Tithiyori tohambana hambana ta nsirhelelo wa swimilani tinyika nkongomiso wa nkoka wa tithiyori eka ku hlamusela swivumbeko swa metabolism yo hlawuleka ya swimilani, kambe ku vhumbha ka tona ka nkoka ka ha fanele ku kamberiwa. Laha, hi tirhise nxopaxopo wa unbiased tandem mass spectrometry (MS/MS) ku lavisisa hi ndlela leyi hlelekeke metabolome ya tinxaka leti hungutiweke hi fole kusuka eka swimilani ha xin’we kuya eka vaaki na tinxaka leti fambelanaka swinene, naswona hi lulamise nhlayo leyikulu ya tithiyori ta swihlawulekisi swa mass spectrometric leswi simekiweke eka ti compound spectra eka mahungu. Rimba ro kambela vuprofeta bya nkoka bya tithiyori ta vusirheleri lebyinene (OD) na xikongomelo lexi fambafambaka (MT). Xiphemu xa mahungu xa metabolomics ya swimilani xi fambisana na OD theory, kambe xi kanetana na ku vhumbha lokukulu ka MT theory eka metabolomics dynamics leyi vangiwaka hi herbivores. Kusuka eka micro kuya eka macro evolutionary scale, jasmonate signal yi tiviwile tani hi xihlawulekisi lexikulu xa OD, kasi ethylene signal yi nyikile fine-tuning ya herbivore-specific response leyi hlamuseriweke hi MS/MS molecular network.
Ti- metabolite to hlawuleka leti nga ni swivumbeko swo hambana-hambana hi tona leti hlanganyelaka ngopfu eku toloveleni ka swimilana ni mbango, ngopfu-ngopfu eku sirheleleni ka valala (1). Ku hambana loku hlamarisaka ka metabolism yo hlawuleka leyi kumekaka eka swimilani swi hlohlotele makume ya malembe ya ndzavisiso lowu dzikeke eka swiyenge swa yona swotala swa mintirho ya ecological, naswona swivumbe nxaxamelo wo leha wa ti theories ta nsirhelelo wa swimilani, letinga nhluvukiso wa evolutionary na ecological wa ku tirhisana ka swimilani na switsotswana. Ndzavisiso wa empiriki wu nyika nkongomiso wa nkoka (2). Hambiswiritano, tithiyori leti ta nsirhelelo wa swimilana a ti landzelelanga ndlela ya normative ya hypothetical deductive reasoning, laha ku vhumbha ka nkoka a ku ri eka xiyimo lexi fanaka xa nxopaxopo (3) naswona ku kamberiwile hi ku ringeta ku yisa emahlweni xirhendzevutani lexi landzelaka xa nhluvukiso wa thiyori (4). Swipimelo swa xithekiniki swi siveriwa nhlengeleto wa datha eka swiyenge swo karhi swa metaboliki naswona swi susa nxopaxopo lowu heleleke wa timetabolite to hlawuleka, xisweswo swi sivela ku pimanisiwa exikarhi ka swiyenge leswi nga swa nkoka eka nhluvukiso wa thiyori (5). Ku pfumaleka ka data ya metabolomics leyi heleleke xikan’we na mali leyi tolovelekeke ku pimanisa maendlelo ya ntirho wa processing ya ndzhawu ya metabolic exikarhi ka mintlawa yohambana hambana ya swimilani swi sivela ku vupfa ka sayense ka nsimu.
Swiendlakalo swa sweswinyana eka xiyenge xa ​​tandem mass spectrometry (MS/MS) metabolomics swinga hlamusela hiku hetiseka ku cinca ka metabolic endzeni nale xikarhi ka tinxaka ta clade ya sisiteme leyi nyikiweke, naswona swinga hlanganisiwa na maendlelo ya xibalo ku hlayela kufana ka xivumbeko exikarhi ka swihlanganisi leswi swo rharhangana. Vutivi bya le mahlweni bya tikhemikhali (5). Ku hlanganisiwa ka thekinoloji ya xiyimo xale henhla eka nxopaxopo na khomphyuta swinyika rimba leri lavekaka eka ku kamberiwa ka nkarhi woleha ka ku vhumbha ko tala loku endliweke hi tithiyori ta ikholoji na nhluvukiso wa ku hambana ka metabolic. Shannon (6) u nghenise thiyori ya mahungu ro sungula eka xitsalwana xa yena xa seminal hi 1948, a veka masungulo ya nxopaxopo wa tinhlayo wa mahungu, lowu tirhisiweke eka tinsimu to tala handle ka ku tirhisiwa ka wona ko sungula. Eka genomics, dyondzo ya mahungu yi tirhisiwile hi ndlela leyi humelelaka ku pima nhlayo ya mahungu ya ku hlayisa ntlhandlamano (7). Eka ndzavisiso wa transcriptomics, thiyori ya mahungu yi xopaxopa ku cinca hinkwako eka transcriptome (8). Eka ndzavisiso wa khale, hi tirhise rimba ra tinhlayo ra thiyori ya mahungu eka metabolomics ku hlamusela vutshila bya metabolic bya levele ya tissue eka swimilani (9). Laha, hi hlanganisa maendlelo ya ntirho lawa ya simekiweke eka MS/MS na rimba ra tinhlayo ra thiyori ya mahungu, leyi tivekaka hi ku hambana ka metabolic eka mali leyi tolovelekeke, ku pimanisa ku vhumbha ka nkoka ka thiyori ya nsirhelelo wa swimilani ya metabolome leyi hlohloteriwaka hi swiharhi leswi dyaka byanyi.
Maendlelo ya thiyori ya nsirhelelo wa swimilani hi ntolovelo ya katsana naswona yanga avanyisiwa hi swiyenge swimbirhi: lawa ya ringetaka ku hlamusela ku hangalaka ka ti metabolites ta swimilani leswi kongomeke leswi simekiweke eka mintirho ya nsirhelelo, kufana na optimal defense (OD) (10), moving target (MT) (11) ) Na ku languteka (12) theory, kasi van’wana va lava tinhlamuselo ta michini ta ndlela leyi ku cinca eka ku kumeka ka switirhisiwa swi khumbaka ha yona ku kula ni ku hlengeletiwa ka ti- metabolite to hlawuleka, to tanihi khaboni: xiringanyeto xa ku ringanisela ka swakudya (13), xiringanyeto xa mpimo wa ku kula (14 ), ni xiringanyeto xa ku ringanisela ka ku kula ni ku hambana (15). Tisethi timbirhi ta tithiyori ti le ka swiyimo swo hambana swa nxopaxopo (4). Hambiswiritano, tithiyori timbirhi leti katsaka mintirho yo sirhelela eka xiyimo xa ntirho ti lawula mbulavurisano mayelana na vusirheleri bya swimilani lebyi vumbiwaka na lebyi hlohloteriwaka: Tiyori ya OD, leyi tekelaka enhlokweni leswaku swimilani swi vekisa eka vusirheleri bya swona byo durha bya tikhemikhali ntsena loko swi laveka, xikombiso, loko swi dyiwa Loko xiharhi xa byanyi xi hlasela, hikokwalaho, hi ku ya hi ku koteka ka nhlaselo wa nkarhi lowu taka, nkatsakanyo lowu nga na ntirho wo sirhelela wa averiwa (10); MT hypothesis yi ringanyeta leswaku akuri na axis ya directional metabolite change, kambe metabolite yi cinca hiku landzelelana, xisweswo yi tumbuluxa ku koteka Ku sivela metabolic “motion target” ya ku hlasela herbivores. Hi marito man’wana, tithiyori leti timbirhi ti endla ku vhumbha loku hambaneke mayelana na ku pfuxetiwa ka metabolic loku humelelaka endzhaku ka nhlaselo wa swiharhi leswi dyaka byanyi: vuxaka exikarhi ka nhlengeleto wa unidirectional ya ti metabolites leti nga na ntirho wo sirhelela (OD) na ku cinca ka metabolic loku nga kongomisiwangiki (MT) (11 ).
Tihypothesis ta OD na MT a ti katsi ntsena ku cinca loku hlohloteriweke eka metabolome, kambe na switandzhaku swa ikholoji na ku hundzuka ka ku hlengeletiwa ka ti metabolite leti, ku fana na ku durha loku pfumelelanaka na ku vuyeriwa ka ku cinca loku ka metaboliki eka ndhawu yo karhi ya ikholoji (16). Hambi leswi ti hypotheses leti hatimbirhi ti lemukaka ntirho wo sirhelela wa ti metabolites to hlawuleka, leti nga durhaka kumbe ku nga durhi, ku vhumbha ka nkoka loku hambanisaka ti hypothesis ta OD na MT ku le ka directionality ya ku cinca ka metabolic loku hlohloteriwaka. Ku vhumbha ka thiyori ya OD ku amukerile nyingiso lowukulu wa ku ringeta ku fikela sweswi. Swikambelo leswi swi katsa dyondzo ya mintirho ya nsirhelelo yo kongoma kumbe leyi nga kongomangiki ya tinyama to hambana ta swihlanganisi swo karhi eka ti greenhouses na swiyimo swa ntumbuluko, xikan’we na ku cinca eka xiteji xa nhluvukiso wa swimilani (17-19). Hambiswiritano, kufikela sweswi, hikokwalaho ka ku pfumaleka ka maendlelo ya ntirho na rimba ra tinhlayo eka nxopaxopo wa misava hinkwayo lowu heleleke wa ku hambana ka metaboliki ya xivumbiwa xin’wana na xin’wana, ku vhumbha lokukulu ka ku hambana exikarhi ka tithiyori letimbirhi (hileswaku, tlhelo ra ku cinca ka metaboliki) swa ha fanele ku kamberiwa. Laha, hi nyika nxopaxopo wo tano.
Xin’wana xa swihlawulekisi swa nkoka swinene swa ti metabolites leti kongomisiweke eka swimilani i ku hambana ka tona lokukulu ka swivumbeko eka swiyimo hinkwaswo kusuka eka swimilani swin’we, nhlayo ya vaaki kuya eka tinxaka leti fanaka (20). Ku cinca ko tala ka nhlayo eka ti metabolites to hlawuleka ku nga voniwa eka xikalo xa vaaki, kasi ku hambana lokukulu ka khwalithi hi ntolovelo ku hlayisiwa eka xiyimo xa muxaka (20). Hikokwalaho, ku hambana ka metabolic ya swimilani i xiphemu lexikulu xa ku hambana ka ntirho, leswi kombisaka ku cinca cinca eka ti niches to hambana, ngopfu ngopfu ti niches toleto letingana ku hlasela ko hambana hi switsotswana swo hlawuleka na ti herbivores leti tolovelekeke (21). Kusukela eka xitsalwana xa Fraenkel (22) lexi pfulekeke eka swivangelo swa vukona bya ti metabolites leti kongomisiweke eka swimilani, ku tirhisana na switsotswana swohambana hambana swi tekiwe tani hi ntshikilelo wa nkoka wo hlawula, naswona ku tshembiwa leswaku ku tirhisana loku ku vumbe swimilani hi nkarhi wa ku hundzuka. Ndlela ya metabolic (23). Ku hambana exikarhi ka tinxaka eka ku hambana ka ti metabolites to hlawuleka swi nga ha tlhela swi kombisa ku ringanisela ka physiological loku fambelanaka na vusirheleri bya swimilani leswi vumbiwaka na leswi hlohlotelaka eka tindlela ta herbivorous, tanihileswi tinxaka letimbirhi ti talaka ku fambelana hi ndlela yo biha na yin’wana na yin’wana (24). Hambi leswi swinga pfunaka ku hlayisa nsirhelelo wa kahle minkarhi hinkwayo, ku cinca ka metabolic hi nkarhi loku fambelanaka na vusirheleri swinyika mbuyelo lowu nga erivaleni eka ku pfumelela swimilani ku avela switirhisiwa swa nkoka eka vuvekisi byin’wana bya physiological (19, 24), naku papalata xilaveko xa symbiosis. Ku onhaka ka xisirhelelo (25). Ku engetela kwalaho, ku hleriwa loku ka ti metabolites to hlawuleka loku vangiwaka hi switsotswana leswi dyaka byanyi swi nga endla leswaku ku va na ku hangalaka loku onhaka eka vaaki (26), naswona swi nga ha kombisa ku hlayiwa loku kongomeke ka ku cinca lokukulu ka ntumbuluko eka xikombiso xa jasmonic acid (JA), lexi nga hlayisiwaka eka vaaki. Swikombiso swa JA swale henhla nale hansi i trade-offs exikarhi ka vusirheleri eka swihadyani leswi dyaka byanyi xikan’we na mphikizano na tinxaka tokarhi (27). Ku engetela kwalaho, tindlela to hlawuleka ta metabolite biosynthetic ti ta lahlekeriwa hi xihatla na ku cinca hi nkarhi wa ku hundzuka, leswi endlaka leswaku ku va na ku hangalasiwa ka metabolic hi swiphemuphemu exikarhi ka tinxaka leti yelanaka swinene ( 28 ). Ti polymorphisms leti tinga simekiwa hiku hatlisa ku hlamula eka ku cinca ka swivumbeko swa herbivore (29), leswivulaka leswaku ku cinca cinca ka miganga ya herbivore i xivangelo xa nkoka lexi fambisaka metabolic heterogeneity.
Laha, hi ku kongoma hi tlhantlhe swiphiqo leswi landzelaka. (I) Xana xitsotswana lexi dyaka byanyi xi tlhela xi hlela njhani metabolome ya ximilani? (Ii) Hi swihi swiphemu leswikulu swa mahungu swa pulasitiki ya metaboliki leswi nga pimiwa ku kambela ku vhumbha ka thiyori ya vusirheleri bya nkarhi wo leha? (Iii) Loko ku fanele ku tlhela ku phurogiremiwa metabolome ya ximilani hi ndlela yo hlawuleka eka muhlaseri, loko swi ri tano, hi xihi xiave lexi hormone ya ximilani yi xi endlaka eku lulamiseni ka nhlamulo yo karhi ya metaboliki, naswona hi tihi ti metabolites leti hoxaka xandla eka ku hlawuleka ka muxaka wa nsirhelelo? (Iv) Tanihi leswi ku vhumbha loku endliweke hi tithiyori to tala ta vusirheleri ku nga andlariwaka eka swiyimo hinkwaswo swa tinyama ta biological, hi vutisile leswaku nhlamulo ya metabolic leyi vangiwaka yi tshamisekile ku fikela kwihi ku suka eka ku pimanisiwa ka le ndzeni ku ya eka ku pimanisiwa exikarhi ka tinxaka? Ku fikelela leswi, hi dyondze hi ndlela leyi hlelekeke metabolome ya matluka ya nicotine ya fole, leyi kunga ximilani xa xikombiso xa ecological lexinga fuwa hi metabolism yo hlawuleka, naswona xitirha eka switsotswana swa ti herbivores timbirhi ta ndzhavuko, Lepidoptera Datura (Ms) ( Very aggressive, mainly eaten) Eka Solanaceae na Spodoptera litoralis (Sl), switsotswana swa matluka ya cotton i muxaka wa “genus”, na swimilani leswi amukelaka swa Solanaceae na swimilani swin’wana swa tinxaka tin’wana na mindyangu Swakudya swa swimilani. Hi hambanise MS/MS metabolomics spectrum na ku humesa swihlamuseri swa tinhlayo swa thiyori ya mahungu ku pimanisa tithiyori ta OD na MT. Endla mimepe ya specificity ku paluxa vutivi bya ti metabolites ta nkoka. Nxopaxopo wu andlariwile eka nhlayo ya ndzhavuko ya N. nasi na tinxaka ta fole leti fambelanaka swinene ku ya emahlweni ku xopaxopa covariance exikarhi ka swikombiso swa ti hormone ta swimilani na OD induction.
Leswaku hi khoma mepe hi ku angarhela mayelana na pulasitiki na xivumbeko xa metabolome ya matluka ya fole leri dyaka byanyi, hi tirhise nxopaxopo lowu tumbuluxiweke khale na ku famba ka ntirho wa xibalo ku hlengeleta hi ku hetiseka na ku deconvolute data ya xiyimo xa le henhla leyi tiyimeleke MS / MS spectra ku suka eka swiphemu swa swimilana ( 9 ). Ndlela leyi yinga hambanisiwangiki (leyi vuriwaka MS/MS) yinga aka ti non-redundant compound spectra, leti endzhaku tinga tirhisiwaka eka ti analyses hinkwato ta compound-level leti hlamuseriweke laha. Ti metabolites leti ta swimilani letinga deconvoluted i ta tinxaka tohambana hambana, letingana madzana kuya eka magidi ya ti metabolites (kwalomu ka 500-1000-s/MS/MS laha). Laha, hi languta plastiki ya metabolic eka rimba ra thiyori ya mahungu, naswona hi pima ku hambana na vutshila bya metabolome leswi simekiweke eka Shannon entropy ya ku hangalasiwa ka frequency ya metabolic. Hi ku tirhisa fomula leyi tirhisiweke khale (8), hi hlayele sete ya swikombiso leswi nga tirhisiwaka ku pima ku hambana ka metabolome (xikombiso xa Hj), ku hlawuleka ka xivumbeko xa metaboliki (xikombiso xa δj) na ku hlawuleka ka metaboliki ya metabolite yin’we (xikombiso xa Si) . Ku engetela kwalaho, hi tirhise Relative Distance Plasticity Index (RDPI) ku pima mpimo wa metabolome inducibility ya herbivores (Xifaniso 1A) (30). Endzeni ka rimba leri ra tinhlayo, hi khoma MS/MS spectrum tanihi yuniti ya mahungu ya masungulo, naswona hi tirhisa ku tala loku ringanaka ka MS/MS eka mepe wa ku hangalasiwa ka frequency, ivi hi tirhisa Shannon entropy ku ringanyeta ku hambana ka metabolome ku suka eka yona. Ku hlawuleka ka metabolome ku pimiwa hi ku hlawuleka ka xikarhi ka spectrum yin’we ya MS/MS. Hikokwalaho, ku tlakuka ka nhlayo ya titlilasi tin’wana ta MS/MS endzhaku ka herbivore induction swi hundzuriwa kuva spectral inducibility, RDPI na specialization, leswivulaka leswaku, ku tlakuka ka δj index, hikuva ku humelerisiwa ti metabolites to hlawuleka swinene naswona ku humelerisiwa Si index yale henhla. Ku hungutiwa ka Hj diversity index swi kombisa leswaku kumbe nhlayo ya MS/MS leyi tumbuluxiweke ya hungutiwile, kumbe ku hangalasiwa ka frequency ya profile ku cinca eka tlhelo leri nga faniki ngopfu, loko ku ri karhi ku hungutiwa ku nga tiyiseki ka yona hi ku angarhela. Hiku tirhisa xibalo xa Si index, swa koteka ku kombisa leswaku hi tihi MS/MS leti hlohleteriwaka hi ti herbivores tokarhi, kuhambana na sweswo, hi tihi MS/MS letinga hlamuriki eka induction, leswinga xikombiso xa nkoka ku hambanyisa ku vhumbha ka MT na OD.
(A) Swihlamuseri swa tinhlayo leswi tirhisiwaka eka herbivorous (H1 kuya eka Hx) MS/MS data-inducibility (RDPI), ku hambana (Hj index), specialization (δj index) na metabolite specificity (Si index). Ku tlakuka ka degree ya specialization (δj) swikomba leswaku, hi xiringaniso, ti herbivorous specific metabolites totala tita humelerisiwa, kasi kuhunguteka ka ku hambana (Hj) swikomba kuhunguteka ka vuhumelerisi bya metabolites kumbe ku hangalasiwa lokunga ringaniki ka ti metabolites eka mepe wa ku hangalaka . Nhlayo ya Si yi kambela loko metabolite yi kongomile eka xiyimo lexi nyikiweke (laha, herbivorous) kumbe hi ndlela yohambana yi hlayisiwa eka xiyimo lexi fanaka. (B) Xifaniso xa miehleketo xa ku vhumbha ka thiyori ya vusirheleri hi ku tirhisa xirhendzevutani xa thiyori ya mahungu. Tiyori ya OD yi vhumbha leswaku nhlaselo wa herbivore wuta engetela ti metabolites ta vusirheleri, hindlela yaleyo ku engetela δj. Hi nkarhi lowu fanaka, Hj ya hunguteka hikuva profile yi hleriwa nakambe kuya eka kuhunguteka ka ku nga tiyiseki ka mahungu ya metabolic. Tiyori ya MT yi vhumbha leswaku nhlaselo wa ti herbivores wuta vanga ku cinca lokunga kongomangiki eka metabolome, hindlela yaleyo ku engeteleriwa Hj tani hi xikombiso xa ku tlakuka ka ku nga tiyiseki ka mahungu ya metabolic xikan’we naku vanga ku hangalasiwa ka random ka Si. Hi tlhele hi ringanyeta modele lowu pfanganisiweke, MT ya kahle swinene, laha ti metabolites tin’wana leti nga na mimpimo ya le henhla yo sirhelela ti nga ta engeteriwa ngopfungopfu (nhlayo ya le henhla ya Si), kasi tin’wana ti kombisa tinhlamulo ta xitshuketa (nhlayo ya le hansi ya Si).
Hi ku tirhisa vahlamuseri va thiyori ya mahungu, hi hlamusela thiyori ya OD ku vhumbha leswaku ku cinca ka metabolite yo hlawuleka leyi hlohloteriwaka hi herbivore eka xiyimo xa vumbiwa lexi nga hlohloteriwangiki swi ta yisa eka (i) ku tlakuka ka metabolic specificity (Si index) leswi fambisaka metabolic specificity (δj index) Ku tlakuka ka) mintlawa yo karhi yo hlawuleka ya metabolite leyi nga na nkoka wa le henhla wa nsirhelelo, na (ii) ku hunguteka ka metabolome diversity (Hj) index . hikwalaho ka ku cinca ka ku hangalasiwa ka frequency ya metabolic kuya eka ku hangalasiwa ka miri wa leptin yotala. Eka levele ya metabolite yin’we, ku languteriwa ku hangalasiwa ka Si loku hlelekeke, laha metabolite yingata engetela nhlayo ya Si kuya hi nkoka wa yona wo sirhelela (Xifaniso 1B). Eka layini leyi, hi hlamusela thiyori ya MT ku vhumbha leswaku ku hlohloteriwa ku ta yisa eka (i) ku cinca loku nga kongomangiki eka ti metabolites leswi endlaka leswaku ku va na ku hunguteka ka δj index, na (ii) ku tlakuka ka Hj index hikwalaho ka ku tlakuka ka ku nga tiyiseki ka metabolic. Kumbe randomness, leyi nga pimiwa hi Shannon entropy hi xivumbeko xa generalized diversity. Loko kuri xivumbeko xa metabolic, MT theory yita vhumbha ku hangalasiwa ka random ka Si. Hi ku tekela enhlokweni leswaku ti metabolite to karhi ti le hansi ka swiyimo swo karhi ehansi ka swiyimo swo karhi, naswona swiyimo swin’wana a swi le hansi ka swiyimo swo karhi, naswona nkoka wa tona wo sirhelela wu titshege hi mbango, hi tlhele hi ringanyeta modele wa nsirhelelo lowu pfanganisiweke, laha δj na Hj swi hangalasiweke hi swimbirhi ku fambisana na Si Ku engeteleka eka matlhelo hinkwawo, i mintlawa yo karhi ntsena ya ti metabolite, leyi nga na mimpimo ya le henhla ya nsirhelelo, ngopfu-ngopfu yi ta engetela Si, kasi yin’wana yi ta va na xirhendzevutani xa xitshuketa ku hangalasiwa (Xifaniso 1B).
Ku kota ku kambela vuprofeta bya thiyori ya vusirheleri lebyi hlamuseriweke nakambe eka axis ya muhlamuseri wa thiyori ya mahungu, hi kurisile switsotswana swa expert (Ms) kumbe generalist (Sl) herbivore eka matluka ya Nepenthes pallens (Xifaniso 2A). Hiku tirhisa MS/MS analysis, hi kumile 599 non-redundant MS/MS spectra (data file S1) kusuka eka methanol extracts ya tissue ya matluka leyi hlengeletiweke endzhaku ka ku phameriwa ka caterpillar. Ku tirhisa ti-index ta RDPI, Hj, na δj ku vona hi mahlo ya mianakanyo ku lulamisiwa nakambe ka nhundzu ya mahungu eka tifayela ta vuhlanganisi bya MS/MS swi paluxa swivumbeko swo tsakisa (Xifaniso 2B). Ntshamiseko hinkwawo hileswaku, tani hileswi swi hlamuseriweke hi muhlamuseri wa mahungu, loko ti caterpillars ti ya emahlweni ti dya matluka, degree ya metabolic reorganization hinkwayo yi tlakuka hiku famba ka nkarhi: 72 wa tiawara endzhaku ka loko herbivore yi dyile, RDPI yi tlakuka swinene. Loko ku pimanyisiwa na vulawuri lebyi nga onhakiki, Hj yi hungutiwile swinene, leswi aswi vangiwa hiku tlakuka ka degree ya specialization ya metabolic profile, leyi ayi pimiwa hi δj index. Ntshamiseko lowu wu vonakaka wu fambisana na ku vhumbha ka thiyori ya OD, kambe a wu fambisani na ku vhumbha lokukulu ka thiyori ya MT, leyi pfumelaka leswaku ku cinca loku nga hlelekangiki (ku nga ri ka nkongomiso) eka swiyimo swa metabolite ku tirhisiwa tanihi ku fihla ku sirhelela (Xifaniso 1B). Hambi leswi nhlayo ya oral secretion (OS) elicitor xikan’we na mahanyelo yo dya ya ti herbivores leti timbirhi swihambanile, ku dya ka swona hiku kongoma swiendle leswaku kuva na ku cinca loku fanaka eka matlhelo ya Hj na δj eka nkarhi wa ntshovelo wa 24 wa tiawara na 72 wa tiawara. Ku hambana ntsena ku humelerile eka tiawara ta 72 ta RDPI. Loko ku pimanyisiwa na leswi vangiwaka hi ku phameriwa ka Ms, metabolism hinkwayo leyi hlohleteriwaka hi ku phameriwa ka Sl a yi ri ehenhla.
(A) Dizayini ya swikambelo: tinguluve leti tolovelekeke (S1) kumbe ti expert (Ms) herbivores ti phameriwa matluka lawa yanga susiwa munyu ya swimilani swa pitcher, kasi eka simulated herbivory, OS ya Ms (W + OSMs) yitirhisiwa ku khoma ku tlhaviwa ka standardized leaf positions wound. S1 (W + OSSl) switsotswana kumbe mati (W + W). Xilawuri (C) i tluka leri nga onhakiki. (B) Inducibility (RDPI loko yi pimanisiwa na chati ya vulawuri), ku hambana (Hj index) na specialization (δj index) index leyi hlayiweke eka mepe wo hlawuleka wa metabolite (599 MS/MS; data file S1). Tinyeleti ti kombisa ku hambana lokukulu exikarhi ka ku phameriwa ka herbivore hi ku kongoma na ntlawa wa vulawuri (Student’s t-test with paired t-test, *P<0.05 na ***P<0.001). ns, a hi swa nkoka. (C) Time resolution index ya main (bokisi ra wasi, amino acid, organic acid na chukele; data file S2) na special metabolite spectrum (bokisi ro tshwuka 443 MS/MS; data file S1) endzhaku ka vutshunguri bya herbivory lebyi tekeleriweke. Band ya mihlovo yi kombetela eka 95% wa nkarhi wa ku tshemba. Nyeleti yi kombisa ku hambana lokukulu exikarhi ka vutshunguri na vulawuri [quadratic analysis of variance (ANOVA), ku landzela ku hambana lokukulu ka Tukey hi ku tshembeka (HSD) eka ku pimanisiwa ko tala ka post hoc, *P<0.05, **P<0.01 Na *** P <0.001]. (D) Ku hlawuleka ka tiploto to hangalaka na tiphrofayili to hlawuleka ta metabolite (tisampulu leti phindha-phindhiweke leti nga na vutshunguri byo hambana).
Ku lavisisa loko herbivore-induced remodeling eka metabolome level yi kombisiwa eka ku cinca eka levele ya metabolites yin’wana na yin’wana, hisungule hi kongomisa eka ti metabolites letinga dyondziwa khale eka matluka ya Nepenthes pallens lawa yanga tiyisisiwa ku lwisana na herbivore. Phenolic amides i hydroxycinnamamide-polyamine conjugates leyi hlengeletanaka hi nkarhi wa herbivory process ya switsotswana naswona yitiveka kuva yi hunguta matirhelo ya switsotswana ( 32 ). Hi lavisisile swikombiso swa MS/MS leyi fambelanaka na ku plota ti cumulative kinetic curves ta tona (Xifaniso xa S1). A swi hlamarisi leswi, phenol derivatives leyinga katsekiki hiku kongoma eka ku sirhelela eka swiharhi leswi dyaka byanyi, kufana na chlorogenic acid (CGA) na rutin, swilawuriwa ehansi endzhaku ka herbivory. Kuhambana na sweswo, swihadyani leswi dyaka byanyi swinga endla leswaku ti phenol amides tiva na matimba swinene. Ku dyiwa loku yaka emahlweni ka ti herbivores letimbirhi swiendle leswaku kuva na kwalomu ka excitation spectrum leyi fanaka ya phenolamides, naswona xivumbeko lexi axiri erivaleni ngopfu eka de novo synthesis ya phenolamides. Xiendlakalo lexi fanaka xi ta voniwa loko ku lavisisiwa ndlela ya 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), leyi humesaka nhlayo leyikulu ya ti acyclic diterpenes leti nga na mintirho leyi tirhaka ya anti-herbivore (33), laha Ms Feeding with Sl yi hlohloteleke xivumbeko xa ku paluxiwa lexi fanaka (Xifaniso S1)).
Ku pfumaleka loku nga vaka kona ka swikambelo swa ku dya swakudya swa herbivore hi ku kongoma i ku hambana ka mpimo wa ku dyiwa ka matluka na nkarhi wo dya wa swiharhi leswi dyaka byanyi, leswi endlaka leswaku swi tika ku herisa switandzhaku swa herbivore-specific leswi vangiwaka hi swilondza na swihadyani leswi dyaka byanyi. Ku kota ku tlhantlha ku antswa ku hlawuleka ka tinxaka ta herbivore ta nhlamulo ya metabolic ya matluka leyi hlohloteriweke, hi tekelele ku phameriwa ka switsotswana swa Ms na Sl hi ku hatlisa hi chela OS leyi ha ku hlengeletiwaka (OSM na OSS1) eka standard puncture W ya swiyimo swa matluka leswi nga cincekiki. Endlelo leri ri vuriwa vutshunguri bya W + OS, naswona ri ringanisa induction hi ku veka nkarhi lowu kongomeke wa ku sungula ka nhlamulo leyi humesiweke hi herbivore handle ko vanga vuyelo lebyi pfilunganyaka bya ku hambana eka mpimo kumbe nhlayo ya ku lahlekeriwa hi tinyama (Xifaniso 2A) (34). Hiku tirhisa phayiphi ya nxopaxopo wa MS/MS na xibalo, hi kumile 443 wa ti MS/MS spectra (data file S1), leswinga hlangana na ti spectra letinga hlengeletiwa khale kusuka eka swikambelo swa swakudya swo kongoma. Nxopaxopo wa thiyori ya mahungu ya sete leyi ya datha ya MS/MS wu kombisile leswaku ku phurogiremiwa nakambe ka ti metabolomes leti hlawulekeke eka matluka hi ku tekelela ti herbivores swi kombisile ku hlohloteriwa ka OS-specific (Xifaniso 2C). Ngopfungopfu, loko ku pimanisiwa na vutshunguri bya OSS1, OSM yi vange ku antswisiwa ka ku hlawuleka ka metabolome eka tiawara ta 4. Swifanele ku xiya leswaku loko ku pimanyisiwa na sete ya data ya experimental ya swakudya swa herbivore yo kongoma, metabolic kinetics leyi voniweke eka ndzhawu ya swiphemu swimbirhi hiku tirhisa Hj na δj tani hi ti coordinates xikan’we na directionality ya metabolome specialization eka ku hlamula eka vutshunguri bya herbivore lebyi simulated hiku famba ka nkarhi Ku engeteleka loku nga cincekiki (Figure 2D). Hi nkarhi lowu fanaka, hi pimile nhlayo ya nhlayo ya ti amino acids, ti organic acids na chukele (data file S2) ku lavisisa loko ku tlakuka loku ku kongomisiweke eka vutshila bya metabolome ku vangiwa hi ku lulamisiwa nakambe ka metabolism ya khaboni ya le xikarhi ku hlamula eka ti herbivores leti simulated ( Figure S2). Ku hlamusela ku antswa xivumbeko lexi, hi tlhele hi veka tihlo eka metabolic accumulation kinetics ya tindlela ta phenolamide na 17-HGL-DTG leti ku buriweke ha tona khale. Induction ya OS-specific ya herbivores yi hundzuriwa kuva differential rearrangement pattern endzeni ka phenolamide metabolism (Xifaniso S3). Phenolic amides leyingana coumarin na caffeoyl moieties yitsakela ku hlohleteriwa hi OSS1, kasi ti OSMs ti hlohletela induction yokarhi ya ferulyl conjugates. Eka ndlela ya 17-HGL-DTG, differential OS induction hi downstream malonylation na swikumiwa swa dimalonylation swi kumiwile (Xifaniso xa S3).
Endzhaku ka sweswo, hi dyondze hi OS-induced transcriptome plasticity hiku tirhisa time-course microarray data set, leyi tekelelaka kutirhisiwa ka ti OSMs ku tshungula matluka ya matluka ya ximilani xa rosette eka ti herbivores. Kinetics ya sampling hi xisekelo yi hlangana na kinetics leyi tirhisiweke eka dyondzo leyi ya metabolomics (35). Loko ku pimanyisiwa na metabolome reconfiguration laha metabolic plasticity yi engetelekaka ngopfu hiku famba ka nkarhi, hi vona ku buluka ka transient transcription eka matluka lawa ya hlohleteriwaka hi Ms, laha transcriptome inducibility (RDPI) na specialization (δj) swinga eka 1 Ku vile na ku tlakuka lokukulu eka tiawara, na ku hambana (Hj) eka nkarhi lowu, ku paluxiwa ka BMP1 ku hungutiwile swinene, ku landzela ku ntshunxeka ya ku hlawuleka ka transcriptome (Xifaniso xa S4). Mindyangu ya ti gene ta metabolic (kufana na P450, glycosyltransferase, na BAHD acyltransferase) yi nghenelela eka endlelo ro hlengeleta ti metabolites to hlawuleka kusuka eka ti structural units leti humaka eka metabolism yosungula, hiku landzelela modele wosungula wa high-specialization lowu boxiweke laha henhla. Tanihi dyondzo ya xiyimo, ndlela ya phenylalanine yi kamberiwile. Nxopaxopo wu tiyisisile leswaku ti core genes eka phenolamide metabolism ti hlohleteriwa swinene hi OS eka ti herbivores loko ku pimanyisiwa na swimilani leswinga kokeriwangiki, naswona ti fambelanisiwa swinene eka ti expression patterns ta tona. Xiaki xa matsalelo MYB8 na tijini ta xivumbeko PAL1, PAL2, C4H na 4CL eka le henhla ka ndlela leyi swi kombisile ku sunguriwa ka matsalelo ka ha ri na nkarhi. Acyltransferases leyi tlangaka xiave eka nhlengeletano yo hetelela ya phenolamide, ku fana na AT1, DH29, na CV86, yi kombisa xivumbeko xa upregulation xo leha (Xifaniso xa S4). Swibumabumelo leswi nga laha henhla swi kombisa leswaku ku sunguriwa ka ha ri emahlweni ka ku hlawuleka ka transcriptome na ku antswisiwa ka le ndzhaku ka ku hlawuleka ka metabolomics i ndlela leyi hlanganisiweke, leswi nga ha vaka swi vangiwa hi sisiteme ya vulawuri bya synchronous leyi sungulaka nhlamulo ya matimba ya vusirheleri.
Ku lulamisiwa nakambe eka swikombiso swa ti hormone ta swimilani switirha tani hi regulatory layer leyi hlanganisaka vuxokoxoko bya herbivorous kuva yi tlhela yi phurogirema physiology ya swimilani. Endzhaku ka ku tekeleriwa ka herbivore, hi pimile cumulative dynamics ya swiyenge swa nkoka swa ti hormone ta swimilani naswona hi vonile hi mahlo ya mianakanyo temporal co-expression exikarhi ka swona [Pearson correlation coefficient (PCC)> 0.4] (Xifaniso 3A). Tani hilaha swi languteriweke hakona, ti hormone ta swimilani leti fambelanaka na biosynthesis ti hlanganisiwa endzeni ka network ya ti hormone ta swimilani co-expression. Ku engetela kwalano, metabolic specificity (Si index) yi mepiwile eka network leyi kuva yi kombisa ti hormones ta swimilani leti hlohleteriwaka hi vutshunguri byohambana hambana. Tindzhawu timbirhi letikulu ta nhlamulo yokarhi ya herbivorous ti dirowiwile: yin’we yile ka JA cluster, laha JA (xivumbeko xa yona xa biologically active JA-Ile) na swin’wana leswi humaka eka JA swikombisaka xikoro xale henhla xa Si; yin’wana i ethylene (ET). Gibberellin yi kombisile ntsena ku tlakuka loku ringaneleke eka herbivore specificity, kasi ti hormones tin’wana ta swimilani, tofana na cytokinin, auxin, na abscisic acid, atiri na low induction specificity eka herbivores. Loko ku pimanisiwa na ku tirhisa W + W ntsena, ku kurisiwa ka nkoka wa nhlohlorhi ya swiaki swa JA hi ku tirhisa xitirhisiwa xa OS (W + OS) hi xisekelo swi nga hundzuriwa ku va xikombiso xo karhi xo tiya xa ti-JA. Swinga languteriwanga, OSM na OSS1 leswingana nhlayo yohambana hambana ya elicitor switiveka kuva swivanga ku hlengeletiwa loku fanaka ka JA na JA-Ile. Ku hambana na OSS1, OSM yi hlohloteriwa hi ku kongoma na hi matimba hi ti-OSM, kasi OSS1 a yi kurisi nhlamulo ya swilondza swa le hansi (Xifaniso 3B).
(A) Nxopaxopo wa netiweke ya co-expression lowu simekiweke eka xibalo xa PCC xa herbivore-induced plant hormone accumulation kinetics simulation. Node yiyimela hormone yin’we ya ximilani, naswona vukulu bya node byi yimela Si index leyi kongomeke eka hormone ya ximilani exikarhi ka vutshunguri. (B) Ku hlengeletiwa ka JA, JA-Ile na ET eka matluka leswi vangiwaka hi vutshunguri byo hambana lebyi kombisiweke hi mihlovo yo hambana: apricot, W + OSM; ya wasi, W + OSSl; ntima, W + W; grey, C (vulawuri) . Tinyeleti ti kombisa ku hambana lokukulu exikarhi ka vutshunguri na vulawuri (2-way ANOVA leyi landzeriwaka hi Tukey HSD post hoc multiple comparison, *** P <0.001). Nxopaxopo wa thiyori ya mahungu ya (C)697 MS/MS (data file S1) eka JA biosynthesis na impaired perception spectrum (irAOC na irCOI1) na (D)585 MS/MS (data file S1) eka ETR1 leyi nga na ku onhaka ka ET signal Vutshunguri byimbirhi lebyi tekeleriweke bya herbivore byi hlohlotele milayeni ya swimilani na swimilani swo lawula movha lowu nga riki na nchumu (EV). Tinyeleti ti kombisa ku hambana lokukulu exikarhi ka vutshunguri bya W+OS na vulawuri lebyi nga onhakiki (ANOVA ya tindlela timbirhi leyi landzeriwaka hi ku pimanisiwa ko tala ka Tukey HSD post hoc, *P<0.05, **P<0.01 na ***P<0.001). (E) Tigirafu leti hangalakeke ta ku kanetana loku hangalakeke na ku hlawuleka. Mihlovo yi yimela tinxaka to hambana-hambana leti cinciweke tijini; swikombiso swi yimela tindlela to hambana ta vutshunguri: triangle, W + OSS1; xirhendzevutani, W + OSM; xirhendzevutana xa C
Endzhaku ka sweswo, hi tirhisa muxaka lowu cinciweke ti gene ta attenuated Nepenthes (irCOI1 na sETR1) eka magoza ya nkoka ya JA na ET biosynthesis (irAOC na irACO) na ku twisisa (irCOI1 na sETR1) ku xopaxopa metabolism ya ti hormones leti timbirhi ta swimilani eka ti herbivores The relative contribution of reprogramming. Ku fambisana na swikambelo swa khale, hi tiyisisile ku nghenisiwa ka herbivore-OS eka swimilani swa empty carrier (EV) (Xifaniso 3, C kuya eka D) xikan’we nakuhunguteka hinkwako eka Hj index leswi vangiwaka hi OSM, kasi δj index yi tlakukile. Nhlamulo yi vonaka ngopfu ku tlula nhlamulo leyi hlohloteriwaka hi OSS1. Girafamu ya milayeni yimbirhi leyi tirhisaka Hj na δj tanihi tikhodixini yi kombisa ku herisiwa ka milawu yo karhi (Xifaniso 3E). Ntshamiseko lowu vonakaka swinene hileswaku eka tinxaka leti pfumalaka xikombiso xa JA, ku hambana ka metabolome na ku cinca ka specialization leswi vangiwaka hi ti herbivores swi lava ku herisiwa hi ku helela (Figure 3C). Kuhambana na sweswo, ku vona ka ET loku nga vulavuriki eka swimilani swa sETR1, hambi leswi mbuyelo hinkwawo eka ku cinca eka metabolism ya herbivorous wunga ehansi swinene kutlula wa JA signaling, wu hunguta ku hambana eka Hj na δj indices exikarhi ka OSM na OSS1 excitations (Figure 3D na Figure S5). . Leswi swikomba leswaku ku engetela eka ntirho wa nkoka wa JA signal transduction, ET signal transduction yitlhela yitirha tani hi fine-tuning ya species-specific metabolic response ya herbivores. Ku fambisana na ntirho lowu wa fine-tuning, akuri hava ku cinca eka metabolome inducibility hinkwayo eka swimilani swa sETR1. Hi tlhelo rin’wana, loko ku pimanyisiwa na swimilani swa sETR1, swimilani swa irACO swi hlohlotele ti amplitudes hinkwato leti fanaka ta ku cinca ka metabolic loku vangiwaka hi ti herbivores, kambe swikombe swikoweto swohambana swinene swa Hj na δj exikarhi ka mintlhontlho ya OSM na OSS1 (Xifaniso xa S5).
Ku kota ku kuma ti metabolites to hlawuleka letingana ku hoxa xandla ka nkoka eka nhlamulo ya tinxaka tokarhi ta herbivores xikan’we naku lulamisa kahle vuhumelerisi bya tona hiku tirhisa swikombiso swa ET, hi tirhise ndlela ya structural MS/MS leyi ayi tumbuluxiwile khale. Ndlela leyi yi titshege hi ndlela ya bi-clustering ku tlhela yi ringanyeta ndyangu wa metabolic kusuka eka swiphemu swa MS/MS [normalized dot product (NDP)] na xikoro xa kufana lexi seketeriweke eka neutral loss (NL). Seti ya datha ya MS/MS leyi akiweke hi ku tirhisa nxopaxopo wa milayeni ya ET transgenic yi humesile 585 MS/MS (fayili ya datha S1), leyi tlhantlhiweke hi ku yi hlengeletiwa eka mimojula ya nkombo leyikulu ya MS/MS (M) (Xifaniso 4A) . Tin’wana ta ti modules leti ti tele ngopfu hi ti metabolites to hlawuleka letinga tiveka khale: xikombiso, M1, M2, M3, M4 na M7 ti fuwile hi ti phenol derivatives to hambana hambana (M1), flavonoid glycosides (M2), chukele ra acyl (M3 Na M4), na 17-HGL-DTG (M7). Ku engetela kwalano, vuxokoxoko bya metabolic specific (Si index) ya metabolite yin’we eka module yin’wana na yin’wana ya hlayeriwa, naswona ku hangalasiwa ka yona ka Si kunga voniwa hindlela yo twisiseka. Hiku komisa, MS/MS spectra leyi kombisaka herbivory yale henhla xikan’we na genotype specificity yitiveka hi mimpimo yale henhla ya Si, naswona tinhlayo ta kurtosis tikomba ku hangalaka ka voya eka khoneni ya ncila wa xinene. Ku hangalasiwa kun’wana ko tano ka lean colloid ku kumiwile eka M1, laha phenol amide yi kombiseke xiphemu xa le henhla xa Si (Xifaniso 4B). Herbivorous inducible 17-HGL-DTG leyi boxiweke khale eka M7 yi kombisile xikoro xa Si xo ringanela, leswi kombisaka mpimo wo ringanela wa vulawuri byo hambana exikarhi ka tinxaka timbirhi ta OS. Kuhambana na sweswo, ngopfu ngopfu constitutively produced specialized metabolites, kufana na rutin, CGA, na acyl sugars, swile xikarhi ka swikoweto swale hansi swa Si. Ku kota ku lavisisa ku antswa ku rharhangana ka xivumbeko na ku hangalasiwa ka Si exikarhi ka ti metabolites to hlawuleka, ku akiwile netiweke ya molekhuli eka modula yin’wana na yin’wana (Xifaniso 4B). Ku vhumbha ka nkoka ka OD theory (leswi katsakanyiweke eka Figure 1B) hileswaku ku hleriwa nakambe ka ti metabolites to hlawuleka endzhaku ka herbivory swifanele ku yisa eka ku cinca ka ndlela yin’we eka ti metabolites letingana nkoka wale henhla wa nsirhelelo, ngopfu ngopfu hiku engetela ku hlawuleka ka tona (kuhambanana na random distribution) Mode) Defensive metabolite leyi vhumbhiweke hi MT theory. Vunyingi bya ti phenol derivatives leti hlengeletiweke eka M1 ti fambelana hi ntirho na ku hunguteka ka matirhelo ya switsotswana ( 32 ). Loko hi pimanisa mimpimo ya Si eka ti metabolites ta M1 exikarhi ka matluka lawa ya hlohleteriweke na matluka lawa ya vumbeke ya swimilani swa vulawuri bya EV eka tiawara ta 24, hi xiye leswaku metabolic specificity ya ti metabolites totala endzhaku ka switsotswana leswi dyaka byanyi yina ntshamiseko lowukulu wo tlakuka (Figure 4C). Ku tlakuka ko karhi ka nkoka wa Si ku kumiwile ntsena eka ti phenolamides to sirhelela, kambe ku hava ku tlakuka ka nkoka wa Si loku kumiweke eka ti phenols tin’wana na ti metabolites leti nga tiviwiki leti hanyaka swin’we eka modula leyi. Leyi i modele wo hlawuleka, lowu fambelanaka na thiyori ya OD. Ku vhumbha lokukulu ka ku cinca ka metabolic loku vangiwaka hi ti herbivores swa tshamiseka. Ku kota ku kambela loko particularity leyi ya phenolamide spectrum yi hlohloteriwile hi OS-specific ET, hi plotile metabolite Si index naswona hi vange ku hambana ka expression value exikarhi ka OSM na OSS1 eka EV na sETR1 genotypes (Xifaniso 4D ). Eka sETR1, ku hambana loku hlohloteriwaka hi phenamide exikarhi ka OSM na OSS1 ku hungutiwile swinene. Ndlela ya bi-clustering yi tlhele yi tirhisiwa eka datha ya MS/MS leyi hlengeletiweke eka tinxaka leti nga na JA leyi nga enelangiki ku ringanyeta mimojula leyikulu ya MS/MS leyi fambelanaka na JA-regulated metabolic specialization (Xifaniso xa S6).
(A) Mimbuyelo ya ku hlengeletiwa ka 585 MS/MS leyi simekiweke eka xiphemu lexi avelaniwa (ku fana ka NDP) na ku lahlekeriwa loku avelaniwa ka vukalatlhelo (ku fana ka NL) swi endla leswaku modyuli (M) yi fambisana na ndyangu wa nkatsakanyo lowu tivekaka, kumbe hi xivumbeko xa Metabolite lexi nga tiviwiki kumbe lexi nga metabolized kahle. Etlhelo ka modula yin’wana na yin’wana, ku kombisiwa ku hangalasiwa ka metabolite (MS/MS) specific (Si). (B) Netiweke ya molekhuli ya modula: Tinotsi ti yimela MS/MS na matlhelo, swikoweto swa NDP (yo tshwuka) na NL (ya wasi) swa MS/MS (ku tsemiwa,> 0.6). Graded metabolite specificity index (Si) yi pendiwile hi mihlovo kuya hi module (exineneni) naswona yi mepiwile kuya eka molecular network (exineneni). (C) Modyuli M1 ya ximilani xa EV eka xiyimo xa constitutive (control) na induced (simulated herbivore) eka tiawara ta 24: molecular network diagram (Si value i node size, defensive phenolamide yi kombisiwile hi muhlovo wa wasi). (D) Xifaniso xa netiweke ya timolekhuli ya M1 xa layini ya spektrum sETR1 leyi nga na ku onhaka ka ku twisisa ka EV na ET: nkatsakanyo wa phenolic lowu yimeriweke hi node ya xirhendzevutana xa rihlaza, na ku hambana lokukulu (nxavo wa P) exikarhi ka vutshunguri bya W + OSM na W + OSS1 tanihi sayizi ya Node. CP, N-kafeyoyili-tyrosine; CS, N-kafeyoyili-spermidini; FP, N-ferulic acid ya ester-asidi ya uric; FS, N-ferulyl-spermidine, ku nga xitsongwatsongwana xa le henhla; CoP, N’, N “-Kuumarolyl-tyrosine;DCS, N’, N”-dikafeyoyili-spermidini; CFS, N’, N”-caffeoyl, feruloyl-spermidine;Lycium barbarum eka n’wana wa wolfberry;Nick.O-AS, chukele ra O-acyl.
Hi tlhele hi ndlandlamuxa nxopaxopo ku suka eka genotype yin’we ya Nepenthes leyi hungutekeke ku ya eka vaaki va ntumbuluko, laha ku cinca lokukulu ka intraspecific eka swiyimo swa JA swa herbivorous na swiyimo swo karhi swa metabolite swi hlamuseriweke khale eka vaaki va ntumbuluko ( 26 ). Tirhisa sete leyi ya data ku funengeta 43 wa ti germplasm. Ti germplasms leti tina 123 wa tinxaka ta swimilani kusuka eka N. pallens. Swimilani leswi swi tekiwile kusuka eka mbewu leyi hlengeletiweke eka tindzhawu tohambana hambana ta ndzhavuko e Utah, Nevada, Arizona, na California (Figure S7), hi hlayele ku hambana ka metabolome (laha swivuriwa population level) β diversity) xikan’we naku hlawuleka loku vangiwaka hi OSM. Ku fambisana na tidyondzo tale mahlweni, hi vonile ku cinca ko anama ka metabolic kuya hi Hj na δj axes, leswi kombaka leswaku ti germplasms tina ku hambana lokukulu eka plasticity ya tinhlamulo ta tona ta metabolic eka ti herbivores (Xifaniso S7). Nhlangano lowu wu tsundzuxa swibumabumelo swa khale mayelana na dynamic range ya ku cinca ka JAs loku vangiwaka hi herbivores, naswona yi hlayise nkoka wa le henhla swinene eka nhlayo yin’we (26, 36). Hi ku tirhisa JA na JA-Ile ku kambela ku yelana ka xiyimo hinkwaxo exikarhi ka Hj na δj, hi kumile leswaku ku na ku yelana lokunene lokukulu exikarhi ka JA na metabolome β diversity na specialization index (Xifaniso S7). Leswi swikomba leswaku heterogeneity leyi hlohleteriwaka hi herbivore eka JAs induction leyi kumiweke eka levele ya vaaki yingava hikokwalaho ka ti key metabolic polymorphisms leti vangiwaka hiku hlawuriwa kusuka eka ti herbivores ta switsotswana.
Mindzavisiso ya khale yi kombisile leswaku tinxaka ta fole ti hambana swinene hi muxaka na ku titshega loku ringanaka eka induced na constitutive metabolic defenses. Kutshembiwa leswaku ku cinca loku eka anti-herbivore signal transduction xikan’we na vuswikoti byo sirhelela swilawuriwa hi ntshikelelo wa nhlayo ya switsotswana, xirhendzevutani xa vutomi bya swimilani, na ntsengo wa vuhumelerisi bya vusirheleri eka niche laha muxaka lowu nyikiweke wu milaka kona. Hi dyondze hi ku ringanana ka ku pfuxetiwa ka metabolome ya matluka loku hlohloteriweke hi swiharhi leswi dyaka byanyi swa tinxaka ta tsevu ta Nicotiana leti tumbulukaka eAmerika N’walungu na Amerika Dzonga. Tinxaka leti ti fambelana swinene na Nepenthes North America, kunga Nicolas Bociflo. La, N. switsongwatsongwana swa switsongwatsongwana, Nicotiana n. byanyi lebyi tsaneke, Nicotiana tabacum, fole ra linear, fole (Nicotiana spegazzinii) na fole ra matluka ya fole (Nicotiana obtusifolia) (Xifaniso 5A) (37). Tsevu wa tinxaka leti, ku katsa na muxaka lowu tivekaka kahle wa N. please, i swimilani swa lembe na lembe swa petunia clade, naswona obtusifolia N. i swimilani leswi nga heriki swa sister clade Trigonophyllae (38). Endzhaku ka sweswo, W + W, W + OSM na W + OSS1 induction swiendliwile eka tinxaka leti ta nkombo ku dyondza hi species-level metabolic rearrangement ya swakudya swa switsotswana.
(A) Murhi wa phylogenetic wa bootstrap lowu sekeriweke eka ku koteka lokukulu [ka ku endliwa ka glutamine ya nyutliya (38)] na ku hangalaka ka ndhawu ya tinxaka ta nkombo ta Nicotiana leti yelanaka swinene (mihlovo yo hambana) (37). (B) Ploto yo hangalaka ya ku hambana loku hlawulekeke eka tiphrofayili ta metaboliki ta tinxaka ta nkombo ta Nicotiana (939 MS/MS; fayili ya datha S1). Eka xiyimo xa tinxaka, ku hambana ka metabolome swi fambelanisiwa hi ndlela yo biha na mpimo wa ku hlawuleka. Nxopaxopo wa ku yelana ka xiyimo xa tinxaka exikarhi ka ku hambana ka metaboliki na ku hlawuleka na ku hlengeletiwa ka JA swi kombisiwile eka Xifaniso 2. S9. Muhlovo, tinxaka to hambana; khumbi-nharhu, W + OSS1; xirhendzevutani, W + OSM; (C) Nicotiana JA na JA-Ile dynamics yi vekiwile hi ku ya hi OS excitation amplitude (tindlela timbirhi ANOVA na Tukey HSD endzhaku ka ku pimanisiwa ko tala, * P <0.05, ** P <0.01 na * ** Ku pimanisiwa ka W + OS na W + W, P <0.001). Ploto ya bokisi ya (D) ku hambana na (E) ku hlawuleka ka muxaka wun’wana na wun’wana endzhaku ko tekelela herbivorous na methyl JA (MeJA). Nyeleti yi kombisa ku hambana lokukulu exikarhi ka W + OS na W + W kumbe lanolin plus W (Lan + W) kumbe Lan plus MeJA (Lan + MeJa) na Lan control (nxopaxopo wa tindlela timbirhi wa ku hambana, lowu landzeriwaka hi Tukey’s HSD post hoc multiple comparison , *P<0.05, **P<0.01 na ***P<0.001).
Hi ku tirhisa ndlela ya dual cluster, hi kumile 9 wa mimojula ya 939 MS/MS (fayili ya data S1). Xivumbeko xa MS/MS lexi lulamisiweke nakambe hi vutshunguri byo hambana xi hambana swinene exikarhi ka mimojula yo hambana exikarhi ka tinxaka (Xifaniso xa S8). Ku vona hi mahlo ya mianakanyo Hj (leyi vuriwaka laha tani hi species-level γ-diversity) na δj swi paluxa leswaku tinxaka to hambana ti hlengeletana eka mintlawa yo hambana swinene eka ndzhawu ya metabolic, laha ku avana ka species-level hi ntolovelo ku vonakaka ngopfu kutlula ku hlohloteriwa. Handle ka N. linear na N. obliquus, ti kombisa nxaxamelo wo anama wa dynamic wa switandzhaku swa induction (Xifaniso 5B). Kuhambana na sweswo, tinxaka tofana na N. purpurea na N. obtusifolia tina nhlamulo ya metabolic leyinga vonakiki ngopfu eka vutshunguri, kambe metabolome yihambanile swinene. Ku hangalasiwa ka tinxaka to karhi ka nhlamulo ya metaboliki leyi hlohloteriweke swi endle leswaku ku va na ku yelana lokukulu ko biha exikarhi ka ku hlawuleka na ku hambana ka gamma (PCC = -0.46, P = 4.9×10-8). Ku cinca loku hlohloteriwaka hi OS eka swiyimo swa JAs swi fambelanisiwa kahle na ku hlawuleka ka metabolome, naswona swi fambelanisiwa hi ndlela yo biha na ku hambana ka metabolic gamma loku kombisiweke hi muxaka wun’wana na wun’wana (Xifaniso 5B na Xifaniso xa S9). Swifanele ku xiya leswaku muxaka lowu hi ririmi rivuriwaka tani hi “signal response” species eka Figure 5C, kufana na Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, na Nepenthes attenuated, yi vange swikombiso leswikulu eka 30 wa timinete. Ku tumbuluka ka sweswinyana ka OS-specific JA na JA-Ile, kasi ti bacteria tin’wana leti vuriwaka “signal unresponsive”, kufana na Nepenthes mills, Nepenthes powdery na N. obtusifolia ti kombisa ntsena JA-Ile Edge induction handle ka OS specificity (Figure 5C). Eka levele ya metabolic, tani hileswi swinga boxiwaka laha henhla, eka attenuated Nepenthes, swilo leswi hlamulaka eka signal swikombe OS specificity naswona swi engetele swinene δj, loko swihunguta Hj. Vuyelo lebyi bya OS-specific priming abyi voniwanga eka tinxaka leti hlayiweke tani hi signal non-reactive species (Xifaniso 5, D na E). OS-specific metabolites yi averiwa ngopfu exikarhi ka tinxaka leti hlamulaka swikombiso, naswona swirhabyani leswi swa swikombiso swi hlengeletana na tinxaka leti nga na tinhlamulo ta swikombiso leti tsaneke, kasi tinxaka leti nga na tinhlamulo ta swikombiso leti tsaneke ti kombisa ku titshega kutsongo (Xifaniso S8 ). Mbuyelo lowu wu kombisa leswaku OS-specific induction ya JAs xikan’we na OS-specific reconfiguration ya downstream metabolome swihlanganisiwa eka xiyimo xa muxaka.
Endzhaku ka sweswo, hi tirhise lanolin paste leyinga na methyl JA (MeJA) ku tshungula swimilani ku lavisisa loko tindlela leti ta coupling ti siveriwa hiku kumeka ka JA leyi tirhisiwaka hi exogenous JA, leyingatava eka cytoplasm ya swimilani. Ku deesterification hi xihatla i JA. Hi kumile ntshamiseko lowu fanaka wa ku cinca hakatsongotsongo ku suka eka tinxaka leti hlamulaka swikombiso ku ya eka tinxaka leti nga hlamuriki swikombiso leswi vangiwaka hi mphakelo lowu yaka emahlweni wa JA (Xifaniso 5, D na E). Hiku komisa, vutshunguri bya MeJA byi tlhele byi phurogirema swinene ti metabolomes ta linear nematodes, N. obliquus, N. aquaticus, N. pallens, na N. mikimotoi, leswinga endla leswaku kuva na ku tlakuka lokukulu ka δj xikan’we naku hunguteka ka Hj. N. purpurea yi kombisile ntsena ku tlakuka ka δj, kambe kungari Hj. N. obtusifolia, leyi khale ayi kombisiwa kuva yi hlengeleta ti levele tale hansi swinene ta JAs, yitlhela yi hlamula kahle eka vutshunguri bya MeJA hi mayelana na metabolome reconfiguration. Mimbuyelo leyi yi kombisa leswaku vuhumelerisi bya JA kumbe ku hundziseriwa ka swikombiso swi siveriwa hi tlhelo ra physiological eka tinxaka leti nga hlamuriki swikombiso. Ku kambela xiringanyeto lexi, hi dyondze tinxaka ta mune (N. pallens, N. mills, N. pink na N. microphylla) leti hlohloteriweke hi W + W, W + OSMs na W + OSS1 Transcriptome (39). Ku fambisana na xivumbeko xa metabolome remodeling, tinxaka ti hambanisiwile kahle eka ndzhawu ya transcriptome, exikarhi ka swona N. attenuated yi kombisile RDPI yale henhla swinene leyi hlohleteriwaka hi OS, kasi N. gracilis ayi ri na yale hansi swinene (Figure 6A). Hambiswiritano, ku kumiwile leswaku ku hambana ka transcriptome loku hlohloteriwaka hi N. oblonga a ku ri ka le hansi swinene eka tinxaka ta mune, ku hambana na ku hambana ka le henhla ka metabonomic ka N. oblonga loku tshameke ku kombisiwa eka tinxaka ta nkombo. Mindzavisiso ya khale yi kombisile leswaku sete ya ti genes leti fambelanaka na swikombiso swa nsirhelelo wosungula, kukatsa na swikombiso swa JA, yi hlamusela ku hlawuleka ka tinhlamulo ta nsirhelelo wosungula leti hlohleteriwaka hi ti herbivore-related elicitors eka tinxaka ta Nicotiana ( 39 ). Ku pimanisa tindlela ta JA signaling exikarhi ka tinxaka leti ta mune swi paluxe xivumbeko xo tsakisa (Figure 6B). Ti genes totala eka ndlela leyi, kufana na AOC, OPR3, ACX na COI1, ti kombisile ti levele tale henhla swinene ta induction eka tinxaka leti ta mune. Hambiswiritano, gene ya nkoka, JAR4, yi hundzula JA kuva xivumbeko xa yona xa biologically active xa JA-Ile accumulated transcripts, naswona levele ya yona ya transcription yile hansi swinene, ngopfu ngopfu eka N. mills, Nepenthes pieris na N. microphylla. Ku engetela kwalano, i ntsena transcript ya gene yin’wana ya AOS leyi ayi voniwanga eka N. bifidum. Ku cinca loku eka ku paluxiwa ka ti gene swinga va na vutihlamuleri eka ti phenotypes to tlula mpimo leti hlohleteriwaka hi vuhumelerisi byale hansi bya JA eka tinxaka ta signal anergic xikan’we naku hlohleteriwa ka N. gracilis.
(A) Nxopaxopo wa thiyori ya mahungu wa ku phurogiremiwa nakambe ka tinhlamulo to sungula ta matsalelo ya tinxaka ta mune ta fole leti fambelanaka swinene leti sampuliweke endzhaku ka timinete ta 30 endzhaku ka ku nghenisiwa ka herbivory. RDPI yi hlayeriwa hiku pimanisa matluka lawa ya hlohleteriwaka hi herbivore OS na vulawuri bya swilondza. Mihlovo yi kombisa tinxaka to hambana-hambana, naswona mimfungho yi kombisa tindlela to hambana-hambana to tshungula. (B) Nxopaxopo wa ku paluxiwa ka tijini eka tindlela ta swikombiso swa JA exikarhi ka tinxaka ta mune. Ndlela ya JA leyi olovisiweke yi kombisiwile ekusuhi na plot ya bokisi. Mihlovo yo hambana-hambana yi kombisa tindlela to hambana-hambana to lulamisa. Nyeleti yi kombisa leswaku ku na ku hambana lokukulu exikarhi ka vutshunguri bya W + OS na vulawuri bya W + W (eka Student’s t-test for pairwise differences, *P<0.05, **P<0.01 na ***P<0.001). OPDA, asidi ya 12-oxophytodienoic; OPC-8: 0,3-oxo-2 (2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Eka xiphemu xohetelela, hi dyondze hilaha ku pfuxetiwa ka tinxaka ta switsotswana hi ku kongoma ka metabolome ya tinxaka to hambana ta swimilani swinga lwisanaka na switsotswana leswi dyaka byanyi. Vulavisisi bya khale byi kandziyise muxaka wa Nicotiana. Ku lwisana ka tona na Ms na switsotswana swa hambana swinene (40). Laha, hi dyondze hi ku yelana exikarhi ka modele lowu na plasticity ya vona ya metabolic. Hiku tirhisa tinxaka ta mune ta fole letinga laha henhla, naku kambela ku yelana exikarhi ka ku hambana na ku hlawuleka ka metabolome leyi vangiwaka hi ti herbivores na ku lwisana ka swimilani eka Ms na Sl, hi kumile ku lwisana, ku hambana na ku hlawuleka eka generalist Sl Hinkwaswo swi fambelana kahle, kasi ku yelana exikarhi ka ku lwisana na vamanana va vutshila na vutshila a swi tsani, naswona ku yelana na ku hambana a ku na nkoka (Xifaniso S10). Malunghana na ku lwisana na S1, havumbirhi bya attenuated N. chinensis na N. gracilis, leswi khale a swi kombisiwa ku kombisa havumbirhi bya JA signal transduction levels na metabolome plasticity, a swi ri na tinhlamulo to hambana swinene eka herbivore induction, naswona ti tlhele ti kombisa ku lwisana ka le henhla loku fanaka. Rimbewu.
Eka malembe ya makume-nhungu lawa ya hundzeke, thiyori ya nsirhelelo wa swimilani yi nyikile rimba ra thiyori, leri sekeriweke eka rona valavisisi va vhumbhe nhlayo leyikulu ya ku hundzuka na mintirho ya ti metabolites to hlawuleka ta swimilani. Vunyingi bya tithiyori leti a ti landzeleli endlelo leri tolovelekeke ra swibumabumelo swo tiya (41). Va ringanyeta vuprofeta bya nkoka (3) eka xiyimo lexi fanaka xa nxopaxopo. Loko ku kamberiwa ka vuprofeta bya nkoka ku pfumelela tithiyori to karhi ku xopaxopiwa, leswi swi ta endla leswaku Nsimu yi ya emahlweni. Ku seketeriwa, kambe ala van'wana (42). Ematshan’wini ya sweswo, thiyori leyintshwa yi endla vuprofeta eka swiyimo swo hambana swa nxopaxopo naswona yi engetela leyara leyintshwa ya ku tekeriwa enhlokweni loku hlamuselaka (42). Hambiswiritano, tithiyori timbirhi leti ringanyetiweke eka xiyimo xa ntirho, MT na OD, tinga hlamuseriwa hiku olova tani hi ku vhumbha ka nkoka ka ku cinca ko hlawuleka ka metabolic loku vangiwaka hi swiharhi leswi dyaka swimilani: Tithiyori ya OD yi tshemba leswaku ku cinca eka “xivandla” xo hlawuleka xa metabolic swi kongomisa swinene . Tiyori ya MT yi tshemba leswaku ku cinca loku kutava lokunga kongomisiwiki naswona kuta kumeka hiku landzelelana eka ndzhawu ya metabolic, naswona switala kuva na ti metabolites ta nkoka wa le henhla wa vusirheleri. Swikambelo swa khale swa ku vhumbha ka OD na MT swi kamberiwile hi ku tirhisa sete yo koma ya swihlanganisi swa “vusirheleri” swa a priori. Swikambelo leswi swa metabolite-centric swi sivela vuswikoti byo xopaxopa mpimo na trajectory ya metabolome reconfiguration hinkarhi wa herbivory, naswona aswi pfumeleli ku kamberiwa endzeni ka rimba ra tinhlayo leri nga cincekiki ku lava ku vhumbha ka nkoka loku nga tekiwaka tani hi hinkwaswo Ku pima ku cinca eka metabolome ya swimilani. Laha, hi tirhisile thekinoloji ya vutumbuluxi eka metabolomics leyi simekiweke eka MS ya xibalo naswona hi endlile nxopaxopo wa deconvolution MS eka mali yo angarhela ya swihlamuseri swa thiyori ya mahungu ku kambela ku hambana exikarhi ka leswimbirhi leswi ringanyetiweke eka xiyimo xa metabolomics ya misava hinkwayo. Ku vhumbha ka nkoka ka dyondzo leyi. Tiyori ya mahungu yi tirhisiwile eka tinsimu to tala, ngopfungopfu eka xiyimo xa ndzavisiso wa biodiversity na ku khuluka ka swakudya (43). Hambiswiritano, kuya hi leswi hi switivaka, leyi i xitirhisiwa xosungula lexi tirhisiwaka ku hlamusela ndzhawu ya mahungu ya metabolic ya swimilani naku tlhantlha swiphiqo swa ecological leswi fambelanaka na ku cinca ka metabolic ka xinkarhana ku hlamula eka swikombiso swa mbango. Ngopfu ngopfu, vuswikoti bya ndlela leyi byi le ka vuswikoti bya yona byo pimanisa swivumbeko endzeni na le xikarhi ka tinxaka ta swimilani ku kambela ndlela leyi ti herbivores ti hundzukeke ha yona kusuka eka tinxaka tohambana hambana kuya eka ti inter-species macroevolutionary patterns eka swiyimo swohambana hambana swa evolution. Ku cinca-cinca ka swiaki swa miri.
Nxopaxopo wa swiphemu swa nkoka (PCA) wu hundzula sete ya datha ya swilo swo hambana-hambana yi va ndhawu yo hunguta tidimenxini leswaku ntshamiseko lowukulu wa datha wu ta hlamuseriwa, hikwalaho hi ntolovelo wu tirhisiwa tanihi thekiniki yo lavisisa ku hlahluva sete ya datha, ku fana na metabolome ya deconvolution. Hambiswiritano, kuhungutiwa ka dimensionality swita lahlekeriwa hi xiphemu xa nhundzu ya mahungu eka sete ya data, naswona PCA ayi nge nyiki vuxokoxoko bya nhlayo mayelana na swihlawulekisi leswi fambelanaka ngopfu na thiyori ya ikholoji, kufana na: ndlela leyi swihadyani leswi dyaka byanyi swi lulamisaka hi vuntshwa ku hambana eka tinsimu to hlawuleka (xikombiso, ku fuwa, ku hangalaka) Na ku tala) ti metabolites? Hi tihi ti metabolites leti vhumbhaka xiyimo lexi hlohloteriweke xa herbivore leyi nyikiweke? Kusuka eka mavonelo ya specificity, diversity na inducibility, vuxokoxoko bya leaf-specific metabolite profile ya bola, naswona swikumeke leswaku ku dyiwa ka herbivores swinga endla leswaku metabolism yokarhi yi tirha. Leswi nga languteriwangiki, hi xiye leswaku, tani hileswi swi hlamuseriweke eka swikombiso swa thiyori ya mahungu leswi tirhisiweke, xiyimo xa metabolic lexi humelelaka xi na ku hlangana lokukulu endzhaku ka minhlaselo ya swihadyani leswimbirhi leswi dyaka byanyi (the night-fed generalist Sl) na mutivi wa Solanaceae Ms. Hambi leswi mahanyelo ya swona yo dya na ku hlangana ka swona swi hambaneke swinene. Xisunguri xa asidi ya mafurha-amino acid conjugate (FAC) eka OS (31). Hiku tirhisa herbivore OS ku tshungula swilondza swa standardized puncture, vutshunguri bya simulated herbivore na byona byi kombisile ntshamiseko lowu fanaka. Endlelo leri ra standardized ro tekelela nhlamulo ya swimilani eka nhlaselo wa herbivore ri herisa swilo leswi pfilunganyaka leswi vangiwaka hi ku cinca eka mahanyelo yo dya ya herbivores, leswi yisaka eka ti degree to hambana ta ku onhaka hi minkarhi yo hambana ( 34 ). FAC, leyi tivekaka tani hi xivangelo lexikulu xa OSM, yi hunguta JAS na tinhlamulo tin’wana ta ti hormone ta swimilani eka OSS1, kasi OSS1 yi hunguta madzana ya minkarhi ( 31 ). Hambiswiritano, OSS1 yi vange swiyimo leswi fanaka swa ku hlengeletiwa ka JA loko ku pimanisiwa na OSM. Khale swikombisiwile leswaku nhlamulo ya JA eka attenuated Nepenthes yiva na vuxiyaxiya swinene eka OSM, laha FAC yinga hlayisaka ntirho wa yona hambi loko yi cheriwa 1:1000 hi mati ( 44 ). Hikwalaho, loko ku pimanisiwa na OSM, hambi leswi FAC eka OSS1 yi nga ehansi swinene, yi ringanerile ku hlohlotela ku tumbuluka ka JA loku eneleke. Mindzavisiso ya khale yi kombisile leswaku ti protein tofana na porin (45) na oligosaccharides (46) tinga tirhisiwa tani hi swikombiso swa molecular ku hlohletela tinhlamulo ta nsirhelelo wa swimilani eka OSS1. Hambiswiritano, swa ha ri erivaleni loko ti elicitors leti eka OSS1 ti ri na vutihlamuleri eka ku hlengeletiwa ka JA leyi voniweke eka ndzavisiso wa sweswi.
Hambi leswi ku nga na tidyondzo ti nga ri tingani leti hlamuselaka ti differential metabolic fingerprints leti vangiwaka hi ku tirhisiwa ka herbivores yo hambana kumbe exogenous JA kumbe SA (salicylic acid) (47), a nga kona loyi a kavanyeteke ku kavanyeteka ka muxaka wa herbivore-specific eka network ya byanyi bya swimilani na vuyelo bya yona eka specific The overall impact of metabolism is systematically studied. vuxokoxoko bya munhu hi xiyexe. Nxopaxopo lowu wu tlhele wu tiyisisa leswaku vuhlanganisi bya le ndzeni bya network ya ti hormone na ti hormone tin’wana ta swimilani handle ka JAs swi vumba ku hlawuleka ka metabolic reorganization leyi vangiwaka hi herbivores. Ngopfungopfu, hi kumile leswaku ET leyi vangiwaka hi OSM a yi ri yikulu swinene ku tlula leyi vangiwaka hi OSS1. Mode leyi yi fambisana na nhundzu yo tala ya FAC eka OSM, leyi nga xiyimo lexi lavekaka na lexi eneleke xo hlohlotela ku buluka ka ET ( 48 ). Eka xiyimo xa ku tirhisana exikarhi ka swimilani na ti herbivores, ntirho wa signaling wa ET eka dynamics ya metabolite leyi kongomeke ya swimilani ya ha ri ya nkarhi na nkarhi naswona wu kongomisa ntsena eka ntlawa wun’we wa compound. Ku engetela kwalaho, tidyondzo to tala ti tirhise ku tirhisiwa ka exogenous ka ET kumbe ti precursors ta yona kumbe ti inhibitors to hambana ku dyondza ku lawuriwa ka ET, exikarhi ka swona ku tirhisiwa loku ka tikhemikhali ta exogenous ku ta humesa switandzhaku swo tala leswi nga kongomangiki. Hiku tiva ka hina, ndzavisiso lowu wuyimela ku kamberiwa kosungula lokukulu ka maendlelo ya xiave xa ET eka kutirhisiwa ka ET ku humelerisa naku vona swimilani swa transgenic leswi onhakeke ku fambisana na dynamics ya metabolome ya swimilani. Herbivore-specific ET induction yinga hetelela yi cinca cinca nhlamulo ya metabolome. Lexi xiyekaka swinene i transgenic manipulation ya ET biosynthesis (ACO) na perception (ETR1) genes leswi paluxeke herbivore-specific de novo accumulation ya phenolamides. Khale swi kombisiwile leswaku ET yi nga lulamisa kahle ku hlengeletiwa ka nicotine loku hlohloteriwaka hi JA hi ku lawula putrescine N-methyltransferase ( 49 ). Hambiswiritano, hi ku ya hi langutelo ra michini, a swi le rivaleni leswaku ET yi lulamisa njhani ku nghenisiwa ka phenamide. Ku engetela eka ntirho wa signal transduction wa ET, metabolic flux yinga tlhela yi shunted kuya eka S-adenosyl-1-methionine ku lawula vuvekisi eka polyaminophenol amides. S-adenosyl-1-methionine i ET na Xikarhi lexi tolovelekeke xa ndlela ya polyamine biosynthetic. Endlelo leri xikombiso xa ET xi lawulaka mpimo wa phenolamide ri lava dyondzo yo ya emahlweni.
Ku ringana nkarhi woleha, hikokwalaho ka nhlayo leyikulu ya ti metabolites to hlawuleka ta xivumbeko lexinga tivekiki, nyingiso lowukulu eka swiyenge swokarhi swa metabolic awu tsandzeka ku kambela hiku hetiseka ku cinca ka nkarhi wa ku hambana ka metabolic endzhaku ka ku tirhisana ka biological. Eka nkarhi wa sweswi, kuya hi nxopaxopo wa information theory, mbuyelo lowukulu wa MS/MS spectrum acquisition leyi seketeriweke eka ti metabolites letinga na xihlawuhlawu hileswaku ti herbivores leti dyaka kumbe ku tekelela ti herbivores ti ya emahlweni ti hunguta ku hambana ka metabolic hinkwayo ya metabolome ya matluka loko yirikarhi yi engetela degree ya yona ya specialization. Ku tlakuka loku ka xinkarhana ka metabolome specificity loku vangiwaka hi herbivores swi fambelanisiwa na ku tlakuka ka synergistic eka transcriptome specificity. Xivumbeko lexi hoxaka xandla swinene eka ku hlawuleka loku lokukulu ka metabolome (leyi nga na ntikelo wa le henhla wa Si) i metabolite yo hlawuleka leyi nga na ntirho wa herbivorous lowu a wu tiveka khale. Modeli leyi yi fambisana na ku vhumbha ka thiyori ya OD, kambe ku vhumbha ka MT loku fambelanaka na ku randomness ka metabolome reprogramming a ku fambisani. Hambiswiritano, datha leyi yi tlhela yi fambisana na ku vhumbha ka modele lowu pfanganisiweke (MT ya kahle swinene; Xifaniso 1B), hikuva ti metabolite tin’wana leti nga tivekiki leti nga na mintirho yo sirhelela leyi nga tiviwiki ti nga ha landzelela ku hangalasiwa ka Si loku nga hlelekangiki.
Xivumbeko lexi xiyekaka lexi yaka emahlweni xi khomiwa hi ndzavisiso lowu hileswaku kusuka eka xiyimo xa micro-evolution (ximilani xin’we na nhlayo ya fole) kuya eka xikalo lexikulu xa evolutionary (tinxaka ta fole leti fambelanaka swinene), swiyimo swohambana hambana swa nhlengeletano ya evolutionary swile ka “vusirheleri lebyinene” Kuna ku hambana lokukulu eka vuswikoti bya swimilani leswi dyaka swimilani. Moore na van’wana. (20) na Kessler na Kalske (1) hi ku tiyimela va ringanyete ku hundzula swiyimo swinharhu swa ntirho swa biodiversity leswi ekusunguleni swi hambanisiweke hi Whittaker (50) ku va ku cinca ka nkarhi loku vumbiwaka na loku hlohloteriweke ka ku hambana ka tikhemikhali; vatsari lava a va katsakanya Maendlelo ya nhlengeleto wa datha ya metabolome hi xikalo lexikulu na wona a ma boxi ndlela yo hlayela ku hambana ka metaboliki ku suka eka datha leyi. Eka dyondzo leyi, ku lulamisiwa lokuntsongo eka ku hlawuriwa ka ntirho ka Whittaker ku ta languta ku hambana ka α-metabolic tani hi ku hambana ka MS/MS spectra eka ximilani lexi nyikiweke, na ku hambana ka β-metabolic tani hi metabolism ya xisekelo ya intraspecific ya ntlawa wa vaaki Space, na γ-metabolic diversity swi ta va ku engeteriwa ka nxopaxopo wa tinxaka leti fanaka.
Xikombiso xa JA i xa nkoka eka tinhlamulo to hambana hambana ta metabolic ta herbivore. Hambiswiritano, ku na ku pfumaleka ka ku kamberiwa ka nhlayo loku tiyeke ka ku hoxa xandla ka vulawuri bya intraspecific bya JA biosynthesis eka ku hambana ka metabolome, naswona loko xikombiso xa JA xi ri ndhawu yo angarhela ya ku hambana ka metabolic loku hlohloteriwaka hi ntshikilelo eka xikalo xa le henhla xa macroevolutionary swa ha ri leswi nga twisisekiki. Hi xiye leswaku ntumbuluko wa herbivorous wa Nepenthes herbivorous wu hlohletela ku hlawuleka ka metabolome xikan’we naku hambana ka metabolome specialization endzeni ka nhlayo ya tinxaka ta Nicotiana na le xikarhi ka tinxaka ta Nicotiana leti fambelanaka swinene swi fambelanisiwa hindlela leyinene na JA signaling. Ku engetela kwalano, loko xikombiso xa JA xi onhakile, metabolic specificity leyi hlohleteriwaka hi genotype herbivore yin’we yita khanseriwa (Figure 3, C na E). Leswi ku cinca ka metabolic spectrum ya ti populations ta Nepenthes letinga hunguteka hi ntumbuluko swinga tala hi nhlayo, ku cinca eka metabolic β diversity na specificity eka nxopaxopo lowu swinga vangiwa ngopfu hiku hlohloteriwa lokukulu ka metabolite-rich compound categories. Titlilasi leti ta compound ti lawula xiphemu xa metabolome profile naswona ti yisa eka ku yelana lokunene na swikombiso swa JA.
Hikuva maendlelo ya biochemical ya muxaka wa fole lowu fambelanaka swinene na wona ya hambanile swinene, ti metabolites ti tiveka hiku kongoma eka xiphemu xa qualitative, hikokwalaho switala kuva na vuxopaxopi. Information theory’s processing ya captured metabolic profile yi paluxa leswaku herbivorous induction yi nyanyisa ku xaviselana exikarhi ka metabolic gamma diversity na specialization. Xikombiso xa JA xi tlanga xiave xa nkoka eka ku cinca loku. Ku tlakuka ka ku hlawuleka ka metabolome swi fambisana na ku vhumbha lokukulu ka OD naswona swi fambelanisiwa kahle na xikombiso xa JA, kasi xikombiso xa JA xi fambelanisiwa hi ndlela yo biha na ku hambana ka metabolic gamma. Ti models leti tikomba leswaku vuswikoti bya OD bya swimilani byi vekiwa ngopfu hi plasticity ya JA, kungava eka xikalo xa microevolutionary kumbe eka xikalo lexikulu xa evolutionary. Exogenous JA application experiments leyi rhendzelekaka JA biosynthesis defects yiya emahlweni yi paluxa leswaku tinxaka ta fole leti fambelanaka swinene tinga hambanisiwa kuva ti signal-responsive na signal-non-responsive species, kufana na ndlela ya tona ya JA na metabolome plasticity leyi hlohleteriwaka hi herbivores. Tinxaka letinga hlamuriki ta signal ati koti ku hlamula hikokwalaho ka ku tsandzeka ka tona ku humelerisa endogenous JA naswona hikokwalaho ti le hansi ka swipimelo swa physiological. Leswi swinga vangiwa hi ku cinca eka ti genes tin’wana ta nkoka eka JA signaling pathway (AOS na JAR4 eka N. crescens) ya. Mbuyelo lowu wu kombisa leswaku swivumbeko leswi swa macroevolutionary swa interspecies swinga hlohloteriwa ngopfu hiku cinca eka mavonelo ya ti hormone tale ndzeni xikan’we naku hlamula.
Ku engetela eka ku tirhisana exikarhi ka swimilani na ti herbivores, ku lavisisa ku hambana ka metabolic swi fambelana na nhluvuko hinkwawo wa nkoka wa theoretical eka ndzavisiso wa biological adaptation eka mbango xikan’we naku cinca ka swihlawulekisi swa phenotypic leswi rharhanganeke. Hi ku tlakuka ka nhlayo ya datha leyi kumiweke hi switirhisiwa swa manguva lawa swa MS, ku kamberiwa ka xiringanyeto eka ku hambana ka metaboliki sweswi ku nga tlula ku hambana ka metabolite ya munhu hi xiyexe/xiyenge na ku endla nxopaxopo wa misava hinkwayo ku paluxa swivumbeko leswi nga languteriwangiki. Eka endlelo ra nxopaxopo lowukulu, xifaniso xa nkoka i mianakanyo yo ehleketa mimepe leyi nga na nhlamuselo leyi nga tirhisiwaka ku lavisisa datha. Hikokwalaho, mbuyelo wa nkoka wa nhlanganelo wa sweswi wa metabolomics ya MS/MS leyi nga na xihlawuhlawu na thiyori ya mahungu hileswaku yi nyika metric yo olova leyi nga tirhisiwaka ku aka mimepe ku pfula ku hambana ka metabolic eka swikalo swo hambana swa taxonomic. I xilaveko xa xisekelo xa endlelo leri. Dyondzo ya micro/macro evolution na ikholoji ya vaaki.
Eka xiyimo xa macro-evolutionary, xisekelo xa thiyori ya plant-insect co-evolution ya Ehrlich na Raven (51) i ku vhumbha leswaku ku hambana ka interspecies metabolic diversity i xivangelo xa ku hambana ka tinxaka ta swimilani. Kambe eka malembe ya makume ntlhanu ku sukela loko ku kandziyisiwile ntirho lowu wa seminal, xiringanyeto lexi a xi kamberiwanga ngopfu (52). Leswi swivangiwa ngopfu hi swihlawulekisi swa phylogenetic swa swihlawulekisi swa metabolic leswi ringanisiwaka eka tinxaka ta swimilani swa mpfhuka woleha. Rarity yinga tirhisiwa ku anchor maendlelo ya nxopaxopo wa swikongomelo. Ntirho wa sweswi wa MS/MS lowu phurosesiweke hi thiyori ya mahungu wu pima ku fana ka xivumbeko xa MS/MS xa ti metabolite leti nga tiviwiki (handle ka ku hlawuriwa ka metabolite ka le mahlweni) naswona wu hundzula ti MS/MS leti ti va sete ya MS/MS, xisweswo eka metabolism ya xiphurofexinali Timodelo leti ta macro-evolutionary ti pimanisiwa eka xikalo xa ku hlawula. Swikombiso swo olova swa tinhlayo. Endlelo leri rifana na nxopaxopo wa phylogenetic, lowu nga tirhisaka ku ringanisiwa ka ntlhandlamano ku pima mpimo wa ku hambana kumbe ku cinca ka swimunhuhatwa handle ko vhumbha ka ha ri emahlweni.
Eka xiyimo xa biochemical, screening hypothesis ya Firn na Jones (53) yi kombisa leswaku ku hambana ka metabolic ku hlayisiwa eka swiyimo swo hambana ku nyika switirhisiwa swo ka swi nga tirhisiwanga ku tirhisa migingiriko ya biological ya ti metabolites leti khale a ti nga fambelani kumbe leti nga siviwa. Maendlelo ya thiyori ya mahungu ya nyika rimba leri eka rona ku cinca loku ka nhluvukiso loku kongomisiweke eka metabolite loku humelelaka hi nkarhi wa ku hlawuleka ka metabolite ku nga pimiwa tanihi xiphemu xa endlelo ro kambela nhluvukiso leri ringanyetiweke: ku cinca loku tirhaka hi biology ku suka eka ku hlawuleka ka le hansi ku ya eka ku hlawuleka ka le henhla Timetabolite leti siveleriweke ta ndhawu leyi nyikiweke.
Hinkwaswo hinkwaswo, emasikwini yosungula ya molecular biology, tithiyori ta nkoka ta nsirhelelo wa swimilani ti endliwile, naswona maendlelo lawa ya fambiwaka hi deductive hypothesis hi xitalo ya tekiwa tani hi wona ntsena ndlela ya nhluvuko wa sayense. Leswi swivangiwa ngopfu hi swipimelo swa xithekiniki swaku pima metabolome hinkwayo. Hambi leswi tindlela leti fambiwaka hi xiringanyeto ti pfunaka ngopfu eku hlawuleni ka tindlela tin’wana ta xivangelo, vuswikoti bya tona byo yisa emahlweni ku twisisa ka hina ka tinetiweki ta tikhemikhali ta vutomi byi ringaniseriwile ku tlula tindlela ta xibalo leti kumekaka sweswi eka sayense ya manguva lawa leyi tirhisaka datha yo tala. Hikwalaho, tithiyori leti nga vhumbhiwangiki ti le kule swinene na mpimo wa datha leyi nga kona, hikwalaho xirhendzevutani xa fomula/xikambelo xa xikambelo xa nhluvuko eka xiyenge xa ​​ndzavisiso a xi nge herisiwi (4). Hi vona ka ha ri emahlweni leswaku ntirho wa xibalo wa metabolomics lowu nghenisiweke laha wu nga tlhela wu pfuxa ku tsakela eka timhaka ta sweswinyana (ndlela) na to hetelela (ha yini) ta ku hambana ka metabolic, naswona wu hoxa xandla eka nguva leyintshwa ya sayense ya datha leyi kongomisiweke hi thiyori. Nguva leyi yi tlhele yi kambisisa mianakanyo ya nkoka leyi hlohloteleke tinxaka leti hundzeke.
Ku phameriwa ka herbivore hiku kongoma swiendliwa hiku kurisa second instar larva kumbe Sl larva eka tluka rin’we ra Pale-colored pitcher plant ya ximilani xin’we xa rose blooming, laha kungana 10 wa ti plant replicates hi ximilani. Ti larvae ta switsotswana ti clamped hi ti clamps, naswona tissue ya matluka leyi saleke yi hlengeletiwile endzhaku ka 24 na 72 wa tiawara endzhaku ka ku tluleriwa naswona yi hatlisa yi titimela, naswona ti metabolites ti humesiwile.
Tekelela vutshunguri bya herbivorous hi ndlela leyi fambisanaka swinene. Ndlela iku tirhisa mavhilwa ya fabric pattern ku tlhava tilayini tinharhu ta mitwa eka tlhelo rin’wana na rin’wana ra midrib ya matluka yanharhu lawa ya andlariweke hiku hetiseka ya ximilani hinkarhi wa xiteji xa kukula ka fabric garland, naswona hiku hatlisa u chela 1:5 Diluted Ms. Kumbe tirhisa tintiho letinga na magilavu ​​ku nghenisa S1 OS eka xilondza lexi tlhaviweke. Ntshovela naku lulamisa tluka tani hileswi swinga hlamuseriwa laha henhla. Tirhisa ndlela leyi hlamuseriweke khale ku humesa ti metabolites to sungula na tihomoni ta swimilani (54).
Eka switirhisiwa swa exogenous JA, matluka yanharhu ya petiole ya xin’wana na xin’wana xa swimilani swa tsevu swa rose blooming swa muxaka wun’wana na wun’wana ya tshunguriwa hi 20μl ya lanolin paste leyingana 150μg MeJA (Lan + MeJA), na 20μl ya lanolin plus wound treatment (Lan + W), kutani ya tirhisa 20μl ya pure lanolin tani hi vulawuri. Matluka ya tshoveriwile endzhaku ka 72 wa tiawara endzhaku ka vutshunguri, ya snap-frozen eka liquid nitrogen, naswona ya hlayisiwa eka -80°C kufikela loko ya tirhisiwa.
Milayeni ya mune ya JA na ET transgenic, ku nga irAOC (36), irCOI1 (55), irACO na sETR1 (48), yi tiviwile eka ntlawa wa hina wa ndzavisiso. irAOC yi kombisile swinene ku hunguteka ka ti levele ta JA na JA-Ile, kasi irCOI1 a yi nga ri na vuxiyaxiya eka JAs. Loko ku pimanisiwa na EV, ku hlengeletiwa ka JA-Ile ku tlakukile. Hilaha ku fanaka, irACO yi ta hunguta vuhumelerisi bya ET, naswona loko yi pimanisiwa na EV, sETR1, leyi nga riki na vuxiyaxiya eka ET, yi ta engetela vuhumelerisi bya ET.
Photoacoustic laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) yi tirhisiwa ku endla ku pima ka ET hi ndlela leyi nga ngheneriki. Hiku hatlisa endzhaku ka vutshunguri, hafu ya matluka ya tsemiwile kutani ya hundziseriwa eka 4-ml sealed glass vial, naswona ndzhawu ya nhloko yi pfumeleriwile ku hlengeletana endzhaku ka 5 wa tiawara. Hi nkarhi wa ku pima, vial yin’wana na yin’wana yi hlantswiwile hi stream ya 2 litres/hour ya moya wo basa kuringana 8 wa timinete, lowu khale a wu hundza eka catalyst leyi nyikiweke hi Sensor Sense ku susa CO2 na mati.
Data ya microarray yi kandziyisiwile ekusunguleni eka (35) naswona yi hlayisiwile eka National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (nomboro ya mfikelelo GSE30287). Data leyi fambelanaka na matluka lawa ya vangiwaka hi vutshunguri bya W + OSMs xikan’we na vulawuri lebyi nga onhakiki yi humesiwile eka ndzavisiso lowu. Ntshikelelo wa raw i log2. Loko ku nga si endliwa nxopaxopo wa tinhlayo, masungulo ya hundzuriwile no normaliseriwa eka phesente ya wona ya vu 75 hi ku tirhisa phakeji ya software ya R.
Data yosungula ya RNA sequencing (RNA-seq) ya tinxaka ta Nicotiana yi tekiwile kusuka eka NCBI Short Reading Archives (SRA), nomboro ya phurojeke i PRJNA301787, leyi vikiweke hi Zhou et al. (39) ivi u ya emahlweni tanihilaha swi hlamuseriweke hakona eka (56). Data ya raw leyi lulamisiweke hi W + W, W + OSM na W + OSS1 leyi fambelanaka na tinxaka ta Nicotiana yi hlawuriwile kuva yi kamberiwa eka ndzavisiso lowu, naswona yi lulamisiwile hindlela leyi landzelaka: Xosungula, ti raw RNA-seq readings ti hundzuriwile eka xivumbeko xa FASTQ. HISAT2 yi hundzuluxa FASTQ ku va SAM, naswona SAMtools yi hundzula tifayela ta SAM ti va tifayela ta BAM leti hleriweke. StringTie yi tirhisiwa ku hlayela ku paluxiwa ka tijini, naswona ndlela ya yona yo paluxiwa hileswaku ku na swiphemu eka gidi ra swiphemu swa xisekelo eka miliyoni ya swiphemu swa matsalelo leswi landzelelanaka.
Kholomo ya Acclaim chromatographic (150 mm x 2.1 mm; vukulu bya swiphemuphemu 2.2μm) leyi tirhisiweke eka nxopaxopo na kholomo ya 4 mm x 4 mm ya murindzi yi vumbiwa hi swilo leswi fanaka. Gradient ya binary leyi landzelaka yitirhisiwa eka sisiteme ya Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): 0 kuya eka 0.5 wa timinete, isocratic 90% A [mati ya deionized, 0.1% (v/v) acetonitrile na 0.05% formic acid], 10% B (Acetonitrile na 0.05% formic acid); 0.5 kuya eka 23.5 wa timinete, xiphemu xa gradient i 10% A na 90% B, hiku landzelelana; 23.5 kuya eka 25 wa timinete, isocratic 10% A na 90% B. Nhlayo ya ku khuluka i 400μl/min. Eka minxopaxopo hinkwayo ya MS, chela column eluent eka quadrupole na time-of-flight (qTOF) analyzer leyi hlomisiweke hi xihlovo xa electrospray lexi tirhaka eka positive ionization mode (capillary voltage, 4500 V; capillary outlet 130 V ; Mahiselo yo omisa 200°C; ku khuluka ka moya wo omisa 10 liters/min).
Endla nxopaxopo wa swiphemu swa MS / MS (laha ku nga ta vuriwa MS / MS) lowu nga riki wa nkoka kumbe lowu nga hambanisiwiki na datha ku kuma vuxokoxoko bya xivumbeko mayelana na phurofayili ya metaboliki leyi voniwaka hinkwayo. Mhaka ya ndlela ya MS/MS leyi nga hlawuriki yi titshege hi mhaka ya leswaku quadrupole yina fasitere lerikulu swinene ro hambanyisa masa [hikwalaho, teka swikombiso hinkwaswo swa mass-to-charge ratio (m/z) tani hi swiphemu]. Hi xivangelo lexi, hikuva xitirhisiwa xa Impact II a xi tsandzeka ku tumbuluxa CE tilt, minxopaxopo yo hlayanyana leyi tiyimeleke yi endliwile hi ku tirhisa mimpimo leyi engetelekeke ya collision-induced dissociation collision energy (CE). Hi ku komisa, rhanga hi ku kambisisa xikombiso hi UHPLC-electrospray ionization/qTOF-MS hi ku tirhisa single mass spectrometry mode (swiyimo swa le hansi swa ku avana leswi endliweke hi ku avana ka le ndzeni ka xihlovo), ku skena ku suka eka m/z 50 ku ya eka 1500 eka frequency yo phindha-phindha ya 5 Hz. Tirhisa nitrogen tani hi gasi ya ku tlumbana eka nxopaxopo wa MS/MS, naswona endla mimpimo leyi tiyimeleke eka ti voltage ta mune to hambana leti landzelaka ta dissociation leti hlohloteriwaka hi ku tlumbana: 20, 30, 40, na 50 eV. Eka endlelo hinkwaro ro pima, quadrupole yina fasitere lerikulu swinene ra mass isolation, kusuka eka m/z 50 kuya eka 1500. Loko front body m/z na isolation width experiment yi vekiwile eka 200, mass range yi tirhisiwa hiku tisungulela hi software yo tirha ya xitirhisiwa na 0 Da. Scan ku kuma swiphemu swa mass tani hi le ka single mass mode. Tirhisa sodium formate (50 ml isopropanol, 200 μl formic acid na 1 ml ya 1M NaOH aqueous solution) eka ku kalibhara ka masa. Hi ku tirhisa algorithm ya Bruker ya high-precision calibration, fayili ya data yi kalibiwa endzhaku ko fambisa spectrum ya xikarhi eka nkarhi lowu nyikiweke. Tirhisa ntirho wo rhumela ehandle wa sofwere ya Data Analysis v4.0 (Brook Dalton, Bremen, Germany) ku hundzuluxa tifayela ta datha leti nga si swekiwaka eka xivumbeko xa NetCDF. Seti ya datha ya MS/MS yi hlayisiwile eka database ya metabolomics leyi pfulekeke MetaboLights (www.ebi.ac.uk) na nomboro ya ku nghena. MTBLS1471.
Nhlengeletano ya MS/MS yi nga endliwa hi ku tirhisa nxopaxopo wa ku yelana exikarhi ka swikombiso swa khwalithi ya MS1 na MS/MS swa eneji ya le hansi na ya le henhla ya ku tlumbana na milawu leyintshwa leyi tirhisiweke. Tsalwa ra R ri tirhisiwa ku lemuka nxopaxopo wa ku yelana ka ku hangalasiwa ka xiletelo xa xiendliwa, naswona tsalwa ra C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) ri tirhisiwa ku tirhisa milawu.
Ku kota ku hunguta swihoxo swa mavunwa swa kahle leswi vangiwaka hi mpfumawulo wa le ndzhaku na ku yelana ka mavunwa loku vangiwaka hi ku kuma swihlawulekisi swo karhi swa m/z eka swikombiso swi nga ri swingani ntsena, hi tirhisa ntirho wa “filled peak” wa R package XCMS (for background noise correction) Yi fanele ku tirhisiwa ku siva “NA” (undetected peak) intensity. Loko ntirho wa nhlohlorhi yo tata wu tirhisiwa, ka ha ri na mimpimo yo tala ya “0′′ ya matimba eka sete ya datha leyi nga ta khumba xibalo xa ku yelana.Kutani, hi pimanisa mbuyelo wa vukorhokeri bya datha lowu kumiweke loko ntirho wa nhlohlorhi leyi tatiweke wu tirhisiwa na loko ntirho wa nhlohlorhi leyi tatiweke wu nga tirhisiwi, naswona hi hlayela ntikelo wa mpfumawulo wa le ndzhaku hi ku ya hi ntikelo lowu ringanyetiweke wa xikarhi lowu lulamisiweke, ivi hi siva mimpimo leyi ya matimba ya 0 hi ntikelo wa le ndzhaku lowu hlayiweke.Hi tlhela hi pimanisa a yi tekela ntsena swivumbeko leswi matimba ya swona a ma tlula mpimo wa le ndzhaku kanharhu naswona a a swi teka tanihi “tinhlohlorhi ta ntiyiso.” Eka swibalo swa PCC, ku tekeriwa enhlokweni ntsena swikombiso swa m/z swa xikombiso xa xikombiso (MS1) na tisethi ta datha ya swiphemu leswi nga na kwalomu ka tinhlohlorhi ta ntiyiso ta tsevu.
Loko matimba ya xivumbeko xa khwalithi ya precursor eka xikombiso hinkwaxo ya fambelanisiwa swinene na ku hunguteka ka matimba ya xivumbeko xa khwalithi lexi fanaka lexi nga ehansi ka eneji ya le hansi kumbe ya le henhla ya ku tlumbana, naswona xivumbeko lexi xi nga lebuliwangiki tanihi nhlohlorhi ya isotope hi CAMERA, xi nga hlamuseriwa ku ya emahlweni. Kutani, hi ku hlayela mimpatswa hinkwayo leyi kotekaka ya precursor-product eka 3 s (fasitere ra nkarhi wo hlayisa leri ringanyetiweke ra ku hlayisiwa ka nhlohlorhi), nxopaxopo wa ku yelana wu endliwa. Ntsena loko nhlayo ya m/z yi ri ehansi ka ntikelo wa precursor naswona ku avana ka MS/MS ku humelela eka ndhawu yin’we ya xikombiso eka sete ya datha na precursor leyi yi humaka eka yona, yi tekiwa tanihi xiphemu.
Hi ku ya hi milawu leyi yimbirhi yo olova, hi susa swiphemu leswi boxiweke leswi nga na mimpimo ya m/z leyikulu ku tlula m/z ya xiletelo lexi hlawuriweke, naswona hi ku ya hi ndhawu ya xikombiso laha xiletelo xi humelelaka kona na xiphemu lexi boxiweke. Swi tlhela swi koteka ku hlawula swihlawulekisi swa khwalithi leswi tumbuluxiweke hi swiphemu swo tala swa le ndzeni ka xihlovo leswi tumbuluxiweke eka movha wa MS1 tanihi swihlawulekisi swa nkandziyiso, hi ndlela yoleyo ku tumbuluxiwa swihlanganisi swa MS/MS leswi nga lavekiki. Ku kota ku hunguta data redundancy leyi, loko ku fana ka NDP ka ti spectra ku tlula 0.6, naswona ti wela eka chromatogram “pcgroup” leyi hlamuseriweke hi CAMERA, hi ta ti hlanganisa. Eku heteleleni, hi hlanganisa mimbuyelo hinkwayo ya mune ya CE leyi fambelanaka na precursor na swiphemu eka spectrum yo hetelela ya deconvoluted composite hi ku hlawula nhlohlorhi ya matimba ya le henhla swinene exikarhi ka tinhlohlorhi hinkwato ta nkandziyiso leti nga na ntikelo wa m/z lowu fanaka eka matimba yo hambana ya ku tlumbana. Magoza ya vukorhokeri lawa ya landzelaka ya sekeriwe eka mhaka ya spectrum leyi hlanganisiweke naswona ya tekela enhlokweni swiyimo swohambana hambana swa CE leswi lavekaka ku kurisa ku koteka ka ku avana, hikuva swiphemu swin’wana swinga voniwa ntsena ehansi ka matimba yokarhi ya ku tlumbana.
RDPI (30) yi tirhisiwile ku hlayela inducibility ya metabolic profile. Ku hambana ka metabolic spectrum (Hj index) swi huma eka vunyingi bya ti MS/MS precursors hiku tirhisa Shannon entropy ya MS/MS frequency distribution hiku tirhisa equation leyi landzelaka leyi hlamuseriweke hi Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) laha Pij yi fambelanaka na nhlayo ya xiringaniso ya i-th MS/MS eka xikombiso xa j-th (j = 1, 2,..., m) (i = 1, 2, ..., m) t).
Metabolic specificity (Si index) yi hlamuseriwa tani hi vutivi bya ku paluxiwa ka MS/MS leyi nyikiweke hi mayelana na nhlayo exikarhi ka swikombiso leswi tekeriwaka enhlokweni. Ku hlawuleka ka MS/MS ku hlayeriwa tanihi Si = 1t (∑j = 1tPijPilog2PijPi) .
Tirhisa fomula leyi landzelaka ku pima metabolome-specific δj index ya xikombiso xin’wana na xin’wana xa j, na avhareji ya MS/MS specificity δj = ∑i = 1mPijSi
MS/MS spectra yi ringanisiwile hi vambirhi-mbirhi, naswona ku fana ku hlayeriwa hi ku ya hi swikoweto leswimbirhi. Xo sungula, hi ku tirhisa NDP ya ntolovelo (leyi tlhelaka yi tiviwa tanihi ndlela ya ku yelana ka cosine), tirhisa xiringaniso lexi landzelaka ku kuma ku fana ka xiphemu exikarhi ka spektra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 laha S1 na S2 Hi ku fambisana, eka spektramu 1 na spektramu 2, xikan’we na WS1, i na WS2, i yi yimela ntiko lowu simekiweke eka matimba ya nhlohlorhi leswaku ku hambana ka nhlohlorhi ya i-th leyi tolovelekeke exikarhi ka ti spectra letimbirhi ku le hansi ka 0.01 Da. Ndzilo wu hlayeriwa hi ndlela leyi landzelaka: W = [nhlohlorhi ya matimba] m [khwaliti] n, m = 0.5, n = 2, tanihilaha swi ringanyetiweke hakona hi MassBank.
Ndlela ya vumbirhi yo nyika swikoweto yi tirhisiwile, leyi katsaka ku xopaxopa NL leyi avelaniwa exikarhi ka MS/MS. Ku fikelela leswi, hi tirhise minxaxamelo ya 52 ya NL leyi talaka ku hlangana na yona hi nkarhi wa endlelo ra ku avana ka MS hi tandem, na NL leyi kongomeke swinene (fayili ya data S1) leyi nga tshama yi hlamuseriwa eka spectrum ya MS/MS ya ti metabolites ta vumbirhi ta muxaka wa Nepenthes lowu tsaneke ( 9, 26). Endla vector ya binary ya 1 na 0 eka MS/MS yin’wana na yin’wana, leyi fambelanaka na sweswi na leyi nga riki kona ya NL yin’wana hi ku landzelelana. Hi ku ya hi ku fana ka mpfhuka wa Euclidean, xikoro xa ku fana ka NL xi hlayeriwa eka mpatswa wun’wana na wun’wana wa ti vector ta binary NL.
Ku endla ku hlengeletiwa ka swiyenge swimbirhi, hi tirhise phakeji ya R DiffCoEx, leyi sekeriweke eka ku engeteriwa ka Weighted Gene Co-expression Analysis (WGCNA). Hi ku tirhisa matirisisi yo nyika swikoweto ya NDP na NL ya swiphemu swa MS/MS, hi tirhise DiffCoEx ku hlayela matiriki ya ku yelana yo pimanisa. Ku hlengeletiwa ka binary ku endliwa hi ku veka parameter ya “cutreeDynamic” eka method = “hybrid”, cutHeight = 0.9999, deepSplit = T, na minClusterSize = 10. Khodi ya xihlovo xa R ya DiffCoEx yi dawunilodiwile ku suka eka fayili yo engetela ya 1 hi Tesson et al. (57) naswona; Phakeji ya sofwere ya R WGCNA leyi lavekaka yi nga kumeka eka https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Leswaku hi endla nxopaxopo wa netiweke ya timolekhuli ta MS/MS, hi hlayele vuhlanganisi bya spectral lebyi hlanganisiweke hi swiphemuphemu lebyi simekiweke eka tinxaka ta ku fana ta NDP na NL, naswona hi tirhise sofwere ya Cytoscape ku vona hi mahlo ya mianakanyo topology ya netiweke hi ku tirhisa layout ya organic eka xitirhisiwa xa ku engetela xa algorithm ya layout ya CyFilescape yFiles.
Tirhisa R version 3.0.1 ku endla nxopaxopo wa tinhlayo eka datha. Nkoka wa tinhlayo wu kamberiwile hi ku tirhisa nxopaxopo wa tindlela timbirhi wa ku hambana (ANOVA), ku landzela xikambelo xa le ndzhaku xa Tukey’s honestly significant difference (HSD). Ku kota ku xopaxopa ku hambana exikarhi ka vutshunguri bya herbivorous na vulawuri, ku hangalasiwa ka misisi yimbirhi ya mintlawa yimbirhi ya swikombiso leswingana ku hambana loku fanaka swikamberiwile hiku tirhisa Student’s t test.
Ku kuma switirhisiwa swo engetela swa xihloko lexi, hi kombela u vona http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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