili (8), sibalele iseti yezalathisi ezinokusetyenziselwa ukulinganisa ukwahluka kwe-metabolome (isalathisi se-Hj), i-metabolic profile specialization (isalathisi se-δj) kunye ne-metabolic specificity ye-single metabolite (isalathisi se-Si). Ukongeza, sisebenzise iRelative Distance Plasticity Index (RDPI) ukuze silinganise i-metabolome inducibility yezilwanyana ezitya utyani (Umfanekiso 1A) (30). Ngaphakathi kwesi sikhokelo sezibalo, siphatha i-MS/MS spectrum njengeyunithi yolwazi olusisiseko, kwaye sicubungule ubuninzi be-MS/MS kwimephu yokusasazwa kwe-frequency, size sisebenzise iShannon entropy ukuqikelela ukungafani kwe-metabolome kuyo. I-metabolome specialization ilinganiswa ngokwe-avareji specificity ye-MS/MS spectrum enye. Ke ngoko, ukwanda kobuninzi bezinye iiklasi ze-MS/MS emva kokungeniswa kwe-herbivore kuguqulwa kube yi-spectral inducibility, i-RDPI kunye ne-specialization, oko kukuthi, ukwanda kwe-δj index, kuba ii-metabolites ezikhethekileyo ngakumbi ziyaveliswa kwaye i-Si index ephezulu iyaveliswa. Ukwehla kwe-Hj diversity index kubonisa ukuba inani le-MS/MS eveliswayo linciphile, okanye ukusasazwa kwe-profile frequency kutshintsha kwicala elingalinganiyo kangako, ngelixa kunciphisa ukungaqiniseki kwayo ngokubanzi. Ngokusetyenziswa kokubala i-Si index, kunokwenzeka ukugqamisa ukuba zeziphi i-MS/MS ezibangelwa zizilwanyana ezithile ezitya utyani, ngokuchaseneyo, zeziphi i-MS/MS ezingaphenduliyo kwi-induction, nto leyo eluphawu oluphambili lokwahlula uqikelelo lwe-MT kunye ne-OD.
(A) Iinkcazo-manani ezisetyenziselwa i-herbivorous (H1 ukuya kwi-Hx) i-MS/MS data-inducibility (RDPI), i-diversity (Hj index), i-specialization (δj index) kunye ne-metabolite specificity (Si index). Ukwanda kwinqanaba le-specialization (δj) kubonisa ukuba, ngokomndilili, kuya kuveliswa ii-metabolites ezithile ze-herbivorous, ngelixa ukwehla kwe-diversity (Hj) kubonisa ukwehla kwemveliso yee-metabolites okanye ukusasazwa okungalinganiyo kwee-metabolites kwimephu yokusasazwa. Ixabiso le-Si livavanya ukuba i-metabolite ikwimeko ethile (apha, i-herbivorous) okanye igcinwe kwinqanaba elifanayo. (B) Umzobo wengcamango yokuqikelela ithiyori yokuzikhusela usebenzisa i-axis yethiyori yolwazi. Ithiyori ye-OD iqikelela ukuba uhlaselo lwe-herbivore luya kwandisa ii-metabolites zokuzikhusela, ngaloo ndlela lwandise i-δj. Kwangaxeshanye, i-Hj iyancipha kuba iprofayili ihlelwe ngokutsha ukuya ekunciphiseni ukungaqiniseki kolwazi lwe-metabolic. Ithiyori ye-MT iqikelela ukuba uhlaselo lwe-herbivores luya kubangela utshintsho olungekho kwicala kwi-metabolome, ngaloo ndlela lwandise i-Hj njengophawu lokwanda kokungaqiniseki kolwazi lwe-metabolic kwaye lubangele ukusasazwa okungahleliwe kwe-Si. Sikwacebise imodeli exutyiweyo, eyona MT ilungileyo, apho ezinye ii-metabolites ezinexabiso eliphezulu lokuzikhusela ziya kwandiswa ngokukodwa (ixabiso eliphezulu le-Si), ngelixa ezinye zibonisa iimpendulo ezingacwangciswanga (ixabiso eliphantsi le-Si).
Sisebenzisa izichazi-magama zethiyori yolwazi, sitolika ithiyori ye-OD ukuqikelela ukuba utshintsho olukhethekileyo lwe-metabolite olubangelwa yi-herbivore kwimeko engabangelwanga luya kukhokelela (i) ukwanda kwe-metabolic specificity (i-Si index) eqhuba i-metabonomic specificity (i-δj index) Ukwanda) kwamaqela athile e-metabolite akhethekileyo anexabiso eliphezulu lokuzikhusela, kunye (ii) ukwehla kwe-metabolome diversity (i-Hj index) ngenxa yotshintsho lokusasazwa kwe-metabolic frequency ukuya kukusasazwa komzimba we-leptin engaphezulu. Kwinqanaba le-metabolite enye, kulindeleke ukusasazwa kwe-Si okucwangcisiweyo, apho i-metabolite iya kunyusa ixabiso le-Si ngokwexabiso layo lokuzikhusela (Umfanekiso 1B). Ngale ndlela, sichaza ithiyori ye-MT ukuqikelela ukuba ukuvuselela kuya kukhokelela (i) kutshintsho olungekho kwicala kwi-metabolites olubangela ukwehla kwe-δj index, kunye (ii) ukwanda kwe-Hj index ngenxa yokwanda kokungaqiniseki kwe-metabolic. Okanye ukungacwangciswanga, okunokulinganiswa yi-Shannon entropy ngendlela yokwahluka ngokubanzi. Ngokuphathelele ukwakheka kwe-metabolic, ithiyori ye-MT iya kuxela ukusasazwa okungacwangciswanga kwe-Si. Xa sicinga ukuba ii-metabolites ezithile ziphantsi kweemeko ezithile phantsi kweemeko ezithile, kwaye ezinye iimeko azikho phantsi kweemeko ezithile, kwaye ixabiso lazo lokuzikhusela lixhomekeke kwindalo esingqongileyo, sikwacebise imodeli yokuzikhusela exutyiweyo, apho i-δj kunye ne-Hj zisasazwa kabini ecaleni kwe-Si Increase kuzo zonke iindlela, kuphela amaqela athile e-metabolite, anamaxabiso aphezulu okuzikhusela, aya kunyusa i-Si ngokukodwa, ngelixa amanye aya kuba nokusasazwa okungacwangciswanga (Umfanekiso 1B).
Ukuze kuvavanywe uqikelelo lwethiyori yokuzikhusela oluchazwe ngokutsha kwi-axis yenkcazo yethiyori yolwazi, siphakamise iingca ezitya utyani (Ms) okanye i-generalist (Sl) ezikwiingqayi zeNepenthes pallens (Umfanekiso 2A). Sisebenzisa uhlalutyo lwe-MS/MS, sifumene ii-spectra ze-MS/MS ezingama-599 ezingezizo ezingafunekiyo (ifayile yedatha S1) ezivela kwi-methanol extracts yezicubu zamagqabi eziqokelelwe emva kokutya iincakuba. Sisebenzisa ii-indexes ze-RDPI, Hj, kunye ne-δj ukuze sibone uhlengahlengiso lomxholo wolwazi kwiifayile zoqwalaselo lwe-MS/MS zityhila iipateni ezinomdla (Umfanekiso 2B). Umkhwa uwonke kukuba, njengoko kuchaziwe yinkcazo yolwazi, njengoko iincakuba ziqhubeka nokutya amagqabi, inqanaba layo yonke i-metabolic reorganization iyanda ngokuhamba kwexesha: iiyure ezingama-72 emva kokuba i-herbivore ityile, i-RDPI iyanda kakhulu. Xa kuthelekiswa nolawulo olungonakalanga, i-Hj yancitshiswa kakhulu, okwabangelwa lizinga elongezelelweyo lobuchwephesha be-metabolic profile, elalinganiswa yi-δj index. Olu hlobo lubonakalayo luhambelana noqikelelo lwethiyori ye-OD, kodwa aluhambelani noqikelelo oluphambili lwethiyori ye-MT, ekholelwa ukuba utshintsho olungacwangciswanga (olungangqalanga) kumanqanaba e-metabolite lusetyenziswa njengesixhobo sokuzikhusela (Umfanekiso 1B). Nangona umxholo we-exicitor womlomo (OS) kunye nokuziphatha kokutya kwezi zilwanyana zimbini ezitya utyani zahlukile, ukutya kwazo ngokuthe ngqo kubangele utshintsho olufanayo kwicala le-Hj kunye ne-δj ngexesha lokuvuna iiyure ezingama-24 kunye neeyure ezingama-72. Umahluko kuphela wenzeke kwiiyure ezingama-72 ze-RDPI. Xa kuthelekiswa noko kubangelwa kukutya kukaMs, i-metabolism iyonke ebangelwa kukutya kwe-Sl yayiphezulu.
(A) Uyilo lovavanyo: iihagu eziqhelekileyo (S1) okanye iingcali (Ms) ezitya utyani zondliwa ngamagqabi asuswe ityuwa kwizityalo zepitcher, ngelixa kwi-herbivory elinganisiweyo, i-OS ye-Ms (W + OSMs) isetyenziselwa ukuphatha ukugqobhozwa kweendawo zamagqabi ezisemgangathweni. I-S1 (W + OSSl) izibungu okanye amanzi (W + W). Ulawulo (C) ligqabi elingonakalanga. (B) Ukungaguquki (RDPI xa kuthelekiswa netshathi yokulawula), ukwahluka (isalathisi se-Hj) kunye nesalathiso sobuchwephesha (isalathisi se-δj) esibalwe kwimephu ekhethekileyo ye-metabolite (599 MS/MS; ifayile yedatha S1). Ii-asterisk zibonisa umahluko omkhulu phakathi kokutya ngokuthe ngqo kwe-herbivore kunye neqela lolawulo (uvavanyo lwe-t lomfundi olune-t-test edibeneyo, *P<0.05 kunye ***P<0.001). ns, akubalulekanga. (C) Isalathisi sexesha lesisombululo sezinto eziphambili (ibhokisi eluhlaza okwesibhakabhaka, i-amino acid, i-organic acid kunye neswekile; ifayile yedatha S2) kunye ne-spectrum ekhethekileyo ye-metabolite (ibhokisi ebomvu 443 MS/MS; ifayile yedatha S1) emva konyango lwe-herbivory olulinganisiweyo. Ibhendi yombala ibhekisa kwisithuba sokuzithemba se-95%. I-asterisk ibonisa umahluko obalulekileyo phakathi konyango kunye nolawulo [uhlalutyo lwe-quadratic of variance (ANOVA), lulandelwa ngumahluko obalulekileyo kaTukey (HSD) wokuthelekiswa okuninzi emva kwe-hoc, *P<0.05, **P<0.01 kunye *** P <0.001]. (D) Ukuchonga iiploti ze-scatter kunye neeprofayili ze-metabolite ezikhethekileyo (iisampulu eziphindaphindwayo ezinonyango olwahlukeneyo).
Ukuze sihlolisise ukuba ukutshintshwa kwezityalo ezitya utyani kwinqanaba le-metabolome kubonakala na kutshintsho kwinqanaba lee-metabolites zomntu ngamnye, siqale sagxila kwi-metabolites ezazifundwe ngaphambili kumagqabi e-Nepenthes pallens ezinokumelana nezityalo ezitya utyani okuqinisekisiweyo. Ii-phenolic amides zii-hydroxycinnamamide-polyamine conjugates eziqokelelana ngexesha lenkqubo yezinambuzane ezitya utyani kwaye zaziwa ngokunciphisa ukusebenza kwezinambuzane (32). Sikhangele ii-precursors ze-MS/MS ehambelanayo saza sadweba ii-cumulative kinetic curves zazo (Umfanekiso S1). Akumangalisi ukuba ii-phenol derivatives ezingabandakanyekanga ngokuthe ngqo ekukhuseleni izilwanyana ezitya utyani, ezifana ne-chlorogenic acid (CGA) kunye ne-rutin, ziphantsi emva kwezityalo ezitya utyani. Ngokwahlukileyo koko, izilwanyana ezitya utyani zinokwenza i-phenol amides ibe namandla kakhulu. Ukondla okuqhubekayo kwezi zilwanyana zitya utyani ezimbini kubangele phantse i-excitation spectrum efanayo yee-phenolamides, kwaye le patheni yayicacile ngakumbi kwi-de novo synthesis ye-phenolamides. Le nto ifanayo iya kubonwa xa kuphononongwa indlela ye-17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), evelisa inani elikhulu le-acyclic diterpenes enemisebenzi esebenzayo yokulwa notyani (33), apho i-Ms Feeding with Sl yabangela iprofayili efanayo yokubonakalisa (Umfanekiso S1)).
Ingxaki enokubakho yovavanyo lokutya imifuno ngqo kukwahluka kwinqanaba lokusetyenziswa kwamagqabi kunye nexesha lokutya imifuno, okwenza kube nzima ukuphelisa iziphumo ezithile zemifuno ezibangelwa ngamanxeba kunye nemifuno. Ukuze sisombulule ngcono ukucaciswa kweentlobo zemifuno kwimpendulo ye-metabolic yamagqabi ebangelwe kukutya, silinganise ukondla i-Ms kunye ne-Sl larvae ngokusebenzisa ngoko nangoko i-OSM eqokelelweyo (OSM kunye ne-OSS1) kwi-standard puncture W yeendawo zamagqabi ezihambelanayo. Le nkqubo ibizwa ngokuba yi-W + OS treatment, kwaye imisela ukungeniswa ngokubeka ixesha elichanekileyo ukuqala kwempendulo ebangelwe yimifuno ngaphandle kokubangela iziphumo ezididayo zomahluko kwizinga okanye ubungakanani bokulahleka kwezicubu (Umfanekiso 2A) (34). Sisebenzisa umbhobho wohlalutyo lwe-MS/MS kunye nokubala, sifumene i-443 MS/MS spectra (ifayile yedatha S1), eyayihambelana ne-spectra eyayiqokelelwe ngaphambili kwi-direct feed experiments. Uhlalutyo lwethiyori yolwazi lwale seti yedatha ye-MS/MS lubonise ukuba ukuhlelwa ngokutsha kwee-metabolomes ezikhethekileyo zamagqabi ngokulinganisa ii-herbivores kubonise i-OS-specific induces (Umfanekiso 2C). Ngokukodwa, xa kuthelekiswa nonyango lwe-OSS1, i-OSM ibangele ukuphuculwa kokuchwetheza kwe-metabolome kwiiyure ezi-4. Kubalulekile ukuqaphela ukuba xa kuthelekiswa neseti yedatha yovavanyo lokutya i-herbivore ngokuthe ngqo, i-metabolism kinetics ebonwe kwindawo enemilinganiselo emibini kusetyenziswa i-Hj kunye ne-δj njengee-coordinates kunye nesikhokelo sokuchwetheza kwe-metabolome ekuphenduleni unyango lwe-herbivore olulinganisiweyo ngokuhamba kwexesha Ukwanda okuqhubekayo (Umfanekiso 2D). Kwangaxeshanye, silinganise umxholo wee-amino acids, ii-organic acids kunye neeswekile (ifayile yedatha S2) ukuphanda ukuba olu nyuko olujoliswe kulo kubuchule be-metabolome lubangelwa kukulungiswa kwakhona kwe-metabolism yekhabhoni ephakathi ekuphenduleni ii-herbivores ezilinganisiweyo (Umfanekiso S2). Ukuze sichaze ngcono le patheni, sijonge ngakumbi i-metabolism accumulation kinetics yeendlela ze-phenolamide kunye ne-17-HGL-DTG ezixoxwe ngazo ngaphambili. Ukungeniswa kwezityalo ezitya utyani kwi-OS kuguqulwa kube yipatheni yokuhlengahlengisa eyahlukileyo ngaphakathi kwe-phenolamide metabolism (Umfanekiso S3). Ii-phenolic amides eziqulethe i-coumarin kunye ne-caffeoyl moieties zikhethwa ngokukhethekileyo yi-OSS1, ngelixa ii-OSM zibangela ukungeniswa okuthile kwe-ferulyl conjugates. Kwindlela ye-17-HGL-DTG, ukungeniswa kwe-OS eyahlukileyo yimveliso ye-malonylation esezantsi kunye ne-dimalonylation kufunyenwe (Umfanekiso S3).
Okulandelayo, sifunde i-OS-induced transcriptome plasticity sisebenzisa i-time-course microarray data set, efanisa ukusetyenziswa kwee-OSM ukunyanga amagqabi amagqabi esityalo se-rosette kwizityalo ezitya utyani. I-sampling kinetics ngokusisiseko ihambelana ne-kinetics esetyenziswe kolu phononongo lwe-metabolomics (35). Xa kuthelekiswa nokulungiswa kwakhona kwe-metabolome apho i-metabolic plasticity iyanda ngakumbi ngokuhamba kwexesha, sibona ukuqhuma kwe-transient transcription kumagqabi okubangelwa yi-Ms, apho i-transcriptome inducibility (RDPI) kunye ne-specialization (δj) zikwi-1 Kwakukho ukwanda okukhulu kwiiyure, kunye nokwahluka (Hj) ngeli xesha, ukubonakaliswa kwe-BMP1 kuncitshiswe kakhulu, kulandelwa kukukhululeka kwe-transcriptome specialization (Umfanekiso S4). Iintsapho ze-metabolic gene (ezifana ne-P450, i-glycosyltransferase, kunye ne-BAHD acyltransferase) zithatha inxaxheba kwinkqubo yokuhlanganisa ii-metabolites ezikhethekileyo ezivela kwiiyunithi zesakhiwo ezivela kwi-metabolism ephambili, kulandela imodeli yokuqala ye-high-specialization. Njengophononongo lwetyala, indlela ye-phenylalanine yahlalutywa. Uhlalutyo luqinisekisile ukuba iijini eziphambili kwi-metabolism ye-phenolamide zibangelwa kakhulu yi-OS kwizilwanyana ezitya utyani xa kuthelekiswa nezityalo ezingatsalwanga, kwaye zihambelana ngokusondeleyo kwiipateni zazo zokubonakalisa. I-transcription factor MYB8 kunye ne-structural genes PAL1, PAL2, C4H kunye ne-4CL kumphezulu wale ndlela zibonise ukuqaliswa kwangoko kokubhalwa kombhalo. Ii-Acyltransferases ezidlala indima ekuhlanganisweni kokugqibela kwe-phenolamide, njenge-AT1, DH29, kunye ne-CV86, zibonisa iphethini yokunyuka okude (Umfanekiso S4). Oku kubonwe ngasentla kubonisa ukuba ukuqaliswa kwangoko kobuchwephesha be-transcriptome kunye nokuphuculwa kamva kobuchwephesha be-metabolomics yindlela edibeneyo, enokubangelwa yinkqubo yokulawula ehambelanayo eqala impendulo enamandla yokuzikhusela.
Uhlengahlengiso kwi-plant hormone signaling lusebenza njengomaleko olawulayo odibanisa ulwazi lwe-herbivorous ukuze kuhlelwe kwakhona i-physiology yezityalo. Emva kokulinganisa i-herbivore, silinganise i-cumulative dynamics yeendidi eziphambili ze-plant hormones kwaye sabona i-temporal co-expression phakathi kwazo [Pearson correlation coefficient (PCC)> 0.4] (Umfanekiso 3A). Njengoko bekulindelekile, ii-plant hormones ezinxulumene ne-biosynthesis zidibene ngaphakathi kwi-plant hormone co-expression network. Ukongeza, i-metabolic specificity (i-Si index) idweliswe kule network ukuze ibonise ii-plant hormones ezibangelwa ziindlela ezahlukeneyo zonyango. Iindawo ezimbini eziphambili ze-herbivorous specific response zidwetshiwe: enye ikwi-JA cluster, apho i-JA (ifomu yayo esebenzayo ngokwebhayoloji i-JA-Ile) kunye nezinye i-JA derivatives zibonisa amanqaku aphezulu e-Si; enye yi-ethylene (ET). I-Gibberellin ibonise ukunyuka okuphakathi kwi-herbivore specificity, ngelixa ezinye ii-plant hormones, ezifana ne-cytokinin, i-auxin, kunye ne-abscisic acid, zazine-induction specificity ephantsi kwi-herbivores. Xa kuthelekiswa nokusebenzisa i-W + W yodwa, ukwandiswa kwexabiso eliphezulu le-JA derivatives nge-OS application (W + OS) ngokusisiseko kunokuguqulwa kube sisalathisi esinamandla se-JAs. Ngokungalindelekanga, i-OSM kunye ne-OSS1 enomxholo owahlukileyo we-exicitor zaziwa ukuba zibangela ukuqokelelana okufanayo kwe-JA kunye ne-JA-Ile. Ngokungafaniyo ne-OSS1, i-OSM ibangelwa ngokukodwa kwaye ngamandla yi-OSMs, ngelixa i-OSM1 ingayandisi impendulo yamanxeba asezantsi (Umfanekiso 3B).
(A) Uhlalutyo lwenethiwekhi yokubonisana ngokudibeneyo olusekwe ekubalweni kwe-PCC kokulinganisa i-herbivore-induced plant hormone accumulation kinetics simulation. I-node imele i-hormone yesityalo esinye, kwaye ubungakanani be-node bumele i-Si index ethile kwi-hormone yesityalo phakathi konyango. (B) Ukuqokelelwa kwe-JA, i-JA-Ile kunye ne-ET kumagqabi abangelwa ziindlela ezahlukeneyo zonyango eziboniswe yimibala eyahlukeneyo: i-apricot, i-W + i-OSM; eluhlaza okwesibhakabhaka, i-W + i-OSL; emnyama, i-W + i-W; ngwevu, i-C (ulawulo). Ii-asterisk zibonisa umahluko omkhulu phakathi konyango kunye nolawulo (i-ANOVA yeendlela ezimbini ilandelwa yiTukey HSD post hoc multiple comparison, *** P <0.001). Uhlalutyo lwethiyori yolwazi lwe-(C)697 MS/MS (ifayile yedatha S1) kwi-JA biosynthesis kunye ne-spectrum yokuqonda okungalunganga (irAOC kunye ne-irCOI1) kunye (D)585 MS/MS (ifayile yedatha S1) kwi-ETR1 enesignali ye-ET engalunganga Unyango lwe-herbivore olulinganisiweyo oluye lwabangela imigca yezityalo kunye nezityalo zokulawula ezingenanto (EV). Ii-asterisk zibonisa umahluko omkhulu phakathi konyango lwe-W+OS kunye nolawulo olungonakalanga (i-ANOVA eneendlela ezimbini ilandelwa yiTukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 kunye ***P<0.001). (E) Iigrafu ezisasazekileyo zokuchasana okuthe tyaba nokuchwetheza. Imibala imele iintlobo ezahlukeneyo eziguqulwe ngokwemfuza; iimpawu zimele iindlela ezahlukeneyo zonyango: unxantathu, u-W + OSS1; uxande, u-W + OSM; isangqa u-C
Okulandelayo, sisebenzisa uhlobo lwe-genetic modified strain ye-attenuated Nepenthes (irCOI1 kunye ne-sETR1) kumanyathelo aphambili e-JA kunye ne-ET biosynthesis (irAOC kunye ne-irACO) kunye ne-perception (irCOI1 kunye ne-sETR1) ukuhlalutya imetabolism yezi hormone zezityalo zimbini kwi-herbivores. Igalelo elihambelanayo lokuprograma kwakhona. Ngokuhambelana novavanyo lwangaphambili, siqinisekisile ukungeniswa kwe-herbivore-OS kwizityalo ezingenanto (EV) (Umfanekiso 3, C ukuya ku-D) kunye nokwehla ngokubanzi kwi-Hj index okubangelwa yi-OSM, ngelixa i-δj index inyukile. Impendulo icace ngakumbi kunempendulo ebangelwe yi-OSS1. Igrafu yemigca emibini esebenzisa i-Hj kunye ne-δj njenge-coordinates ibonisa ukurhoxiswa kwemithetho ethile (Umfanekiso 3E). Eyona ndlela icacileyo kukuba kwiintlobo ezingenayo i-JA signal, utshintsho lwe-metabolome kunye notshintsho olubangelwa yi-herbivores luphantse lupheliswe ngokupheleleyo (Umfanekiso 3C). Ngokwahlukileyo koko, umbono we-ET ongathethiyo kwizityalo ze-SETR1, nangona isiphumo sisonke kutshintsho kwi-metabolism yezityalo ezitya utyani siphantsi kakhulu kuneso se-JA signaling, sinciphisa umahluko kwi-Hj kunye ne-δj indices phakathi kwe-OSM kunye ne-OSS1 excitations (Umfanekiso 3D kunye noMfanekiso S5). . Oku kubonisa ukuba ukongeza kumsebenzi oyintloko we-JA signal transduction, i-ET signal transduction ikwasebenza njengohlengahlengiso lwempendulo ye-metabolic yeentlobo ezithile zezilwanyana ezitya utyani. Ngokuhambelana nalo msebenzi wohlengahlengiso, bekungekho tshintsho kwi-metabolome inducibility iyonke kwizityalo ze-SETR1. Kwelinye icala, xa kuthelekiswa nezityalo ze-sETR1, izityalo ze-irACO zibangele ubuninzi obufanayo botshintsho lwe-metabolic olubangelwa zizilwanyana ezitya utyani, kodwa zibonise amanqaku ahlukeneyo kakhulu e-Hj kunye ne-δj phakathi kwemingeni ye-OSM kunye ne-OSS1 (Umfanekiso S5).
Ukuze sichonge ii-metabolites ezikhethekileyo ezinegalelo elibalulekileyo kwimpendulo yezilwanyana ezitya utyani kunye nokulungisa imveliso yazo ngemiqondiso ye-ET, sisebenzise indlela ye-MS/MS eyakhiwe ngaphambili. Le ndlela ixhomekeke kwindlela ye-bi-clustering ukuze iphinde ifumane usapho lwe-metabolic kwi-MS/MS fragments [normalized dot product (NDP)] kunye ne-similarity score esekelwe kwi-neutral loss (NL). Iseti yedatha ye-MS/MS eyakhiwe ngohlalutyo lwemigca ye-ET transgenic ivelise i-585 MS/MS (ifayile yedatha S1), eyasonjululwa ngokuzihlanganisa kwiimodyuli ezisixhenxe eziphambili ze-MS/MS (M) (Umfanekiso 4A). Ezinye zezi modyuli zigcwele kakhulu ii-metabolites ezikhethekileyo ezazichazwe ngaphambili: umzekelo, i-M1, i-M2, i-M3, i-M4 kunye ne-M7 zizityebi kwi-phenol derivatives ezahlukeneyo (M1), i-flavonoid glycosides (M2), i-acyl sugar (M3 kunye ne-M4), kunye ne-17-HGL-DTG (M7). Ukongeza, ulwazi oluthile lwe-metabolic (i-Si index) ye-metabolite enye kwimodyuli nganye luyabalwa, kwaye ukusasazwa kwayo yi-Si kunokubonwa ngendlela engabonakaliyo. Ngamafutshane, ii-spectra ze-MS/MS ezibonisa ubuninzi be-herbivory kunye ne-genotype specificity zibonakaliswa ngamaxabiso aphezulu e-Si, kwaye izibalo ze-kurtosis zibonisa ukusasazwa koboya kwikona yomsila wasekunene. Olunye usasazo lwe-colloid oluthambileyo lufunyenwe kwi-M1, apho i-phenol amide ibonise i-Si fraction ephezulu (Umfanekiso 4B). I-herbivorous inducible 17-HGL-DTG ekhankanyiweyo ngaphambili kwi-M7 ibonise i-Si score ephakathi, ebonisa inqanaba eliphakathi lolawulo lokwahluka phakathi kweentlobo ezimbini ze-OS. Ngokwahlukileyo koko, uninzi lwe-metabolites ezikhethekileyo eziveliswe ngokukodwa, ezifana ne-rutin, i-CGA, kunye ne-acyl sugar, ziphakathi kwamanqaku aphantsi e-Si. Ukuze kuhlolwe ngcono ubunzima besakhiwo kunye nokusasazwa kwe-Si phakathi kwe-metabolites ezikhethekileyo, kwakhiwe inethiwekhi ye-molecular kwimodyuli nganye (Umfanekiso 4B). Ingqikelelo ebalulekileyo yethiyori ye-OD (eshwankathelwe kuMfanekiso 1B) kukuba ukuhlelwa kwakhona kwee-metabolites ezikhethekileyo emva kwe-herbivory kufanele kukhokelele kutshintsho lwendlela enye kwii-metabolites ezinexabiso eliphezulu lokuzikhusela, ngakumbi ngokwandisa ukucaciswa kwazo (ngokuchasene nokusasazwa okungacwangciswanga) I-Mode) I-metabolite yokuzikhusela eqikelelwe yithiyori ye-MT. Uninzi lwee-phenol derivatives eziqokelelwe kwi-M1 zinxulumene nokusebenza ngokudibeneyo nokwehla kokusebenza kwezinambuzane (32). Xa sithelekisa amaxabiso e-Si ​​kwi-metabolites ye-M1 phakathi kwamagqabi abangelwayo kunye namagqabi ayinxalenye yezityalo zokulawula i-EV kwiiyure ezingama-24, siqaphele ukuba ukucaciswa kwe-metabolism yee-metabolites ezininzi emva kwezinambuzane ze-herbivory kunendlela ekhulayo kakhulu (Umfanekiso 4C). Ukunyuka okuthe ngqo kwexabiso le-Si kufunyenwe kuphela kwii-phenolamides zokuzikhusela, kodwa akukho kunyuka kwexabiso le-Si kufunyenwe kwezinye ii-phenols kunye nee-metabolites ezingaziwayo ezihlala kule modyuli. Le yimodeli ekhethekileyo, enxulumene nethiyori ye-OD. Izibikezelo eziphambili zotshintsho lwe-metabolic olubangelwa zii-herbivores ziyahambelana. Ukuze kuvavanywe ukuba olu hlobo lwe-phenolamide spectrum lubangelwe yi-OS-specific ET, sidwebe i-metabolite Si index kwaye sabangela ixabiso lokubonisa umahluko phakathi kwe-OSM kunye ne-OSS1 kwii-genotypes ze-EV kunye ne-sETR1 (Umfanekiso 4D). Kwi-sETR1, umahluko obangelwe yi-phenamide phakathi kwe-OSM kunye ne-OSS1 wehlisiwe kakhulu. Indlela ye-bi-clustering ikwasetyenziswe kwidatha ye-MS/MS eqokelelwe kwiintlobo ezingenayo i-JA eyaneleyo yokugqiba iimodyuli eziphambili ze-MS/MS ezinxulumene ne-JA-regulated metabolic specialization (Umfanekiso S6).
(A) Iziphumo zokudibanisa i-585 MS/MS ngokusekelwe kwi-shared fragment (NDP similarity) kunye ne-shared neutral loss (NL similarity) ziphumela ekubeni i-module (M) ihambelane nosapho lwe-compound eyaziwayo, okanye ngokwakhiwa kwe-Metabolite engaziwayo okanye engalungiswanga kakuhle. Ecaleni kwe-module nganye, ukusasazwa kwe-metabolite (MS/MS) specific (Si) kuboniswa. (B) I-Modular molecular network: Ii-Nodes zimele i-MS/MS kunye nemiphetho, i-NDP (ebomvu) kunye ne-NL (eluhlaza okwesibhakabhaka) amanqaku e-MS/MS (anqunyulwe,> 0.6). I-graded metabolite specificity index (Si) efakwe umbala ngokusekelwe kwi-module (ekhohlo) kwaye idweliswe kwi-molecular network (ekunene). (C) I-Module M1 yesityalo se-EV kwimeko equlathe (ulawulo) kunye ne-induced (i-herbivore eyenziweyo) kwiiyure ezingama-24: i-molecular network diagram (Ixabiso le-Si likhulu le-node, i-defensive phenolamide igqanyiswe ngombala oluhlaza okwesibhakabhaka). (D) Umzobo wenethiwekhi ye-M1 molecular yomgca we-spectrum sETR1 one-EV kunye ne-ET ephazamisekileyo: i-phenolic compound emelwe yi-node yesangqa esiluhlaza, kunye nomahluko obalulekileyo (ixabiso le-P) phakathi konyango lwe-W + OSM kunye ne-W + OSS1 njengobungakanani beNode. 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 in wolfberry Son; Nick. O-AS, O-acyl sugar.
Siphinde sandisa uhlalutyo ukusuka kwi-genotype enye ye-Nepenthes enciphileyo ukuya kwi-natural populations, apho utshintsho olunamandla lwangaphakathi kwizinga le-JA ezitya utyani kunye namanqanaba athile e-metabolite achazwe ngaphambili kwi-natural populations (26). Sebenzisa le seti yedatha ukugubungela ii-germplasm ezingama-43. Ezi germplasm ziquka iintlobo ezili-123 zezityalo ezivela kwi-N. pallens. Ezi zityalo zithathwe kwimbewu eqokelelwe kwiindawo ezahlukeneyo zokuhlala e-Utah, eNevada, eArizona, naseCalifornia (Umfanekiso S7), sibale ulwahluko lwe-metabolome (apha olubizwa ngokuba linqanaba labemi) i-β diversity) kunye nobuchwephesha obubangelwa yi-OSM. Ngokuhambelana nezifundo zangaphambili, sibonile uluhlu olubanzi lotshintsho lwe-metabolic kwi-Hj kunye ne-δj axes, nto leyo ebonisa ukuba ii-germplasms zinomahluko omkhulu kwi-plasticity yempendulo yazo ye-metabolic kwi-herbivores (Umfanekiso S7). Le ntlangano ikhumbuza izinto ezibonwe ngaphambili malunga noluhlu oluguquguqukayo lotshintsho lwe-JA olubangelwa zi-herbivores, kwaye igcine ixabiso eliphezulu kakhulu kwi-natural populations enye (26, 36). Ngokusebenzisa i-JA kunye ne-JA-Ile ukuvavanya ulwalamano olupheleleyo phakathi kwe-Hj kunye ne-δj, sifumanise ukuba kukho ulwalamano oluhle phakathi kwe-JA kunye ne-metabolome β diversity kunye ne-specialization index (Umfanekiso S7). Oku kubonisa ukuba ukungafani okubangelwa yi-herbivore kwi-JAs induction efunyenwe kwinqanaba labantu kusenokuba kungenxa ye-polymorphisms ephambili ye-metabolic ebangelwa kukukhethwa kwizinambuzane ezitya utyani.
Izifundo zangaphambili zibonise ukuba iintlobo zecuba zahlukile kakhulu kuhlobo kunye nokuxhomekeka okunxulumene nokuzikhusela okubangelwayo kunye nokwakha i-metabolism. Kukholelwa ukuba olu tshintsho ekudlulisweni kwesignali yokulwa nezityalo kunye nokukwazi ukuzikhusela lulawulwa luxinzelelo lwabemi bezinambuzane, umjikelo wobomi bezityalo, kunye neendleko zemveliso yokuzikhusela kwindawo apho uhlobo oluthile lukhula khona. Sifunde ukuhambelana kokuguqulwa kwe-metabolome yamagqabi okubangelwa zizityalo ezitya utyani zeentlobo ezintandathu zeNicotiana ezivela eMntla Melika naseMzantsi Melika. Ezi ntlobo zihlobene kakhulu neNepenthes eMntla Melika, ezizezi Nicolas Bociflo. La, N. nicotinis, Nicotiana n. ingca encitshisiweyo, Nicotiana tabacum, linear tobacco, icuba (Nicotiana spegazzinii) kunye necuba legqabi legwayi (Nicotiana obtusifolia) (Umfanekiso 5A) (37). Ezi ntlobo zintandathu kwezi, kuquka uhlobo oluphawulwe kakuhle lwe-N. nceda, zizityalo zonyaka ze-petunia clade, kwaye i-obtusifolia N. zizityalo ezihlala ixesha elide ze-sister clade Trigonophyllae (38). Emva koko, ukungeniswa kwe-W + W, W + OSM kunye ne-W + OSS1 kwenziwa kwezi ntlobo zisixhenxe ukuze kufundwe uhlengahlengiso lwe-metabolic kwinqanaba leentlobo zokutya kwezinambuzane.
(A) Umthi we-phylogenetic we-bootstrap osekelwe kubungakanani obukhulu [bokwenziwa kwe-glutamine yenyukliya (38)] kunye nokusasazwa kweendawo zeentlobo ezisixhenxe zeNicotiana ezinxulumene ngokusondeleyo (imibala eyahlukileyo) (37). (B) Isicwangciso sokusasazeka kokwahluka okukhethekileyo kwiiprofayili ze-metabolic zeentlobo ezisixhenxe zeNicotiana (939 MS/MS; ifayile yedatha S1). Kwinqanaba leentlobo, ukwahluka kwe-metabolome kunxulunyaniswa kakubi nomgangatho wobuchwephesha. Uhlalutyo lolwalamano lwenqanaba leentlobo phakathi kokwahluka kwe-metabolic kunye nobuchwephesha kunye nokuqokelelwa kwe-JA kuboniswe kuMfanekiso 2. S9. Umbala, iintlobo ezahlukeneyo; unxantathu, W + OSS1; uxande, W + OSM; (C) I-Nicotiana JA kunye ne-JA-Ile dynamics zibekwe ngokwe-OS excitation amplitude (i-ANOVA eneendlela ezimbini kunye ne-Tukey HSD emva kokuthelekiswa okuninzi, * P <0.05, ** P <0.01 kunye * ** Ukuthelekisa i-W + OS kunye ne-W + W, P <0.001). Ibhokisi yesicwangciso se (D) sokwahluka kunye (E) nokuchonga uhlobo ngalunye emva kokulinganisa i-herbivorous kunye ne-methyl JA (MeJA). I-asterisk ibonisa umahluko obalulekileyo phakathi kwe-W + OS kunye ne-W + W okanye i-lanolin kunye ne-W (Lan + W) okanye i-Lan kunye ne-MeJA (Lan + MeJa) kunye nolawulo lwe-Lan (uhlalutyo lweendlela ezimbini zokwahluka, kulandelwa yi-Tukey's HSD post hoc multiple comparison, *P<0.05, **P<0.01 kunye ***P<0.001).
Sisebenzisa indlela yeqela elibini, sichonge iimodyuli ezili-9 ze-939 MS/MS (ifayile yedatha S1). Ukwakhiwa kwe-MS/MS okuhlengahlengiswe ngonyango olwahlukeneyo kwahluka kakhulu phakathi kweemodyuli ezahlukeneyo phakathi kweentlobo (Umfanekiso S8). Ukubona i-Hj (ebizwa apha njenge-species-level γ-diversity) kunye ne-δj kubonisa ukuba iintlobo ezahlukeneyo zihlangana zibe ngamaqela ahlukeneyo kakhulu kwindawo ye-metabolic, apho ulwahlulo lwenqanaba leentlobo ludla ngokubonakala ngakumbi kune-excitation. Ngaphandle kwe-N. linear kunye ne-N. obliquus, zibonisa uluhlu olubanzi lweziphumo zokungenisa (Umfanekiso 5B). Ngokwahlukileyo koko, iintlobo ezifana ne-N. purpurea kunye ne-N. obtusifolia zinempendulo ye-metabolic engacacanga kangako kunyango, kodwa i-metabolome yahlukile ngakumbi. Ukusasazwa kwempendulo ye-metabolic ebangelwayo kubangele ulwalamano olubi olubalulekileyo phakathi kokuchwephesha kunye nokwahluka kwe-gamma (PCC = -0.46, P = 4.9×10-8). Utshintsho olubangelwa yi-OS kumanqanaba e-JA luhambelana kakuhle nobuchwephesha be-metabolome, kwaye luhambelana kakubi nokwahluka kwe-gamma ye-metabolic eboniswa yintlobo nganye (Umfanekiso 5B kunye noMfanekiso S9). Kubalulekile ukuqaphela ukuba iintlobo ezibizwa ngokuba ziintlobo "zempendulo yesignali" kuMfanekiso 5C, ezifana neNepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, kunye neNepenthes ezincitshisiweyo, zibangele iimpawu ezibalulekileyo kwimizuzu engama-30. Ukuqhambuka kwe-JA kunye ne-JA-Ile yakutshanje kwi-OS, ngelixa ezinye iibhaktheriya ezibizwa ngokuba "ziyi-signal responsive", ezifana neNepenthes mills, Nepenthes powdery kunye ne-N. obtusifolia zibonisa kuphela i-JA-Ile Edge induction ngaphandle kobunye ubunyani be-OS (Umfanekiso 5C). Kwinqanaba le-metabolic, njengoko kukhankanyiwe ngasentla, kwiNepenthes ezincitshisiweyo, izinto eziphendula isignali zibonise ubunyani be-OS kwaye zandisa kakhulu i-δj, ngelixa zinciphisa i-Hj. Esi siphumo se-OS-specific priming asizange sibonwe kwiintlobo ezihlelwe njengeentlobo zesignali ezingasebenziyo (Umfanekiso 5, D kunye no-E). Iimetabolites ezithile ze-OS zabelwana rhoqo phakathi kweentlobo ezisabela kwisignali, kwaye la maqela eesignali aqokelelana neentlobo ezineempendulo zesignali ezibuthathaka, ngelixa iintlobo ezineempendulo zesignali ezibuthathaka zibonisa ukuxhomekeka okuncinci (Umfanekiso S8). Esi siphumo sibonisa ukuba ukungeniswa kwe-OS ethile yee-JA kunye nokulungiswa kwakhona kwe-OS ethile ye-metabolome esezantsi kudityaniswe kwinqanaba leentlobo.
Okulandelayo, sisebenzise intlama yelanolin equlethe i-methyl JA (MeJA) ukunyanga izityalo ukuphanda ukuba ezi ndlela zokudibanisa zithintelwe na kukufumaneka kwe-JA esetyenziswa yi-JA yangaphandle, eya kuba kwi-cytoplasm yezityalo. Ukuchithwa ngokukhawuleza yi-JA. Sifumene umkhwa ofanayo wotshintsho oluhamba kancinci ukusuka kwiintlobo eziphendula imiqondiso ukuya kwiintlobo ezingaphenduliyo imiqondiso ezibangelwa kukunikezelwa okuqhubekayo kwe-JA (Umfanekiso 5, D kunye no-E). Ngamafutshane, unyango lwe-MeJA luhlengahlengise kakhulu ii-metabolomes ze-linear nematodes, i-N. obliquus, i-N. aquaticus, i-N. pallens, kunye ne-N. mikimotoi, nto leyo ebangele ukwanda okukhulu kwe-δj kunye nokwehla kwe-Hj. N. purpurea kubonise kuphela ukwanda kwe-δj, kodwa hayi i-Hj. N. obtusifolia, ebikade ibonakalisiwe ukuba iqokelela amanqanaba aphantsi kakhulu e-JA, nayo ayiphenduli kakuhle kunyango lwe-MeJA ngokubhekiselele ekuhlengahlengisweni kwe-metabolome. Ezi ziphumo zibonisa ukuba ukuveliswa kwe-JA okanye ukudluliselwa kwemiqondiso kuthintelwe ngokwengqondo kwiintlobo ezingaphenduliyo imiqondiso. Ukuvavanya le ngcamango, sifunde iintlobo ezine (ii-N. pallens, ii-N. mills, ii-N. pink kunye nee-N. microphylla) ezibangelwa yi-W + W, ii-W + OSM kunye ne-W + OSS1 Transcriptome (39). Ngokuhambelana nepateni yokuguqulwa kwe-metabolome, iintlobo zahlulwe kakuhle kwindawo ye-transcriptome, phakathi kwazo i-N. attenuated ibonise i-RDPI ephezulu ebangelwa yi-OS, ngelixa i-N. gracilis yayineyona iphantsi (Umfanekiso 6A). Nangona kunjalo, kufunyenwe ukuba umahluko we-transcriptome obangelwe yi-N. oblonga wawungowona uphantsi phakathi kweentlobo ezine, ngokuchaseneyo nomahluko ophezulu we-metabonomic we-N. oblonga oboniswe ngaphambili kwiintlobo ezisixhenxe. Izifundo zangaphambili zibonise ukuba iseti yezakhi zofuzo ezinxulumene neempawu zokuzikhusela zakuqala, kuquka nezibonakaliso ze-JA, ichaza ukuchaneka kweempendulo zokuzikhusela zakuqala ezibangelwa zi-elicitors ezinxulumene ne-herbivore kwiintlobo zeNicotiana (39). Ukuthelekisa iindlela zokubonisa ze-JA phakathi kwezi ntlobo zine kutyhile iphethini enomdla (Umfanekiso 6B). Uninzi lweejini kule ndlela, ezifana ne-AOC, i-OPR3, i-ACX kunye ne-COI1, zibonise amanqanaba aphezulu okungeniswa kwezi ntlobo zine. Nangona kunjalo, ijini ephambili, i-JAR4, iguqula i-JA ibe yifom yayo esebenzayo ngokwebhayoloji yee-transcripts eziqokelelweyo ze-JA-Ile, kwaye inqanaba layo lokubhalwa liphantsi kakhulu, ngakumbi kwi-N. mills, i-Nepenthes pieris kunye ne-N. microphylla. Ukongeza, kuphela i-transcript yesinye ijini i-AOS engakhange ifunyanwe kwi-N. bifidum. Olu tshintsho kwimbonakalo yejini lunokuba noxanduva lwe-phenotypes egqithisileyo ebangelwa yimveliso ephantsi ye-JA kwiintlobo ze-signal anergic kunye nokungeniswa kwe-N. gracilis.
(A) Uhlalutyo lwethiyori yolwazi malunga nokuhlelwa ngokutsha kweempendulo zokuqala zokubhalwa kweentlobo ezine zecuba ezinxulumene kakhulu ezithathwe iisampulu emva kwemizuzu engama-30 emva kokungeniswa kwezityalo ezitya utyani. I-RDPI ibalwa ngokuthelekisa amagqabi abangelwa yi-OS yezityalo ezitya utyani kunye nolawulo lwamanxeba. Imibala ibonisa iintlobo ezahlukeneyo, kwaye iimpawu zibonisa iindlela ezahlukeneyo zonyango. (B) Uhlalutyo lokubonakaliswa kwezakhi zofuzo kwiindlela zokubonisa i-JA phakathi kweentlobo ezine. Indlela ye-JA elula iboniswe ecaleni kweploti yebhokisi. Imibala eyahlukeneyo ibonisa iindlela ezahlukeneyo zokucubungula. I-asterisk ibonisa ukuba kukho umahluko omkhulu phakathi konyango lwe-W + OS kunye nolawulo lwe-W + W (kuvavanyo lwe-t loMfundi lokwahluka kwezibini, *P<0.05, **P<0.01 kunye ***P<0.001). I-OPDA, i-12-oxophytodienoic acid; I-OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Kwinxalenye yokugqibela, sifunde indlela ukutshintshwa kweentlobo zezinambuzane ezithile kwi-metabolome yeentlobo ezahlukeneyo zezityalo okunokumelana ngayo nezilwanyana ezitya utyani. Uphando lwangaphambili lugxininise uhlobo lweNicotiana. Ukumelana kwazo ne-Ms kunye nee-larvae kwahluka kakhulu (40). Apha, sifunde unxibelelwano phakathi kwale modeli kunye nokuguquguquka kwazo kwe-metabolic. Sisebenzisa iintlobo ezine zecuba ezingasentla, kwaye sivavanya unxibelelwano phakathi kokungafani kunye nokuzikhethela kwe-metabolome ebangelwa zizilwanyana ezitya utyani kunye nokumelana kwezityalo kwi-Ms kunye ne-Sl, sifumanise ukuba ukumelana, ukungafani kunye nokuzikhethela kwi-generalist Sl All kunxulumene kakuhle, ngelixa ulwalamano phakathi kokumelana nabafazi abaziingcali kunye nokuzikhethela lubuthathaka, kwaye ulwalamano nokungafani alubalulekanga (Umfanekiso S10). Ngokuphathelele ukumelana ne-S1, zombini i-N. chinensis kunye ne-N. gracilis ezinciphileyo, ezaziboniswe ngaphambili ukuba zibonisa amanqanaba okudluliselwa kwesignali ye-JA kunye nokuguquguquka kwe-metabolome, zazineempendulo ezahlukeneyo kakhulu ekungenisweni kwe-herbivore, kwaye zikwabonise ukumelana okuphezulu okufanayo. Isondo.
Kwiminyaka engamashumi amathandathu edlulileyo, ithiyori yokukhusela izityalo ibonelele ngesakhelo sethiyori, esisekelwe kuyo abaphandi baqikelele inani elikhulu lokuvela kunye nemisebenzi ye-metabolites ekhethekileyo yezityalo. Uninzi lwezi thiyori azilandeli inkqubo eqhelekileyo yezigqibo ezinamandla (41). Zicebisa izibikezelo eziphambili (3) kwinqanaba elifanayo lohlalutyo. Xa uvavanyo lwezibikezelo eziphambili luvumela ukuba iingcamango ezithile zihlalutywe, oku kuya kwenza ukuba intsimi iqhubeke phambili. Xhaswa, kodwa ungamkeli ezinye (42). Endaweni yoko, ithiyori entsha yenza izibikezelo kumanqanaba ahlukeneyo ohlalutyo kwaye yongeza umaleko omtsha wezinto ezichazayo (42). Nangona kunjalo, iingcamango ezimbini ezicetywayo kwinqanaba lokusebenza, i-MT kunye ne-OD, zinokuchazwa ngokulula njengezibikezelo ezibalulekileyo zotshintsho olukhethekileyo lwe-metabolic olubangelwa zizilwanyana ezitya utyani: ithiyori ye-OD ikholelwa ukuba utshintsho "kwindawo" ekhethekileyo ye-metabolic lujolise kakhulu. Ithiyori ye-MT ikholelwa ukuba olu tshintsho aluyi kuba kwicala kwaye lubekwe ngokungacwangciswanga kwindawo ye-metabolic, kwaye ludla ngokuba ne-metabolites enexabiso eliphezulu lokuzikhusela. Uvavanyo lwangaphambili lwezibikezelo ze-OD kunye ne-MT luye lwavavanywa kusetyenziswa iseti encinci yeekhompawundi "zokuzikhusela" zangaphambili. Olu vavanyo lwe-metabolite-centric luthintela amandla okuhlalutya ubungakanani kunye nendlela yokulungiswa kwakhona kwe-metabolome ngexesha lokutya izityalo, kwaye aluvumeli uvavanyo ngaphakathi kwesakhelo sezibalo esihambelanayo ukuba lufune izibikezelo eziphambili ezinokuqwalaselwa ngokubanzi. Ukulinganisa utshintsho kwi-metabolome yezityalo. Apha, sisebenzise itekhnoloji entsha kwi-metabolomics esekelwe kwi-MS yokubala kwaye senza uhlalutyo lwe-deconvolution MS kwi-currency jikelele yeenkcazo zethiyori yolwazi ukuvavanya umahluko phakathi kwezi zimbini zicetywayo kwinqanaba le-metabolomics yehlabathi. Uqikelelo oluphambili lwale thiyori. Ithiyori yolwazi isetyenziswe kwiindawo ezininzi, ngakumbi kumxholo wophando lwezinto eziphilayo kunye nokuhamba kwezondlo (43). Nangona kunjalo, ngokokwazi kwethu, esi sisicelo sokuqala esisetyenziselwa ukuchaza indawo yolwazi lwe-metabolic yezityalo kunye nokusombulula iingxaki zendalo ezinxulumene notshintsho lwe-metabolic okwethutyana ekuphenduleni izikhokelo zendalo. Ngokukodwa, amandla ale ndlela akwikhono layo lokuthelekisa iipateni ngaphakathi naphakathi kweentlobo zezityalo ukuhlola indlela i-herbivores eziguquke ngayo ukusuka kwiintlobo ezahlukeneyo ukuya kwiipateni ze-macroevolutionary eziphakathi kweentlobo ezahlukeneyo kumanqanaba ahlukeneyo okuzivelela. I-Metabolism.
Uhlalutyo lwecandelo eliphambili (i-PCA) luguqula iseti yedatha ye-multivariate ibe yindawo yokunciphisa ubukhulu ukuze indlela ephambili yedatha ichazwe, ngoko ke idla ngokusetyenziswa njengendlela yokuhlola ukuvavanya iseti yedatha, njenge-deconvolution metabolome. Nangona kunjalo, ukunciphisa ubukhulu kuya kulahlekelwa yinxalenye yomxholo wolwazi kwiseti yedatha, kwaye i-PCA ayinakubonelela ngolwazi oluninzi malunga neempawu ezihambelana ngokukodwa nethiyori ye-ecological, ezinje: indlela ii-herbivores ezilungisa ngayo ukwahluka kwiindawo ezikhethekileyo (umzekelo, ubutyebi, ukusasazwa) kunye nobuninzi) ii-metabolites? Zeziphi ii-metabolites ezixela kwangaphambili imeko ebangelwa yi-herbivore ethile? Ngokwembono yokuchazwa, ukwahluka kunye nokungaguquki, umxholo wolwazi weprofayili ye-metabolite ethile yegqabi uyabola, kwaye kufunyaniswe ukuba ukutya ii-herbivores kunokuvuselela i-metabolism ethile. Ngokungalindelekanga, siqaphele ukuba, njengoko kuchaziwe kwizalathisi zethiyori yolwazi ezisetyenzisiweyo, imeko ye-metabolic ephumayo inokudibana okukhulu emva kokuhlaselwa kwezilwanyana ezimbini ezitya utyani (i-generalist etya ubusuku bonke i-Sl) kunye nengcali yeSolanaceae uNks. Nangona indlela ezitya ngayo kunye nokugxila kwazo kwahlukile kakhulu. I-Fatty acid-amino acid conjugate (FAC) initiator kwi-OS (31). Ngokusebenzisa i-herbivore OS ukunyanga amanxeba aqhelekileyo okubhoboza, unyango lwe-herbivore olulinganisiweyo nalo lubonise umkhwa ofanayo. Le nkqubo imiselweyo yokulinganisa impendulo yezityalo kuhlaselo lwe-herbivore isusa izinto ezididayo ezibangelwa lutshintsho kwindlela yokutya yezilwanyana ezitya utyani, ezikhokelela kumanqanaba ahlukeneyo omonakalo ngamaxesha ahlukeneyo (34). I-FAC, eyaziwa ngokuba yimbangela ephambili ye-OSM, inciphisa i-JAS kunye nezinye iimpendulo zehomoni yezityalo kwi-OSS1, ngelixa i-OSS1 inciphisa amaxesha amaninzi (31). Nangona kunjalo, i-OSS1 ibangele amanqanaba afanayo okuqokelelwa kwe-JA xa kuthelekiswa ne-OSM. Kuye kwaboniswa ngaphambili ukuba impendulo ye-JA kwi-Nepenthes enciphileyo inovelwano kakhulu kwi-OSM, apho i-FAC inokugcina umsebenzi wayo nokuba ixutywe nge-1:1000 ngamanzi (44). Ke ngoko, xa kuthelekiswa ne-OSM, nangona i-FAC kwi-OSS1 iphantsi kakhulu, yanele ukubangela ukuqhambuka kwe-JA okwaneleyo. Izifundo zangaphambili zibonise ukuba iiproteni ezifana ne-porin (45) kunye ne-oligosaccharides (46) zingasetyenziswa njengeempawu zemolekyuli ukuqala iimpendulo zokhuselo lwezityalo kwi-OSS1. Nangona kunjalo, akukacaci ukuba ezi zinto zibangela ukuqokelelwa kwe-JA ezibonwe kuphononongo lwangoku.
Nangona zimbalwa izifundo ezichaza iminwe ye-metabolic eyahlukileyo ebangelwa kukusetyenziswa kwezityalo ezahlukeneyo ezitya utyani okanye i-JA okanye i-SA yangaphandle (i-salicylic acid) (47), akukho mntu uphazamise ukuphazamiseka kweentlobo ezithile zezityalo ezitya utyani kwinethiwekhi yengca yezityalo kunye nemiphumo yako kwi-specific Impembelelo iyonke ye-metabolism ifundwa ngokucwangcisiweyo. Olu hlalutyo luqinisekisile ngakumbi ukuba unxibelelwano lwenethiwekhi yehomoni yangaphakathi kunye nezinye iihomoni zezityalo ngaphandle kwe-JAs lubumba ukucaciswa kokuhlelwa kwakhona kwe-metabolic okubangelwa zizilwanyana ezitya utyani. Ngokukodwa, sifumanise ukuba i-ET ebangelwa yi-OSM yayinkulu kakhulu kunoko kubangelwa yi-OSS1. Le ndlela ihambelana nomxholo we-FAC ongaphezulu kwi-OSM, oyimfuneko kwaye owaneleyo wokubangela ukuqhuma kwe-ET (48). Kwimeko yokusebenzisana phakathi kwezityalo kunye nezilwanyana ezitya utyani, umsebenzi wokubonisa we-ET kwi-metabolite dynamics yezityalo ezithile usaqhubeka kwaye ujolise kuphela kwiqela elinye elihlanganisiweyo. Ukongeza, uninzi lwezifundo lusebenzise ukusetyenziswa kwe-ET okanye izinto ezandulelayo okanye izithinteli ezahlukeneyo ukuze kufundwe ukulawulwa kwe-ET, apho ezi zicelo zeekhemikhali zangaphandle ziya kuvelisa iziphumo ebezingalindelekanga ezininzi. Ngokolwazi lwethu, olu phononongo lumele uvavanyo lokuqala olukhulu olucwangcisiweyo lwendima ye-ET ekusebenziseni i-ET ukuvelisa nokubona izityalo eziguqulweyo ezingasebenzi kakuhle ukuze kuququzelelwe i-metabolome dynamics yezityalo. Ukungeniswa kwe-ET ethile ye-Herbivore ekugqibeleni kunokuguqula impendulo ye-metabolome. Eyona nto ibalulekileyo kukuguqulwa kwe-ET biosynthesis (ACO) kunye ne-perception (ETR1) genes ezityhile ukuqokelelwa kwe-phenolamides ethile ye-herbivore. Kuye kwaboniswa ngaphambili ukuba i-ET inokulungisa ukuqokelelwa kwe-nicotine okubangelwe yi-JA ngokulawula i-putrescine N-methyltransferase (49). Nangona kunjalo, ngokwembono yoomatshini, akucaci ukuba i-ET ilungisa njani ukuqokelelwa kwe-phenamide. Ukongeza kumsebenzi wokudluliselwa kwesignali ye-ET, i-metabolic flux ingathunyelwa kwi-S-adenosyl-1-methionine ukulawula utyalo-mali kwi-polyaminophenol amides. I-S-adenosyl-1-methionine yi-ET kwaye yindlela eqhelekileyo ye-polyamine biosynthetic pathway. Indlela i-ET signal elawula ngayo inqanaba le-phenolamide idinga uphando olongezelelweyo.
Kangangexesha elide, ngenxa yenani elikhulu lee-metabolites ezikhethekileyo zesakhiwo esingaziwayo, ingqwalasela enzulu kwiindidi ezithile ze-metabolic ayizange ikwazi ukuvavanya ngokungqongqo utshintsho lwexesha lokwahluka kwe-metabolic emva kokunxibelelana kwezinto eziphilayo. Okwangoku, ngokusekelwe kuhlalutyo lwethiyori yolwazi, isiphumo esiphambili sokufunyanwa kwe-MS/MS spectrum ngokusekelwe kwi-metabolites ezingakhethiyo kukuba ii-herbivores ezitya okanye ezilinganisa ii-herbivores ziyaqhubeka nokunciphisa ukwahluka kwe-metabolic iyonke ye-metabolome yamagqabi ngelixa zinyusa inqanaba lazo lobuchwephesha. Oku kwanda okwethutyana kwi-metabolome specificity ebangelwa zii-herbivores kunxulunyaniswa nokwanda kwe-synergistic kwi-transcriptome specificity. Uphawu olunegalelo kakhulu kule metabolome specialization inkulu (enexabiso eliphezulu le-Si) yi-metabolite ekhethekileyo enomsebenzi we-herbivorous ochazwe ngaphambili. Le modeli iyahambelana nokuqikelela kwethiyori ye-OD, kodwa ukuqikelela kwe-MT enxulumene nokungacwangciswanga kwe-metabolome reprogramming akuhambelani. Nangona kunjalo, le datha ikwahambelana nokuqikelela kwemodeli exutyiweyo (i-MT engcono kakhulu; Umfanekiso 1B), kuba ezinye ii-metabolites ezingachazwanga ezinemisebenzi yokuzikhusela engaziwayo zisenokulandela ukusasazwa kwe-Si okungacwangciswanga.
Ipateni ephawulekayo efunyenwe ngakumbi lolu phando kukuba ukusuka kwinqanaba le-micro-evolution (isityalo esinye kunye nenani lecuba) ukuya kwinqanaba elikhulu le-evolution (iintlobo zecuba ezinxulumeneyo), amanqanaba ahlukeneyo olungelelwaniso lwe-evolution "akwindlela engcono yokuzikhusela." Kukho umahluko omkhulu kubuchule bezilwanyana ezitya utyani. UMoore et al. (20) kunye noKessler kunye noKalske (1) bacebise ngokuzimeleyo ukuguqula amanqanaba amathathu okusebenza kwe-biodiversity ahlukaniswe nguWhittaker (50) ekuqaleni abe lutshintsho oluqulunqwe kwaye lubangelwe yi-temporal diversity ye-chemical diversity; aba babhali abazange bashwankathele. Iinkqubo zokuqokelelwa kwedatha ye-metabolome enkulu azichazi indlela yokubala i-metabolism diversity kwezi datha. Kolu phononongo, uhlengahlengiso oluncinci kuhlelo lwe-functional lukaWhittaker luya kuqwalasela i-α-metabolic diversity njengokwahluka kwe-MS/MS spectra kwisityalo esithile, kunye ne-β-metabolic diversity njenge-basic intraspecific metabolism yeqela le-populations Space, kunye ne-γ-metabolic diversity iya kuba lulwandiso lohlalutyo lweentlobo ezifanayo.
Isignali ye-JA ibalulekile kwiindlela ezahlukeneyo zokuphendula kwi-metabolism yezityalo ezitya utyani. Nangona kunjalo, akukho vavanyo oluqinileyo lobungakanani lwegalelo lokulawulwa kwe-JA biosynthesis kwi-metabolome variation, kwaye ukuba isignali ye-JA yindawo eqhelekileyo yokwahlukahluka kwe-metabolic okubangelwa luxinzelelo kwinqanaba eliphezulu lokuguquguquka kwe-macroevolution akukacaci. Siqaphele ukuba indlela i-Nepenthes herbivorous etya utyani ibangela i-metabolome specialization kwaye ukwahluka kwe-metabolome specialization ngaphakathi kwenani leentlobo zeNicotiana kunye neentlobo zeNicotiana ezinxulumeneyo ngokusondeleyo kunxulumene kakuhle ne-JA signaling. Ukongeza, xa isignali ye-JA yonakele, i-metabolic specificity ebangelwa yi-genotype herbivore enye iya kurhoxiswa (Umfanekiso 3, C kunye no-E). Ekubeni utshintsho lwe-metabolic spectrum lwamaqela e-Nepenthes ancipiated ngokwendalo luninzi kakhulu, utshintsho kwi-metabolic β diversity kunye ne-specificity kolu hlalutyo lunokubangelwa kakhulu kukuvuseleleka okukhulu kweendidi ze-metabolite-rich compound. Ezi klasi ze-compound zilawula inxalenye yeprofayili ye-metabolome kwaye zikhokelela kulwalamano oluhle neempawu ze-JA.
Ngenxa yokuba iindlela zebhayoloji zeentlobo zecuba ezinxulumene kakhulu nayo zahlukile kakhulu, ii-metabolites zichongiwe ngokukodwa kwicala lomgangatho, ngoko ke luhlalutyo ngakumbi. Ukucubungula kwethiyori yolwazi kwiprofayili ye-metabolic ebanjiweyo kubonisa ukuba ukungeniswa kwe-herbivorous kuyandisa urhwebo phakathi kokwahluka kwe-gamma ye-metabolic kunye nobuchwephesha. Isignali ye-JA idlala indima ephambili kolu rhwebo. Ukwanda kokuchwephesha kwe-metabolome kuhambelana noqikelelo oluphambili lwe-OD kwaye kunxulumene kakuhle nesignali ye-JA, ngelixa isignali ye-JA inxulumene kakubi nokwahluka kwe-gamma ye-metabolic. Ezi modeli zibonisa ukuba amandla e-OD ezityalo amiselwa kakhulu yi-plasticity ye-JA, nokuba kukwizinga le-microevolutionary okanye kwinqanaba elikhulu le-evolution. Uvavanyo lwesicelo se-JA yangaphandle oluthintela iziphene ze-JA biosynthesis lubonisa ngakumbi ukuba iintlobo zecuba ezinxulumeneyo zinokwahlulwa zibe ziintlobo eziphendulayo kwisignali kunye neentlobo ezingaphenduliyo kwisignali, njengendlela yazo ye-JA kunye ne-metabolome plasticity ebangelwa zizilwanyana ezitya utyani. Iintlobo ezingaphenduliyo kwisignali azinakuphendula ngenxa yokungakwazi kwazo ukuvelisa i-JA yendalo kwaye ke ngoko ziphantsi kwemida ye-physiological. Oku kunokubangelwa kukuguquka kwezinye iijini eziphambili kwindlela yokubonisa i-JA (i-AOS kunye ne-JAR4 kwi-N. crescens). Esi siphumo sigxininisa ukuba ezi patheni ze-interspecies macroevolutionary zinokuqhutywa ikakhulu lutshintsho kwindlela yokuqonda kunye nokuphendula kwehomoni yangaphakathi.
Ukongeza kwintsebenziswano phakathi kwezityalo kunye nezilwanyana ezitya utyani, ukuphononongwa kokwahluka kwe-metabolic kunxulumene nayo yonke inkqubela phambili ebalulekileyo yethiyori kuphando lokuziqhelanisa nebhayoloji nokusingqongileyo kunye nokuguquka kweempawu ezintsonkothileyo ze-phenotypic. Ngokwanda kwenani ledatha efunyenwe zizixhobo ze-MS zanamhlanje, uvavanyo lwe-hypothesis malunga nokwahluka kwe-metabolic ngoku lunokudlula umahluko we-metabolite yomntu ngamnye/udidi kwaye lwenze uhlalutyo lwehlabathi jikelele ukuze kutyhile iipatheni ezingalindelekanga. Kwinkqubo yohlalutyo olukhulu, umfanekiso obalulekileyo yingcinga yokucinga ngeemaphu ezinentsingiselo ezinokusetyenziselwa ukuhlola idatha. Ke ngoko, isiphumo esibalulekileyo sokudibanisa kwangoku kwe-metabolomics ye-MS/MS engakhethi cala kunye nethiyori yolwazi kukuba ibonelela nge-metric elula enokusetyenziswa ukwakha iimaphu zokukhangela ukwahluka kwe-metabolic kwiikali ezahlukeneyo ze-taxonomic. Yimfuneko esisiseko yale ndlela. Uphononongo lwe-micro/macro evolution kunye ne-ecology yoluntu.
Kwinqanaba le-macro-evolutionary, isiseko sethiyori ye-co-evolution yezityalo nezinambuzane zika-Ehrlich noRaven (51) kukuqikelela ukuba umahluko phakathi kweentlobo ezahlukeneyo ze-metabolic diversity yimbangela yokwahlukahlukana kweentlobo zezityalo. Nangona kunjalo, kwiminyaka engamashumi amahlanu ukusukela ekupapashweni kwalo msebenzi ubalulekileyo, le ngcamango ayikaze ivavanywe (52). Oku kungenxa kakhulu yeempawu ze-phylogenetic zeempawu ze-metabolic ezifanayo kuzo zonke iintlobo zezityalo ezikude. Ukunqaba kunokusetyenziswa ukuqinisa iindlela zohlalutyo olujoliswe kuzo. Umsebenzi wangoku we-MS/MS ocutshungulwa yithiyori yolwazi ulinganisa ukufana kwesakhiwo se-MS/MS see-metabolites ezingaziwayo (ngaphandle kokukhethwa kwe-metabolite kwangaphambili) kwaye uguqula ezi MS/MS zibe yiseti ye-MS/MS, ngoko ke kwi-metabolism yobungcali Ezi modeli ze-macro-evolutionary zithelekiswa kwisikali sohlulo. Izalathisi zezibalo ezilula. Inkqubo ifana nohlalutyo lwe-phylogenetic, olunokusebenzisa ulungelelwaniso lolandelelwano ukulinganisa izinga lokwahlukahlukana okanye ukuguquka kwabalinganiswa ngaphandle kokuqikelela kwangaphambili.
Kwinqanaba le-biochemical, ingcamango yokuvavanya kaFirn noJones (53) ibonisa ukuba ukwahluka kwe-metabolic kugcinwa kumanqanaba ahlukeneyo ukuze kubonelelwe ngezinto eziluhlaza ukuze kusetyenziswe imisebenzi ye-biological yee-metabolites ezingadibaniyo ngaphambili okanye ezitshintshiweyo. Iindlela zethiyori yolwazi zibonelela ngesakhelo apho olu tshintsho oluthile lwe-metabolite oluvela ngexesha lokuchonga i-metabolite lunokulinganiswa njengenxalenye yenkqubo yokuvavanya ecetywayo: uhlengahlengiso olusebenzayo lwe-biological ukusuka kwi-specificity ephantsi ukuya kwi-specificity ephezulu Ii-metabolites ezithintelweyo zendalo enikiweyo.
Lilonke, kwiintsuku zokuqala zebhayoloji yeemolekyuli, kwaveliswa iingcamango ezibalulekileyo zokukhusela izityalo, kwaye iindlela ezisekelwe kwingcamango ezisekelwe kwingcamango zithathwa njengezona ndlela zodwa zokuqhubela phambili kwezesayensi. Oku kungenxa kakhulu yemida yobuchwepheshe yokulinganisa i-metabolome yonke. Nangona iindlela ezisekelwe kwingcamango ziluncedo kakhulu ekukhetheni ezinye iindlela ezibangela iingxaki, amandla azo okuphucula ukuqonda kwethu iinethiwekhi zebhayoloji anqongophele ngakumbi kuneendlela zokubala ezikhoyo kwisayensi yanamhlanje enolwazi olupheleleyo. Ke ngoko, iingcamango ezingenakuqikelelwa zingaphaya kobubanzi bedatha ekhoyo, ngoko ke umjikelo wefomula/uvavanyo oluqikelelwayo lwenkqubela kwicandelo lophando awunakupheliswa (4). Sibona kwangaphambili ukuba indlela yokusebenza yokubala ye-metabolomics engeniswe apha inokuvuselela umdla kwimiba yamva nje (njani) kunye neyokugqibela (kutheni) yokwahluka kwe-metabolic, kwaye inegalelo kwixesha elitsha lesayensi yedatha ekhokelwa yingcamango. Eli xesha liphinde lahlola iingcamango ezibalulekileyo ezaphefumlela izizukulwana zangaphambili.
Ukondla ngokuthe ngqo izinambuzane ezitya utyani kwenziwa ngokuphakamisa i-instar larva yesibini okanye i-Sl larva kwigqabi lesityalo elinye elinombala ongwevu lesityalo esinye esidubula irozi, kunye nezityalo ezili-10 eziphindaphindayo kwisityalo ngasinye. Izinambuzane ezibungu zabanjwa ngee-clamps, kwaye izicubu zegqabi eziseleyo zaqokelelwa emva kweeyure ezingama-24 kunye nama-72 emva kokosuleleka kwaye zaqandiswa ngokukhawuleza, kwaye ii-metabolites zakhutshwa.
Lingisa unyango olutya utyani ngendlela ehambelanayo kakhulu. Le ndlela kukusebenzisa amavili eepateni zelaphu ukugqobhoza imiqolo emithathu yameva kumacala omabini embambo ephakathi yamagqabi amathathu akhule ngokupheleleyo esityalo ngexesha lokukhula kwe-garland yelaphu, kwaye ngoko nangoko ufake i-1:5 Diluted Ms. Okanye sebenzisa iminwe eneglavu ukufaka i-S1 OS kwinxeba lokugqobhoza. Vuna kwaye ulungise igqabi njengoko kuchaziwe apha ngasentla. Sebenzisa indlela echazwe ngaphambili ukukhupha ii-metabolites eziphambili kunye neehomoni zezityalo (54).
Kwizicelo ze-JA zangaphandle, amagqabi amathathu e-petiole kwisityalo ngasinye kwezintandathu eziqhakazayo ze-rose zohlobo ngalunye aphathwa nge-20μl ye-lanolin paste equlethe i-150μg MeJA (Lan + MeJA), kunye ne-20μl ye-lanolin kunye nonyango lwamanxeba (Lan + W), okanye sebenzisa i-20μl ye-lanolin ecocekileyo njengolawulo. Amagqabi avunwa emva kweeyure ezingama-72 emva konyango, aqanduselwa kwi-nitrogen engamanzi, aze agcinwe kwi--80°C de asetyenziswe.
Imigca emine ye-JA kunye ne-ET transgenic, eyile i-irAOC (36), i-irCOI1 (55), i-irACO kunye ne-sETR1 (48), ichongiwe kwiqela lethu lophando. I-irAOC ibonise kakhulu ukwehla kwamanqanaba e-JA kunye ne-JA-Ile, ngelixa i-irCOI1 yayingenazo ii-JA. Xa kuthelekiswa ne-EV, ukuqokelelwa kwe-JA-Ile kwanda. Ngokufanayo, i-irACO iya kunciphisa imveliso ye-ET, kwaye xa ithelekiswa ne-EV, i-sETR1, engakhathaliyo kwi-ET, iya kwandisa imveliso ye-ET.
I-photoacoustic laser spectrometer (i-Sensor Sense ET-300 real-time ET sensor) isetyenziselwa ukwenza umlinganiselo we-ET ngaphandle kokungenelela. Emva nje konyango, isiqingatha samagqabi sanqunyulwa saza sadluliselwa kwi-vial yeglasi evaliweyo eyi-4-ml, kwaye indawo yentloko yavunyelwa ukuba iqokeleleke kwiiyure ezi-5. Ngexesha lokulinganisa, i-vial nganye yahlanjwa ngomjelo weelitha ezi-2/iyure yomoya ococekileyo kangangemizuzu esi-8, eyayidlule ngaphambili kwi-catalyst enikwe yi-Sensor Sense ukususa i-CO2 namanzi.
Idatha ye-microarray yapapashwa okokuqala kwi-(35) yaza yagcinwa kwi-National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (inombolo yokufikelela kwi-GSE30287). Idatha ehambelana namagqabi abangelwa lunyango lwe-W + OSMs kunye nolawulo olungonakalanga ikhutshiwe kolu phononongo. Ubunzulu obungavuthwanga yi-log2. Ngaphambi kohlalutyo lwezibalo, isiseko saguqulwa saza sahlengahlengiswa saya kwi-75th percentile kusetyenziswa iphakheji yesoftware ye-R.
Idatha yokuqala ye-RNA sequencing (RNA-seq) yeentlobo zeNicotiana ifunyenwe kwi-NCBI Short Reading Archives (SRA), inombolo yeprojekthi yi-PRJNA301787, eyaxelwa nguZhou et al. (39) kwaye iyaqhubeka njengoko kuchaziwe kwi-(56). Idatha eluhlaza ecutshungulwe yi-W + W, W + OSM kunye ne-W + OSS1 ehambelana neentlobo zeNicotiana ikhethwe ukuba ihlalutywe kolu phononongo, kwaye icutshungulwe ngale ndlela ilandelayo: Okokuqala, ukufundwa kwe-RNA-seq eluhlaza kuguqulwe kwaba yifomathi ye-FASTQ. I-HISAT2 iguqula i-FASTQ ibe yi-SAM, kwaye i-SAMtools iguqula iifayile ze-SAM zibe ziifayile ze-BAM ezihleliweyo. I-StringTie isetyenziselwa ukubala ukubonakaliswa kwe-gene, kwaye indlela yayo yokubonakaliswa kukuba kukho iziqwenga ngeziqwenga eziliwaka ngeziqwenga ze-transcription ezilandelelanayo ezizizigidi.
Ikholamu ye-Acclaim chromatographic (150 mm x 2.1 mm; ubungakanani be-particle yi-2.2μm) esetyenzisiweyo kuhlalutyo kunye nekholamu yokulinda ye-4 mm x 4 mm iqulethe izinto ezifanayo. Le gradient ye-binary ilandelayo isetyenziswa kwinkqubo ye-Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): imizuzu eyi-0 ukuya kweyi-0.5, isocratic 90% A [amanzi anyibilikisiweyo, 0.1% (v/v) acetonitrile kunye ne-0.05% formic acid], 10% B (Acetonitrile kunye ne-0.05% formic acid); imizuzu eyi-0.5 ukuya kweyi-23.5, isigaba se-gradient yi-10% A kunye ne-90% B, ngokulandelelana; imizuzu engama-23.5 ukuya kweyi-25, isocratic 10% A kunye ne-90% B. Isantya sokuhamba yi-400μl/min. Kuzo zonke ii-MS analysis, faka i-column eluent kwi-quadrupole kunye ne-time-of-flight analyzer (qTOF) analyzer exhotyiswe nge-electrospray source esebenza kwi-positive ionization mode (i-capillary voltage, 4500 V; i-capillary outlet 130 V; Ubushushu bokomisa 200°C; ukomisa umoya 10 litres/min).
Yenza uhlalutyo lwe-MS / MS fragment (ebizwa ngokuba yi-MS / MS) olungabalulekanga okanye olungahlukaniyo nedatha ukuze ufumane ulwazi lwesakhiwo malunga neprofayili iyonke ye-metabolic enokubonwa. Ingcamango yendlela ye-MS/MS engakhethiyo ixhomekeke kwinto yokuba i-quadrupole ine-mass solidation window enkulu kakhulu [ngoko ke, cinga zonke iimpawu ze-mass-to-charge ratio (m/z) njengeziqwenga]. Ngenxa yesi sizathu, ngenxa yokuba isixhobo se-Impact II asikwazanga ukwenza i-CE tilt, kwenziwa uhlalutyo oluzimeleyo oluninzi kusetyenziswa amaxabiso anyusiweyo e-collision-induced dissociation collision energy (CE) values. Ngamafutshane, qala uhlalutye isampuli nge-UHPLC-electrospray ionization/qTOF-MS usebenzisa i-single mass spectrometry mode (iimeko eziphantsi zokuqhekeka eziveliswa kukuqhekeka komthombo), ukuskena ukusuka kwi-m/z 50 ukuya kwi-1500 kwi-repetition frequency ye-5 Hz. Sebenzisa i-nitrogen njengegesi yokungqubana kuhlalutyo lwe-MS/MS, kwaye wenze imilinganiselo ezimeleyo kwezi voltage zine zahlukeneyo zokungqubana ezibangelwa kukungqubana: 20, 30, 40, kunye ne-50 eV. Kuyo yonke inkqubo yokulinganisa, i-quadrupole inefestile enkulu yokuhluza ubunzima, ukusuka kwi-m/z 50 ukuya kwi-1500. Xa uvavanyo lomzimba wangaphambili m/z kunye nobubanzi bokuhluza lumiselwe kwi-200, uluhlu lobunzima luvuselelwa ngokuzenzekelayo yisoftware yokusebenza yesixhobo kunye ne-0 Da. Skena iziqwenga zobunzima njengakwimo yobunzima obunye. Sebenzisa i-sodium formate (i-50 ml isopropanol, i-200 μl formic acid kunye ne-1 ml 1M NaOH aqueous solution) yokulinganisa ubunzima. Usebenzisa i-algorithm yokulinganisa echanekileyo kaBruker, ifayile yedatha iyalinganiswa emva kokusebenzisa i-avareji ye-spectrum kwixesha elithile. Sebenzisa umsebenzi wokuthumela ngaphandle wesoftware ye-Data Analysis v4.0 (Brook Dalton, Bremen, Germany) ukuguqula iifayile zedatha eluhlaza zibe yifomathi yeNetCDF. Iseti yedatha ye-MS/MS igcinwe kwisiseko sedatha se-metabolomics esivulekileyo iMetaboLights (www.ebi.ac.uk) kunye nenombolo yokungena. MTBLS1471.
Indibano ye-MS/MS inokufezekiswa ngohlalutyo lokuhambelana phakathi kweempawu zomgangatho we-MS1 kunye ne-MS/MS zamandla aphantsi naphezulu okungqubana kunye nemithetho emitsha esetyenzisiweyo. Iskripthi se-R sisetyenziselwa ukuqonda uhlalutyo lokuhambelana kokusasazwa kwesandulela kwimveliso, kwaye iskripthi se-C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) sisetyenziselwa ukuphumeza imithetho.
Ukuze kuncitshiswe iimpazamo ezingalunganga ezibangelwa yingxolo yangasemva kunye nolwalamano olungelulo olubangelwa kukufumanisa iimpawu ezithile ze-m/z kwiisampuli ezimbalwa kuphela, sisebenzisa umsebenzi we-"filled peak" wephakheji ye-R XCMS (yokulungisa ingxolo yangasemva). Kufuneka isetyenziswe endaweni yobunzulu be-"NA" (incopho engabonwanga). Xa kusetyenziswa umsebenzi we-fill peak, kusekho amaxabiso amaninzi e-"0″ intensity kwiseti yedatha aya kuchaphazela ukubalwa kolwalamano. Emva koko, sithelekisa iziphumo zokucubungula idatha ezifunyenweyo xa kusetyenziswa umsebenzi we-filled peak kwaye xa umsebenzi we-filled peak ungasetyenziswa, kwaye sibale ixabiso lengxolo yangasemva ngokusekelwe kwixabiso eliqikelelweyo elilungisiweyo, size sitshintshe la maxabiso e-0 intensity ngexabiso lemvelaphi elibaliweyo. Sikwaqwalasele kuphela iimpawu ezinobunzulu obugqithe kathathu kwixabiso langasemva kwaye sizijonga njenge "true peaks." Kubalo lwe-PCC, kuphela imiqondiso ye-m/z yesampulu yomzekelo (MS1) kunye neeseti zedatha eziqhekezayo ezineencopho ezisibhozo zokwenyani eziqwalaselwayo.
Ukuba amandla ophawu lomgangatho we-precursor kwisampulu yonke ahambelana kakhulu namandla ancitshisiweyo ophawu lomgangatho ofanayo oluphantsi kwamandla aphantsi okanye aphezulu okungqubana, kwaye uphawu alubhalwanga njengencochoyi ye-isotope yi-CAMERA, lunokuchazwa ngakumbi. Emva koko, ngokubala zonke iimbini ezinokwenzeka ze-precursor-product ngaphakathi kwemizuzwana emi-3 (iwindow yexesha eliqikelelweyo lokugcina incochoyi), uhlalutyo lolwalamano lwenziwa. Kuphela xa ixabiso le-m/z liphantsi kunexabiso le-precursor kwaye ukuqhekeka kwe-MS/MS kwenzeka kwindawo efanayo yesampulu kwiseti yedatha njenge-precursor apho ithathwe khona, apho ithathwa njenge-fragment.
Ngokusekelwe kule mithetho mibini ilula, sizikhuphela ngaphandle iziqwenga ezichaziweyo ezinexabiso le-m/z elikhulu kune-m/z yesandulela esichongiweyo, kwaye ngokusekwe kwindawo yesampulu apho isandulela sivela khona kunye nesiqwenga esichaziweyo. Kukwanokwenzeka ukukhetha iimpawu zomgangatho eziveliswe ziziqwenga ezininzi ezikwimvelaphi eziveliswe kwimo ye-MS1 ​​njengezandulela ezikhethiweyo, ngaloo ndlela zivelisa iikhompawundi ze-MS/MS ezingafunekiyo. Ukuze kuncitshiswe olu phindaphindo lwedatha, ukuba ukufana kwe-NDP kwe-spectra kudlula i-0.6, kwaye zeze-chromatogram "pcgroup" echazwe yi-CAMERA, siza kuzidibanisa. Okokugqibela, sidibanisa zonke iziphumo ezine ze-CE ezinxulumene nesandulela kunye neziqwenga kwi-spectrum yokugqibela ye-composite deconvoluted ngokukhetha i-peak ephezulu kakhulu phakathi kwazo zonke iincopho ezikhethiweyo ezinexabiso elifanayo le-m/z kumandla ahlukeneyo e-collision. Amanyathelo okucubungula alandelayo asekelwe kwingcamango ye-composite spectrum kwaye athathela ingqalelo iimeko ezahlukeneyo ze-CE ezifunekayo ukwandisa amathuba okuqhekeka, kuba ezinye iziqwenga zinokufunyanwa kuphela phantsi kwamandla athile e-collision.
I-RDPI (30) isetyenzisiwe ukubala i-inducibility yeprofayili ye-metabolic. Ukwahluka kwe-metabolic spectrum (i-Hj index) kuvela kubuninzi be-MS/MS precursors besebenzisa i-Shannon entropy yokusasazwa kwe-MS/MS frequency besebenzisa i-equation elandelayo echazwe nguMartínez et al. (8). I-Hj = −∑i = 1mPijlog2(Pij) apho i-Pij ihambelana ne-relative frequency ye-i-th MS/MS kwisampulu ye-j-th (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Ukucaciswa kweMetabolic (i-Si index) kuchazwa njengophawu lokubonakaliswa kwe-MS/MS enikiweyo ngokunxulumene nemvamisa phakathi kweesampuli eziqwalaselwayo. Ukucaciswa kwe-MS/MS kubalwa njengo-Si = 1t (∑j = 1tPijPilog2PijPi)
Sebenzisa le fomyula ilandelayo ukulinganisa i-metabolome-specific δj index yesampulu nganye ye-j, kunye nomndilili we-MS/MS specificity δj = ∑i = 1mPijSi
Iispectra ze-MS/MS zilungelelaniswe ngababini, kwaye ukufana kubalwa ngokusekelwe kumanqaku amabini. Okokuqala, usebenzisa i-standard NDP (ekwaziwa ngokuba yindlela yokudibanisa i-cosine), sebenzisa le equation ilandelayo ukufumana ukufana kwecandelo phakathi kwe-spectra NDP = (∑iS1 kunye ne-S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 apho i-S1 kunye ne-S2 Ngokuhambelanayo, kwi-spectrum 1 kunye ne-spectrum 2, kunye ne-WS1, i kunye ne-WS2, i imele ubunzima obusekelwe kubunzulu bencochoyi yokuba umahluko wencochoyi eqhelekileyo ye-i-th phakathi kwee-spectra ezimbini ungaphantsi kwe-0.01 Da. Ubunzima bubalwa ngolu hlobo lulandelayo: W = [ubunzulu bencochoyi] m [umgangatho] n, m = 0.5, n = 2, njengoko kucetyisiwe yiMassBank.
Indlela yesibini yokufumana amanqaku iye yasetyenziswa, eyayiquka ukuhlalutya i-NL ekwabelwana ngayo phakathi kwe-MS/MS. Ngenxa yesi sizathu, sisebenzise uluhlu lwe-NL ezingama-52 oluhlala lufunyanwa ngexesha lenkqubo yokuqhekeka kwe-MS ngaxeshanye, kunye ne-NL ecacileyo ngakumbi (ifayile yedatha i-S1) ebikade ichaziwe ngaphambili kwi-MS/MS spectrum yee-metabolites zesibini ze-Nepenthes species ezibuthathaka (9, 26). Yenza i-binary vector ye-1 kunye ne-0 kwi-MS/MS nganye, ehambelana ne-current kunye ne-non-areal ye-NL ethile ngokulandelelana. Ngokusekelwe kufana komgama we-Euclidean, amanqaku okufana kwe-NL abalwa kwisibini ngasinye se-binary NL vectors.
Ukwenza i-dual clustering, sisebenzise iphakheji ye-R i-DiffCoEx, esekelwe kulwandiso lwe-Weighted Gene Co-expression Analysis (WGCNA). Sisebenzisa i-NDP kunye ne-NL scoring matrices ze-MS/MS spectra, sisebenzise i-DiffCoEx ukubala i-comparative correlation matrix. I-binary clustering yenziwa ngokuseta i-“cutreeDynamic” parameter kwi-method = “hybrid”, cutHeight = 0.9999, deepSplit = T, kunye ne-minClusterSize = 10. Ikhowudi yomthombo we-R ye-DiffCoEx ikhutshelwe kwifayile eyongezelelweyo 1 nguTesson et al. (57); Iphakheji yesoftware ye-R WGCNA efunekayo inokufumaneka kwi-https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Ukuze senze uhlalutyo lwenethiwekhi ye-MS/MS molecular, sibale uqhagamshelo lwe-spectral oludibeneyo olusekelwe kwiintlobo zokufana kwe-NDP kunye ne-NL, saza sasebenzisa isoftware yeCytoscape ukuze sibone i-topology yenethiwekhi sisebenzisa uyilo lwendalo kwisicelo sokwandiswa kwe-algorithm yoyilo lweCyFilescape yFiles.
Sebenzisa i-R version 3.0.1 ukwenza uhlalutyo lwezibalo kwidatha. Ukubaluleka kwezibalo kuhlolwe kusetyenziswa uhlalutyo lweendlela ezimbini zokwahluka (ANOVA), kulandele uvavanyo lwe-post-hoc lukaTukey olunentsingiselo ecacileyo (HSD). Ukuze kuhlalutywe umahluko phakathi konyango lwe-herbivorous kunye nolawulo, ukusasazwa kwe-two-tailed kwamaqela amabini eesampuli ezine-variance efanayo kuhlalutywe kusetyenziswa uvavanyo lwe-Student's t.
Ukuze ufumane ezinye izinto ezongezelelweyo kweli nqaku, nceda ujonge ku-http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Eli linqaku elivulelekileyo elisasazwa phantsi kwemigaqo yeLayisensi yeCreative Commons Attribution-Non-Commercial, evumela ukusetyenziswa, ukusasazwa kunye nokuveliswa kwakhona kuyo nayiphi na indlela, lo gama nje ukusetyenziswa kokugqibela kungekuko ukuzuza kwezorhwebo kwaye ingqikelelo kukuba umsebenzi wokuqala uchanekile. Isalathiso.
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