Deng miyayaliwang teorya ning depensa da reng tanaman mamye lang importanting tuntunan king teorya para king pamagpalino kareng pattern ning specialized metabolism da reng tanaman, dapot deng karelang susi a ula e la pa subukan. Keni, ginamit mi ing unbiased tandem mass spectrometry (MS/MS) analysis para sistematikung abalu ing metabolome da reng tabaku a attenuated strains ibat kareng indibidwal a tanaman angga na kareng populasyun ampo kareng malapit a species, ampo miprosesu ing maragul a bilang da reng mass spectrometric feature theories base kareng compound spectra keng impormasyun. Framework para subukan ing susi a pamaghula ning optimal defense (OD) ampo moving target (MT) a teorya. Ing impormasyun a sangkap ning metabolomics ning tanaman makayagpang ya keng OD theory, pero sasalangsang ya keng main prediction ning MT theory keng metabolomics dynamics a sangkan da reng herbivores. Manibat keng micro angga keng macro evolutionary scale, ing jasmonate signal mekilala ya bilang peka-determinant ning OD, kabang ing ethylene signal mamye yang fine-tuning ning herbivore-specific response a me-annotate keng MS/MS molecular network.
Deng espesyal a metabolites a miyayaliwang istruktura ila reng manimunang makiabe king pamagbayu ning tanaman king kapaligiran, lalu na king pamagdipensa kareng kalaban (1). Ing makapagmulalang pamiyaliwa-liwa da reng espesyal a metabolismu a mayayakit kareng tanaman, meging sangkan ning dekada ning malalam a pamanyaliksik kareng dakal nang aspetu ning ecological functions, at mebuu yang makabang listahan da reng teorya king pamagdipensa da reng tanaman, a ilapin deng evolutionary ampong ecological a panyulung da reng pamiugnayan da reng tanaman-insektu. Ing empirical a pamanyaliksik mamye yang importanting tuntunan (2). Makanyan man, deng teorya ning depensa da reng tanaman e la tinuki keng normative path ning hypothetical deductive reasoning, nung nukarin deng key predictions atyu la keng pareung antas ning pamagsuri (3) at me-test la experimentally para i-advance ing tutuking cycle ning theoretical development (4). Deng limitasyun a teknikal babawal da ing pamagkolekta data kareng partikular a metabolic a kategorya at e la kayabe deng komprehensibung pamagsuri kareng espesyalisadung metabolites, inya pin makaiwas la kareng pamiyaliwa da reng kategorya a kailangan para king panyulung ning teorya (5). Ing kakulangan ning komprehensibung data ning metabolomics ampo ing karaniwan a salapi para ikumpara ing pamagprosesu ning workflow ning metabolic space pilatan da reng miyayaliwang grupu da reng tanaman makasabat ya keng scientific maturity ning larangan.
Ing bayung panyulung keng larangan ning tandem mass spectrometry (MS/MS) metabolomics malyari yang komprehensibung i-characterize ing metabolic a pamagbayu kilub ampo pilatan da reng species ning metung a sistemang clade, ampo malyari yang iyabe kareng computational a paralan para makalkula ing pamikakatulad da reng komplikadung timpla. minunang beluan keng chemistry (5). Ing pamisanmetung da reng advanced a teknolohiya keng pamagsuri ampo pamagcompute mamye yang kailangan a balangkas para keng makabang panaun a pamagsuri kareng dakal a pamaghula a gewa da reng ecological ampo evolutionary a teorya ning metabolic diversity. I Shannon (6) pepakilala ne ing teorya ning impormasyun keng mumunang besis keng kayang seminal a artikulu inyang 1948, a miglalang pundasyun para keng mathematical analysis ning impormasyun, na megamit kareng dakal a larangan bukud keng orihinal nang aplikasyun. keng genomics, ing teorya ning impormasyun matagumpe yang megamit para abalu ing sequence conservative a impormasyun (7). keng pamanyaliksik keng transcriptomics, ing teorya ning impormasyun susuri na ing kabilugan a pamagbayu keng transcriptome (8). keng minunang pamanyaliksik, ginamit mi ing statistical framework ning teorya ning impormasyun keng metabolomics para ilarawan ing metabolic expertise ning tissue level kareng tanaman (9). Keni, pisamut mi ing MS/MS-based workflow keng statistical framework ning teorya ning impormasyun, a mipakilala keng metabolic diversity keng karaniwan a salapi, para ikumpara la reng susi a ula ning teorya ning depensa da reng tanaman ning metabolome a gewa da reng herbivores.
Deng theoretical frameworks ning depensa da reng tanaman keraklan parehu lang miyabe-abe at malyari lang miyabe kareng adwang kategorya: detang susubukan dang ipaliwanag ing pamikalat da reng metabolites a partikular king tanaman base kareng depensang function, antimo ing optimal defense (OD) (10), moving target (MT) (11) ) at appearance (12) theory, kabang deng aliwa manintun lang mechanical a paliwanag nung makananu lang makaapektu deng mechanical a pamagbayu king resource ing pamandagul ning tanaman ampo ing pamitipun da reng specialized metabolites, antimo ing carbon: nutrient balance hypothesis (13), growth rate hypothesis (14 ), ampo ing growth and differentiation equilibrium hypothesis (15). Deng adwang set da reng teorya atyu la keng miyayaliwang antas ning pamagsuri (4). Makanyan man, adwa lang teorya a maki kaugnayan kareng defensive functions keng functional level ing mangibabaw keng pamisabi-sabi tungkul kareng plant constitutive ampo inducible defenses: OD theory, na mag-assume na deng tanaman mamuhunan la keng karelang mamasang chemical defenses nung kailangan la, alimbawa, nung ila ing ingest Nung ing animal a dikut ataki ne, inya, agpang keng posibilidad ning hinaharap a compound a maki defensive a function metung yang depensa (10); ing MT hypothesis mumusuk ya na alang axis ning pamagbayu ning direksyun ning metabolite, pero ing metabolite magbayu yang random, inya sa’t gagawang posibilidad ing Obstruct ing metabolic “motion target” ning pamaglusub kareng herbivores. keng aliwang salita, deng adwang teorya gagawang salangsang a ula tungkul keng metabolic remodeling a malilyari kaybat ning pangaatake da reng herbivores: ing relasyun ning unidirectional accumulation da reng metabolites a maki defensive function (OD) ampo reng non-directed metabolic changes (MT) (11 ).
Ing OD ampo MT hypotheses e mu kabud ing induced a pamagbayu keng metabolome, nune pati na ing ecological ampo evolutionary a bunga ning pamitipun da reng metabolites a reni, antimo reng adaptive costs ampo benepisyu da reng metabolic a pamagbayu keng metung a partikular a ecological a kapaligiran (16). Agyang parehu lang akilala deng hypothesis ing defensive function da reng specialized metabolites, na malyari o ali maging mamasa, ing susi a pamaghula na makayaliwa kareng OD ampo MT hypotheses atyu ya keng direksyun da reng induced metabolic changes. Ing pamaghula ning OD theory ing tinggap na ing peka-experimental a pansin angga ngeni. Deng test a reni kayabe la reng pamagaral kareng direkta o e direktang depensa da reng miyayaliwang tissue da reng partikular a compound kareng greenhouse ampo kareng natural a kundisyun, ampo reng pamagbayu keng yugtu ning pamangawang tanaman (17-19). Makanyan man, angga ngeni, pauli ning kakulangan ning workflow ampo statistical framework para keng global a komprehensibung pamagsuri keng metabolic diversity ning nanu mang organismu, ing pekamaulaga a pamiyaliwa ning pamaghula kareng adwang teorya (yapin ing direksyun da reng pamagbayu keng metabolic) manatili yang e masubukan. keni, mamye kaming makanining pamagsuri.
Metung kareng pekamaulaga a katangian da reng tanaman-specific metabolites yapin ing karelang sobrang pamiyaliwa-liwa ning istruktura kareng sablang antas manibat kareng metung a tanaman, populasyun angga na kareng parehung species (20). Dakal a pamagbayu keng dagul da reng specialized metabolites ing mayayakit keng populasyun, kabang deng masikan a pamiyaliwa keng kalidad keraklan manatili la keng species level (20). Uli na niti, ing metabolic diversity da reng tanaman ing pekamaulaga nang aspetu ning functional diversity, a magpasyag king pamagbayu kareng miyayaliwang niches, lalu na deng niches a ating miyayaliwang posibilidad ning pamaglusub da reng espesyal a insektu ampong karaniwan a herbivores (21). Manibat keng Fraenkel's (22) groundbreaking article tungkul kareng sangkan ning panga-atyu da reng plant-specific metabolites, ing pamiugnayan kareng miyayaliwang insektu metung yang importanting selection pressures, at deng pamiugnayan a reni paniwalan dang meging sangkan da reng tanaman kabang mag-evolve la. Metabolic pathway (23). Ing pamiyaliwa da reng species keng pamiyaliwa-liwa da reng specialized metabolites malyari ya namang magreflect keng physiological balance a makasuglung keng constitutive ampo inducible a depensa da reng tanaman laban kareng herbivorous strategies, uling deng adwang species maralas lang negatibung makaugne king metung at metung (24). Agyang malyari yang maging kapakibatan ing pamagpanatili king masanting a depensa king sablang panaun, ing panaun a pamagbayu king metabolic a makasuglung king depensa mamye yang malino pakinabang king pamagpaintulut kareng tanaman a maglaan kareng maulagang pibandyan kareng aliwang puhunan king katawan (19, 24), at makaiwas king pamangailangan king simbiosis. Collateral damage (25). Dagdag pa, deng reorganisasyun da reng specialized metabolites a sangkan da reng insektung herbivores malyari lang magdalang makasira a pamikalat keng populasyun (26), at malyari lang magreflect kareng direktang pamamasa da reng maragul a natural a pamagbayu keng jasmonic acid (JA) signal, na malyaring manatili keng populasyun. Deng matas ampo mababang JA signals ila reng trade-offs ning depensa laban kareng herbivores ampo ing kompetisyon kareng specific species (27). Dagdag pa, deng specialized metabolite biosynthetic pathways dumalan lang mabilis a pangawala ampo pamagbayu kabang mag evolution la, a magresulta keng patchy metabolic distribution kareng malapit a species (28). Deting polymorphisms mabilis lang mitatag bilang pakibat keng pamagbayu da reng herbivore patterns (29), na mangabaldugan na ing pamagbayu da reng herbivore communities metung yang susi a sangkan ning metabolic heterogeneity.
keni, a-solve mi la reng makatuking problema. (I) makananu neng i-reconfigure ning herbivorous a insektu ing metabolome ning tanaman? (Ii) Nanu la reng peka-impormasyun a sangkap ning metabolic plasticity a malyaring i-quantify para asubukan ing pamag-predict ning long-term defense theory? (Iii) Nung i-reprogram ya ing metabolome ning tanaman king paralan a makabukud king attacker, nung makanyan, nanu ing papil ning hormone ning tanaman king pamag-tailored king metung a partikular a metabolic a pakibat, at nanu la reng metabolites a mag-ambag king species specificity ning depensa? (Iv) Uling deng predictions a gewa da reng dakal a defense theories malyari lang mipalapad kareng eganaganang antas ning biological tissues, kitnan mi nung makananu ya ka-consistent ing metabolic response a ibat keng internal a pamagkumpara keng interspecies a pamagkumpara? Para kaniti, sistematiku ming abalu ing metabolome ning bulung ning nikotina ning tabako, a metung a ecological a modelung tanaman a maki mabandi a specialized metabolism, at epektibo ya laban kareng larvae da reng adwang katutubung herbivore, Lepidoptera Datura (Ms) ( Very aggressive, mainly eaten) King Solanaceae ampong Spodoptera (Spodopteral), metung lang uri ning bulung a worm “genus”, kareng tanaman a host da reng Solanaceae ampo reng aliwang host da reng aliwang genera ampo pamilya Pamangan da reng tanaman. linawe mi ing MS/MS metabolomics spectrum ampo kinua mi ing information theory statistical descriptors para ikumpara la reng OD ampo MT theories. Gawa kang specificity maps para abalu mu ing pangatau da reng key metabolites. Ing pamagsuri mipalapad ya keng katutubung populasyun ning N. nasi ampo kareng malapit a species ning tabaku para mas masuri ing covariance pilatan ning plant hormone signaling ampo ing OD induction.
Para akit mi ing kabilugan a mapa tungkul keng plasticity ampo istruktura ning metabolome ning bulung ning herbivorous tobacco, ginamit mi ing minunang megawa nang analysis ampo calculation workflow para komprehensibung makakolekta ampo maka-deconvolute kareng high-resolution data independent MS/MS spectra ibat kareng plant extracts ( 9). Iting undifferentiated a paralan (ausan dang MS/MS) malyari yang gawang non-redundant compound spectra, na malyaring gamitan para kareng eganaganang compound-level a pamagsuri a makalarawan keni. Deting deconvoluted plant metabolites miyayaliwa la uri, atin lang dinalan anggang libu-libung metabolites (mga 500-1000-s/MS/MS keni). keni, isipan mi ing metabolic plasticity keng framework ning teorya ning impormasyun, ampo i-quantify ing diversity ampo professionalism ning metabolome base keng Shannon entropy ning metabolic frequency distribution. Gamit ing minunang mepatupad a formula (8), kinalkula mi ing metung a set da reng indicator a malyaring gamitan para abalu ing metabolome diversity (Hj indicator), metabolic profile specialization (δj indicator) ampo ing metabolic specificity ning metung a metabolite (Si indicator). Dagdag pa, ginamit mi ing Relative Distance Plasticity Index (RDPI) para abalu mi ing metabolome inducibility da reng herbivores (Figure 1A) (30). Kilub na niting statistical framework, tuturing mi ing MS/MS spectrum bilang ing basic information unit, at iprosesu mi ing relatibong karakal ning MS/MS king metung a frequency distribution map, at kaybat gamitan mi ing Shannon entropy para abalu mi ing metabolome diversity ibat kaniti. Ing metabolome specialization susukat ya keng average specificity ning metung a MS/MS spectrum. Uli na niti, ing pamagtas ning karakal da reng mapilan a MS/MS classes kaybat ning herbivore induction mibayu ya keng spectral inducibility, RDPI ampo specialization, yapin ing pamagtas ning δj index, uling mas dakal a specialized metabolites ing gagawan da ampo ing matas a Si index ing gagawan da. Ing pamagbaba ning Hj diversity index papakit na ing bilang da reng MS/MS a me-generate mabawas ya, o ing profile frequency distribution magbayu ya king e masyadung pare-pareung direksyun, kabang mabawas ing kayang kabilugan a e kasiguraduan. Kapamilatan ning Si index calculation, malyari yang i-highlight nung sanung MS/MS ing mipapalwal kareng mapilan a herbivores, king kabaligtaran, nung sanung MS/MS ing e makibat king induction, a metung a susi a indicator para akilala ing MT ampong OD prediction.
(A) Statistical descriptors a ginamit para kareng herbivorous (H1 anggang Hx) MS/MS data-inducibility (RDPI), diversity (Hj index), specialization (δj index) ampo metabolite specificity (Si index). Ing pamagtas ning degree ning specialization (δj) papakit na, keng karaniwan, mas dakal a herbivorous specific metabolites ing magawa, kabang ing pamagbaba keng diversity (Hj) papakit na ing pamagbaba keng pamaglalang da reng metabolites o e pantas a pamikalat da reng metabolites keng distribution map. Ing Si value tatas ne nung ing metabolite specific ya keng metung a kundisyun (keni, herbivorous) o kaya naman manatili ya keng pareung antas. (B) Conceptual diagram ning defense theory prediction gamit ing information theory axis. Ing OD theory sasabyan na ing herbivore attack dagdagan na ing defense metabolites, inya sa’t dagdagan ne ing δj. At ing parehung oras, ing Hj bababa ya uling ing profile mi-reorganisa ya papunta keng mabawas a uncertainty ning metabolic information. Ing MT theory sasabyan na ing pangaatake da reng herbivores magdalang non-directional a pamagbayu keng metabolome, inya sa’t daragul ing Hj bilang indicator ning daragul a metabolic information uncertainty at magdalang random a pamikalat ning Si. Migmungkahi kami muring metung a mixed model, ing pekamasanting a MT, nung nukarin deng mapilan a metabolites a maki mas matas a defensive values ​​partikular lang miragdagan (matas a Si value), kabang deng aliwa magpakit lang random a pakibat (mas mababa a Si value).
Gamit ing information theory descriptors, aintindian mi ing OD theory para abalu mi na ing herbivore-induced special metabolite changes keng e induced constitutive state magdalang (i) pamagtas keng metabolic specificity (Si index) a magmaneho keng metabolic specificity (δj index) Ing pamagtas da reng) mapilan a special metabolite groups a maki mas matas a ulaga ning depensa, ampo (ii) ing pamagbaba ning diversity (Hj) ning metabolite uling keng index pamagbayu ning metabolic frequency distribution para mas dakal a leptin body distribution. King antas ning metung a metabolite, metung a maka-order a Si distribution ing asahan, nung nukarin ing metabolite dagdagan ne ing Si value agpang king kayang defense value (Figure 1B). King linya ayni, ipaliwanag mi ing MT theory para abalu mi na ing excitation magdalang (i) non-directional a pamagbayu kareng metabolites a magresulta keng pamagbaba ning δj index, ampo (ii) pamagtas ning Hj index pauli ning pamagtas ning metabolic uncertainty. O kaya ing randomness, na malyaring i-quantify kapamilatan ning Shannon entropy keng porma ning generalized diversity. Para keng metabolic composition, ing MT theory ing magpredict keng random distribution ning Si. Nung isipan tamung deng mapilan a metabolites atyu la lalam ning partikular a kundisyun lalam ning partikular a kundisyun, at deng aliwang kundisyun e la lalam ning partikular a kundisyun, at ing karelang ulaga ning depensa makayagpang ya king kapaligiran, migmungkahi kami muring metung a modelung depensa a maki mixed defense, nung nukarin ing δj ampong Hj mipamye la king adwang along Si Increase king sablang direksyun, deng mapilan mung grupu da reng metabolite, a maki mas matas a ulaga ning depensa, partikular lang miragdagan kabang deng aliwang Siure atin lang random a pamikalat (Figure a 1B).
Para asubukan ing me-redefined a defense theory prediction keng axis ning information theory descriptor, peparagul mi la reng expert (Ms) o generalist (Sl) herbivore larvae kareng bulung ning Nepenthes pallens (Figure 2A). Gamit ing MS/MS analysis, menakit kaming 599 non-redundant MS/MS spectra (data file S1) ibat kareng methanol extracts ning leaf tissue a tinipun kaybat dang pepakan deng caterpillar. Ing pamangamit kareng RDPI, Hj, ampo δj indexes para akit ing pamagbayu ning laman ning impormasyun keng MS/MS configuration files papakit na ing makawili a pattern (Figure 2B). Ing pangkabilugan a trend yapin ing, antimo ing makalarawan keng information descriptor, kabang deng ulad patuloy lang mamangan bulung, ing degree ning eganaganang metabolic reorganisation daragul ya keng panaun: 72 oras kaybat nang mengan ning herbivore, ing RDPI daragul yang maragul. Nung ikumpara me keng undamaged control, ing Hj mebawas yang maragul, na meging sangkan ning daragul a degree ning specialization ning metabolic profile, na me-quantify ya keng δj index. Ing malilyaring trend ayni makayagpang ya kareng predictions ning OD theory, pero ali ya makayagpang kareng main predictions ning MT theory, na maniwala na ing random (non-directional) a pamagbayu keng metabolite levels magagamit ya bilang defensive camouflage (Figure 1B). Agyang miyayaliwa ing oral secretion (OS) elicitor content ampo ing pamangan da reng adwang herbivore, ing karelang direktang pamangan meging sangkan ning parehung pamagbayu keng direksyun ning Hj ampo δj kilub ning 24-oras ampo 72-oras a panaun ning pamamupul. Ing metung mung pamiyaliwa milyari ya keng 72 oras ning RDPI. Nung ikumpara me keng Ms feeding, ing kabilugan a metabolism a gewa ning Sl feeding mas matas ya.
(A) Experimental design: deng karaniwan a babi (S1) o expert (Ms) herbivores pakanan do reng desalinated a bulung da reng tanaman a pitsel, kabang para keng simulated herbivory, ing OS ning Ms (W + OSMs) gagamitan de para asikasu ing pamanuktuk kareng standardized leaf positions a sugat. S1 (W + OSSl) larvae o danum (W + W). Ing control (C) metung yang bulung a e mesira. (B) Inducibility (RDPI kumpara keng control chart), diversity (Hj index) ampo specialization (δj index) index a mekwa para keng special metabolite map (599 MS/MS; data file S1). Deng asterisks papakit da ing makabaldugan a pamiyaliwa da reng direct herbivore feeding ampo ing control group (Student's t-test with paired t-test, *P<0.05 and ***P<0.001). ns, ali importanti. (C) Time resolution index ning main (blue box, amino acid, organic acid ampo asukal; data file S2) ampo special metabolite spectrum (red box 443 MS/MS; data file S1) kaybat ning simulated herbivory treatment. Ing kule band mangabaldugan yang 95% confidence interval. Ing asterisk papakit na ing makabaldugan a pamiyaliwa ning panulu ampo ing control [quadratic analysis of variance (ANOVA), kaybat ning Tukey's honestly significant difference (HSD) para kareng post hoc multiple comparisons, *P<0.05, **P<0.01 At *** P <0.001]. (D) Specialization da reng scatter plots ampo reng special a metabolite profiles (paulit-ulit a sample a maki miyayaliwang pamanulu).
Para abalu nung ing herbivore-induced remodeling keng metabolome level mayayakit ya keng pamagbayu keng level da reng indibidwal a metabolites, mumuna mi lang tinuknang deng metabolites a minunang me-aral kareng bulung ning Nepenthes pallens na ating proven herbivore resistance. Deng phenolic amides ila reng hydroxycinnamamide-polyamine conjugates na titipun keng herbivory process da reng insektu ampo balu dang babawasan ing performance da reng insektu (32). Penintun mi la reng precursors ning katumbas a MS/MS at linawe mi la reng karelang cumulative kinetic curves (Figure S1). E makatula, deng phenol derivatives na ali direktang makiabe keng depensa laban kareng herbivores, antimo ing chlorogenic acid (CGA) ampo ing rutin, mibaba la kaybat ning herbivory. Ing pamiyaliwa, deng herbivores malyari lang gawang phenol amides a masikan. Ing patuloy a pamangan da reng adwang herbivores meging sangkan ning halus pareung excitation spectrum da reng phenolamides, at iting pattern malino ya para keng de novo synthesis da reng phenolamides. Ing parehung phenomenon mayayakit ya nung mag explore ya keng 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs) pathway, na magproduce keng maragul a bilang da reng acyclic diterpenes a maki epektibung anti-herbivore functions (33), nung nukarin i Ms Feeding with Sl triggered ing kalupa nang profile (Figure S1)).
Ing posibling disbentaha ning direct herbivore feeding experiments yapin ing pamiyaliwa ning leaf consumption rate ampo ing oras ning pamangan da reng herbivore, na gagawang masakit ing pamaglako kareng herbivore-specific a epektu a darala da reng sugat ampo herbivore. Para mas masanting a solusyunan ing herbivore species specificity ning induced leaf metabolic response, simulate mi ing pamangan da reng Ms ampo Sl larvae kapamilatan ning agad a pamag-apply kareng bayung tinipun a OS (OSM ampo OSS1) keng standard puncture W da reng pare-pareung posisyun da reng bulung. Ing pamamaralan ayni awsan deng W + OS treatment, at iti standardize ne ing induction kapamilatan ning eksaktung timing ning pamagumpisa ning pakibat a pepalwal ning herbivore a alang sangkan a makalitu a epektu ning pamiyaliwa king rate o dagul ning pangawala ning tissue (Figure 2A) (34). Gamit ing MS/MS analysis ampo calculation pipeline, menakit kaming 443 MS/MS spectra (data file S1), na mipapalibut kareng spectra a minunang me-assemble ibat kareng direct feed experiments. Ing information theory analysis na niting MS/MS data set pepakit na ing pamag-reprogram kareng leaf-specialized metabolomes kapamilatan ning pamag-simulate kareng herbivores pepakit na ing OS-specific inducements (Figure 2C). Partikular, kumpara keng OSS1 a pamanulu, ing OSM meging sangkan ning pamagpaunlad ning metabolome specialization kilub ning 4 oras. Dapat papansinan na nung ikumpara me keng direct herbivore feeding experimental data set, ing metabolic kinetics a akakit keng two-dimensional a espasyu gamit ing Hj ampo δj bilang coordinates ampo ing directionality ning metabolome specialization bilang pakibat keng simulated herbivore treatment keng panaun. Dagdagan ing consistent (Figure 2D). Kasabe na niti, me-quantify mi ing laman da reng amino acids, organic acids ampo sugars (data file S2) para abalu mi nung ining targeted a pamagtas keng metabolome expertise uli ning pamagbayu ning central carbon metabolism bilang pakibat kareng simulated herbivores ( Figure S2). Para mas masanting a apaliwanag ining pattern, mas linawe mi ing metabolic accumulation kinetics da reng minunang mesabing phenolamide ampo 17-HGL-DTG pathways. Ing OS-specific induction da reng herbivores mibayu ya keng differential rearrangement pattern kilub ning phenolamide metabolism (Figure S3). Deng phenolic amides a maki coumarin ampo caffeoyl moieties mas buri da ing OSS1, kabang deng OSMs mag trigger lang metung a specific induction da reng ferulyl conjugates. Para keng 17-HGL-DTG pathway, ing differential OS induction kapamilatan ning downstream malonylation ampo dimalonylation products meyakit ya (Figure S3).
Kaybat, inaral mi ing OS-induced transcriptome plasticity gamit ing time-course microarray data set, a magsimulate king pamangamit kareng OSMs para gamutan la reng bulung da reng bulung ning tanaman a rosette kareng herbivores. Ing sampling kinetics basically overlap ya keng kinetics a ginamit kening metabolomics study (35). Nung ikumpara me ing metabolome reconfiguration nung nukarin ing metabolic plasticity partikular yang daragul keng panaun, akit tamu ing transient transcription bursts kareng bulung a pepalwal ning Ms, nung nukarin ing transcriptome inducibility (RDPI) ampo ing specialization (δj) atyu la keng 1 Atin yang makabaldugan a pamagtas kareng oras, ampo diversity (Hj) keng oras ayni, ing pamagpahayag ning BMP1, mebaba ya keng pamagrelaks ning pamagrelaks transcriptome specialization (Figure S4). Deng pamilyang metabolic genes (antimo ing P450, glycosyltransferase, ampo BAHD acyltransferase) makiabe la keng prosesu ning pamagtipun kareng espesyal a metabolites ibat kareng structural units a menibatan keng mumunang metabolismo, agpang keng minunang high-specialization model. Bilang case study, ing phenylalanine pathway me-analisa ya. Ing pamagsuri mekumpirma na ing core genes keng phenolamide metabolism matas lang OS-induced kareng herbivores kumpara kareng e maka-akit a tanaman, ampo malapit lang maka-align keng karelang expression patterns. Ing transcription factor MYB8 ampo reng structural genes PAL1, PAL2, C4H ampo 4CL keng babo ning pathway ayni pepakit da ing mabilis a pamagumpisa ning transcription. Deng acyltransferases na maki papel keng tauling pamisanmetung ning phenolamide, antimo ing AT1, DH29, ampo CV86, magpakit lang makabang upregulation pattern (Figure S4). Ing makatuking obserbasyun papakit na ing mumunang pamagumpisa ning transcriptome specialization ampo ing tauling pamagpaunlad ning metabolomics specialization metung lang coupled mode, na malyaring uli ning synchronous regulatory system na magumpisa keng masikan a defense response.
Ing pamagbayu keng plant hormone signaling gagawan neng regulatory layer na mag integrate kareng herbivorous a impormasyun para i-reprogram ing physiology da reng tanaman. Kaybat ning herbivore simulation, sinukat mi ing cumulative dynamics da reng key plant hormone categories at ikit mi ing temporal co-expression pilatan da [Pearson correlation coefficient (PCC)> 0.4] (Figure 3A). Antimo ing inaasahan, deng hormone ning tanaman a maki kaugnayan keng biosynthesis makasuglung la keng kilub ning network ning hormone ning tanaman a co-expression. Dagdag pa, ing metabolic specificity (Si index) maka-map ya keng network ayni para i-highlight la reng tanaman a hormones a ibat kareng miyayaliwang panulu. Adwa lang main areas ning herbivorous specific response ing makaguhit: ing metung yapin ing JA cluster, nung nukarin ing JA (ing biologically active form JA-Ile) ampo reng aliwang JA derivatives papakit da ing pekamatas a Si score; ing metung yapin ing ethylene (ET). Ing gibberellin pepakit na ing katamtaman a pamagtas keng herbivore specificity, kabang deng aliwang tanaman a hormone, antimo ing cytokinin, auxin, ampo abscisic acid, atin lang mababang induction specificity para kareng herbivore. Nung ikumpara me keng pamangamit W + W mu, ing pamagdagul ning peak value da reng JA derivatives kapamilatan ning OS application (W + OS) malyari yang maging masikan a specific indicator da reng JAs. E asahan, ing OSM ampo ing OSS1 a ating miyayaliwang elicitor content balu da na maging sangkan ning parehung pamitipun ning JA ampo ing JA-Ile. Ing pamiyaliwa ning OSS1, ing OSM partikular ya at masikan a mipapalwal kareng OSMs, kabang ing OSS1 e na amplify ing pakibat da reng basal a sugat (Figure 3B).
(A) Co-expression network analysis base keng PCC calculation ning herbivore-induced plant hormone accumulation kinetics simulation. Ing node mangabaldugan yang metung a hormone ning tanaman, at ing sukad ning node mangabaldugan yang Si index a partikular keng hormone ning tanaman pilatan da reng pamanulu. (B) Ing pamitipun ning JA, JA-Ile ampo ET kareng bulung a sangkan ning miyayaliwang pamanulu a pepakit da reng miyayaliwang kule: apricot, W + OSM; asul, W + OSSl; kule kule, W + W; gray, C (control) . Deng asterisks papakit da ing makabaldugan a pamiyaliwa ning pamanulu ampo ing pamagkontrol (two-way ANOVA a tinuki king Tukey HSD post hoc multiple comparison, *** P <0.001). Information theory analysis ning (C)697 MS/MS (data file S1) keng JA biosynthesis ampo ing impaired perception spectrum (irAOC ampo irCOI1) ampo ing (D)585 MS/MS (data file S1) keng ETR1 a maki impaired ET signal Adwa lang simulated herbivore treatments a mekapag trigger plant line ampo empty vehicle control (EV) plants. Deng asterisk papakit da ing makabaldugan a pamiyaliwa ning W+OS a pamanulu ampo ing e mesira a kontrol (two-way ANOVA a tinuki king Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 ampo ***P<0.001). (E) Makalat a graph ning makalat a oposisyon keng pamag-espesyalisa. Deng kule mangabaldugan la kareng miyayaliwang genetically modified a strain; deng symbol mangabaldugan la kareng miyayaliwang paralan ning pamanulu: triangle, W + OSS1; rectangle, W + OSM; gulis C
Kaybat, gamitan mi ing genetically modified strain ning attenuated Nepenthes (irCOI1 ampo sETR1) keng susi a lakbang ning JA ampo ET biosynthesis (irAOC ampo irACO) ampo pamangilala (irCOI1 ampo sETR1) para asuri ing metabolism da reng adwang tanaman a hormone kareng herbivores Ing ambag ning pamag-reprogram. Agpang kareng minunang eksperimentu, kinumpirma mi ing induction ning herbivore-OS kareng empty carrier (EV) a tanaman (Figure 3, C to D) ampo ing kabilugan a pamagbaba ning Hj index a sangkan ning OSM, kabang ing δj index meragul ya. Ing pakibat mas maragul ya kesa keng pakibat a pepalwal ning OSS1. Ing adwang linya ning graph gamit ing Hj ampo δj bilang coordinates papakit ne ing specific deregulation (Figure 3E). Ing peka-malino a trend yapin ing kareng strain a alang JA signal, ing metabolome diversity ampo ing pamagbayu king specialization a sangkan da reng herbivores halus lubus lang mewala (Figure 3C). Ing pamiyaliwa, ing silent ET perception kareng sETR1 a tanaman, agyang ing kabilugan a epektu kareng pamagbayu keng herbivorous metabolism mas mababa ya kesa keng JA signaling, babawasan na ing pamiyaliwa da reng Hj ampo δj indices pilatan ning OSM ampo OSS1 excitations (Figure 3D ampo Figure S5). . Iti papakit na dagdag keng core function ning JA signal transduction, ing ET signal transduction magsilbi ya namang fine-tuning ning species-specific metabolic response da reng herbivores. Agpang keng fine-tuning function, ala yang pamagbayu ing kabilugan a metabolome inducibility kareng tanaman a sETR1. King aliwang banda, nung ikumpara la kareng tanaman a sETR1, deng tanaman a irACO mika parehu lang pangkabilugan a amplitudes ning pamagbayu king metabolic a sangkan da reng herbivores, dapot pepakit da ing makabaldugan a pamiyaliwa da reng Hj ampong δj scores pilatan da reng OSM ampong OSS1 a hamon (Figure S5).
Para akilala la reng specialized metabolites na atin importanting ambag keng species-specific a pakibat da reng herbivores ampo a-fine-tune ing karelang produksyun kapamilatan da reng ET signals, ginamit mi ing minunang megawa nang structural MS/MS a paralan. Ing paralan ayni magdependi ya keng bi-clustering a paralan para abalu ing metabolic family ibat kareng MS/MS fragments [normalized dot product (NDP)] ampo ing similarity score base keng neutral loss (NL). Ing MS/MS data set a megawa kapamilatan ning pamagsuri kareng ET transgenic lines mekagawa yang 585 MS/MS (data file S1), a meresolba kapamilatan ning pamag-cluster kareti kareng pitung main MS/MS modules (M) (Figure 4A) . Deng mapilan kareng modules a reni masikan lang makabalut kareng minunang mekilalang special metabolites: alimbawa, M1, M2, M3, M4 ampo M7 dakal la kareng miyayaliwang phenol derivatives (M1), flavonoid glycosides (M2), acyl sugars (M3 ampo M4), ampo 17-HGL-DTG (M7). Dagdag pa, ing metabolic specific information (Si index) ning metung a metabolite keng balang module makalkula ya, at ing kayang Si distribution akit ya keng intuitively. king makuyad a salita, ing MS/MS spectra a magpakit matas a herbivory ampo genotype specificity maki katangian ya kareng matas a Si values, ampo ing kurtosis statistics papakit na ing pamikalat ning balahibo keng wanan a sulok ning buntuk. Metung kareng lean colloid distribution ing ikit da keng M1, nung nukarin ing phenol amide pepakit ne ing pekamatas a Si fraction (Figure 4B). Ing minunang mebanggit a herbivorous inducible 17-HGL-DTG keng M7 pepakit na ing katamtaman a Si score, a magpakit keng katamtaman a degree ning differential regulation pilatan da reng adwang uri ning OS. Ing pamiyaliwa, keraklan kareng constitutively produced specialized metabolites, antimo ing rutin, CGA, ampo reng acyl sugars, ila reng pekamalating Si scores. Para mas masanting a abalu ing structural complexity ampo ing Si distribution pilatan da reng special metabolites, metung a molecular network ing megawa para kareng balang module (Figure 4B). Ing metung a importanting pamaghula ning OD theory (me-summarize ya keng Figure 1B) yapin ing ing pamag-reorganisa kareng special metabolites kaybat ning herbivory dapat magdalang one-way a pamagbayu kareng metabolites a maki matas a ulaga ning depensa, lalu na kapamilatan ning pamagdake king karelang specificity (as opposed to random distribution) Mode) Defensive metabolite a me-predict ning MT theory. keraklan kareng phenol derivatives a mitipun keng M1 makaugne la keng pamagbaba ning performance da reng insektu (32). Nung ikumpara la reng Si values ​​kareng M1 metabolites pilatan da reng induced leaves ampo reng constituent leaves da reng EV control plants kilub ning 24 oras, ikit mi na ing metabolic specificity da reng dakal a metabolites kaybat da reng herbivory insects atin yang makabaldugan a pamagtas a trend (Figure 4C). Ing specific a pamagtas ning Si value meyakit yamu kareng defensive phenolamides, pero alang pamagtas keng Si value a meyakit kareng aliwang phenols ampo kareng e balu a metabolites a kayabe keng module ayni. Iti metung yang specialized model, na maki kaugnayan keng OD theory. Deng manimunang ula da reng pamagbayu ning metabolic a sangkan da reng herbivore parehu la. Para asuri nung ining partikularidad ning phenolamide spectrum me-induce ya keng OS-specific ET, linawe mi ya ing metabolite Si index at meging sangkan ning differential expression value pilatan ning OSM ampo OSS1 keng EV ampo sETR1 genotypes (Figure 4D ). keng sETR1, ing phenamide-induced a pamiyaliwa ning OSM ampo OSS1 mebawas yang maragul. Ing bi-clustering a paralan megamit ya naman kareng MS/MS data a tinipun kareng strain a alang sapat a JA para abalu ing main MS/MS modules a maki kaugnayan keng JA-regulated metabolic specialization (Figure S6).
(A) Ing resulta ning pamag-cluster ning 585 MS/MS base keng shared fragment (NDP similarity) ampo ing shared neutral loss (NL similarity) magresulta ya keng module (M) na consistent ya keng balu nang pamilya ning compound, o keng e balu o e masanting a metabolisyung Metabolite composition. king siping ning balang module, ing metabolite (MS/MS) specific (Si) distribution mipalabas ya. (B) Modular molecular network: Deng nodes mangabaldugan lang MS/MS ampo deng gilid, NDP (pula) ampo NL (asul) MS/MS scores (cut-off,> 0.6). Ing graded metabolite specificity index (Si) mekulay ya base keng module (kayli) ampo me-map ya keng molecular network (wanan). (C) Module M1 ning EV plant keng constitutive (control) ampo induced state (simulated herbivore) keng 24 oras: molecular network diagram (Si value ing node size, defensive phenolamide maka highlight ya keng blue). (D) Ing M1 molecular network diagram ning spectrum line sETR1 a maki impaired EV ampo ET perception: ing phenolic compound a mipaintagun keng green circle node, ampo ing makabaldugan a pamiyaliwa (P value) pilatan ning W + OSM ampo W + OSS1 a pamanulu bilang Node size. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, N-ferulic acid ester-uric acid; FS, N-ferulyl-spermidine; CoP, N', N “-Coumarolyl-tyrosine; DCS, N’, N”-dicaffeoyl-spermidine; CFS, N', N”-caffeoyl, feruloyl-spermidine; Lycium barbarum keng wolfberry Son; Nick. O-AS, O-acyl sugar.
Pepalapad mi pa ing pamagsuri ibat keng metung a attenuated Nepenthes genotype papunta kareng natural a populasyun, nung nukarin ing masikan a intraspecific a pamagbayu kareng herbivorous JA levels ampo reng specific metabolite levels minunang misulat la kareng natural a populasyun (26). Gamitan me ining data set para akit me ing 43 germplasms. Deting germplasm bibilugan de reng 123 species da reng tanaman ibat king N. pallens. Deting tanaman mekwa la kareng bini a tinipun kareng miyayaliwang katutubung tuknangan keng Utah, Nevada, Arizona, ampo California (Figure S7), kinalkula mi ing metabolome diversity (keni ausan deng population level) β diversity) ampo ing specialization a sangkan ning OSM. Agpang kareng minunang pamagaral, menakit kaming maragul a pamagbayu keng metabolic kareng Hj ampo δj axes, a papakit na deng germplasms atin lang makabaldugan a pamiyaliwa keng plasticity da reng karelang metabolic a pakibat kareng herbivores (Figure S7). Ing organisasyun ayni makaganaka ya kareng minunang obserbasyun tungkul keng dynamic range da reng pamagbayu da reng JAs a sangkan da reng herbivores, at menatili yang matas a ulaga keng metung a populasyun (26, 36). Gamit ing JA ampo JA-Ile para subukan ing kabilugan a lebel ning correlation pilatan ning Hj ampo δj, ikit mi na atin yang makabaldugan a positibong correlation ing pilatan ning JA ampo ing metabolome β diversity ampo specialization index (Figure S7). Iti magpasyag na ing herbivore-induced heterogeneity keng JAs induction a akit keng populasyun malyari yang maging sangkan ning key metabolic polymorphisms a sangkan ning pamamili kareng insektung herbivores.
Deng minunang pamagaral pepakit da na deng uri ning tabaku miyayaliwa la keng uri ampo keng relatibung pamagdependi keng induced ampo constitutive metabolic defenses. Paniwalan da na deng pamagbayu keng anti-herbivore signal transduction ampo defense capabilities miregulate la keng pressure ning populasyun da reng insektu, siklo ning bie da reng tanaman, ampo gastus keng pamaglalang depensa keng niche nung nukarin ya daragul ing metung a species. Inaral mi ing consistency ning leaf metabolome remodeling a gewa da reng herbivores da reng anam a species ning Nicotiana a katutubu king North America ampo South America. Deng species a reni malapit lang mikakamaganak keng Nepenthes North America, yapin ing Nicolas Bociflo. La, N. nicotinis, Nicotiana n. attenuated grass, Nicotiana tabacum, linear tobacco, tobacco (Nicotiana spegazzinii) ampo tobacco leaf tobacco (Nicotiana obtusifolia) (Figure 5A) (37). Anam kareng species a reni, kayabe na ing well-characterized species N. please, ila reng annual plants ning petunia clade, ampo ing obtusifolia N. ila reng perennials ning sister clade Trigonophyllae (38). Kaybat, ing W + W, W + OSM ampo W + OSS1 induction megawa la kareng pitung species para abalu ing species-level metabolic rearrangement ning pamangan da reng insektu.
(A) Metung a bootstrap phylogenetic tree base keng maximum likelihood [para keng nuclear glutamine synthesis (38)] ampo ing geographical distribution da reng pitung malapit a Nicotiana species (miyayaliwa kule) (37). (B) Metung a scatter plot ning specialized diversity para kareng metabolic profiles da reng pitung Nicotiana species (939 MS/MS; data file S1). keng species level, ing metabolome diversity negatibu yang makaugne keng degree ning specialization. Ing pamagsuri keng species-level correlation pilatan ning metabolic diversity ampo ing specialization ampo ing JA accumulation mayayakit ya keng Figure 2. S9. Kule, miyayaliwang uri; triangle, W + OSS1; rectangle, W + OSM; (C) Nicotiana JA ampo JA-Ile dynamics maka-ranggo la agpang keng OS excitation amplitude (two-way ANOVA ampo Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 ampo * ** Para keng pamagkumpara ning W + OS ampo W + W, P <0.001). Box plot ning (D) diversity ampo (E) specialization ning balang species kaybat dang me-simulate herbivorous ampo methyl JA (MeJA). Ing asterisk papakit na ing makabaldugan a pamiyaliwa ning W + OS ampo W + W o lanolin plus W (Lan + W) o Lan plus MeJA (Lan + MeJa) ampo Lan control (two-way analysis of variance, kaybat ning Tukey's HSD post hoc multiple comparison , *P<0.05, **P<0.01 ampo ***P<0.01).
Gamit ing dual cluster method, akilala mi la reng 9 modules ning 939 MS/MS (data file S1). Ing komposisyun ning MS/MS a me-reconfigure kareng miyayaliwang panulu miyayaliwa yang maragul kareng miyayaliwang modules kareng species (Figure S8). Ing pamag-visualize king Hj (ausan dang species-level γ-diversity) ampo δj papakit na ing miyayaliwang species mag-aggregate la kareng miyayaliwang grupu keng metabolic space, nung nukarin ing species-level division mas prominenti ya kesa keng excitation. Maliban kareng N. linear ampo N. obliquus, magpakit lang maragul a dynamic range ning induction effects (Figure 5B). Ing pamiyaliwa, deng species antimo ing N. purpurea ampo ing N. obtusifolia atin lang e masyadung malino metabolic response keng pamanulu, pero mas miyayaliwa ing metabolome. Ing species-specific distribution ning induced metabolic response meging sangkan ning metung a makabaldugan a negatibong correlation king pilatan ning specialization ampo ing gamma diversity (PCC = -0.46, P = 4.9×10-8). Deng pamagbayu ning OS keng JAs levels positibong makaugne la keng metabolome specialization, ampo negatibo lang makaugne keng metabolic gamma diversity a pepakit da reng balang species (Figure 5B ampo Figure S9). Dapat papansinan na ing species a awsan dang “signal response” species keng Figure 5C, antimo reng Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, ampo Nepenthes attenuated, mika makabaldugan a tanda keng 30 minutus. Ing bayung OS-specific JA ampo JA-Ile outbreaks, kabang deng aliwang bacteria a ausan dang "signal unresponsive", antimo reng Nepenthes mills, Nepenthes powdery ampo N. obtusifolia papakit da mu ing JA-Ile Edge induction a alang OS specificity (Figure 5C). keng metabolic level, antimo ing mebanggit babo, para keng attenuated Nepenthes, ing signal-responsive substances pepakit na ing OS specificity ampo makabaldugan ing pamagtas ning δj, kabang babawasan ne ing Hj. Ing OS-specific priming effect ali ya meyakit kareng species a me-classify bilang signal non-reactive species (Figure 5, D ampo E). Deng OS-specific metabolites mas maralas lang miyayabe kareng signal-responsive species, at deng signal clusters a reni mag cluster la kareng species a mas mahina ing signal responses, kabang deng species a mas mahina ing signal responses mas ditak la interdependence (Figure S8 ). Ing resulta ayni papakit na ing OS-specific induction da reng JAs ampo ing OS-specific reconfiguration ning downstream metabolome makayabe la keng species level.
Kaybat, ginamit kaming lanolin paste a maki methyl JA (MeJA) para gamutan la reng tanaman para abalu nung deng coupling modes a reni makabawal la keng availability ning JA a mipatupad keng exogenous JA, na atyu keng cytoplasm da reng tanaman. Ing mabilis a deesterification yapin ing JA. Ikit mi ing parehung trend ning bagya-bagyang pamagbayu ibat kareng signal-responsive species papunta kareng signal-non-responsive species a sangkan ning patuloy a suplay ning JA (Figure 5, D ampo E). king makuyad a salita, ing MeJA treatment masikan yang me-reprogram kareng metabolomes da reng linear nematodes, N. obliquus, N. aquaticus, N. pallens, ampo N. mikimotoi, a magresulta keng maragul a pamagtas ning δj ampo pamagbaba ning Hj. N. purpurea pepakit na ing pamagtas ning δj, pero ali ing Hj. Ing N. obtusifolia, na minunang pepakit na makatipun yang sobrang mababang antas ning JAs, e ya naman masanting ing pakibat na keng MeJA treatment keng metabolome reconfiguration. Deng resulta papakit da na ing JA production o signal transduction physiologically restricted ya kareng signal-unresponsive species. Para asubukan ing hypothesis ayni, inaral mi la reng apat a species (N. pallens, N. mills, N. pink ampo N. microphylla) a me-induce keng W + W, W + OSMs ampo W + OSS1 Transcriptome (39). Agpang keng pattern ning metabolome remodeling, deng species makabukud la keng transcriptome space, nung nukarin ing N. attenuated pepakit na ing pekamatas a OS-induced RDPI, kabang ing N. gracilis atin yang pekababang (Figure 6A). Makanyan man, ikit da na ing transcriptome diversity a pepalwal ning N. oblonga ing pekamababa kareng apat a species, salungat king pekamatas a metabonomic diversity ning N. oblonga a minunang pepakit kareng pitung species. Deng minunang pamagaral pepakit da na ing metung a set da reng genes a maki kaugnayan kareng early defense signals, kayabe nala reng JA signals, ing magpaliwanag king specificity da reng early defense responses a gewa da reng herbivore-related elicitors kareng Nicotiana species (39). Ing pamagkumpara kareng JA signaling pathways kareng apat a species a reni pepakit na ing metung a makawili a pattern (Figure 6B). keraklan kareng genes keng pathway ayni, antimo reng AOC, OPR3, ACX ampo COI1, pepakit da ing matas a lebel ning induction kareng apat a species a reni. Makanyan man, ing metung a gene, JAR4, magbayu ya keng JA keng kayang biologically active form ning JA-Ile accumulated transcripts, ampo ing kayang transcription level mababa ya, lalu na keng N. mills, Nepenthes pieris ampo N. microphylla. Dagdag pa, ing transcript mung aliwang gene AOS ing e meyakit keng N. bifidum. Deting pamagbayu keng gene expression malyari lang maging sangkan ning sobrang phenotypes a sangkan ning mababang JA production kareng signal anergic species ampo ing induction ning N. gracilis.
(A) Ing pamagsuri king teorya ning impormasyun ning pamag-reprogram kareng minunang transcriptional a pakibat da reng apat a malapit a kamag-anak a species ning tabako a me-sample 30 minutu kaybat ning herbivory induction. Ing RDPI makalkula ya kapamilatan ning pamagkumpara kareng bulung a gewa ning herbivore OS ampo ing wound control. Deng kule mangabaldugan lang miyayaliwang species, at deng symbol mangabaldugan lang miyayaliwang paralan ning pamanulu. (B) Pamagsuri keng gene expression keng JA signaling pathways kareng apat a species. Ing simplified JA path makakit ya keng siping ning box plot. Deng miyayaliwang kule papakit da ing miyayaliwang paralan ning pamagprosesu. Ing asterisk papakit na atin yang makabaldugan a pamiyaliwa ing W + OS treatment ampo ing W + W control (para keng Student's t-test para kareng pairwise differences, *P<0.05, **P<0.01 ampo ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
keng tauling dake, inaral mi nung makananu ing pamagbayu da reng species da reng insektu keng metabolome da reng miyayaliwang species ning tanaman malyari yang maging resistant kareng herbivores. Ing minunang pamanyaliksik dininan neng emphasis ing Nicotiana genus. Ing karelang resistensya kareng Ms ampo kareng larvae miyayaliwa la (40). keni, inaral mi ing koneksyun na ning model ayni ampo ing karelang metabolic plasticity. Gamit la reng apat a species ning tabako, at ing pamagsuri king pamiugnayan ning pamiyaliwa-liwa at pamag-espesyalisadu ning metabolome a sangkan da reng herbivore ampo ing pamaglaban da reng tanaman kareng Ms ampong Sl, ikit mi ing pamaglaban, pamiyaliwa-liwa ampong pamag-espesyalisa king generalist Sl All positibong maka-correlate, kabang ing pamiugnayan king pilatan ning pamaglaban kareng expert ladies ampong specialization mahina ya, at ing pamiugnayan king pamiyaliwa-liwa e ya makabaldugan (Figure 100). Patungkul keng S1 resistance, parehu lang attenuated N. chinensis ampo N. gracilis, na minunang pepakit na parehu lang JA signal transduction levels ampo metabolome plasticity, atin lang miyayaliwang pakibat keng herbivore induction, ampo parehu lang matas a resistensya. Sex.
keng milabas a anam a pulung banua, ing teorya ning depensa ning tanaman dininan neng balangkas ning teorya, nung nukarin ding talapanaliksik mekahula lang maragul a bilang ning ebolusyun ampo gagawan da reng tanaman a espesyalisadung metabolites. keraklan kareng teorya a reni e la tutuki keng normal a paralan ning masikan a pamag-infer (41). Magpropose lang key predictions (3) keng pareung antas ning pamagsuri. Nung ing pamagsuri kareng susi a pamaghula paintulutan na ing pamag-analisa kareng partikular a teorya, iti gawan na ing Ing larangan sumulung ya. Maging suportadu ka, pero tanggian mo reng aliwa (42). Bagkus, ing bayung teorya gagawang ula kareng miyayaliwang antas ning pamagsuri at daragdagan na ing bayung layer ning pamaglarawan a pamisip (42). Makanyan man, deng adwang teoryang mipanukala keng functional level, MT ampo OD, malyari lang ipaliwanag bilang importanting pamaghula kareng specialized metabolic changes a sangkan da reng herbivores: ing OD theory maniwala ya na ing pamagbayu keng specialized metabolic “space” matas yang direksyun. Ing MT theory maniwala ya na deng pamagbayu a reni e la direksyunal ampo random lang makatuknang keng metabolic space, ampo atin lang matas a defense value a metabolites. Deng minunang pamagsuri kareng OD ampo MT predictions me-test la gamit ing makitid a set da reng a priori “defense” compounds. Deting metabolite-centric tests e la malyaring mag-analisa keng lakbang ampo ing trajectory ning metabolome reconfiguration kabang mag-herbivory, ampo e la paintulutan ing pamag-test kilub ning metung a consistent a statistical framework para mangailangan kareng key predictions na malyaring ituring bilang metung a kabilugan Quantify changes in plant metabolome. keni, ginamit mi ing makabayung teknolohiya keng metabolomics base keng computational MS ampo ginawa mi ing deconvolution MS analysis keng pangkabilugan a salapi da reng descriptors ning teorya ning impormasyun para subukan ing pamiyaliwa da reng adwang mipanukala keng global metabolomics level. Ing susi keng pamaghula na niting teorya. Ing teorya ning impormasyun megamit ya kareng dakal a larangan, lalu na keng kontekstu ning pamanyaliksik keng biodiversity ampo nutrient flow (43). Makanyan man, agpang keng balu mi, ini ing mumunang aplikasyun a ginamit para ilarawan ing metabolic information space da reng tanaman ampo asolve la reng problemang ecological a maki kaugnayan kareng pansamantalang pamagbayu keng metabolic bilang pakibat kareng environmental cues. Ing partikular, ing abilidad na niting paralan atiu keng kayang abilidad a magkumpara kareng pattern kilub ampo pilatan da reng species ning tanaman para asuri nung makananu lang mibayu deng herbivores ibat kareng miyayaliwang species papunta kareng inter-species macroevolutionary patterns kareng miyayaliwang antas ning pamagbayu. Metabolism.
Ing principal component analysis (PCA) mag convert yang multivariate data set keng dimensionality reduction space para ing main trend ning data malyari yang ipaliwanag, inya keraklan gagamitan de bilang exploratory technique para i-parse ing data set, antimo ing deconvolution metabolome. Makanyan man, ing pamagbawas king dimensionality mawala ya ing dake ning impormasyun a laman ning data set, at ing PCA e ya malyaring mamye quantitative a impormasyun tungkul kareng katangian a partikular a maki kaugnayan king teorya ning ekolohiya, antimo: makananu lang i-reconfigure deng herbivores ing pamiyaliwa-liwa kareng espesyalistang larangan (alimbawa, kayamanan, pamikalat) at kasaganaan) metabolites? Nanu la reng metabolites a magpredictor keng induced state ning metung a herbivore? Manibat keng panaun ning specificity, diversity ampo inducibility, ing impormasyun a laman ning leaf-specific metabolite profile mesira ya, at mayayakit na ing pamangan da reng herbivores malyari yang mag-activate specific metabolism. E mi asahan, ikit mi na, antimo ing misulat kareng information theory indicators a mipatupad, ing resulta ning metabolic a sitwasyun atin yang maragul a overlap kaybat ning panga-atake da reng adwang herbivores (ing night-fed generalist Sl) ampo ing Solanaceae expert Ms. Agyang ing karelang pamamangan a pamibiebie at konsentrasyun miyayaliwa la. Fatty acid-amino acid conjugate (FAC) initiator keng OS (31). Gamit ing herbivore OS para gamutan ing standardized puncture wounds, ing simulated herbivore treatment pepakit na ing parehung trend. Iting standardized a paralan para king pamag-simulate king pakibat da reng tanaman kareng panga-atake da reng herbivore, lalako na la reng makalitu a sangkan a sangkan ning pamagbayu king pamangan da reng herbivore, a magdalang miyayaliwang degree ning pangasira king miyayaliwang panaun (34). Ing FAC, na balu dang peka-sangkan ning OSM, babawasan ne ing JAS ampo reng aliwang pakibat da reng hormone ning tanaman keng OSS1, kabang ing OSS1 babawasan na ing dinalan a besis (31). Makanyan man, ing OSS1 meging sangkan ning pareung antas ning pamitipun ning JA kumpara king OSM. Metung yang minunang pepakit na ing JA response keng attenuated Nepenthes sensitibo ya keng OSM, nung nukarin ing FAC malyari yang manatili keng kayang aktibidad agyang mi-dilute yang 1:1000 keng danum (44). Inya, nung ikumpara me keng OSM, agyang mababa ya ing FAC keng OSS1, sapat na para mika sapat a JA outbreak. Deng minunang pamagaral pepakit da na deng porin-like proteins (45) ampo oligosaccharides (46) malyari lang gamitan bilang molecular clues para mag-trigger kareng plant defense responses keng OSS1. Makanyan man, e pa malino nung deng elicitors a reni keng OSS1 ila reng sangkan ning pamitipun ning JA a ikit keng kasalukuyan a pamagaral.
Agyang ditak la mu reng pamagaral a maglarawan kareng differential metabolic fingerprints a sangkan ning pamangamit kareng miyayaliwang herbivore o exogenous JA o SA (salicylic acid) (47), ala pang mekapag-perturb keng herbivore species-specific perturbation keng plant grass network ampo reng epektu na keng specific. personal a impormasyun. Ing pamagsuri ayni lalu yang mekumpirma na ing internal hormone network a koneksyun kareng aliwang tanaman a hormone bukud kareng JAs ing magshape keng specificity ning metabolic reorganisation a sangkan da reng herbivores. Partikular, ikit mi na ing ET a sangkan ning OSM mas maragul ya kesa keng sangkan ning OSS1. Ing mode ayni makayagpang ya keng mas dakal a FAC content keng OSM, na metung yang kailangan at sapat a kondisyun para keng pamag-trigger ning ET burst (48). keng context ning pamiugnayan da reng tanaman ampo reng herbivores, ing signaling function ning ET keng plant-specific metabolite dynamics sporadic ya pa mu rin at metung ya mung compound group ing target na. Dagdag pa, keraklan kareng pamagaral ginamit lang exogenous application ning ET o deng precursors na o miyayaliwang inhibitors para abalu ing regulasyun ning ET, nung nukarin deng exogenous chemical applications magproduce lang dakal a e specific a side effects. keng kekaming beluan, ining pamagaral ing mumunang maragul a sistematikung pamagsuri keng papil ning ET keng pamangamit ning ET para makagawa at maka-perceive kareng transgenic a tanaman para maka-coordinate keng plant metabolome dynamics. Ing Herbivore-specific ET induction malyari yang mag modulate keng metabolome response. Ing pekamaulaga yapin ing transgenic manipulation ning ET biosynthesis (ACO) ampo perception (ETR1) genes na pepakit na ing herbivore-specific de novo accumulation da reng phenolamides. Dati nang pepakit na ing ET malyari yang mag-fine-tune king JA-induced nicotine accumulation kapamilatan ning pamag-regulate king putrescine N-methyltransferase (49). Makanyan man, ibat keng mechanical a panaun, ali malino nung makananu yang i-fine-tune ning ET ing induction ning phenamide. Dagdag pa keng signal transduction function ning ET, ing metabolic flux malyari ya namang i-shunt keng S-adenosyl-1-methionine para i-regulate ing investment keng polyaminophenol amides. Ing S-adenosyl-1-methionine metung yang ET ampo ing karaniwan a intermediate ning polyamine biosynthetic pathway. Ing mekanismo nung makananu yang magregulate ing ET signal keng level ning phenolamide kailangan yang karagdagang pamagaral.
King makabang panaun, pauli ning maragul a bilang da reng espesyal a metabolites a e balu ing istruktura, ing masikan a pansin kareng partikular a metabolic a kategorya e na agyung i-assess ing temporal a pamagbayu ning metabolic diversity kaybat ning biological interactions. Ngeni, base keng information theory analysis, ing peka-resulta ning MS/MS spectrum acquisition base kareng unbiased metabolites yapin ing herbivores a mamangan o mag-simulate kareng herbivores patuloy lang babawasan ing kabilugan a metabolic diversity ning leaf metabolome kabang daragul ing kayang degree ning specialization. Ing pansamantalang pamagtas ning metabolome specificity a sangkan da reng herbivores makasuglung ya keng synergistic a pamagtas ning transcriptome specificity. Ing tampok na makaambag keng mas maragul a metabolome specialization (a atin mas matas a Si value) yapin ing espesyal a metabolite na ating minunang mekilalang herbivorous function. Ing model ayni makayagpang ya keng pamaghula ning OD theory, pero ing pamaghula ning MT a maki kaugnayan keng randomness ning metabolome reprogramming ali ya consistent. Makanyan man, ing data ayni makayagpang ya keng pamaghula ning mixed model (best MT; Figure 1B), uling deng aliwang e maki katangian a metabolites a alang balu a defense functions malyari la pa muring tuki keng random a Si distribution.
Ing metung a makabaldugan a pattern a akit na ning pamanyaliksik ayni yapin ing manibat keng micro-evolution level (metung a tanaman ampo populasyun ning tabaku) angga na keng mas maragul a evolutionary scale (malapit a species ning tabako), miyayaliwang antas ning organisasyun ning ebolusyun ing "pekamasanting a depensa" Atin yang makabaldugan a pamiyaliwa ing abilidad da reng herbivore. Moore et al. (20) ampo i Kessler ampo i Kalske (1) independenti lang migmungkahi para i-convert la reng atlung functional levels ning biodiversity a minunang pepakit nang Whittaker (50) keng constitutive ampo induced a temporal a pamagbayu ning chemical diversity; deng talasulat a reni e la me-summarize Deng paralan para keng maragul a metabolome data collection e la naman maka-outline nung makananu yang makalkula ing metabolic diversity ibat kareng data a reni. Kening pamagaral ayni, deng mangalating pamagbayu keng functional classification nang Whittaker ikonsidera de ing α-metabolic diversity bilang pamiyaliwa da reng MS/MS spectra keng metung a tanaman, ampo ing β-metabolic diversity bilang ing basic intraspecific metabolism ning metung a grupu da reng populasyun Space, ampo ing γ-metabolic diversity maging yang extension ning pamagsuri kareng pareung species.
Ing JA signal importanti ya para kareng dakal a herbivore metabolic responses. Makanyan man, kulang ya ing masikan a quantitative testing ning ambag ning intraspecific regulation ning JA biosynthesis king metabolome diversity, at nung ing JA signal metung yang pangkabilugan a lugal para king stress-induced metabolic diversification king mas matas a macroevolutionary scale, e pa malino. ikit mi na ing herbivorous a kalikasan ning Nepenthes herbivorous magdalang metabolome specialization ampo ing pamiyaliwa ning metabolome specialization kilub ning populasyun da reng Nicotiana species ampo kareng malapit a Nicotiana species sistematikung positibong makaugne keng JA signaling. Dagdag pa, nung ing JA signal mesira ya, ing metabolic specificity a pepalwal ning metung a genotype herbivore ma-cancel ya (Figure 3, C ampo E). Uling ing pamagbayu keng metabolic spectrum da reng natural a attenuated a populasyun ning Nepenthes keraklan quantitative la, ing pamagbayu keng metabolic β diversity ampo specificity keng pamagsuri ayni malyari yang maragul a sangkan ning masikan a pamag-excite da reng metabolite-rich compound categories. Deting compound classes ila reng mangibabaw keng metabolome profile ampo magdalang positibong correlation kareng JA signals.
Uling miyayaliwa la reng biochemical mechanisms da reng species ning tabaku a malapit kaya, deng metabolites partikular lang mekilala keng qualitative aspect, inya mas analytical ya. Ing pamagprosesu ning teorya ning impormasyun kareng mekwa nang metabolic profile papakit na ing herbivorous induction papaslag ne ing trade-off pilatan ning metabolic gamma diversity ampo ing specialization. Ing JA signal atin yang sentrung papil keng trade-off ayni. Ing pamagtas ning metabolome specialization makayagpang ya keng main OD prediction at positibong makasuglung ya keng JA signal, kabang ing JA signal negatibu yang makasuglung keng metabolic gamma diversity. Deng modelung deni papakit da na ing OD capacity da reng tanaman keraklan makabasi ya keng plasticity ning JA, nung keng microevolutionary scale o keng mas maragul a evolutionary scale. Deng exogenous JA application experiments na mag circumvent kareng JA biosynthesis defects lalu lang papakit na deng malapit a species ning tabako malyari lang akilala kareng signal-responsive ampo signal-non-responsive species, kalupa ning karelang mode ning JA ampo metabolome plasticity a gewa da reng herbivores. Deng signalal non-responsive species e la malyaring makibat uling e la makagawa keng endogenous JA at uli na niti, atin lang physiological a limitasyun. Malyari yang maging sangkan iti ning mutation kareng mapilan a key genes keng JA signaling pathway (AOS ampo JAR4 keng N. crescens) ning. Ing resulta ayni papakit na na deng interspecies macroevolutionary patterns malyari lang maging sangkan ning pamagbayu keng internal hormone perception ampo responsiveness.
Dagdag pa keng pamiugnayan da reng tanaman ampo reng herbivores, ing pamag explore keng metabolic diversity makasuglung ya kareng eganaganang importanting theoretical advances keng pamanyaliksik keng biological adaptation keng kapaligiran ampo ing evolution da reng complex phenotypic traits. Uling keng pamagdakal ning data a akit da reng modernung MS instruments, ing hypothesis testing keng metabolic diversity malyari neng lampasan ing individual/category metabolite differences ampo gawan ing global analysis para akit la reng e asahan a pattern. keng prosesu ning maragul a pamagsuri, metung a importanting metapora yapin ing ideya ning pamaglalang makabaldugan a mapa na malyaring gamitan para abalu ing data. Uli na niti, ing metung a importanting resulta ning kasalukuyan a pamisanmetung ning alang pamiyaliwa ning MS/MS metabolomics ampo ing teorya ning impormasyun ya pin ing mamye yang simpleng metric a malyaring gamitan para gawang mapa para abalu ing metabolic diversity kareng miyayaliwang taxonomic scales. Iti ing basic a kailangan na niting paralan. Ing pamagaral keng micro/macro evolution ampo ing community ecology.
King macro-evolutionary level, ing core ning plant-insect co-evolution theory da ri Ehrlich ampo Raven (51) yapin ing pamaghula na ing pamiyaliwa ning interspecies metabolic diversity ing sangkan ning pamiyaliwa da reng linya da reng tanaman. Makanyan man, kilub ning limang pulung banua manibat inyang mibulalag ya ining seminal a obra, ing hypothesis a ini bihira yang me-test (52). Iti keraklan uli ning phylogenetic a katangian da reng makayagpang a metabolic a katangian kareng makarayung linya da reng tanaman. Ing rarity malyari yang gamitan para i-anchor ing target analysis methods. Ing kasalukuyan a MS/MS workflow a miprosesu kapamilatan ning teorya ning impormasyun, quantify ne ing MS/MS structural similarity da reng e balu a metabolites (a alang minunang pamili kareng metabolite) at babagutan no reng MS/MSs a reni king metung a set ning MS/MS, inya sa’t king propesyonal a metabolism Deting macro-evolutionary models mipapagkumpara la king classification scale. Simpleng statistical indicators. Ing prosesu kalupa ne ning phylogenetic analysis, nung nu magagamit ing sequence alignment ba yang abalu ing rate ning diversification o character evolution a alang pauna nang pamag-predict.
King biochemical level, ing screening hypothesis nang Firn ampo Jones (53) papakit na ing metabolic diversity mipanatili ya kareng miyayaliwang antas para mamye raw materials para gawan ing biological activities da reng minunang e mikaugne o mipalitan a metabolites. Deng paralan ning teorya ning impormasyun mamye lang balangkas nung nukarin deng metabolite-specific evolutionary transitions a malilyari keng metabolite specialization malyari lang ma-quantify bilang dake ning mipanukalang evolutionary screening process: biologically active adaptation ibat keng mababang specificity papunta keng matas a specificity Inhibited metabolites ning metung a kapaligiran.
Ing kabilugan, kanitang minunang panaun ning molecular biology, mika importanting teorya king pamagdipensa da reng tanaman, at deng deductive hypothesis-driven a paralan ila reng tuturing dang bukud mung paralan ning pamagprogresu ning siyensya. Iti keraklan uli ning limitasyun ning teknikal a pamanuknang king mabilug a metabolome. Agyang deng hypothesis-driven a paralan partikular lang magamit keng pamili kareng aliwang causal mechanisms, ing karelang abilidad para isulong ing kekatamung pamanintindi kareng biochemical networks mas limitadu ya kesa kareng computational a paralan a kasalukuyan a magagamit keng contemporary data-intensive science. Uli na niti, deng teorya na e malyaring hulaan marayu la keng sakup ning available a data, inya ing hypothetical formula/test cycle ning pamagprogresu keng larangan ning pamanyaliksik e malyaring ilako (4). akakit mi na ing computational workflow ning metabolomics a mipakilala keni malyari yang magbalik interes kareng bayung (makananu) ampo tauling (bakit) isyu ning metabolic diversity, ampo makaambag ya keng bayung panaun ning theoretically guided data science. Ing panaun sinuri nong pasibayu deng importanting teorya a meging inspirasyun da reng minunang henerasyun.
Ing direct herbivore feeding gagawan de kapamilatan ning pamagpalaki keng second instar larva o Sl larva keng metung a Pale-colored pitcher plant leaf ning metung a rose blooming plant, a maki 10 plant replicates balang tanaman. Deng larvae da reng insektu me-clamp la kareng clamps, at ing mitagan a tissue ning bulung mekolekta ya 24 ampo 72 oras kaybat ning impeksyun at mabilis yang me-frozen, at ding metabolites me-extract la.
I-simulate ing herbivorous treatment keng matas a synchronized a paralan. Ing paralan yapin ing gamitan ing fabric pattern wheels para butasan ing atlung hilera da reng suksuk keng balang gilid ning midrib da reng atlung ganap a bulung ning tanaman kabang daragul ya ing tela garland, at agad yang gamitan ing 1:5 Diluted Ms. O gamitan ing gloved fingers para isulud ing S1 OS keng sugat. Puni at iprosesu ing bulung antimo ing makasulat babo. Gamitan me ing minunang makasulat a paralan para makakwa kareng mumunang metabolites ampo kareng hormone da reng tanaman (54).
Para kareng exogenous JA applications, deng atlung petiole leaves ning balang metung kareng anam a rose blooming plants ning balang species me-treat la keng 20μl ning lanolin paste a maki 150μg MeJA (Lan + MeJA), ampo 20μl ning lanolin plus wound treatment (Lan + W), o gamitan de ing 20μl a pure lanolin bilang control. Deng bulung me-ani la 72 oras kaybat ning pamag-treat, me-snap-frozen la keng liquid nitrogen, ampo mi-store la keng -80°C anggang gamitan la.
Apat a JA ampo ET transgenic lines, ilapin deng irAOC (36), irCOI1 (55), irACO ampo sETR1 (48), ing mekilala keng kekaming grupung pamanyaliksik. ing irAOC masikan yang pepakit a pamagbaba keng JA ampo JA-Ile levels, kabang ing irCOI1 ali ya sensitibo kareng JAs. Nung ikumpara me keng EV, ing JA-Ile accumulation meragdagan ya. makanyan mu naman, ing irACO babawasan na ing pamaglalang ning ET, at nung ikumpara me keng EV, ing sETR1, na e sensitive keng ET, daragdagan na ing pamaglalang ning ET.
Ing photoacoustic laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) magagamit ya para gawan ing ET a sukad a alang invasive. Kaybat ning pamagpaulu, ing kapitna da reng bulung meputut la at mipalitan la keng 4-ml a sealed glass vial, at ing headspace pepaintulutan deng mitipun kilub ning 5 oras. Kabang sukad ya, ing balang vial me-flush ya keng 2 litru/oras ning purung angin kilub ning 8 minutu, na minunang dinalan keng catalyst a binie ning Sensor Sense para ilako ing CO2 ampo ing danum.
Ing microarray data minunang mibulalag ya keng (35) at mi-save ya keng National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (accession number GSE30287). Ing data a makayagpang kareng bulung a sangkan ning W + OSMs a pamanulu ampo ing e mesira a kontrol mekwa la para kening pamagaral. Ing raw intensity log2 ya. Bayu ing statistical analysis, ing baseline me-convert ya at me-normalize ya keng 75th percentile gamit ing R software package.
Ing orihinal a RNA sequencing (RNA-seq) data ning Nicotiana species mekwa ya keng NCBI Short Reading Archives (SRA), ing project number yapin ing PRJNA301787, a mibalita nang Zhou et al. (39) at isundu ing makasulat keng (56). Ing raw data a meprosesu ning W + W, W + OSM ampo W + OSS1 a maki kaugnayan kareng species ning Nicotiana mepili ya para keng pamagsuri keng pamagaral ayni, ampo meprosesu ya keng makatuking paralan: Mumuna, ing raw RNA-seq readings me-convert ya keng FASTQ format. Ing HISAT2 mag convert yang FASTQ keng SAM, ampo ing SAMtools mag convert yang SAM files keng me-sort a BAM files. Ing StringTie magagamit ya para makalkula ing gene expression, at ing kayang paralan ning pamag-expression yapin ing atin yang fragments balang libung base fragments balang milyung sequenced transcription fragments.
Ing Acclaim chromatographic column (150 mm x 2.1 mm; particle size 2.2μm) a ginamit keng pamagsuri ampo ing 4 mm x 4 mm guard column parehu lang materyalis. Ing makatuking binary gradient magagamit ya keng Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system: 0 anggang 0.5 minutu, isocratic 90% A [deionized water, 0.1% (v/v) acetonitrile ampo 0.05% formic acid], 10% B, ampo Acetonitrile 0.05% formic acid); 0.5 anggang 23.5 minutu, ing gradient phase 10% A ampo 90% B, agpang; 23.5 anggang 25 minutus, isocratic 10% A ampo 90% B. Ing flow rate 400μl/min ya. Para kareng eganaganang MS analyses, i-inject me ing column eluent keng quadrupole ampo time-of-flight (qTOF) analyzer a maki electrospray source a mag-operate keng positibong ionization mode (capillary voltage, 4500 V; capillary outlet 130 V ; Drying temperature 200°C; drying airflow 10 liter/min).
Gawa kang MS / MS fragment analysis (keni mayayaus yang MS / MS) na alang kaugnayan o e malyaring akilala keng data para makakwa kang structural a impormasyun tungkul keng kabilugan a malyaring akit a metabolic profile. Ing konsepto ning indiscriminate MS/MS method magdependi ya keng katutwan na ing quadrupole atin yang maragul a mass isolation window [nya, isipan mu la ngan deng mass-to-charge ratio (m/z) signals bilang fragments]. Uli na niti, uling ing Impact II instrument e ya mekagawa CE tilt, mapilang independenting pamagsuri ing megawa gamit ing daragul a collision-induced dissociation collision energy (CE) values. king makuyad a salita, mumuna yang analisa ing sample kapamilatan ning UHPLC-electrospray ionization/qTOF-MS gamit ing single mass spectrometry mode (mababang fragmentation conditions a gewa ning in-source fragmentation), mag-scan manibat m/z 50 anggang 1500 king repetition frequency a 5 Hz. Gamitan me ing nitrogen bilang collision gas para keng MS/MS analysis, ampo gawa kang independenting sukad kareng tutuking apat a miyayaliwang collision-induced dissociation voltages: 20, 30, 40, ampo 50 eV. King mabilug a prosesu ning pamanuknang, ing quadrupole atin yang pekamaragul a mass isolation window, manibat m/z 50 anggang 1500. Nung ing front body m/z ampo ing isolation width experiment maka set ya keng 200, ing mass range automatic yang mi-activate ning operating software ning instrumentu ampo 0 Da. I-scan la reng mass fragments antimo ing single mass mode. Gamitan ing sodium formate (50 ml isopropanol, 200 μl formic acid ampo 1 ml 1M NaOH aqueous solution) para keng mass calibration. Gamit ing high-precision calibration algorithm nang Bruker, ing data file me-calibrate ya kaybat nang me-run ing average spectrum keng metung a panaun. Gamitan me ing export function ning Data Analysis v4.0 software (Brook Dalton, Bremen, Germany) para i-convert la reng raw data files keng NetCDF format. Ing MS/MS data set maka-save ya keng open metabolomics database MetaboLights (www.ebi.ac.uk) ampo ing accession number. MTBLS1471.
Ing MS/MS assembly malyari yang abalu kapamilatan ning correlation analysis pilatan ning MS1 ampo MS/MS quality signals para keng mababa ampo matas a collision energy ampo bayung mipatupad a patakaran. Ing R script magagamit ya para abalu ing correlation analysis ning pamikalat ning precursor keng produktu, ampo ing C# script (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) magagamit ya para ipatupad ing patakaran.
Para mabawas ing false positive errors a sangkan ning background noise ampo ing false correlation a sangkan ning pamangilala kareng mapilan a m/z features kareng mapilan mung sample, gamitan mi ing “filled peak” function ning R package XCMS (para king background noise correction) Dapat yang gamitan para palitan ing “NA” (undetected peak) intensity. Nung gamitan ya ing fill peak function, dakal la pa reng “0′′ intensity values ​​keng data set na makaapektu keng correlation calculation. Kaybat, ikumpara mi la reng data processing results a mekwa nung gamitan ya ing filled peak function ampo nung e ya gamitan ing filled peak function, at kalkulan mi ing background noise value base keng average a me-correct, kaybat palitan mi la reng intensity values ​​a reni kinalkula mi la reng features na ing karelang intensity migit yang atlung besis keng background value ampo tinggap mi la bilang "tune peaks." Para kareng PCC a kalkulasyun, ing m/z signals mu ning sample precursor (MS1) ampo ing fragment data sets a ating at least walung tune peaks ing mikonsidera.
nung ing intensity ning precursor quality feature keng mabilug a sample makabaldugan yang makaugne keng mabawas a intensity ning pareung kalidad a feature na mipailalam keng mababang o matas a collision energy, at ing feature ali ya me-label bilang isotope peak ning CAMERA, malyari yang maging mas maragul a kabaldugan. Kaybat, kapamilatan ning pamangulkula kareng eganaganang posibling precursor-product pairs kilub ning 3 s (ing estimated retention time window para keng peak retention), ing correlation analysis gagawan de. nung ing m/z value mas mababa ya kesa keng precursor value ampo ing MS/MS fragmentation malilyari ya keng pareung sample location keng data set antimo ing precursor nung nukarin ya menibatan, ita ing metung a fragment.
Base kareng adwang simpleng tuntunan a reni, e mi la kayabe deng metung a fragment a maki m/z values ​​a mas maragul kesa keng m/z ning mekilalang precursor, ampo base keng sample position nung nukarin ya linwal ing precursor ampo ing metung a fragment. Malyari ya namang mamili kareng kalidad a katangian a gewa da reng dakal a in-source fragments a gewa da reng MS1 mode bilang kandidatung precursors, inya sa’t makagawa lang redundant MS/MS compounds. Para mabawas ing data redundancy, nung ing NDP similarity da reng spectra migit ya keng 0.6, at kayabe la keng chromatogram "pcgroup" a me-annotate keng CAMERA, isamut mi la. Katataulian, isamut mi la ngan deng apat a resulta ning CE a makasuglung keng precursor ampo reng pirasu keng tauling deconvoluted composite spectrum kapamilatan ning pamamili keng pekamatas a intensity peak kareng eganaganang kandidatung peak a maki pareung m/z value kareng miyayaliwang collision energies. Deng tutuking lakbang ning pamagprosesu makabasi la keng konsepto ning composite spectrum at kukunsidera la reng miyayaliwang kundisyun ning CE a kailangan para mas maragul ing probability ning fragmentation, uling deng mapilan a fragments malyari lang akit keng lalam ning metung a specific collision energy.
RDPI (30) ing ginamit para makalkula ing inducibility ning metabolic profile. Ing metabolic spectrum diversity (Hj index) menibatan ya keng karakal da reng MS/MS precursors gamit ing Shannon entropy ning MS/MS frequency distribution gamit ing makatuking equation a milarawan nang Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) nung nukarin ing Pij makayagpang ya keng relatibung frequency ning i-th MS/MS keng j-th sample (j = 1, 2,..., m) (i = 1, 2, ..., m) t).
Ing metabolic specificity (Si index) metung yang expression identity ning metung a MS/MS a maki kaugnayan keng frequency da reng sample a kukunsidera. Ing MS/MS specificity makalkula ya bilang Si = 1t (∑j = 1tPijPilog2PijPi)
Gamitan me ing makatuking formula para sukatan ing metabolome-specific δj index ning balang j sample, ampo ing average ning MS/MS specificity δj = ∑i = 1mPijSi
Deng MS/MS spectra makalinya la keng pares, at ing pamikakatulad makalkula ya base kareng adwang score. Mumuna, gamit ing standard NDP (ausan dang cosine correlation method), gamitan me ing makatuking equation para a-score ing segment similarity pilatan da reng spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 nung nukarin S1 ampo S2 Katumbas, para keng spectrum 1 ampo 2, WS1, ampo iWS2, mangabaldugan yang timbang base keng peak intensity na ing pamiyaliwa ning i-th common peak pilatan da reng adwang spectra mas mababa ya keng 0.01 Da. Ing timbang makalkula ya anti kanini: W = [peak intensity] m [quality] n, m = 0.5, n = 2, antimo ing sinabi ning MassBank.
Ing kaduang paralan ning pamag-score mipatupad ya, nung nukarin ing pamag-analisa king miyabe-abe NL pilatan da reng MS/MS. Para kaniti, ginamit mi la reng 52 a listahan ning NL a maralas a akatagmu king prosesu ning MS fragmentation king tandem, ampo ing mas partikular a NL (data file S1) a minunang me-annotate para king MS/MS spectrum da reng secondary metabolites ning mehina nang Nepenthes species ( 9, 26). Gawa kang binary vector a 1 ampo 0 para keng balang MS/MS, a makayagpang keng kasalukuyan ampo ing alang NL. Base keng Euclidean distance similarity, ing NL similarity score mekwa ya para kareng balang pares da reng binary NL vectors.
Para gawan ing dual clustering, ginamit mi ing R package DiffCoEx, na makabasi keng extension ning Weighted Gene Co-expression Analysis (WGCNA). Gamit ing NDP ampo NL scoring matrices ning MS/MS spectra, ginamit mi ing DiffCoEx para makalkula ing comparative correlation matrix. Ing binary clustering gagawan de kapamilatan ning pamag-set king “cutreeDynamic” parameter king method = “hybrid”, cutHeight = 0.9999, deepSplit = T, at minClusterSize = 10. Ing R source code ning DiffCoEx me-download ya ibat king karagdagang file 1 ibat kang Tesson et al. (57); Ing kailangan a R WGCNA software package akit ya keng https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Para gawan ing MS/MS molecular network analysis, kinalkula mi ing paired spectral connectivity base keng NDP ampo NL similarity types, ampo ginamit mi ing Cytoscape software para akit ing network topology gamit ing organic layout keng CyFilescape yFiles layout algorithm extension application.
Gamitan me ing R version 3.0.1 para gawan ing statistical analysis keng data. Ing statistical significance me-assess ya gamit ing two-way analysis of variance (ANOVA), kaybat ing Tukey's honestly significant difference (HSD) post-hoc test. Para abalu ing pamiyaliwa ning herbivorous treatment ampo ing control, ing two-tailed distribution da reng adwang grupu da reng sample a parehu ing variance me-analisa ya gamit ing Student's t test.
Para kareng karagdagang materyales para kening artikulu, lawan me ing http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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Ing teorya ning impormasyun mamye yang universal a salapi para keng pamagkumpara kareng espesyal a metabolomes ampo ing pamaghula kareng teorya ning pamagdipensa keng test.
Ing teorya ning impormasyun mamye yang universal a salapi para keng pamagkumpara kareng espesyal a metabolomes ampo ing pamaghula kareng teorya ning pamagdipensa keng test.
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