Saray nanduruman teorya nipaakar ed depensa na tanaman so mangiiter na importantin teoretikal a giya parad pangipaliwawa ed saray padron na espisyal a metabolismo na tanaman, balet saray manunan prediksion da et kaukolan nin nasubok. Dia, inusar mi so unbiased tandem mass spectrometry (MS/MS) analysis pian sistematikon nasukimat so metabolome na saray tabako ya attenuated strains manlapud indibidual iran tanaman ya anggad populasyon tan maapit a klase na tanaman, tan inproseso mi so baleg a bilang na mass spectrometric feature theories base ed compound spectra ed impormasyon. Framework pian nasubok iray manunan prediksion na optimal defense (OD) tan moving target (MT) iran teorya. Say impormasyon ya kabiangan na metabolomics na tanaman et mipara ed teorya na OD, balet ta onkontra ed manunan prediksion na teorya na MT nipaakar ed metabolomics dynamics ya sengegan na saray herbivore. Manlapud micro anggad macro ya sukat na ebolusyon, say jasmonate signal so abitla bilang manunan determinante na OD, bangta say ethylene signal so angiter na fine-tuning na herbivore-specific ya ebat ya in-annotate na MS/MS molecular network.
Saray espisyal a metabolites ya walaan na nanduruman istruktura so manunan mibibiang ed iyaadapta na tanaman ed kaliberliber, lalo la ed pangidepensa ed saray kalaban (1). Say makapakelkelaw ya pandurumaan na espisyal a metabolismo ya naromog ed tanaman so amakiwas ed pigaran dekada na aralem a panagsukimat ed dakel ya aspekto na ekolohikon kimey to, tan akaporma na andukey a listaan ​​na saray teorya nipaakar ed depensa na tanaman, salanti, say ebolusyonaryo tan ekolohikon iyaaligwas na pidedeneng na tanaman tan insekto. Say empirical research et mangiiter na importantin giya (2). Balet, sarayan teorya nipaakar ed depensa na tanaman et ag-ontutumbok ed normatibon dalan na hypothetical deductive reasoning, ya saray manunan prediksion et walad parehon lebel na panagsukimat (3) tan nasubok ed eksperimento pian napaaligwas so ontumbok a siklo na teorya (4). Saray limitasyon ed teknikal et mamapaarap ed panagkolekta na datos ed espisipikon kategorya na metaboliko tan ag mangilaktip ed komprehensibon panagsukimat ed saray espisyal a metabolite, kanian naamper so panangikompara ed baetan na saray kategorya ya makanakana parad teorya (5). Say kakulangan na komprehensibon datos nipaakar ed metabolomics tan say parehon paraan pian nakompara so proseso na panagproseso na metabolic space ed baetan na nanduruman grupo na tanaman so mangaamper ed inkatatken na siensia na lawak.
Saray sankabaloan ya iyaaligwas ed lawak na tandem mass spectrometry (MS/MS) metabolomics et nayarin komprehensibon mangibiig ed saray pananguman ed metabolismo ed loob tan baetan na saray klase na sakey a klase na sistema, tan nayarin ikombina ed saray paraan na panagkuenta pian nakalkula so pampareho na istruktura na sarayan komplikadon laok. Say akaunan pikakabat ed chemistry (5). Say kombinasyon na saray maaligwas a teknolohiya ed panagsukimat tan panagkuenta so mangiiter na nakaukolan a balangkas parad andukey a panagsukimat ed dakel a prediksion ya ginawa na saray teorya na ekolohiya tan ebolusyon nipaakar ed pandurumaan na metabolismo. Si Shannon (6) so angiparungtal na teorya na impormasyon ed primeron pankanawnawa diad seminal ya artikulo to nen 1948, ya angiletneg na pundasyon parad mathematical analysis na impormasyon, ya inusar ed dakel a lawak nilikud ed orihinal ya aplikasyon to. Diad genomics, say teorya na impormasyon et matalonan inyaplika pian nabilang so konserbatibon impormasyon (7). Diad panagsukimat ed transcriptomics, say teorya na impormasyon et manguusisa ed amin ya pananguman ed transcriptome (8). Diad akaunan research, inyaplika mi so statistical framework na information theory ed metabolomics pian nideskribe so metabolic expertise na tissue level ed tanaman (9). Dia, ikombina mi so MS/MS-based workflow tan say statistical framework na teorya na impormasyon, ya nabibitar ed pandurumaan na metabolismo ed kaslakan a kuarta, pian ikompara iray manunan prediksion na teorya na depensa na tanaman na metabolome ya sengegan na saray herbivore.
Saray teoretikal a balangkas na depensa na tanaman et kaslakan ya mankakasakey tan nayarin naapag ed duaran kategorya: saramay mansasagpot ya mangipaliwawa ed inkaapag-apag na saray metabolite ya espisipiko ed tanaman base ed saray kimey na depensa, a singa say teorya na optimal defense (OD) (10), moving target (MT) (11) ) tan itsura (12), bangta say arum et manaanap na eksplinasyon no panon ya makaapekto so pakawalaan na rekurso say idakel na tanaman tan say idakel na saray espisyal ya metabolite, a singa say carbon: nutrient balance hypothesis (13), growth rate hypothesis (14 ), tan say growth tan differentiation equilibrium hypothesis (15). Say duaran set na teorya et walad nanduruman lebel na panagsukimat (4). Balet, duaran teorya ya mangilalanor ed saray depensan kimey ed lebel na kimey so mangokontrol ed tongtongan nipaakar ed saray depensa na tanaman ya constitutive tan inducible: say teorya na OD, ya mangipapasen ya saray tanaman et manpuonan ed saray mablin depensa ra ed kemikal no nakaukolan labat, alimbawa, no naakan iratan. say MT hypothesis et mangiproproponin anggapoy axis na direksion na pananguman na metabolite, balet say metabolite et manguuman ya ag-inisip, kanian walay posibilidad ya Naamper so metabolic ya “motion target” ya onataki ed saray herbivore. Diad arum a salita, sarayan duaran teorya so manggagawa na mankontraan iran prediksion nipaakar ed metabolic remodeling ya nagagawa kayari na panangataki na saray herbivore: say siglaotan na unidirectional ya panagtipon na metabolites ya walay defensive function (OD) tan saray non-directed metabolic changes (MT) (11 ).
Say OD tan MT ya hypotheses et aglabat mangilalanor ed saray pananguman ed metabolome, no ag ingen pati saray ekolohikal tan ebolusyonaryon pansumpalan na idakel na sarayan metabolite, a singa saray gastos tan benepisyo na sarayan metaboliko ed sakey ya espisipikon kaliberliber (16). Anggaman parehon bibidbiren na sarayan hypothesis so depensa na saray espisyal ya metabolites, ya nayarin mabmabli odino andi, say manunan prediksion ya mangibiig ed saray hypothesis na OD tan MT et walad direksion na saray pananguman ed metabolismo. Say prediksion na OD theory so akaawat na sankarakelan ya imano ed eksperimento anggad natan. Kaiba ed sarayan test so panaral ed direkta odino agdirektan depensa na nanduruman tisyu na espisipikon compound ed greenhouse tan natural iran kondisyon, ontan met ed saray pananguman ed yugtan na itatakken na tanaman (17-19). Balet, anggad natan, lapud anggapoy workflow tan statistical framework parad global a komprehensibon panagsukimat ed metabolic diversity na antokaman ya organismo, say manunan pandumaan na prediksion ed baetan na duaran teorya (salanti, say direksion na metabolic changes) et kaukolan nin nasubok. Dia, mangiiter kami na ontan ya panagsukimat.
Sakey ed saray sankaimportantian a katangian na saray metabolite ya espisipiko ed tanaman et say alablabas a pandurumaan na istruktura ra ed amin a lebel manlapud saksakey a tanaman, populasyon anggad mipadparan klase (20). Dakel a pananguman ed karakel na saray espisyal a metabolite so nanengneng ed sukat na populasyon, bangbalet saray mabiskeg a pandumaan ed kalidad so kaslakan a mansiansia ed lebel na klase (20). Kanian, say pandurumaan na metabolismo na tanaman so manunan aspekto na pandurumaan na kimey, ya mangipapatnag na pakayarin makaadapta ed nanduruman pasen, lalo la ed saramay pasen ya walaan na nanduruman posibilidad na panlusob na saray espisyal ya insekto tan kaslakan ya herbivore (21). Manlapu nen Fraenkel (22) ya artikulo to nipaakar ed saray rason ed kiwawala na saray metabolite ya espisipiko ed tanaman, say pidedeneng ed nanduruman insekto so ipapasen ya importantin presyon na panagpili, tan sarayan pidedeneng so papanisiaan ya amawala na tanaman legan na ebolusyon. Metabolic pathway (23). Say pandumaan na saray species ed pandurumaan na saray espisyal ya metabolites et nayarin mangipatnag met na physiological balance ya misiglaotan ed constitutive tan inducible ya depensa na tanaman sumpad saray estratehiya na herbivorous, lapud saray duaran species et mabetbet ya negatibo so koneksion da ed balang sakey (24). Anggaman makagunggona so pamasiansia na maong a depensa ed amin a panaon, saray nipanpanaon a pananguman ed metabolismo a misiglaotan ed depensa so mangiter na malinew iran gunggona diad pangiter na pankanawnawa ed saray tanaman a mangiter na makanakanan kayamanan ed arum niran pinansyal a pinansyal (19, 24), tan napaliisan so pankaukolan na simbiosis. Collateral damage (25). Niarum ni, sarayan pananguman na saray espisyal ya metabolites ya sengegan na saray insekto ya mankakakan na tanaman et nayarin manresulta ed makaderal ya inkaapag-apag ed populasyon (26), tan nayarin mangipatnag na direktan panagbasa na baleg iran natural ya pananguman ed signal na jasmonic acid (JA), ya nayarin mansiansia ed populasyon. Say atagey tan abeban JA signals et pansasaliwan na depensa sumpad saray herbivore tan say kompetisyon ed espisipikon klase na ayayep (27). Niarum ni, saray espisyal a metabolite biosynthetic pathways et maples ya naandi tan nauman legan na ebolusyon, ya manresulta ed patchy metabolic distribution ed limog na maapit a klase na ayep (28). Sarayan polymorphism et maples ya naestablisa bilang ebat ed manguuman iran pattern na herbivore (29), ya mankabaliksan ya say panguuman na komunidad na herbivore et manunan sengegan na metabolic heterogeneity.
Dia, espisipikon nasolbar mi iray onggendan a problema. (I) Panon ya uumanen na insekto ya mangan na tanaman so metabolome na tanaman? (Ii) Anto ray manunan impormasyon ya kabiangan na metabolic plasticity ya nayarin na-quantify pian nasubok iray prediksion na long-term defense theory? (Iii) No kasin i-reprogram so metabolome na tanaman ed paraan ya nikadkaduma ed manag-atake, no ontan, antoy papel na hormone na tanaman ed pangiparaan na espisipikon metabolikon ebat, tan dinan iran metabolite so makatulong ed inka-espesipiko na depensa na species? (Iv) Lapud saray prediksion ya ginawa na dakel a teorya na depensa et nayarin napalaknab ed amin a lebel na biolohikon tisyu, tinepetan mi no panon kapareho so metabolikon ebat ya sengegan manlapud internal a panangikompara ed interspecies ya panangikompara? Diad sayan gagala, sistematikon inaral mi so metabolome na bulong na tobacco nicotine, ya sakey ya ekolohikon modelo na tanaman ya walaan na mayaman ya espisyal ya metabolismo, tan epektibo sumpad larvae na duaran katutubon herbivore, Lepidoptera Datura (Ms) ( Very aggressive, mainly eaten) Diad Solanaceae tan Spodoptera (Spodoptera), et sakey a klase na bulong na worm “genus”, kaiba iray tanaman ya Solanaceae tan arum niran tanaman ya arum ya genera tan pamilya. Inaral mi so MS/MS metabolomics spectrum tan inala mi ray statistical descriptors na teorya na impormasyon pian ikompara iray teorya na OD tan MT. Manggawa na saray mapa na detalye pian nanengneng so pakabidbiran na saray manunan metabolites. Say panagsukimat et inlaknab ed katutubon populasyon na N. nasi tan saray asingger a klase na tabako pian lalon naanalisa so pandurumaan na plant hormone signaling tan OD induction.
Pian naala so inkalapagan a mapa nipaakar ed plasticity tan structure na leaf metabolome na herbivorous tobacco, inusar mi so akaunan ginawa lan analysis tan calculation workflow pian komprehensibon nakolekta tan na-deconvolute so high-resolution data ya independent MS/MS spectra manlapud saray ekstrak na tanaman ( 9). Sayan undifferentiated method (tatawagen ya MS/MS) et makapanggawa na non-redundant compound spectra, ya nayarin usaren parad amin ya compound-level analyses ya adeskribe dia. Sarayan deconvoluted plant metabolites et nanduruman klase, ya tugyopen na nilasus anggad nilibon metabolites (manga 500-1000-s/MS/MS dia). Dia, ikonsidera tayo so metabolic plasticity diad balangkas na teorya na impormasyon, tan kuentaen tayo so pandurumaan tan propesyonalismo na metabolome base ed Shannon entropy na metabolic frequency distribution. Diad impangusar ed datin inyaplikan pormula (8), kinalkula mi so sakey a set na saray indicator ya nayarin usaren pian nabilang so metabolome diversity (Hj indicator), metabolic profile specialization (δj indicator) tan metabolic specificity na sakey a metabolite (Si indicator). Niarum ni, inyaplika mi so Relative Distance Plasticity Index (RDPI) pian nabilang so metabolome inducibility na saray herbivore (Figure 1A) (30). Diad loob na sayan estadistikal a balangkas, ipapasen mi so MS/MS spectrum bilang manunan yunit na impormasyon, tan iproseso mi so relatibon karakel na MS/MS ed sakey a mapa na panagbunog na frequency, insan usaren so Shannon entropy pian nakalkula so pandurumaan na metabolome manlapud satan. Say metabolome specialization et nasukat ed average ya specificity na sakey ya MS/MS spectrum. Kanian, say idarakel na pigaran klase na MS/MS kayari na herbivore induction et nauman ed spectral inducibility, RDPI tan specialization, salanti, say idarakel na δj index, lapud mas dakel ya espisyal ya metabolites so napapawala tan atagey ya Si index so napapawala. Say iyabeba na Hj diversity index et mangipapatnag ya balanglan say bilang na napapawala ya MS/MS et nabawasan, odino say profile frequency distribution et manguman ed aliwan pareparehon direksion, legan ya nabawasan so inkalapagan ya agpakaseguroan to. Diad panamegley na kalkulasyon na Si index, posiblin nipabitar no dinan iran MS/MS so napapawala na pigaran herbivore, diad kasunian, no dinan iran MS/MS so ag-onkiwas ed induksion, ya saya so manunan pakapatnagan pian nibiig so MT tan OD ya prediksion.
(A) Saray estadistika ya deskriptor ya inusar parad herbivorous (H1 anggad Hx) MS/MS data-inducibility (RDPI), diversity (Hj index), specialization (δj index) tan metabolite specificity (Si index). Say idakel na degree of specialization (δj) et mangipapanengneng ya, diad promedyo, mas dakel so herbivorous ya espisipikon metabolites ya nagawa, bangta say ibawas na diversity (Hj) et mangipapanengneng na ibawas na produksyon na metabolites odino ag-inkaparpareho na distribution na metabolites ed distribution map. Say Si value et man-assess no kasin say metabolite et espisipiko ed sakey a kondisyon (dia, herbivorous) odino diad kabaliktaran et mansiansia ed parehon lebel. (B) Konseptual a diagram na prediksion na teorya na depensa ya uusaren so axis na teorya na impormasyon. Ipapasakbay na teorya na OD ya say panangataki na herbivore et mamarakep ed saray metabolite na depensa, kanian mamarakep ed δj. Diad saman met a panaon, onbabawas so Hj lapud say profile et naorganisa lamet ed nabawasan ya agpakaseguroan na impormasyon nipaakar ed metabolismo. Say teorya na MT et mangibabaga ya say panangataki na saray herbivore et pansengegan na ag-ontutundan pananguman ed metabolome, kanian ondarakel so Hj bilang indikasyon na ondarakel ya ag-inkaseguro na impormasyon nipaakar ed metabolismo tan pansengegan na random ya inkaapag-apag na Si. Inproponin mi met so sakey a mixed model, say sankaabigan ya MT, ya arum ya metabolites ya walaan na mas atagey ya defensive values ​​et lalon ondarakel (atagey so Si value), bangta say arum et mangipapatnag na random iran ebat (abeba so Si value).
Diad pangusar ed saray deskriptor na teorya na impormasyon, ipatalos mi so teorya na OD pian nahulaan ya saray pananguman ed espisyal a metabolite ya sengegan na herbivore diad ag-agawaan a konstitutibon estado et mangitonton ed (i) idakel na metabolic specificity (Si index) ya mangitonton ed metabonomic specificity (δj index) Say idakel na) pigaran espisyal a grupo na metabolite ya walaan na mas atagey a balor na depensa, tan (ii) say ibawas na indeks na metabolite (Hj) lapud panguman na metabolic frequency distribution ed mas dakel ya leptin body distribution. Diad lebel na sakey a metabolite, nailaloan so inkauksoy na Si distribution, ya say metabolite et mamarakep ed Si value unong ed defense value to (Figure 1B). Diad sayan linya, ipapaliwawa mi so teorya na MT pian naisip ya say excitation et mangitonton ed (i) ag-ontutundan pananguman ed saray metabolite ya manresulta ed ibawas na δj index, tan (ii) idakel na Hj index lapud idakel na metabolic uncertainty. Odino say randomness, ya nayarin nabilang na Shannon entropy diad nengneng na generalized diversity. Nipaakar ed komposisyon na metabolismo, say teorya na MT so mangibaga ed random ya inkaapag-apag na Si. No ikonsidera ya wala ray metabolites ya walad espisipikon kondisyon, tan arum ya kondisyon et walad espisipikon kondisyon, tan say balor na depensa ra et mandedepende ed kaliberliber, inproponin mi met so mixed defense model, ya say δj tan Hj et nibunog ed duara diad Si Increase ed amin a direksion, pigaran grupo labat na metabolite, ya walaan na mas atagey ya balor na depensa, so partikular ya ondarakel bangta say arum ya Si, et walaan na random distribution (Figure a 1B).
Pian nasubok so nauman ya prediksion na teorya na depensa ed axis na deskriptor na teorya na impormasyon, pinabaleg mi so eksperto (Ms) odino generalist (Sl) iran larvae na herbivore ed saray bulong na Nepenthes pallens (Figure 2A). Diad impangusar na MS/MS analysis, akaala kami na 599 ya aliwan redundant ya MS/MS spectra (data file S1) manlapud methanol extracts na leaf tissue ya naala kayari na impamakan ed caterpillar. Say pangusar na RDPI, Hj, tan δj indexes pian nanengneng so pananguman na impormasyon ed MS/MS configuration files et mangipapatnag na makapainteres iran padron (Figure 2B). Say inkalapagan ya uso et, unong ed indeskribe na information descriptor, legan ya mantutultuloy ya mangan na bulong iray ulod, say laknab na amin ya metabolic reorganisation et ondarakel legay ilalabas na panaon: 72 oras kayari na pangan na herbivore, say RDPI et ondarakel a maong. No ikompara ed ag-aderal ya kontrol, say Hj et signipikantin abawasan, ya lapud indaak na grado na espisyalisasyon na metabolic profile, ya nabilang panamegley na δj index. Sayan ompatnag ya uso et mitunosan ed saray prediksion na teorya na OD, balet agmitunosan ed saray manunan prediksion na teorya na MT, ya manisia ya saray random (aliwan direksion) ya pananguman ed lebel na metabolite et uusaren bilang depensan panag-amot (Figure 1B). Anggaman mandumaan so oral secretion (OS) elicitor content tan say ugali na panangan na sarayan duaran herbivore, say direktan panangan da et nanresulta ed mipadparan pananguman ed direksion na Hj tan δj legan na 24-oras tan 72-oras ya panaon na panag-ani. Say pandumaan labat et agawa ed 72 oras na RDPI. No ikompara ed samay sengegan na Ms feeding, say inkalapagan ya metabolismo ya sengegan na Sl feeding et mas atagey.
(A) Eksperimental a desinyo: saray ordinaryon baboy (S1) odino eksperto (Ms) ya herbivores et napapakan na saray desalinated leaves na pitcher plants, bangbalet parad simulated herbivory, say OS na Ms (W + OSMs) so uusaren pian naasikaso so pan-puncture na standardized leaf positions wound. S1 (W + OSSl) larvae odino danum (W + W). Say kontrol (C) et sakey ya ag-aderal a bulong. (B) Inducibility (RDPI no ikompara ed control chart), diversity (Hj index) tan specialization (δj index) index ya kinalkula parad espisyal ya metabolite map (599 MS/MS; data file S1). Saray asterisk so mangipapanengneng na signipikantin pandumaan na direktan panangan na herbivore tan say grupon kontrol (Student's t-test ya walay paired t-test, *P<0.05 tan ***P<0.001). ns, aliwan importante. (C) Time resolution index na manunan (asul a kahon, amino acid, organic acid tan asukar; data file S2) tan espisyal ya metabolite spectrum (red box 443 MS/MS; data file S1) kayari na simulated herbivory treatment. Say kolor na banda et manutukoy ed 95% ya kompiyansa. Say asterisk et mangipapanengneng na signipikantin pandumaan na panagtambal tan say kontrol [quadratic analysis of variance (ANOVA), ya tumbokan na Tukey's honestly significant difference (HSD) parad post hoc multiple comparisons, *P<0.05, **P<0.01 Tan *** P <0.001]. (D) Espesyalisasyon na saray scatter plot tan espisyal iran profile na metabolite (paulit-ulit iran sample ya walaan na nanduruman panagtambal).
Pian naamtaan no kasin say herbivore-induced remodeling ed metabolome level et napatnagan ed saray pananguman ed lebel na indibidual iran metabolites, inmuna min inimano iray metabolites ya datin inaral ed saray bulong na Nepenthes pallens ya walaan na apaneknekan ya resistensya na herbivore. Saray phenolic amides et hydroxycinnamamide-polyamine conjugates ya mantitipon legan na proseso na panangan na tanaman na saray insekto tan kabkabat ya mamapaabeba ed pakayari na insekto (32). Inanap mi ray precursor na mipetekan ya MS/MS tan in-plot mi ray cumulative kinetic curves da (Figure S1). Agla pankelawan, saray phenol derivatives ya ag direktan nalalanor ed depensa sumpad saray herbivore, a singa say chlorogenic acid (CGA) tan rutin, et onbabawas kayari na herbivory. Diad pidumaan, saray herbivore et sarag dan gawaen ya mabiskeg so phenol amides. Say tuloytuloy ya panagpakan na duaran herbivore et nanresulta ed ngalngalin parehon excitation spectrum na phenolamides, tan sayan pattern et lalon mapatnag parad de novo synthesis na phenolamides. Ontan met so naobserbaan no usisaen so 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs) ya dalan, ya mamapawala na baleg a bilang na acyclic diterpenes ya epektibon anti-herbivore functions (33), ya say Ms Feeding ya walay Sl so angigapo na mipadparan profile (Figure S1)).
Say posiblin disbentaha na saray eksperimento ed direktan panagpakan ed herbivore et say pandumaan na karakel na panangan na bulong tan panaon na panagpakan na saray herbivore, ya mamapairap ed pangekal ed saray epekto na herbivore ya sengegan na saray sugat tan herbivore. Pian mas nasolbar so inka-espesipiko na herbivore species na induced leaf metabolic response, in-simulate mi so panagpakan na Ms tan Sl larvae diad pangiyaplika min tampol na balon nakolekta ya OS (OSM tan OSS1) ed standard puncture W na pareparehon posisyon na bulong. Sayan pamaakaran et tatawagen ya W + OS treatment, tan saya so man-standardize ed induction diad eksakton pangi-time na gapoan na ebat ya naala na herbivore ya ag-onsengegan na makapawetwet iran epekto na pandumaan na rate odino karakel na tissue loss (Figure 2A) (34). Diad impangusar ed MS/MS analysis tan calculation pipeline, akaala kami na 443 MS/MS spectra (data file S1), ya nan-overlap ed saray spectra ya datin intipon manlapud direct feed experiments. Say information theory analysis na sayan MS/MS data set et angipatnag ya say pangi-reprogram ed saray leaf-specialized metabolomes diad panamegley na pangi-simulate ed saray herbivore et angipatnag na OS-specific inducements (Figure 2C). Diad partikular, no ikompara ed OSS1 ya panagtambal, say OSM so sengegan na inyaligwas na metabolome specialization ed loob na 4 oras. Nepeg ya imanoen ya no ikompara ed direktan panangan na herbivore ya eksperimento, say metabolic kinetics ya nanengneng ed duaran dimensional ya espasyo ya uusaren so Hj tan δj bilang coordinates tan say direksion na metabolome specialization bilang ebat ed simulated herbivore treatment legay ilalabas na panaon et ondarakel so parepareho (Figure 2D). Diad saman met a panaon, inkuantipika mi so karga na amino acids, organic acids tan asukar (data file S2) pian nausisa no kasin sayan target ya idakel na metabolome expertise et lapud pananguman na central carbon metabolism bilang ebat ed saray simulated herbivores ( Figure S2). Pian mas nipaliwawa iyan padron, lalon inbantayan mi so metabolic accumulation kinetics na saray akauna lan apantongtongan ya phenolamide tan 17-HGL-DTG pathways. Say OS-specific induction na saray herbivore et nauman ed sakey a differential rearrangement pattern ed loob na metabolismo na phenolamide (Figure S3). Saray phenolic amides ya walay coumarin tan caffeoyl moieties et mas labay ya na-induce na OSS1, bangta saray OSMs et mangi-trigger na espisipikon induction na ferulyl conjugates. Parad 17-HGL-DTG pathway, naimano so pandumaan na OS induction panamegley na downstream malonylation tan dimalonylation products (Figure S3).
Insan, inaral mi so OS-induced transcriptome plasticity ya inusar so time-course microarray data set, ya mangi-simulate ed pangusar na OSMs pian tambalen iray bulong na bulong na rosette plant ed saray herbivore. Say sampling kinetics et manunan mipara ed saray kinetics ya inusar ed sayan metabolomics ya impanaral (35). No ikompara ed metabolome reconfiguration ya say metabolic plasticity et lalon ondarakel legay ilalabas na panaon, naobserbaan tayo so transient transcription bursts ed saray bulong ya in-induce na Ms, ya say transcriptome inducibility (RDPI) tan specialization (δj) et walad 1 Walay signipikantin indaak na oras, tan diversity (Hj) ed sayan panaon, say ekspresyon na BMP1 et signipikantin inmabeba, ya tinumbokan na relaxation transcriptome specialization (Figure S4). Saray pamilya na gene na metabolismo (singa say P450, glycosyltransferase, tan BAHD acyltransferase) et mibibiang ed proseso na panagtipon na saray espisyal a metabolite manlapud saray yunit na istruktura ya nanlapud manunan metabolismo, unong ed asalambit lan masakbay ya high-specialization model. Bilang sakey a kaso, say phenylalanine pathway so inaral. Say analysis et akumpirma ya saray manunan genes ed metabolismo na phenolamide et atagey so OS-induced to ed saray herbivore no ikompara ed saray tanaman ya ag-aakit, tan maapit iran mankakasakey ed saray pattern na panangibalikas da. Say transcription factor MYB8 tan saray structural genes ya PAL1, PAL2, C4H tan 4CL ed tagey na sayan dalan so angipatnag na masakbay ya inggapo na transcription. Saray acyltransferases ya walay betang da ed pinal ya pantitipon na phenolamide, singa say AT1, DH29, tan CV86, et mangipapatnag na andukey ya upregulation pattern (Figure S4). Ipapanengneng na saray obserbasyon ed tagey ya say masakbay ya gapoan na transcriptome specialization tan say saginonor ya iyaligwas na metabolomics specialization et nankakasakey a paraan, ya nayarin lapud synchronous regulatory system ya mangigapo na mabiskeg a depensa.
Say pananguman ed signaling na hormone na tanaman et onkikiwas bilang sakey a regulatory layer ya mangiiter na impormasyon nipaakar ed herbivorous pian na-reprogram so physiology na tanaman. Kayari na herbivore simulation, sinukat mi so cumulative dynamics na saray manunan kategorya na hormone na tanaman tan anengneng mi so temporal co-expression ed baetan da [Pearson correlation coefficient (PCC)> 0.4] (Figure 3A). Unong ed iilaloan, saray hormone na tanaman ya misiglaotan ed biosynthesis et mankakasakey ed loob na plant hormone co-expression network. Niarum ni, say metabolic specificity (Si index) et aka-map ed sayan network pian nipabitar iray hormone na tanaman ya nanlapud nanduruman panagtambal. Duaran manunan lugar na herbivorous ya espisipikon ebat so nidrowing: sakey et diad JA cluster, ya ditan et say JA (say biologically active form to JA-Ile) tan arum niran JA derivatives so mangipapanengneng na sankatageyan ya Si score; say sananey et ethylene (ET). Say Gibberellin et walaan labat na kalkalnan indakel na herbivore, bangbalet saray arum ya hormone na tanaman, a singa say cytokinin, auxin, tan abscisic acid, et walaan na abeban induction specificity parad saray herbivore. No ikompara ed pangusar na W + W labat, say pamalaknab na peak value na JA derivatives panamegley na OS application (W + OS) et nayarin magmaliw ya mabiskeg ya espisipikon indicator na JAs. Ag-iilaloan, say OSM tan OSS1 ya walaan na nanduruman elicitor content et kabkabat ya pansengegan na parehon panagtipon na JA tan JA-Ile. Diad pidumaan na OSS1, say OSM et espisipikon tan mabiskeg ya napapakiwas na OSMs, bangta say OSS1 et agto papabiskegen so ebat na basal iran sugat (Figure 3B).
(A) Co-expression network analysis base ed PCC ya kalkulasyon na herbivore-induced plant hormone accumulation kinetics simulation. Say node et mangirerepresenta na sakey a hormone na tanaman, tan say kabaleg na node et mangirerepresenta ed Si index ya espisipiko ed hormone na tanaman ed baetan na saray panagtambal. (B) Say idakel na JA, JA-Ile tan ET ed saray bulong ya sengegan na nanduruman panagtambal ya ipapanengneng na nanduruman kolor: apricot, W + OSM; asul, W + OSSl; andeket, W + W; gray, C (control) . Saray asterisk so mangipapanengneng na signipikantin pandumaan na panagtambal tan kontrol (two-way ANOVA ya tinumbokan na Tukey HSD post hoc multiple comparison, *** P <0.001). Information theory analysis na (C)697 MS/MS (data file S1) ed JA biosynthesis tan impaired perception spectrum (irAOC tan irCOI1) tan (D)585 MS/MS (data file S1) ed ETR1 ya walay impaired ET signal Duaran simulated herbivore treatments so angitonton ed plant line tan empty vehicle control (EV) plants. Saray asterisk so mangipapanengneng na signipikantin pandumaan na W+OS ya panagtambal tan say ag-aderal ya kontrol (two-way ANOVA ya tinumbokan na Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 tan ***P<0.001). (E) Saray nitaytayak ya graph na nitaytayak ya isusumpa ed espesyalisasyon. Saray kolor et mangirerepresenta na nanduruman genetically modified strains; saray simbolo et mangirerepresenta na nanduruman paraan na panagtambal: trianggulo, W + OSS1; rektanggulo, W + OSM; sirkulo C
Kayari to, mangusar kami na genetically modified strain na attenuated Nepenthes (irCOI1 tan sETR1) diad saray manunan kundang na JA tan ET biosynthesis (irAOC tan irACO) tan pakatebek (irCOI1 tan sETR1) pian naanalisa so metabolismo na sarayan duaran hormone na tanaman ed saray herbivore Say kontribusyon na reprogramming. Mitunosan ed saray akaunan eksperimento, akumpirma mi so induksion na herbivore-OS ed saray tanaman ya anggapoy karga (EV) (Figure 3, C anggad D) tan say inkalapagan ya ibabawas na Hj index ya sengegan na OSM, bangta say δj index et dinmaak. Say ebat et mas malinew nen say ebat ya pinakiwas na OSS1. Say duaran linya na graph ya manguusar na Hj tan δj bilang coordinates et mangipapanengneng na espisipikon deregulation (Figure 3E). Say sankapatnagan ya uso et diad saray strain ya anggapoy JA signal, say metabolome diversity tan saray pananguman ed specialization ya sengegan na saray herbivore et ngalngalin sigpot lan naandi (Figure 3C). Diad pidumaan, say silent ET perception ed sETR1 ya tanaman, anggaman say inkalapagan ya epekto ed pananguman ed metabolismo na herbivorous et mas abeba nen say JA signaling, et mamalemew ed pandumaan na Hj tan δj indices ed baetan na OSM tan OSS1 excitations (Figure 3D tan Figure S5). . Ipapanengneng na saya ya nilikud ed manunan kimey na JA signal transduction, say ET signal transduction et manseserbi met ya fine-tuning na species-specific metabolic response na saray herbivore. Mitunosan ed sayan fine-tuning function, anggapoy anguman ed inkalapagan ya metabolome inducibility ed sETR1 ya tanaman. Diad biek a dapag, no ikompara ed saray tanaman ya sETR1, saray tanaman ya irACO et walaan na mipadparan amplitude na pananguman ed metabolismo ya sengegan na saray herbivore, balet ta angipatnag na signipikantin pandumaan na Hj tan δj scores ed baetan na OSM tan OSS1 iran angat (Figure S5).
Pian nabirbir iray espisyal a metabolites ya walaan na importantin kontribusyon ed species-specific response na saray herbivore tan na-fine-tune so produksyon da panamegley na ET signals, inusar mi so akaunan ginawan structural MS/MS method. Sayan paraan et mandedependi ed bi-clustering a paraan pian nanengneng lamet so metabolic family manlapud MS/MS fragments [normalized dot product (NDP)] tan similarity score base ed neutral loss (NL). Say MS/MS data set ya ginawa panamegley na analysis na ET transgenic lines et akagawa na 585 MS/MS (data file S1), ya naresolbe diad impangitipon ed saratan ed pitoran manunan MS/MS modules (M) (Figure 4A) . Arum ed sarayan module et napno na saray datin nibiig ya espisyal iran metabolite: alimbawa, say M1, M2, M3, M4 tan M7 et mayaman ed nanduruman phenol derivatives (M1), flavonoid glycosides (M2), acyl sugars (M3 Tan M4), tan 17-HGL-DTG (M7). Niarum ni, say metabolic specific information (Si index) na sakey a metabolite ed kada module et nakalkula, tan say Si distribution to et nanengneng ya intuitive. Diad antikey, saray MS/MS spectra ya mangipapatnag na atagey a herbivory tan genotype specificity et nabidbir ed atagey iran Si values, tan say kurtosis statistics so mangipapanengneng na inkaapag-apag na bago ed kawanan a suyok na ikol. Sakey ed saratan ya lean colloid distribution so anengneng ed M1, ya ditan et say phenol amide so walaan na sankatageyan a Si fraction (Figure 4B). Say akaunan asalambit ya herbivorous inducible 17-HGL-DTG ed M7 et angipatnag na moderate ya Si score, ya mangipapanengneng na moderate ya degree na differential regulation ed baetan na duaran klase na OS. Diad pidumaan, maslak ed saray espisyal ya metabolite ya napapawala, singa say rutin, CGA, tan acyl sugars, et kaiba ed saray sankaabebaan ya Si scores. Pian mas naeksplor so inkakomplikado na istruktura tan say inkaapag-apag na Si ed baetan na saray espisyal a metabolite, sakey a molecular network so ginawa parad kada module (Figure 4B). Say importantin prediksion na OD theory (ya insumaryo ed Figure 1B) et say pangiorganisa lamet ed saray espisyal a metabolite kayari na herbivory et nepeg ya mangitonton ed sakey a paraan na pananguman ed saray metabolite ya walaan na atagey a balor na depensa, lalo la diad pamarakep ed inka-espesipiko ra (aliwan random distribution) Mode) Defensive metabolite ya impasakbay na MT theory. Maslak ed saray phenol derivatives ya natipon ed M1 et walay koneksion to ed ibabawas na performance na insekto (32). No ikompara so Si values ​​ed saray M1 metabolites ed baetan na saray induced leaves tan saray constituent leaves na EV control plants ed loob na 24 oras, anengneng mi ya say metabolic specificity na dakel a metabolites kayari na saray herbivory insects et walaan na signipikantin itatagey na trend (Figure 4C). Say espisipikon indakel na Si value et anengneng labat ed saray defensive phenolamides, balet anggapoy anengneng ya indaak na Si value ed arum ya phenols tan ag kabat ya metabolites ya kaiba ed sayan module. Saya so sakey ya espisyal a modelo, ya misiglaotan ed teorya na OD. Saray manunan prediksion na metabolic changes ya sengegan na saray herbivore et parepareho. Pian nasubok no kasin sayan partikularidad na phenolamide spectrum et nanlapud OS-specific ET, in-plot mi so metabolite Si index tan nansengegan na differential expression value ed baetan na OSM tan OSS1 diad EV tan sETR1 genotypes (Figure 4D ). Diad sETR1, say pandumaan na OSM tan OSS1 ya sengegan na phenamide et abawasan a maong. Say bi-clustering method et inyaplika met ed MS/MS data ya naala ed saray strain ya kulang so JA pian naisip iray manunan MS/MS modules ya misiglaotan ed JA-regulated metabolic specialization (Figure S6).
(A) Say resulta na panagtipon na 585 MS/MS base ed shared fragment (NDP similarity) tan shared neutral loss (NL similarity) et manresulta ed module (M) ya mipara ed kabkabat ya pamilya na compound, odino diad ag-amta odino aliwan maong ya metabolismo na Metabolite composition. Diad abay na kada module, say metabolite (MS/MS) ya espisipiko (Si) ya distribusyon et nipanengneng. (B) Modular molecular network: Saray node et mangirerepresenta ed MS/MS tan saray gilig, NDP (ambalanga) tan NL (asul) MS/MS scores (cut-off,> 0.6). Say graded metabolite specificity index (Si) ya inkolor base ed module (kawanan) tan in-map ed molecular network (kawanan). (C) Module M1 na EV plant ed constitutive (control) tan induced state (simulated herbivore) ed loob na 24 oras: molecular network diagram (Si value et say kabaleg na node, say defensive phenolamide et aka-highlight ed asul). (D) Say M1 molecular network diagram na spectrum line sETR1 ya walay deperensya ed pakatebek na EV tan ET: say phenolic compound ya irerepresenta na green circle node, tan say signipikantin pandumaan (P value) ed baetan na W + OSM tan W + OSS1 ya panagtambal bilang kabaleg na Node. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, N-ferulic acid ester-uric acid; FS, N-ferulyl-spermidine; CoP, N', N "-Coumarolyl-tyrosine; DCS, N', N"-dicaffeoyl-spermidine; CFS, N', N”-caffeoyl, feruloyl-spermidine; Lycium barbarum ed wolfberry Son; Nick. O-AS, O-acyl sugar.
Inlaknab mi ni so panagsukimat manlapud sakey ya attenuated Nepenthes genotype ya anggad natural iran populasyon, ya ditan et saray mabiskeg ya intraspecific ya pananguman ed herbivorous JA levels tan espisipikon metabolite levels so akauna lan adeskribe ed natural iran populasyon (26). Usaren iyan data set pian nakobrian so 43 ya germplasm. Sarayan germplasm et tugyopen na 123 a klase na tanaman a nanlapud N. pallens. Sarayan tanaman et inala ed saray bukel ya naala ed nanduruman katutubon ayaman ed Utah, Nevada, Arizona, tan California (Figure S7), kinalkula mi so metabolome diversity (tatawagen dia ya population level) β diversity) tan say specialisation ya sengegan na OSM. Mitunosan ed saray akaunan impanaral, anengneng mi so malaknab a pananguman ed metabolismo ed Hj tan δj axes, ya mangipapanengneng ya saray germplasms et walaan na signipikantin pandumaan ed plasticity na metabolic responses da ed saray herbivore (Figure S7). Sayan organisasyon et mipara ed saray akaunan obserbasyon nipaakar ed dinamikon laknab na pananguman na JAs ya sengegan na saray herbivore, tan nansiansia so atagey a balor to ed sakey a populasyon (26, 36). Diad impangusar ed JA tan JA-Ile pian nasubok so inkalapagan ya lebel na korelasyon na Hj tan δj, aromog mi ya walay signipikantin positibon korelasyon na JA tan say metabolome β diversity tan specialization index (Figure S7). Ipapanengneng na saya ya say herbivore-induced heterogeneity ed JAs induction ya anengneng ed lebel na populasyon et nayarin lapud manunan metabolic polymorphisms ya sengegan na pinilin insekto ya herbivore.
Impanengneng na saray akaunan impanaral ya saray klase na tabako et mandumaan a maong ed klase tan say relatibon pandepende ra ed saray depensa na metabolismo. Manisia ya sarayan pananguman ed anti-herbivore signal transduction tan pakayarin mandepensa et nareregulate na pressure na populasyon na insekto, siklo na bilay na tanaman, tan gastos ed produksyon na depensa diad niche ya ombabaleg so sakey a species. Inaral mi so inkapara na pananguman na metabolome na bulong ya sengegan na saray herbivore na anemiran klase na Nicotiana ya nanlapud North America tan South America. Sarayan klase na ayep et maapit a kanayon na Nepenthes North America, salanti, si Nicolas Bociflo. La, N. nicotinis, Nicotiana n. attenuated grass, Nicotiana tabacum, linear tobacco, tobacco (Nicotiana spegazzinii) tan tobacco leaf tobacco (Nicotiana obtusifolia) (Figure 5A) (37). Anem ed sarayan klase, kaiba lay maong ya klase na N. please, et tinaon ya tanaman na petunia clade, tan say obtusifolia N. et perennial na sister clade ya Trigonophyllae (38). Kayari to, ginawa so W + W, W + OSM tan W + OSS1 induction ed sarayan pitoran klase na ayayep pian aralen so species-level metabolic rearrangement na panangan na insekto.
(A) Sakey a bootstrap phylogenetic tree base ed sankatageyan a posibilidad [parad nuclear glutamine synthesis (38)] tan say geograpikon inkaapag-apag na pitoran maapit a klase na Nicotiana (nanduruman kolor) (37). (B) Sakey a scatter plot na espisyal a pandurumaan parad saray metabolikon profile na pitoran klase na Nicotiana (939 MS/MS; data file S1). Diad lebel na species, say pandurumaan na metabolome et negatibon misiglaotan ed laknab na inka-espesyalista. Say analysis na species-level correlation ed baetan na metabolic diversity tan specialisation tan JA accumulation et nanengneng ed Figure 2. S9. Kolor, nanduruman klase; trianggulo, W + OSS1; rektanggulo, W + OSM; (C) Nicotiana JA tan JA-Ile dynamics et niranggo unong ed OS excitation amplitude (two-way ANOVA tan Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 tan * ** Parad panangikompara na W + OS tan W + W, P <0.001). Box plot na (D) pandurumaan tan (E) inka-espesyalisasyon na kada klase kayari na pangi-simulate na herbivorous tan methyl JA (MeJA). Say asterisk et mangipapanengneng na signipikantin pandumaan na W + OS tan W + W odino lanolin plus W (Lan + W) odino Lan plus MeJA (Lan + MeJa) tan Lan control (two-way analysis of variance, ya tinumbokan na Tukey's HSD post hoc multiple comparison , *P<0.05, **P<0.01 tan ***P<0.01).
Diad pangusar na dual cluster method, akaromog kami na 9 ya modules na 939 MS/MS (data file S1). Say komposisyon na MS/MS ya in-reconfigure na nanduruman panagtambal et manduruman tuloy ed nanduruman modules ed baetan na saray species (Figure S8). No nanengneng so Hj (ya tatawagen dia ya species-level γ-diversity) tan δj, nanengneng ya saray nanduruman species et mantitipon ed nanduruman grupo diad metabolic space, ya say species-level division et mas prominenti nen say excitation. Likud ed N. linear tan N. obliquus, walaan ira na malaknab a dinamikon epekto na induction (Figure 5B). Diad pidumaan, saray klase na ayayep a singa say N. purpurea tan N. obtusifolia et walaan na agtanton mapatnag a metabolikon ebat ed panagtambal, balet say metabolome et mas nanduruma. Say species-specific distribution na induced metabolic response et nanresulta ed signipikantin negatibon siglaotan na specialization tan gamma diversity (PCC = -0.46, P = 4.9×10-8). Say OS-induced ya pananguman ed JAs levels et positibon mikonektaan ed metabolome specialization, tan negatibon mikonektaan ed metabolic gamma diversity ya ipapanengneng na kada species (Figure 5B tan Figure S9). Nepeg ya imanoen ya saray klase na ayayep ya tatawagen ya “signal response” ya klase na ayep ed Figure 5C, a singa say Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, tan Nepenthes attenuated, so sengegan na makabkabaliksan iran tanda ed loob na 30 minutos. Say agano nin OS-specific JA tan JA-Ile outbreaks, bangta arum ya bakterya ya tatawagen ya “signal unresponsive”, singa say Nepenthes mills, Nepenthes powdery tan N. obtusifolia et mangipapanengneng labat na JA-Ile Edge induction ya anggapoy antokaman ya OS specificity (Figure 5C). Diad metabolic level, singa asalambit ed tagey, parad attenuated Nepenthes, saray signal-responsive substances so angipatnag na OS specificity tan signipikantin indaak na δj, legan ya babawasan so Hj. Sayan OS-specific priming effect et ag anengneng ed saray species ya niklasipika bilang signal non-reactive species (Figure 5, D tan E). Saray OS-specific metabolites et mas mabetbet ya naapag ed baetan na saray species ya onebat ed signal, tan sarayan signal clusters et mantitipon ed saray species ya mas makapuy so signal responses da, bangta saray species ya mas makapuy so signal responses da et agtanton mankakasakey (Figure S8 ). Ipapanengneng na sayan resulta ya say OS-specific induction na JAs tan say OS-specific reconfiguration na downstream metabolome et nankakasakey ed lebel na species.
Insan, angusar kami na lanolin paste ya walay methyl JA (MeJA) pian tambalen iray tanaman pian nausisa no kasin sarayan coupling modes et limitado lapud pakawalaan na JA ya inyaplika na exogenous JA, ya walad cytoplasm na tanaman. Say maples ya deesterification et JA. Nalmoan mi so parehon ugali na kalkalnan pananguman manlapud signal-responsive species ya anggad signal-non-responsive species ya sengegan na tuloytuloy ya suplay na JA (Figure 5, D tan E). Diad antikey, say panagtambal na MeJA et mabiskeg ya angi-reprogram ed saray metabolomes na linear nematodes, N. obliquus, N. aquaticus, N. pallens, tan N. mikimotoi, ya nanresulta ed signipikantin indaak na δj tan indaak na Hj. Say N. purpurea et angipatnag labat na indakel na δj, balet aliwan say Hj. Say N. obtusifolia, ya akauna lan nipanengneng ya mantitipon na abeban tuloy iran lebel na JAs, et agmet onkiwas ed panagtambal na MeJA no nipaakar ed metabolome reconfiguration. Ipapanengneng na sarayan resulta ya say produksyon na JA odino say pangiyalis na signal et limitado ed saray klase na ayayep ya ag-onkikiwas ed signal. Pian nasubok iyan hypothesis, inaral mi so apatiran klase (N. pallens, N. mills, N. pink tan N. microphylla) ya in-induce na W + W, W + OSMs tan W + OSS1 Transcriptome (39). Mitunosan ed pattern na metabolome remodeling, saray species et maong ya insian ed transcriptome space, ya say N. attenuated so angipatnag na sankatageyan ya OS-induced RDPI, bangta say N. gracilis so walaan na sankaabebaan (Figure 6A). Balet, aromog ya say transcriptome diversity ya sengegan na N. oblonga et sankaabebaan ed apatiran klase, sumlang ed sankatageyan ya metabonomic diversity na N. oblonga ya datin nipanengneng ed pitoran klase. Impanengneng na saray akaunan impanaral ya say sakey a grupo na genes ya misiglaotan ed saray signal na masakbay a depensa, kaiba lay signal na JA, so mangipapaliwawa ed inka-espesipiko na saray masakbay a depensa ya ebat na saray herbivore-related elicitors ed Nicotiana species (39). No ikompara so JA signaling pathways ed baetan na sarayan apatiran klase, walay makapainteres ya padron (Figure 6B). Maslak ed saray genes ed sayan dalan, singa say AOC, OPR3, ACX tan COI1, so angipatnag na medyo atagey ya lebel na induction ed sarayan apatiran klase. Balet, sakey a manunan gene, say JAR4, so manguuman ed JA ed biologically active form na JA-Ile accumulated transcripts, tan say transcription level to et abeba, lalo la ed N. mills, Nepenthes pieris tan N. microphylla. Niarum ni, say transcript labat na sananey a gene ya AOS so ag-anengneng ed N. bifidum. Sarayan pananguman ed gene expression et nayarin sengegan na arawrawin phenotypes ya sengegan na abeban JA production ed signal anergic species tan say induction na N. gracilis.
(A) Information theory analysis na reprogramming na saray masakbay ya transcriptional responses na apatiran maapit a klase na tabako ya inala 30 minutos kayari na herbivory induction. Say RDPI et nakalkula diad pangikompara ed saray bulong ya pinawala na herbivore OS tan say wound control. Saray kolor et mangipapanengneng na nanduruman klase, tan saray simbolo et mangipapanengneng na nanduruman paraan na panagtambal. (B) Panagsukimat ed gene expression ed JA signaling pathways ed baetan na apatiran klase. Say pinasimplin dalan na JA et nipanengneng ed abay na box plot. Say nanduruman kolor et mangipapanengneng na nanduruman paraan na panagproseso. Say asterisk et mangipapanengneng ya walay signipikantin pandumaan na W + OS ya panagtambal tan say W + W ya kontrol (parad Student's t-test parad pairwise differences, *P<0.05, **P<0.01 tan ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Diad unor a parte, inaral mi no panon ya say pananguman na metabolome na nanduruman klase na tanaman ed saray klase na insekto et makaresisti ed saray herbivore. Say akaunan research et indanet to so Nicotiana genus. Say resistensya ra ed Ms tan larvae et mandumaan a maong (40). Dia, inaral mi so koneksion na sayan modelo tan say metabolic plasticity da. Diad impangusar ed apatiran klase na tabako ya asalambit ed tagey, tan say panubok ed siglaotan na pandurumaan tan inka-espesyalista na metabolome ya sengegan na saray herbivore tan say resistensya na tanaman ed Ms tan Sl, aromog mi ya say resistensya, inka-espesyalista tan inka-espesyalisasyon ed generalist Sl All et positibon mikonektaan, bangta say siglaotan na resistensya ed saray eksperton bibii tan say espisyalisasyon et makapuy, tan say siglaotan ed pandurumaan ed Sl et aliwan makabkabaliksan (Figure 10). Nipaakar ed S1 ya resistensya, parehon say attenuated N. chinensis tan N. gracilis, ya akauna lan nipanengneng ya walaan na JA signal transduction levels tan metabolome plasticity, et walaan na nanduruman tuloy ya ebat ed herbivore induction, tan pareho met so atagey ya resistensya ra. Sex.
Diad apalabas ya anemaplon taon, say teorya na depensa na tanaman so angiter na teoretikal a balangkas, a base ed satan et naisip na saray managsukimat so baleg a bilang na ebolusyon tan kimey na saray espisyal a metabolite na tanaman. Maslak ed sarayan teorya et ag-ontutumbok ed normal a paraan na mabiskeg iran konklusyon (41). Mangiproproponin manunan prediksion (3) diad parehon lebel na panagsukimat. No say panubok ed saray manunan prediksion et mangiter na pankanawnawan naanalisa iray espisipikon teorya, saya so manggawa ed The field is moving forward. Mansuporta, balet ipulisay so arum (42). Imbes, say balon teorya et manggagawa na saray prediksion ed nanduruman lebel na panagsukimat tan mangaarum na balon layer na saray deskriptibon konsiderasyon (42). Balet, say duaran teorya ya inproponin walad functional level, say MT tan OD, et mainomay ya ipaliwawa bilang importantin prediksion na saray espisyal ya pananguman ed metabolismo ya sengegan na saray herbivore: Manisia so teorya na OD ya saray pananguman ed espisyal ya metaboliko ya “espasyo” et walay direksion da. Manisia so MT theory ya sarayan pananguman et aliwan direksion tan random ya walad metabolic space, tan walaan na atagey ya defense value ya metabolites. Saray akaunan eksaminasyon ed saray prediksion na OD tan MT et sinubok ya inusar so mainget a set na a priori “defense” compounds. Sarayan metabolite-centric tests et ag mangiiter na abilidad ya analisaen so kalaknab tan trajectory na metabolome reconfiguration legan na herbivory, tan ag da aabuloyan so testing ed loob na sakey a mantutunosan ya statistical framework ya mankaukolan na saray manunan prediksion ya nayarin ikonsidera bilang sakey a kabiangan. Dia, inusar mi so makabayon teknolohiya ed metabolomics base ed computational MS tan nanggawa kami na deconvolution MS analysis ed kaslakan ya deskriptor na teorya na impormasyon pian nasubok so pandumaan na duaran niproponin lebel na global metabolomics. Say manunan prediksion na sayan teorya. Say teorya na impormasyon et inyaplika ed dakel a lawak, lalo la ed konteksto na panagsukimat ed biodiversity tan nutrient flow (43). Balet, anggad amta tayo, saya so sankaunaan ya aplikasyon ya inusar pian deskribien so metabolic information space na tanaman tan solbaren iray problema ed ekolohiya ya misiglaotan ed temporaryon pananguman ed metabolismo bilang ebat ed saray signal na kaliberliber. Diad partikular, say abilidad na sayan paraan et walad abilidad ton ikompara iray pattern ed loob tan baetan na saray klase na tanaman pian nausisa no panon ya inmaligwas iray herbivore manlapud nanduruman klase ya anggad inter-species macroevolutionary patterns ed nanduruman lebel na ebolusyon. Metabolismo.
Say Principal component analysis (PCA) et manguuman ed sakey a multivariate data set ed sakey a dimensionality reduction space pian nipaliwawa so manunan uso na data, kanian kaslakan ya uusaren itan bilang sakey a eksplorasyon a teknik pian na-parse so data set, a singa say deconvolution metabolome. Balet, say pambawas ed dimensionality et naandi so kabiangan na impormasyon ed data set, tan say PCA et ag makapangiter na quantitative ya impormasyon nipaakar ed saray katangian ya partikular ya makanakana ed teorya na ekolohiya, a singa say: panon ya i-reconfigure na saray herbivore so pandurumaan ed saray espisyal a lawak (alimbawa, kayamanan, panagbunog) Tan karakel) metabolites? Anto ran metabolites so makapangibaga na induced state na sakey a herbivore? Diad panmoria na inka-espesipiko, pandurumaan tan inka-inducibility, say impormasyon ya walad profile na metabolite ya espisipiko ed bulong et nabulok, tan naromog ya say pangan na saray herbivore et makapangiter na espisipikon metabolismo. Diad ag inilaloan, naobserbaan mi ya, unong ed adeskribe ed saray indicator na teorya na impormasyon ya inyaplika, say resulta na metabolic situation et walaan na baleg ya overlap kayari na impangataki na duaran herbivore (say night-fed generalist Sl) tan say Solanaceae expert Ms. Anggaman say ugali tan konsentrasyon da ed panangan et signipikantin mandumaan. Fatty acid-amino acid conjugate (FAC) initiator ed OS (31). Diad impangusar na herbivore OS pian tambalen iray standardized puncture wounds, say simulated herbivore treatment et angipatnag met na mipadparan uso. Sayan standardized a paraan na pangi-simulate ed ebat na tanaman ed saray atake na herbivore et mamaandi ed saray makapawetwet a bengatla ya sengegan na pananguman ed ugali na panangan na saray herbivore, ya pansengegan na nanduruman grado na pakaderal ed nanduruman panaon (34). Say FAC, ya kabkabat ya manunan sengegan na OSM, et mamapaabeba ed JAS tan arum niran ebat na hormone na tanaman ed OSS1, bangta say OSS1 et mamapaabeba na nilasus a danay (31). Balet, say OSS1 so sengegan na mipadparan lebel na panagtipon na JA no ikompara ed OSM. Napatnagan la nensaman ya say JA response ed attenuated Nepenthes et sensitibo ed OSM, ya ditan et napansiansia na FAC so aktibidad to anggano na-dilute na 1:1000 ed danum (44). Kanian, no ikompara ed OSM, anggaman abeba so FAC ed OSS1, magenap la itan pian nawalaan na magenap ya JA outbreak. Impanengneng na saray akaunan impanaral ya saray porin-like proteins (45) tan oligosaccharides (46) et nayarin usaren bilang molecular clues pian na-trigger iray depensa na tanaman ed OSS1. Balet, agni malinew no sarayan elicitors ed OSS1 so sengegan na idakel na JA ya naobserbaan ed kaplesan ya impanaral.
Anggaman pigpigara so impanaral ya mangidedeskribe ed pandumaan na metabolic fingerprints ya sengegan na pangiyaplika na nanduruman herbivore odino exogenous JA o SA (salicylic acid) (47), anggapoy toon akaapekto ed herbivore species-specific perturbation ed plant grass network tan saray epekto to ed espisipikon Say inkalapagan ya epekto na metabolismo et sistematikon inaral. personal ya impormasyon. Sayan analysis so lalon akumpirma ya say koneksion na internal hormone network ed arum ya hormone na tanaman nilikud ed JAs so mamorma ed inka-espesipiko na metabolic reorganisation ya sengegan na saray herbivore. Diad partikular, anengneng mi ya say ET ya sengegan na OSM et mas baleg nen say sengegan na OSS1. Sayan mode et mipara ed mas dakel ya FAC content ed OSM, ya sakey a nakaukolan tan magenap a kondisyon pian na-trigger so ET burst (48). Diad konteksto na pidedeneng na tanaman tan herbivores, say signaling function na ET ed tanaman-specific metabolite dynamics et siansia nin sporadic tan manpupuntirya labat ed sakey a grupo na compound. Niarum ni, maslak ed saray impanaral so angusar na exogenous ya aplikasyon na ET odino saray precursor to odino nanduruman inhibitor pian aralen so regulasyon na ET, a diad saraya et sarayan exogenous chemical applications so mamawala na dakel ya aliwan espisipikon epekto. Diad pakaamta mi, sayan impanaral so mangirerepresenta ed sankaunaan ya baleg a sistematikon panangusisa ed papel na ET ed pangusar na ET pian makapawala tan makatebek ed saray deperensya ya transgenic ya tanaman pian nakoordina so metabolome na tanaman. Say herbivore-specific ET induction et nayarin mamasimbalo ed ebat na metabolome. Say sankaimportantian et say transgenic manipulation na ET biosynthesis (ACO) tan perception (ETR1) genes ya angipatnag na herbivore-specific de novo accumulation na phenolamides. Napatnagan la nensaman ya say ET et sarag ton i-fine-tune so JA-induced nicotine accumulation diad panamegley na pangiregula ed putrescine N-methyltransferase (49). Balet, manlapud mekanikal a punto de bista, aliwan malinew no panon ya i-fine-tune na ET so induction na phenamide. Nilikud ed signal transduction function na ET, say metabolic flux et nayarin ipaarap met ed S-adenosyl-1-methionine pian naregula so pinansyal ya pundo ed polyaminophenol amides. Say S-adenosyl-1-methionine et ET tan kaslakan ya intermediate na polyamine biosynthetic pathway. Say mekanismo no panon ya nareregula na ET signal so lebel na phenolamide et kaukolan ya aralen ni.
Diad andukey a panaon, lapud karakel na espisyal iran metabolites ya ag kabat so istruktura ra, say mabiskeg ya imano ed espisipikon kategorya na metaboliko et agmakayarin istrikton nasuri so temporal iran pananguman na pandurumaan na metaboliko kayari na biolohikon pidedeneng. Diad kaplesan, base ed panagsukimat na teorya na impormasyon, say manunan resulta na pakagamor na MS/MS spectrum base ed saray ag-inka-biased metabolites et saray herbivore ya mangan odino man-simulate ed saray herbivore et mantutultuloy ya mamapaabeba ed inkalapagan a metaboliko na metabolome na bulong legan ya ondarakel so grado na espisyalisasyon to. Sayan temporaryon itatagey na metabolome specificity ya sengegan na saray herbivore et misiglaotan ed synergistic ya itatagey na transcriptome specificity. Say feature ya makatulong ed sayan mas baleg ya metabolome ya espisyalisasyon (ya walaan na mas atagey ya Si value) et say espisyal ya metabolite ya walaan na datin karakter ya herbivorous function. Sayan modelo et mipara ed prediksion na teorya na OD, balet say prediksion na MT ya misiglaotan ed inka-random na metabolome reprogramming et aliwan mipara. Balet, sayan datos et mipara met ed prediksion na mixed model (best MT; Figure 1B), lapud arum ya ag-amta iran metabolite ya ag-amta so depensa da et nayarin ontumbok ni ed random ya Si distribution.
Say makatantandan padron ya lalon naala na sayan research et manlapud micro-evolution level (saksakey a tanaman tan populasyon na tabako) anggad mas baleg ya evolutionary scale (maapit a mankakasakey a klase na tabako), nanduruman lebel na ebolusyonaryon organisasyon so walad “sankaabigan a depensa” Wala ray makatantandan pandumaan ed abilidad na saray herbivore. Moore et al. (20) tan si Kessler tan Kalske (1) so independentin angiproponin manguman ed taloran functional levels na biodiversity ya inmuna ya inbiig nen Whittaker (50) ed saray constitutive tan induced temporal changes na chemical diversity; sarayan autor et ag da met insumaryo. Diad sayan impanaral, saray angkekelag ya pananguman ed functional classification nen Whittaker et ikonsidera so α-metabolic diversity bilang say pandurumaan na MS/MS spectra ed sakey a tanaman, tan say β-metabolic diversity bilang say manunan intraspecific metabolism na sakey a grupo na populasyon Space, tan say γ-metabolic diversity et magmaliw ya ekstensyon na panagsukimat ed mipadparan species.
Say JA signal et importante parad nanduruman metabolic responses na herbivore. Balet, wala so kakulangan na malet a quantitative testing ed kontribusyon na intraspecific regulation na JA biosynthesis ed metabolome diversity, tan no kasin say JA signal et sakey a pangkabilugan a pasen parad stress-induced metabolic diversification diad mas atagey a macroevolutionary scale et ag ni natalosan. Naobserbaan mi ya say inka-herbivorous na Nepenthes herbivorous et mamapawala na metabolome specialization tan say pandurumaan na metabolome specialization ed loob na populasyon na Nicotiana species tan ed limog na maapit a Nicotiana species et sistematikon positibon misiglaotan ed JA signaling. Niarum ni, no say JA signal et naapektoan, say metabolic specificity ya sengegan na sakey a genotype herbivore et nakansela (Figure 3, C tan E). Lapud saray pananguman ed metabolic spectrum na saray natural ya attenuated Nepenthes ya populasyon et maslak et quantitative, saray pananguman ed metabolic β diversity tan specificity ed sayan analysis et nayarin manunan sengegan na mabiskeg ya excitation na saray metabolite-rich compound categories. Sarayan klase na compound so mangokontrol ed kabiangan na metabolome profile tan mangitonton ed positibon korelasyon ed saray signal na JA.
Lapud saray biochemical mechanisms na klase na tabako ya asingger ed satan et mandumaan, saray metabolites et espisipikon nabirbir ed kwalitatibo ya aspekto, kanian mas analitikal itan. Say panagproseso na teorya na impormasyon ed naala ya metabolic profile et mangipapatnag ya say herbivorous induction et mamapaawet ed trade-off na metabolic gamma diversity tan specialization. Say JA signal so walaan na manunan papel ed sayan trade-off. Say idarakel na metabolome specialization et mipara ed manunan prediksion na OD tan positibo so pisiglaotan to ed JA signal, bangta say JA signal et negatibon pisiglaotan to ed metabolic gamma diversity. Ipapanengneng na sarayan modelo ya say OD capacity na tanaman et manunan nadetermina ed plasticity na JA, balanglan diad microevolutionary scale odino diad mas baleg ya evolutionary scale. Saray eksperimento ed aplikasyon na exogenous JA ya makaliktar ed saray depekto na JA biosynthesis so lalon mangipapatnag ya saray maapit a klase na tabako et nayarin ibiig ed signal-responsive tan signal-non-responsive species, a singa met ed paraan na JA tan metabolome plasticity ya sengegan na saray herbivore. Saray signal non-responsive species et ag makaebat lapud ag da sarag ya makapawala na endogenous JA kanian wala ray limitasyon ed laman da. Nayarin lapud saya et saray mutation ed pigaran manunan genes ed JA signaling pathway (AOS tan JAR4 ed N. crescens) na. Ipapabitar na sayan resulta ya sarayan interspecies macroevolutionary patterns et nayarin manunan nanlapud saray pananguman ed pakatebek tan pakayari na hormone ed loob.
Nilikud ed pidedeneng na tanaman tan herbivores, say panagsukimat ed metabolic diversity et misiglaotan ed amin ya importantin teoretikal ya iyaaligwas ed panagsukimat na biolohikon adaptasyon ed kaliberliber tan say ebolusyon na komplikadon phenotypic traits. Lapud idarakel na datos ya naala na saray modernon instrumento na MS, say panagsukimat ed hypothesis ed pandurumaan na metabolite et nayarin onlampas ed pandumaan na indibidual/kategorya na metabolite tan manggawa na sankamundoan ya panagsukimat pian nanengneng iray ag-iilaloan iran padron. Diad proseso na baleg ya panagsukimat, sakey ya importantin metapora et say ideya na panggawa na makabaliksan iran mapa ya nayarin usaren pian nasukimat so datos. Kanian, say importantin resulta na kaplesan ya kombinasyon na anggapoy panangidumaduma na MS/MS metabolomics tan teorya na impormasyon et mangiiter itan na simplin sukat ya nayarin usaren ed panggawa na saray mapa pian nanengneng so metabolic diversity ed nanduruman taxonomic scales. Saya so manunan kakaukolanen ed sayan paraan. Say panaral ed micro/macro ya ebolusyon tan ekolohiya na komunidad.
Diad macro-evolutionary level, say manunan kabiangan na teorya na plant-insect co-evolution nen Ehrlich tan Raven (51) et say pangibaga ya say pandurumaan na interspecies metabolic diversity so sengegan na pandurumaan na linya na tanaman. Balet, diad loob na limamplon taon manlapu nen nipalapag iyan manunan kimey, sayan hypothesis et matalag ya nasubok (52). Saya so manunan lapud saray katangian na phylogenetic na mipara iran katangian na metabolismo ed saray andukey a linya na tanaman. Say rarity et nayarin usaren pian i-angkla iray paraan na panagsukimat ed puntirya. Say kaplesan ya MS/MS workflow ya iproseso na teorya na impormasyon et mankukuenta na MS/MS structural similarity na ag kabat ya metabolites (ya anggapoy akaunan pinilin metabolite) tan manguuman ed sarayan MS/MS ed sakey a set na MS/MS, kanian diad propesyonal a metabolismo Sarayan macro-evolutionary models et ikompara ed sukat na klasipikasyon. Simplin estadistikal iran indikasyon. Say proseso et mipara ed phylogenetic analysis, ya nayarin mangusar na sequence alignment pian nabilang so rate na diversification odino character evolution ya anggapoy akauna ya prediksion.
Diad biochemical ya lebel, say screening hypothesis nen Firn tan Jones (53) et mangipapanengneng ya say pandurumaan na metabolismo et mansiasiansia ed nanduruman lebel pian nawalaan na saray hilaw ya materyales ya pangusar ed saray biological ya aktibidad na saray datin ag-akakonekta odino sinalatan iran metabolites. Saray paraan na teorya na impormasyon so mangiiter na sakey a balangkas a ditan et sarayan metabolite-specific evolutionary transitions ya nagagawa legan na metabolite specialization et nayarin nabilang bilang kabiangan na iproproponin proseso na evolutionary screening: biologically active adaptation manlapud abeban specificity ya anggad atagey ya specificity Inhibited metabolites na sakey a kaliberliber.
Diad amin, nen kagapo na molecular biology, wala ray importantin teorya nipaakar ed depensa na tanaman, tan saray deductive hypothesis-driven ya paraan so ipapasen ya saksakey a paraan na iyaaligwas na siensia. Saya so manunan lapud saray teknikal a limitasyon ed panagsukat ed interon metabolome. Anggaman saray paraan ya base ed hypothesis et mausar ed pampili na arum niran mekanismo na sengegan, say pakayari ran mamaaligwas ed pakatalos tayo ed saray biochemical network et mas limitado nen saray paraan na panagkuenta ya wala natan ed contemporary data-intensive science. Kanian, saray teorya ya ag nayarin ipasakbay et arawi ed sakop na walan datos, kanian say hypothetical formula/test cycle na progreso ed lawak na panagsukimat et ag nayarin ekalen (4). Nanengneng tayo ya say computational workflow na metabolomics ya inloob dia et makapangiter lamet na interes ed saray agano nin (panon) tan unor (akin) ya isyu na metabolic diversity, tan makatulong ed balon panaon na teorya ya mangigiya na data science. Inusisa lamet na panaon iray importantin teorya ya angipuyan ed apalabas iran kailalakan.
Say direktan panagpakan ed herbivore et gagawaen diad pamabaleg na second instar larva odino Sl larva ed sakey a Pale-colored pitcher plant leaf na sakey a rose blooming plant, ya walay 10 plant replicates ed kada tanaman. Saray larvae na insekto et in-clamp panamegley na clamp, tan say nakekeraan ya tissue na bulong et inala 24 tan 72 oras kayari na impeksion tan in-quick-frozen, tan saray metabolites et inala.
I-simulate so herbivorous treatment diad paraan ya mankakasakey. Say paraan et say pangusar na fabric pattern wheels pian natusok so taloran hilera na sabisabit ed kada diking na pegley na taloran sigpot ya inlaknab a bulong na tanaman legan ya ombabaleg so tela, tan tampol ya iyaplika so 1:5 Diluted Ms. Odino mangusar na gloved fingers pian isuksok so S1 OS ed sugat. Anien tan iproseso so sakey a bulong unong ed adeskribe ed tagey. Usaren so akauna lan adeskribe a paraan pian naala iray manunan metabolite tan hormone na tanaman (54).
Parad exogenous JA applications, say taloran petiole leaves na kada sakey ed anemiran rose blooming plants na kada species et tratoen na 20μl na lanolin paste ya walay 150μg MeJA (Lan + MeJA), tan 20μl na lanolin plus wound treatment (Lan + W), odino usaren so 20μl ya puron lanolin bilang kontrol. Saray bulong et inani 72 oras kayari na panagtambal, in-snap-frozen ed liquid nitrogen, tan insimpen ed -80°C anggad nausar.
Apatiran JA tan ET transgenic lines, salanti, irAOC (36), irCOI1 (55), irACO tan sETR1 (48), so abitla ed research group mi. Say irAOC et mabiskeg ya angipatnag na ibabawas na JA tan JA-Ile, bangbalet say irCOI1 et aliwan sensitibo ed JAs. No ikompara ed EV, dinmaak so JA-Ile ya akumulasyon. Ontan met, say irACO et mamawas ed produksyon na ET, tan no ikompara ed EV, say sETR1, ya agmakalikna ed ET, et mamarakep ed produksyon na ET.
Say photoacoustic laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) so uusaren pian nagawaan so panagsukat na ET ya ag-onsasakit. Kayari na panagtambal, kapaldua na saray bulong so pinuter tan inyalis ed 4-ml ya akasara ya salming a vial, tan say headspace et inabuloyan ya mantipon ed loob na 5 oras. Legan na panagsukat, kada vial et inurasan na 2 litro/oras na puron dagem ed loob na 8 minuto, ya akauna lan linmabas ed catalyst ya inter na Sensor Sense pian naekal so CO2 tan danum.
Say microarray data et inmunan nipalapag ed (35) tan in-save ed National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (accession number GSE30287). Say datos ya mipara ed saray bulong ya sengegan na W + OSMs ya panagtambal tan say ag-aderal ya kontrol so inala parad sayan impanaral. Say raw intensity et log2. Antis na statistical analysis, say baseline et inuman tan in-normalize ed koma-75 ya porsiento to ya inusar so R software package.
Say orihinal ya RNA sequencing (RNA-seq) ya datos na Nicotiana species et naala ed NCBI Short Reading Archives (SRA), say numero na proyekto et PRJNA301787, ya inreport nen Zhou et al. (39) tan ituloy unong ed nideskribe ed (56). Say raw data ya inproseso na W + W, W + OSM tan W + OSS1 ya mipara ed Nicotiana species so pinili parad panagsukimat ed sayan impanaral, tan inproseso ed onggendan a paraan: Unona, saray raw RNA-seq readings so inuman ed FASTQ format. Say HISAT2 et manguuman ed FASTQ ya magmaliw ya SAM, tan say SAMtools et manguuman ed saray SAM file ya magmaliw ya inurnos ya BAM file. Say StringTie et uusaren pian kalkulaen so gene expression, tan say paraan na pangibalikas to et walay fragments ed kada sanlibon base fragments ed kada milyon ya sequenced transcription fragments.
Say Acclaim chromatographic column (150 mm x 2.1 mm; particle size 2.2μm) ya inusar ed panagsukimat tan say 4 mm x 4 mm guard column et parehon materyales. Say onggendan ya binary gradient so inusar ed Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system: 0 anggad 0.5 minutos, isocratic 90% A [deionized water, 0.1% (v/v) acetonitrile tan 0.05% formic acid], 10% B, tan cetonitrile 0.05% formic acid); 0.5 anggad 23.5 minutos, say gradient phase et 10% A tan 90% B, unong ed inkauksoy da; 23.5 anggad 25 minutos, isocratic 10% A tan 90% B. Say flow rate et 400μl/min. Parad amin ya MS analyses, i-inject so column eluent ed quadrupole tan time-of-flight (qTOF) analyzer ya walay electrospray source ya mankikimey ed positibon ionization mode (capillary voltage, 4500 V; capillary outlet 130 V ; Drying temperature 200°C; drying airflow 10 liter/min).
Manggawa na MS / MS fragment analysis (ya tatawagen ya MS / MS) ya ag makanakana o ag nabidbir ed data pian naala so impormasyon nipaakar ed inkalapagan ya nanengneng ya metabolic profile. Say konsepto na indiscriminate MS/MS method et mandedependi ed katuaan ya say quadrupole et walaan na baleg a mass isolation window [kanian, ikonsidera so amin ya mass-to-charge ratio (m/z) signals bilang fragments]. Lapud saya, lapud ag akagawa na CE tilt so instrumenton Impact II, pigaran independentin panagsukimat so ginawa ya inusar so indaak na collision-induced dissociation collision energy (CE) values. Diad antikey, analisaen nin unona so sample panamegley na UHPLC-electrospray ionization/qTOF-MS ya uusaren so single mass spectrometry mode (saray kondisyon na abeban fragmentation ya ginawa na in-source fragmentation), ya i-scan manlapud m/z 50 anggad 1500 diad repetition frequency ya 5 Hz. Usaren so nitroheno bilang collision gas parad MS/MS analysis, tan manggawa na independentin sukat ed onggendan ya apatiran nanduruman collision-induced dissociation voltages: 20, 30, 40, tan 50 eV. Diad interon proseso na panagsukat, say quadrupole so walaan na sankabalgan ya mass isolation window, manlapud m/z 50 anggad 1500. No say front body m/z tan isolation width experiment et aka-set ed 200, say mass range et automatikon na-activate na operating software na instrumento tan 0 Da. I-scan iray piraso na masa a singa diad single mass mode. Mangusar na sodium formate (50 ml isopropanol, 200 μl formic acid tan 1 ml 1M NaOH aqueous solution) parad mass calibration. Diad pangusar ed high-precision calibration algorithm nen Bruker, say data file et na-calibrate kayari na pangipaandar ed average spectrum ed sakey a panaon. Usaren so export function na Data Analysis v4.0 software (Brook Dalton, Bremen, Germany) pian na-convert iray raw data files ed NetCDF format. Say MS/MS data set et in-save ed open metabolomics database MetaboLights (www.ebi.ac.uk) ya walay accession number to. MTBLS1471.
Say MS/MS assembly et nayarin nagawa panamegley na correlation analysis ed baetan na MS1 tan MS/MS ya kalidad na signal parad abeba tan atagey ya enerhiya na panbabanggaan tan saray balon ipapaakseb ya totontonen. Say R script et uusaren pian nagawaan so correlation analysis na distribution na precursor ed produkto, tan say C# script (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) et uusaren pian nagawaan iray totontonen.
Pian nabawasan so false positive errors ya sengegan na background noise tan false correlation ya sengegan na pakanengneng ed pigaran m/z features ed pigaran sample labat, inusar mi so “filled peak” function na R package XCMS (parad background noise correction) Nepeg ya usaren pian salatan so “NA” (undetected peak) intensity. No usaren so fill peak function, dakel ni so “0′′ ya intensity values ​​ed data set ya mangapekta ed kalkulasyon na correlation. Insan, ikompara tayo ray resulta na data processing ya naala no usaren so filled peak function tan no ag-usaren so filled peak function, tan kalkulaen tayo so background noise value base ed average ya inkorehi ya inkalkula, insan tayo salatan irayan values ​​na 0′′ intensity values kalkulado so balor na background. Inkonsidera mi met labat iray features ya say intensity da et onlampas ed taloran danay ya background value tan impasen mi iratan bilang “tuan pantok.” Parad saray kalkulasyon na PCC, saray m/z signal labat na sample precursor (MS1) tan saray fragment data sets ya walaan na ag-onkulang ed waloran tua iran peak so ikonsidera.
No say inkagrabe na precursor quality feature ed interon sample et signipikantin mikonektaan ed inkabawas na inkagrabe na parehon kalidad na feature ya nipaarap ed abeba odino atagey ya collision energy, tan say feature et ag-inlabel bilang isotope peak na CAMERA, nayarin lalon nadepinsa itan. Insan, diad pankuenta ed amin ya posiblin precursor-product pairs ed loob na 3 s (say kalkuladon retention time window parad peak retention), say correlation analysis et gawaen. No say m/z value et abeba nen say precursor value tan say MS/MS fragmentation et nagagawa ed parehon sample location ed data set bilang precursor ya nanlapuan to, et ipapasen itan ya fragment.
Base ed sarayan duaran simplin totontonen, ipulisay mi ray nibaga ya fragment ya walaan na m/z values ​​ya mas baleg nen say m/z na abitlan precursor, tan base ed posisyon na sample ya kawalaan na precursor tan say nibaga ya fragment. Posible met ya pilien iray kalidad na features ya ginawa na dakel ya in-source fragments ya ginawa ed MS1 mode bilang kandidaton precursors, kanian makagawa na redundant MS/MS compounds. Pian nabawasan so data redundancy, no say NDP similarity na saray spectra et onlampas ed 0.6, tan kabiangan iratan ed chromatogram ya “pcgroup” ya in-annotate na CAMERA, pankasakeyen tayo iratan. Diad kaunoran, pankasakeyen mi so amin ya apatiran resulta na CE ya misiglaotan ed precursor tan saray fragment ed pinal ya deconvoluted composite spectrum diad pampili na sankatageyan ya intensity peak ed amin ya kandidaton peak ya walaan na parehon m/z value ed nanduruman enerhiya na panbabanggaan. Saray ontumbok ya kundang ed panagproseso et nibase ed konsepto na composite spectrum tan ikonsidera so nanduruman kondisyon na CE ya nakaukolan pian napalaknab so posibilidad na inkaapag-apag, lapud arum a piraso so nanengneng labat ed silong na sakey ya espisipikon enerhiya na panbabanggaan.
Say RDPI (30) so inusar pian kalkulaen so inducibility na metabolic profile. Say metabolic spectrum diversity (Hj index) et nanlapud karakel na MS/MS precursors ya uusaren so Shannon entropy na MS/MS frequency distribution ya uusaren so onggendan ya equation ya indeskribe nen Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) no iner say Pij et mipara ed relatibon frequency na i-th MS/MS ed j-th sample (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Say metabolic specificity (Si index) et nidepinsa bilang say expression identity na sakey a MS/MS base ed frequency na saray sample ya ikokonsidera. Say MS/MS specificity et nakalkula bilang Si = 1t (∑j = 1tPijPilog2PijPi)
Usaren so onggendan a pormula pian nasukat so metabolome-specific δj index na kada j sample, tan say average na MS/MS specificity δj = ∑i = 1mPijSi
Saray MS/MS spectra et aka-align ed paris, tan say pamparehoan da et nakalkula base ed duaran iskor. Unona, diad pangusar na standard NDP (ya tatawagen met ya cosine correlation method), usaren so onggendan ya equation pian na-iskor so segment similarity ed baetan na spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 no iner S1 tan S2 Mipara, parad spectrum 1 tan 2, ontan met ed WS1 tan iWS2, irerepresenta toy belat base ed peak intensity ya say pandumaan na i-th common peak ed baetan na duaran spectra et kulang ed 0.01 Da. Say belat et nakalkula unong ed onggendan: W = [peak intensity] m [quality] n, m = 0.5, n = 2, unong ed insuheri na MassBank.
Say komaduan paraan na panagmarka so inusar, ya inlaktip toy panagsukimat ed inabang ya NL na MS/MS. Diad sayan gagala, inusar mi so 52 ya listaan ​​na NL ya mabetbet ya naeksperiensya legan na proseso na MS fragmentation ya mankakasakey, tan say mas espisipikon NL (data file S1) ya akauna lan in-annotate parad MS/MS spectrum na saray segundaryon metabolite na kinmapuy ya Nepenthes species ( 9, 26). Manggawa na binary vector ya 1 tan 0 parad kada MS/MS, ya mipara ed kaplesan tan anggapo na pigaran NL. Base ed Euclidean distance similarity, say NL similarity score et nakalkula parad kada paris na binary NL vectors.
Pian nagawaan so dual clustering, inusar mi so R package DiffCoEx, ya nibase ed extension na Weighted Gene Co-expression Analysis (WGCNA). Diad pangusar ed NDP tan NL scoring matrices na MS/MS spectra, inusar mi so DiffCoEx pian nakalkula so comparative correlation matrix. Say binary clustering et gagawaen diad pangi-set na “cutreeDynamic” parameter ed method = “hybrid”, cutHeight = 0.9999, deepSplit = T, tan minClusterSize = 10. Say R source code na DiffCoEx et in-download manlapud kaaruman ya file 1 nen Tesson et al. (57); Say nakaukolan ya R WGCNA software package et naromog ed https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Pian nagawaan so MS/MS molecular network analysis, kinalkula mi so paired spectral connectivity base ed NDP tan NL similarity types, tan inusar mi so Cytoscape software pian nanengneng so network topology ya uusaren so organic layout diad CyFilescape yFiles layout algorithm extension application.
Usaren so R version 3.0.1 pian nagawaan so statistical analysis ed data. Say statistical significance et inusisa ya inusar so two-way analysis of variance (ANOVA), ya tinumbokan na Tukey's honestly significant difference (HSD) post-hoc test. Pian naanalisa so pandumaan na herbivorous treatment tan say control, say two-tailed distribution na duaran grupo na sample ya parehoy variance et inaral ya inusar so Student's t test.
Parad kaaruman iran materyales parad sayan artikulo, nengnengen so http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Saya so open access ya artikulo ya ibubunog unong ed saray kondisyon na Creative Commons Attribution-Non-Commercial License, ya mangiiter na pankanawnawan mangusar, mangibunog tan manpawala lamet ed antokaman a medium, basta say unor ya pangusar et aliwan parad komersyal a gunggona tan say premis et duga so orihinal ya kimey. Reperensya.
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