Theories matung tung mi gwoko yen miyo telowic ma pire tek pi gonyo kit ma yen tiyo kudu, ento paru migi ma pire tek podi jubi temo. Kae, watiyo ku unbiased tandem mass spectrometry (MS/MS) pi ni ngiyo metabolome pa taba ma jucwalo kud i yen acel acel ni dhanu man lei ma rwate kugi, man watiyo ku wel madwong pa mass spectrometric feature theories ma lubere ku compound spectra i lembe. Yore mi temo lembe ma pire tek ma jular jukoro ikum gwoko maber (OD) man lembe ma juporo (MT). Lembe ma jukoro ikum metabolomics pa yen urombo ku OD theory, ento ukwero lembe ma pire tek pa MT theory ikum metabolomics dynamics ma wok kud i lei ma camu lum. Nicaku i rwom ma nok nituc i rwom ma dit, lanyuth pa jasmonate ungiyure ni piny madit ma nyayu OD, man lanyuth pa ethylene ungiyu aloka loka pa dwokowang pa lei ma camu lum ma jukwanu ku MS/MS molecular network.
Metabolites ma tung tung ku kit ma tung tung gibedo piny ma pire tek i dongo pa yen ni kwo pa piny, asaga pi gwoko judhogi (1). Kit ma tung tung pa metabolism ma nwangre i yen uketho jutimo sayusac ma thuc ikum lembe mapol ikum tic pa yen, man uketho lembe madwong ikum gwoko yen, ma en e dongo man dongo pa yen ku lei makwiny. Ponjo lembe ma jutimo mio telowic ma pire tek (2). Ento, lembe maeni mi gwoko yen ulubo ngo yo ma juketho i cik mi hypothetical deductive reasoning, ma i iye lembe ma pire tek ma jular jukoro ubedo i rwom acel mi nyang ikum lembe (3) man jutemo gi ku yore mi temo pi medo rwom mi dongo pa lembe (4). Peko mi tic uketho yub mi coko lembe ikum dul moko mi metabolic man uketho timo sayusac ikum metabolites moko, eno uketho juporo ngo dul matung tung ma pire tek pi dongo pa theoretical (5). Bedo mbe pa lembe ma dongo dongo mi metabolomics man sente ma rom mi poro tic mi tic mi metabolic space ikind ungu mi yen matung tung nyayu peko i dongo pa ngec mi ngec mi ng'om.
Lembe manyen ma juyubu i thenge mi tandem mass spectrometry (MS/MS) metabolomics copo nyutho aloka loka mi metabolic ikind lei man ikind lei moko, man ecopu bedu karacelu ku yore mi kwan pi kwanu kit ma rom pa piny ma tung tung eni. Ngec macon pa chemistry (5). Dikiri pa diro ma malu i nyang ikum lembe man kwanu piny miyo yore maber mi pimo lembe madwong ma jular jukoro ma jutimo ku theori mi ecological man evolutionary pa metabolic diversity. Shannon (6) ukelo theori mi lembe pi tyen acel i lembe pare ma pire tek i 1948, ma eketho thek pi nyang ikum lembe, ma jutiyo kudu i lembe madwong mukende ma epe ku tic pare macon. I genomics, theory mi lembe jutiyo kudu maber pi kwanu lembe ma jugwoko (7). I lembe mi transcriptomics, theori mi ngec nyang ikum aloka loka mi transcriptome (8). I lembe mukadho, watiyo ku lembe ma juporo ku lembe ma jukoro ikum metabolomics ni nyuthu ngec pa metabolic pa dul kum i lum (9). Kae, wabe ketho tic pa MS/MS ku yub mi kwan pa theory mi ngec, ma nyutho tung tung pa metabolic i sente ma dhanu zo gi tiyo kudu, mi poro lembe ma pire tek ma juporo ku theory mi gwoko yen pa metabolome ma wok kud i lei ma camu lum.
Lembe ma jukwanu ikum gwoko yen pol kare gi rwate man jupoko gi i dul aryo: ju ma tembo ni gonyo poko pa jamb kum yen malubiri ku tic mi gwoko, calu gwoko maber (OD) (10), piny ma wotho (MT) (11) ) man nen (12) theory, man ju mukende gibe sayu gonyo kite ma piny ma jutiyo kudu copo loko kudu dongo pa yen man coko pa piny ma tung tung, calu carbon: paru pa rwom pa cam (13), paru pa dongo pa yen (14), man paru pa dongo man tung tung (15). Theory ario eni gitiye i rwom matung tung mi nyang ikum lembe (4). Ento, paru ario ma mako tic mi gwoko kum i rwom mi tic ucungo iwi wec ikum gwoko kum pa yen: OD theory, ma nyutho nia yen ketho lim i gwoko kum migi ma wel migi tek kende kende ka mitire, calu, ka jucamu gi. MT hypothesis uketho nia epe ku axis mi aloka loka pa metabolite, ento metabolite lokore kende kende, ma uketho ebedo ku copo mi juku metabolic "motion target" mi lwiny ikum lei ma camu lum. I wec mange, lembe ario eni timo lembe ma tung tung ikum aloka loka pa piny ma timore ingey lwiny pa lei ma camu lum: wat ma ni ikin rwom acel pa piny ma kwo ku tic mi gwoko kum (OD) man aloka loka pa piny ma mako kum (MT) (11).
OD ku MT hypotheses uketho i iye aloka loka ma wok kende ngo i metabolome, ento bende adwogi pa kwo man dongo pa piny eni, calu ve bey pa piny man bero pa aloka loka eni i kabedu moko (16). Kadi bende lembe ario eno ungiyu tic mi gwoko kum pa metabolites ma pire tek, ma copo bedu tek nyo epe, lembe ma pire tek ma poko kind OD ku MT hypotheses utiye i kite ma aloka loka mi metabolic timo kudu. Lembe ma jular jukoro ikum OD theory unwangu nen pa lembe madwong akeca nitundu kawoni. Tembe eni uketho i iye somo pa tic mi gwoko kum matung tung pa jamb kum ma tung tung i ot ma jugwoku i iye lum man i kabedo ma leng, man aloka loka ma timere i dongo pa yen (17-19). Ento, nitundu kawoni, pi bedo pe pa tic man lembe mi kwan pi nyang ikum kit ma piny moko ci timo kudu tic, tung tung madit ma jukoro ikind lembe aryo eni (niwacu, yore mi aloka loka pa piny) podi jubi temo. Kany, wabe miyo nyang ma kumeno.
acel ikin kit ma pire tek pa metabolites ma tung tung pa yen utiye tung tung pa kit migi i rwom zo nicaku i yen acel acel, wel pa yen nitundu ikum kit yen ma rom (20). Aloka loka madwong i wel pa metabolites copo nenre i wel pa dhanu, man tung tung ma tek nenre i wel pa lei (20). Pieno, kit tic pa yen ubedo piny madit pa tung tung pa tic, ma nyutho bedo yot ni kwo i kabedo matung tung, asaga kabedo ma tung tung ma copo wok i iye ku lei ma tung tung man lei ma camu lum (21). Nicaku i Fraenkel's (22) lembe ma pire tek ikum the lembe ma nyayu bedo pa piny ma wok kud i yen, wec ku lei makwiny matung tung jubedu ka neno ni piny ma pire tek, man wec eni juyio nia eketho yen ubedo maber i kare mi dongo. Yore mi loko kum (23). Tung tung mi lei matung tung ikind kit piny ma tung tung copo nyutho bedo maber pa piny ma kwo ma rwate ku yub pa lei ma camu lum, calu ma lei ario eni gibedo ku winjiri marac ikindgi (24). Kadok ecopu bedu ber ni gwoko gwok maber kare ku kare, aloka loka mi metabolic ma rwate ku gwok mio bero ma yot ni weko yen ni poko piny ma pigi tek i tic mange (19, 24), man jwigo mitu pa symbiosis. Peko ma rac (25). Mi medo, aloka loka eni pa piny ma jucwalo ma wok kud i lei ma camu lum copo telu poko pa piny ma rac ikind dhanu (26), man copo nyutho aloka loka madit mi jasmonic acid (JA) signal, ma copo bedu ikind dhanu. JA ma malu man ma piny utiye lembe ma juloko ikind gwoko kum lei ma camu lum man pyem ku lei moko (27). Mi medo, yore mi yubu piny ma kwo ma tung tung bi rweny pio pio man bi lokore i saa mi dongo, ma bi kelo poko pa piny ma kwo ikind lei ma gi rwate (28). Polymorphisms eni copu bedu pio pio ni dwokowang ikum aloka loka pa kit lei ma camu lum (29), ma nyutho nya aloka loka pa dul pa lei ma camu lum ubedo piny ma pire tek ma kelo aloka loka pa metabolic.
Kany, wabe timo lembe ma e. (I) Lei ma camu lum loko metabolome pa yen nenedi? (Ii) Lembe ango ma pire tek ma mako metabolic plasticity ma juromo pimo pi temo lembe ma jular jukoro ikum long-term defense theory? (Iii) Ka nyo jubi dwoko metabolome pa yen i yo ma ukoc ku jajok, ka kumeno, tic ango ma hormone pa yen timo i yiko dwokowang pa metabolic moko, man metabolites makani ma konyo i kit pa lei? (Iv) Calu ma lembe ma jular jukoro ma jutimo ku theory mapol mi gwoko kum copo bedu i rwom ceke mi dul kum, wapenjo nia adwogi pa metabolic ma jukelo utiye marom rom nenedi kud i poro piny ma iye ku poro piny ma tung tung? Pi timo eni, wabeponjo lembe ikum lum pa nicotine mi taba, ma en e lum ma nen maber ku metabolism maber, man etiye maber ikum larvae pa lei ario ma camu lum, Lepidoptera Datura (Ms) (ma tek mandha, ma jucamu) I kum Solanaceae man Spodoptera (Storalist), gitiye kit thwol moko "genus", ku yen ma kwo pa Solanaceae man yen mange ma kwo pa genera mange man ju pacu. Cam pa yen. Wapoko MS/MS metabolomics spectrum man wawodho lembe ma jukoro ikum lembe ma jukoro pi poro OD ku MT theories. Yub map mi lembe ma pire tek mi nyuthu kit pa metabolites ma pire tek. Lembe ma jutimo ne umedire ni dhanu ma nwangire i N. nasi man kit taba ma rwate kugi pi medo nyang ikum covariance ikind hormone pa yen man OD induction.
Pi ni mako map ma dongo dongo ikum plasticity man structure pa leaf metabolome pa herbivorous tobacco, watiyo ku lembe ma jular juyubu con man tic mi kwanu pi coko man poko lembe ma rwom pare lac ma mako MS/MS spectra kud i lum ( 9). Yore maeni ma jupoko ngo (ma julwongo MS/MS) ecopu yubu non-redundant compound spectra, ma jucopo tiyo kudu pi compound-level analyses ma juloko keni. Piny ma nyai pa yen eni utiye kit matung tung, ma uketho i iye piny ma romo dak ku miya acel (ma romo 500-1000-s/MS/MS keni). Kae, wabeparu pi metabolic plasticity i thenge mi ngec, man wapimo tung tung man tic pa metabolome malubiri ku Shannon entropy pa metabolic frequency distribution. Ni tiyo ku formula macon (8), wa kwanu dul pa lanyuth ma juromo tiyo kudu pi pimo tung tung pa metabolome (Hj indicator), metabolic profile specialization (δj indicator) man metabolic specificity pa metabolite acel (Si indicator). Mi medo, watiyo ku Relative Distance Plasticity Index (RDPI) pi pimo metabolome inducibility pa lei ma camu lum (lanyut 1A) (30). Ikind lembe maeni mi kwan, watiyo ku MS/MS spectrum calu piny ma pire tek, man watiyo ku wel pa MS/MS i frequency distribution map, man watiyo ku Shannon entropy ni pimo metabolome diversity kud i iye. Metabolome specialization jupimu ku rwom pa MS/MS spectrum acel. Pieno, medu pa wel pa MS/MS moko ingey ketho lei ma camu lum lokore ni spectral inducibility, RDPI man specialization, niwacu, medu pa δj index, kum juyubu metabolites mapol man Si index ma malu juyubu. Dwoko piny pa Hj diversity index nyutho nya wel pa MS/MS ma juyubu udok piny, nyo ke rwom pa frequency mi profile ulokore i yo ma rwinyo ngo, man edwoko piny lembe ma dhanu ungiyu ngo. Ni kadhu kud i kwan pa Si index, ecopu bedu yot ni nyutho MS/MS makani ma wok kud i lei moko ma camu lum, ento, MS/MS makani ma dwoko ngo ikum nyayu, ma en e lanyuth ma pire tek mi poko kind MT ku OD.
(A) Lembe ma jukwanu ma jutiyo kudu pi camu lum (H1 nicidho Hx) MS/MS data-inducibility (RDPI), tung tung (Hj index), specialization (δj index) man metabolite specificity (Si index). Medu pa rwom mi ngec (δj) nyutho nia, i wel, piny mapol ma camu lum bibedo nuti, man dwoko piny pa tung tung (Hj) nyutho dwoko piny pa yiku pa piny nyo poko piny ma rom ungo i map mi poko piny. Si value pimo ka nyo metabolite utiye kende kende pi lembe moko (kani, herbivorous) nyo ke jugwoko i rwom marom. (B) Cal pa lembe ma juporo ku theori mi gwoko ku tiyo ku theori mi ngec. OD theory uwacu nia lwiny pa lei ma camu lum bi medo kero mi gwoko kum, man bi medo δj. I saa marom, Hj dwoko piny pilembe rwom pa piny judwoko piny ni dwoko piny pa lembe ma nyayu anyonga pa piny. The MT theory uwacu nia lwiny pa lei ma camu lum bi kelo aloka loka ma rwinyo ngo i metabolome, ma bi medo Hj calu lanyuth pa mediri pa ngec mi metabolome man bi kelo poko pa Si. Waketho bende yub matung tung, MT maber loyo, ma i iye metabolites moko ma gitiye ku rwom ma malu mi gwoko kum bibedo malu (Si ma malu), man mukende nyutho adwogi ma rwinyo (Si ma piny).
Ni tiyo ku theori mi ngec, wa gonyo theori mi OD ni nyutho nia aloka loka pa metabolite ma jucamu lei ma camu lum kelo (i) medu pa metabolic specificity (Si index) ma telo wi metabonomic specificity (δj index) Medu pa) dul pa metabolite moko ma pire tek ma gitiye ku rwom ma malu mi gwoko kum, man (ii) dwoko piny pa index pa metabolite (Hj) aloka loka pa poko pa metabolic frequency ni poko pa leptin ikum kum. I rwom pa metabolite acel, poko pa Si ma juyiko jukuro, kama metabolite bi medo Si value nimakere ku value pare mi gwoko (lanyut 1B). I lembe maeni, wa gonyo the MT theory ni nyutho nia nyayu anyonga bi kelo (i) aloka loka ma rwinyo ngo i metabolites ma bi kelo dwoko piny pa δj index, man (ii) medo pa Hj index pi medo pa metabolic uncertainty. Nyo randomness, ma jucopo pimu ku Shannon entropy i ayi pa generalized diversity. Pi lembe pa dul pa piny, MT theory bi nyuthu poko pa Si. Ka wakwanu nia metabolites moko utiye i the lembe moko i thenge moko, man lembe mange epe i thenge moko, man bero migi mi gwoko kwo ucungo iwi kabedu, wabe ketho yub mi gwoko piny ma tung tung, ma δj ku Hj jupoko i ario i Si Increase i yore zo, dul pa metabolite moko kende, ma gitiye ku wel mi gwoko piny ma malu, re ma bi medo rwom ma malu, ento Siture mange bi bedu ku poko ma rwinyo (Figure a 1B).
Pi ni temo thek mi gwoko kwo ma judwoko manyen iwi thenge mi thek mi ngec, wa tingo larvae pa lei ma camu lum (Ms) nyo generalist (Sl) iwi ith pa Nepenthes pallens (lanyut 2A). Ni tiyo ku MS/MS analysis, wanwango 599 non-redundant MS/MS spectra (data file S1) kud i methanol ma juwodho kud i lum ma jucoko ingey cam pa caterpillar. Tiyu ku RDPI, Hj, man δj indexes ni neno kit ma juloko ko lembe i MS/MS configuration files nyutho kit ma ber (lanyut 2B). Lembe ma ubetimere utiye nya, calu ma jukoro i lembe ma jukoro, ka caterpillars umediri ku camu ith, kadhiri pa metabolic reorganization umedire ku kare: 72 hours ingeye ma lei ma camu lum ucamu, RDPI umedire lee. Ka juporo ku ju ma timo ngo marac, Hj udoko piny lee, ma ubedu pi medo rwom pa tic pa metabolic profile, ma jupimo ku δj index. Lembe maeni nen ni rwate ku lembe ma jukoro i OD theory, ento epe ku lembe ma jukoro i MT theory, ma yio nia aloka loka ma rwinyo (ma rwinyo ngo) i kadhiri pa metabolite jutiyo kudu calu camouflage (Figure 1B). Kadi bende lembe ma wok kud i dog (OS) man kit ma gicamu ko lei ario eni gi tung tung, camu migi ukelo aloka loka marom i yore pa Hj ku δj i kind sawa 24 ku 72 mi kac. Tung tung acel kende utimere i sawa 72 mi RDPI. Ka juporo ku ma wok kud i cam pa Ms, metabolism ma wok kud i cam pa Sl ubedo malo.
(A) Yub mi temo: lei ma camu lum (S1) nyo ngati ma ngeyo (Ms) jumiyo igi cam ku ith lum ma juyiko ku lum, man pi lei ma camu lum, OS pa Ms (W + OSMs) jutiyo kudu pi timo tic mi poko lum ma juyiko. S1 (W + OSSl) larvae nyoke pii (W + W). Control (C) utiye ith ma unyothere ngo. (B) Inducibility (RDPI compared with the control chart), diversity (Hj index) and specialization (δj index) index calculated for the special metabolite map (599 MS/MS; data file S1). Asterisks nyutho tung tung madit ikum cam pa lei ma camu lum ku dul pa ju ma camu ngo (Student's t-test ku paired t-test, *P<0.05 man ***P<0.001). ns, piretek ungo. (C) Lembe ma pire tek (sanduku macol, amino acid, acid ma kwo man sukari; data file S2) man special metabolite spectrum (red box 443 MS/MS; data file S1) ingey simulated herbivory treatment. Rangi mi rangi nyuthu 95% mi gen. Asterisk nyutho tung tung madit ikum yath ku yath [quadratic analysis of variance (ANOVA), ma ulubre ku Tukey's honestly significant difference (HSD) pi post hoc multiple comparisons, *P<0.05, **P<0.01 And *** P <0.001]. (D) Specialization pa scatter plots man special metabolite profiles (repeated samples ku treatments matung tung).
Mi ngiyo ka nyo aloka loka pa lei ma camu lum i rwom pa metabolome nenre i aloka loka pa rwom pa metabolites acel acel, wacaku ketho wigi ikum metabolites ma jular juponjo i ith pa Nepenthes pallens ku rwom pa lei ma camu lum. Phenolic amides ubedo hydroxycinnamamide-polyamine conjugates ma cokiri i kare ma lei moko camu lum man ungeyere ni dwoko piny tic pa lei moko (32). Wa sayu lembe ma wok iwang MS/MS man wagoro cumulative kinetic curves migi (Figure S1). Ma ukwero ngo, piny ma wok kud i phenol ma tiye ngo ikum gwoko lei ma camu lum, calu chlorogenic acid (CGA) man rutin, judwokogi piny ingey camu lum. I tung ocele, lei ma camu lum copo ketho phenol amides bedu tek. Camu cam ma nja pa lei ario ma camu lum ukelo rwom marom pa phenolamides, man kit ma eni nenre kamaleng pi de novo synthesis pa phenolamides. Lembe marom bino nen ka jube ngiyo 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs) yo, ma nyayu wel madit pa acyclic diterpenes ku tic maber mi juku lum (33), ma Ms Feeding ku Sl ukelo lanyuth marom (Figure S1)).
Peko ma copo bedu i kum cam pa lei ma camu lum utiye tung tung pa rwom pa camu pa lum man kare mi cam pa lei ma camu lum, ma weko bedo tek ni daru adwogi pa lei ma camu lum ma wok kud i ret man lei ma camu lum. Pi ni nyang maber pa kit lei ma camu lum ma nyayu adwogi pa ith, watimo lanyuth pa cam pa larvae pa Ms ku Sl ku tiyo ku OS ma jucoko nyen (OSM man OSS1) iwi puncture W ma rwom pa ith ubedo marom. Lembe maeni julwongo ni W + OS treatment, man edwoko rwom pa tic ku kare ma rwate ku acaki pa dwokowang ma lei ma camu lum ucaku kudu manangu eketho ngo adwogi marac pa tung tung mi kadhiri nyo wel pa rweny pa ler (lanyut 2A) (34). Ni tiyo ku MS/MS analysis man calculation pipeline, wanwango 443 MS/MS spectra (data file S1), ma urombo ku spectra ma jucoko con kud i direct feed experiments. Lembe ma jukoro ikum lembe pa MS/MS onyuthu nia dwoko piny pa leaf-specialized metabolomes ku simulating herbivores unyutho OS-specific inducements (Figure 2C). I andha, ka juporo ku yath pa OSS1, OSM ukelo dongo pa metabolome i sawa angwen. Ber ni ngeyo ni ka juporo ku cam pa lei ma camu lum, kit ma tic pa lei ma camu lum nen kudu i thenge aryo ma jutiyo kudu ku Hj man δj calu coordinates man yore pa metabolome ma jutiyo kudu pi dwoko wang ikum yath pa lei ma camu lum i kare ma ukadhu (Figure 2D). I saa marom, wapimo wel pa amino acids, organic acids man sugars (data file S2) mi ngiyo ka nyo medo pa ngec mi metabolome utiye pi dwoko piny pa carbon metabolism ma juporo ku lei ma camu lum ( cal S2). Mi gonyo lembe eni maber, wamedo lubo tok lembe ma juweco pire con ikum phenolamide man 17-HGL-DTG. OS-specific induction pa herbivores juloko ni differential rearrangement pattern ikind phenolamide metabolism (Figure S3). Phenolic amides ma tye ku coumarin man caffeoyl moieties juyike ku OSS1, man OSMs nyayu ferulyl conjugates. Pi 17-HGL-DTG pathway, tung tung mi OS induction ku downstream malonylation man dimalonylation products junwange (Figure S3).
I ngeye, waponjo OS-induced transcriptome plasticity ku tic ku time-course microarray data set, ma nyutho tic pa OSMs pi timo yath pa lum pa rosette plant i lei ma camu lum. Kinetics mi sampling urombo ku kinetics ma jutiyo kudu i metabolomics study eni (35). Ka juporo ku metabolome reconfiguration ma metabolic plasticity umedire ku kare, waneno transient transcription bursts i lum ma Ms ukelo, kama transcriptome inducibility (RDPI) man specialization (δj) utiye i 1. transcriptome specialization (lanyut S4). Juruot pa gene ma loko kum (macalu P450, glycosyltransferase, man BAHD acyltransferase) gikonyo i yub mi coko metabolites ma pire tek kud i dul ma wok kud i primary metabolism, nimakere ku lapor ma malu ma juweco pire. Calu lanyuth, yore mi phenylalanine jutimo sayusac iwie. Lembe ma jutimo unyutho nia genes ma pire tek i phenolamide metabolism utiye OS-induced lee i lei ma camu lum ka juporo ku lum ma jucamu ungo, man gi rwate i kite ma gi nyuthu ko piny. The transcription factor MYB8 man structural genes PAL1, PAL2, C4H man 4CL in upstream of the pathway onyuthu acaki mi transcription con. Acyltransferases ma timo tic i yik pa phenolamide, calu AT1, DH29, man CV86, nyutho rwom ma malu (lanyut S4). Lembe ma waneno malu eni nyutho nia acaki pa transcriptome man medo pa metabolomics utiye yore ma rwate, ma copo bedu ni kum synchronous regulatory system ma caku dwokowang matek.
Lokruok manyen i lanyuth pa hormone pa yen timo tic calu dul ma doro lembe ma mako lum ma camu lum pi loko kit ma yen tiyo kudu. I ngei lanyuth pa lei ma camu lum, wapimo rwom pa dul ma pire tek pa hormone pa yen man waneno rwom pa timo migi [Pearson correlation coefficient (PCC)> 0.4] (Figure 3A). Calu ma jutekuru, hormone pa yen ma mako biosynthesis gi rwate ikind hormone pa yen. Mi medo, metabolic specificity (Si index) juporo ku network eni ni nyuthu hormones pa yen ma wok kud i yath matung tung. Kabedo ario madongo pa dwokowang lembe pa lei ma camu lum ju goyo: acel utiye i JA cluster, kama JA (kit kwo pare JA-Ile) man JA derivatives mukende nyuthu Si score ma malu; mange ubedo ethylene (ET). Gibberellin unyutho kende kende mediri ma nok i rwom pa lei ma camu lum, ento hormone mange pa yen, calu cytokinin, auxin, man abscisic acid, unyutho rwom ma piny pa rwom pa lei ma camu lum. Ka juporo ku tic ku W + W kende, medo pa wel ma malu pa JA derivatives nikadhu kud i tic pa OS (W + OS) ecopu loke ni lanyuth ma tek pa JAs. Ma dhanu ungiyu ngo, OSM ku OSS1 ku elicitor content matung tung ungeyere ni nyayu rwom marom pa JA ku JA-Ile. I tung tung ku OSS1, OSM utiye ma pire tek man ma tek ma OSMs kelo, man OSS1 eketho ngo dwokowang pa ret ma piny (lanyut 3B).
(A) Co-expression network analysis based on PCC calculation of herbivore-induced plant hormone accumulation kinetics simulation. Node nyuthu hormone acel pa yen, man dit pa node nyuthu Si index ma rwate ku hormone pa yen ikind yath. (B) Coko pa JA, JA-Ile man ET i ith ma wok kud i tic matung tung ma nyutho ku rangi matung tung: apricot, W + OSM; bulu, W + OSSl; macol, W + W; grey, C (control) . Asterisks nyutho tung tung madit ikum yath ku yath (two-way ANOVA followed by Tukey HSD post hoc multiple comparison, *** P <0.001). Lembe ma jukoro ikum (C)697 MS/MS (data file S1) in JA biosynthesis man impaired perception spectrum (irAOC man irCOI1) man (D)585 MS/MS (data file S1) in ETR1 ku impaired ET signal Two simulated herbimore treatments triggered plant lines and vehicle control plants (EV). Asterisks nyutho tung tung madit ikum W+OS treatment ku undamaged control (two-way ANOVA followed by Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 and ***P<0.001). (E) Graph ma nyai pa apoka poka ma nyai ikum tic ma pire tek. Rangi nyuthu kit ma tung tung ma juloko ku gene; lanyuth nyuthu yore mi yath matung tung: triangle, W + OSS1; rectangle, W + OSM; guro C
I ngeye, watiyo ku kit Nepenthes ma juloko (irCOI1 man sETR1) i thenge ma pire tek mi JA man ET biosynthesis (irAOC man irACO) man nen (irCOI1 man sETR1) pi nyang ikum kit ma hormone ario eni gi tiyo kudu ikum lei ma camu lum The relative of reprogramming. Malube ku lembe mukadho, wa nwango nya herbivore-OS ubedo nuti i EV (lanyut 3, C ku D) man dwoko piny pa Hj index ma OSM ukelo, man δj index umedire. Adwogi ne nen maleng uloyu adwogi ma OSS1 ukelo. Graph ma rangi aryo ma tiyo ku Hj ku δj calu coordinates nyutho deregulation ma pire tek (lanyut 3E). Lembe ma nen kamaleng utiye nia i kind kit ma JA signal ope i iye, apoka poka pa metabolome man aloka loka ma wok kud i lei ma camu lum (lanyut 3C). I ayi pa tung tung, nen pa ET ma ling i sETR1 lum, kadi nang adwogi pa aloka loka pa kit ma cam pa lum utiye piny uloyu pa JA signaling, edwoko piny tung tung pa Hj ku δj indices ikind OSM ku OSS1 (lanyut 3D man cal S5). . Eni nyutho nia ikum tic ma pire tek pa JA signal transduction, ET signal transduction de tiyo calu fine-tuning pa kit lei ma camu lum. Malube ku tic mi yubu piny eni, epe aloka loka moku ikum metabolome inducibility in sETR1 plants. Icing ling acel, ka juporo ku yen sETR1, yen irACO ukelo alokaloka marom rom pa aloka loka pa lei ma camu lum, ento unyutho Hj man δj ma tung tung ikind OSM ku OSS1 (Figure S5).
Pi ni nyang ikum metabolites ma pire tek ma konyogi ni dwoko wang lembe pa lei ma camu lum man ni yubu tic migi nikadhu kud i ET signals, watiyo ku yore macon mi structural MS/MS. Yore eni ucungo iwi yore mi bi-clustering pi dwoko rwom pa metabolic family kud i MS/MS fragments [normalized dot product (NDP)] man similarity score based on neutral loss (NL). MS/MS data set ma juyubu nikadhu kud i nyang ikum ET transgenic lines unyayu 585 MS/MS (data file S1), ma juyubu ku poko gi i MS/MS modules abiro (M) (Figure 4A). Module moko mi module eni gipong ku jam piny ma juyiko con: calu, M1, M2, M3, M4 man M7 gitiye ku jam piny matung tung (M1), flavonoid glycosides (M2), acyl sugars (M3 And M4), man 17-HGL-DTG (M7). Mi medo, lembe ma mako metabolic (Si index) pa metabolite acel acel i module acel acel ju kwanu, man poko Si pare jucopu nenu. I lembe ma nok, MS/MS spectra ma nyutho herbivory ma lee man genotype specificity ungiyure ku Si values ​​ma malu, man kurtosis statistics nyutho poko pa yuk wic i kor cinge ma yor acam. Pol pa colloid acel ma kumeno junwango i M1, ma i iye phenol amide unyutho Si fraction ma malu (lanyut 4B). Herbivorous inducible 17-HGL-DTG ma juweco pire con i M7 unyutho Si score ma rwinyo, ma nyutho rwom ma rwinyo pa poko kind kit OS aryo eno. I ayi pa tung tung, pol pa piny ma juyubu maber, calu rutin, CGA, man acyl sugars, gitiye ikind Si scores ma piny. Pi ni nyang maber ikum kit ma piny nen kudu man poko pa Si ikind metabolites ma pire tek, juyike dul pa molecule pi module acel acel (lanyut 4B). Lembe ma pire tek ma jukoro i OD theory (ma jukwanu i cal mir 1B) utiye nia dwoko piny ma pire tek i ngei camu lum ucikere ni kelo aloka loka acel i metabolites ma ku rwom ma malu mi gwoko kum, asaga ni medo kero migi (ma tung tung ku random distribution) Mode) Defensive metabolite ma jukoro i MT theory. Pol pa phenol derivatives ma jucoko i M1 gitiye ku winjiri ku rwinyu pa tic pa lei makwiny (32). Ka waporo wel pa Si in M1 metabolites ikind ith ma juyiko ku ith ma juyiko pa EV control plants i sawa 24, waneno ni metabolic specificity pa metabolites mapol ingey lei ma camu lum utiye ku rwom ma malu (lanyut 4C). Medu pa Si value junwange kende i defensive phenolamides, ento medu pa Si unwangre ngo i phenols mange man metabolites ma jung'eyo ngo ma gikwo karacelu i module eni. Eni ubedo lanyuth ma pire tek, ma rwate ku OD theory. Lembe madongo ma jukoro ikum aloka loka pa piny ma dhanu ma camu lum kelo utiye marom. Pi ni temo ka nyo kit pa phenolamide spectrum eni ukelo OS-specific ET, wa goyo metabolite Si index man wa kelo tung tung pa OSM ku OSS1 i EV man sETR1 genotypes (Figure 4D ). I sETR1, tung tung ma phenamide ukelo ikind OSM ku OSS1 udoko piny akeca. Yore mi bi-clustering de jutiyo kudu pi MS/MS data ma jucoko i strains ma JA migi nok pi ni nyang ikum MS/MS modules madongo ma mako JA-regulated metabolic specialization (Figure S6).
(A) Adwogi mi poko 585 MS/MS malubiri ku dul ma jupoko (NDP marom) man rweny ma jupoko (NL marom) uketho module (M) ubedo marom ku dul pa jam piny ma jung'eyo, nyo ku dul pa Metabolite ma jung'eyo ngo nyo ma juloko ngo maber. I ngete pa module acel acel, poko pa metabolite (MS/MS) ma pire tek (Si) junyutho. (B) Modular molecular network: Nodes represent MS/MS and edges, NDP (red) and NL (blue) MS/MS scores (cut-off,> 0.6). Lanyuth pa metabolite ma jupoko (Si) ma juporo ku module (kor cing) man juporo ku molecular network (kor cing). (C) Module M1 pa EV plant in constitutive (control) man induced state (simulated herbivore) i sawa 24: molecular network diagram (Si value utiye node size, defensive phenolamide is highlighted in blue). (D) M1 molecular network diagram pa spectrum line sETR1 ku EV man ET ma nen ungo: the phenolic compound ma ju nyuthu ku green circle node, man tung tung madit (P value) ikind W + OSM man W + OSS1 ma jutiyo kudu calu dit pa 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 in wolfberry Son; Nick. O-AS, O-acyl sugar.
Wamedo medo nyang ikum genotype acel pa Nepenthes ni dul pa dhanu, kama aloka loka madit ikum lei ma camu lum man rwom pa metabolite ma juweco pire con i dul pa dhanu (26). Ti ku lembe maeni pi poko 43 germplasms. Germplasms eni utiye ku kit yen 123 ma aa kud i N. pallens. Yath eni juwodho kud i kodhi ma jucoko i kabedo matung tung i Utah, Nevada, Arizona, man California (lanyut S7), wa kwanu metabolome diversity (ma julwongo population level) β diversity) man specialization ma OSM ukelo. Malube ku somo mukadho, waneno aloka loka madwong ikum Hj man δj axes, ma nyutho nia germplasms utiye ku tung tung madit ikum kit ma gi dwoko kudu ikum lei ma camu lum (lanyut S7). Kanica eni poyo wiwa ikum lembe mukadho ikum aloka loka pa JAs ma wok kud i lei ma camu lum, man edaru bedo ku wel ma malu ikind dhanu acel (26, 36). Ni tiyo ku JA man JA-Ile pi temo rwom pa rwom pa Hj ku δj, wanwango ni rwom maber utiye ikind JA ku metabolome β diversity man specialization index (Figure S7). Eni nyutho nia tung tung ma wok kud i lei ma camu lum i JAs ma junwango i wel pa dhanu copo bedu ni kum polymorphisms ma pire tek ma wok kud i kwir pa lei ma camu lum.
Somo mukadho unyutho nia kit taba opokiri lee dit i kit man kite ma gicungo kudu ikum gwoko kum. Juyio nia aloka loka eni i rwom pa lanyuth pa lei ma camu lum man kero migi mi gwoko kum juyike ku rwom pa lei makwiny, kwo pa yen, man wel mi gwoko lei i kabedo ma lei moko dongo i iye. Wasomo ikum kit ma lum lokore kudu ma wok kud i lei ma camu lum pa kit Nicotiana abusiel ma nwangire i North America ku South America. Lei eni gi rwate ku Nepenthes North America, ma julwongo ni Nicolas Bociflo. La, N. nicotinis, Nicotiana n. lum ma nyap, Nicotiana tabacum, taba, taba (Nicotiana spegazzinii) man taba ma lum (Nicotiana obtusifolia) (lanyut 5A) (37). Abic pa kit yen eni, ikinde utiye N. please, gitiye yen ma dongo kubang oro pa petunia clade, man obtusifolia N. gitiye yen ma dongo pi oro acel pa Trigonophyllae (38). I ngeye, W + W, W + OSM man W + OSS1 induction jutimo ikum lei abiro eni pi niponjo kit ma lei moko loko ko cam pa lei moko.
(A) The bootstrap phylogenetic tree based on maximum likelihood [for nuclear glutamine synthesis (38)] and the geographical distribution of seven closely closely Nicotiana species (different rangis) (37). (B) Scatter plot pa kit ma tung tung pa kit kwo pa lei abiro pa Nicotiana (939 MS/MS; data file S1). I rwom pa lei, apoka poka pa metabolome utiye ku winjiri marac ku kadhiri pa ngec. Nyamu lembe ikum dul pa lei ma tung tung man rwom pa piny man rwom pa JA nen i cal mir 2. S9. Rangi, kit matung tung; triangle, W + OSS1; rectangle, W + OSM; (C) Nicotiana JA man JA-Ile dynamics jupoko gi nimakere ku OS excitation amplitude (two-way ANOVA man Tukey HSD i ngei poro mapol, * P <0.05, ** P <0.01 man * ** Pi poro pa W + OS man W + W, P <0.001). Box plot pa (D) tung tung man (E) poko pa lei acel acel ingey timo simulation pa lei ma camu lum man methyl JA (MeJA). Asterisk nyutho tung tung madit ikum W + OS ku W + W nyo lanolin plus W (Lan + W) nyo Lan plus MeJA (Lan + MeJa) man Lan control (two-way analysis of variance, followed by Tukey's HSD post hoc multiple comparison , *P<0.05, **P<0.01 and ***P<0.01).
Ni tiyo ku yore mi dual cluster, wanwango 9 modules pa 939 MS/MS (data file S1). Kit pa MS/MS ma juloko ku yore matung tung tung tung tung tung ikind kit lei (lanyut S8). Nen Hj (ma julwongo keni ni species-level γ-diversity) man δj nyutho nia species matung tung gicokiri i ungu matung tung i kabedo mi metabolic, kama apoka poka mi species ubedo piny ma pire tek uloyu ma sagu excitation. Ku weko N. linear ku N. obliquus, ginyutho adwogi matung tung mi kelo aloka loka (lanyut 5B). I ayi pa tung tung, lei ma calu N. purpurea man N. obtusifolia gitiye ku dwokowang ma nen ungo ikum yath, ento metabolome utiye matung tung. Poko pa kit lei ma tung tung pa dwokowang ikum piny ma jucwalo uketho apoka poka marac ikum kit ma jutimo kudu tic ku gamma diversity (PCC = -0.46, P = 4.9×10-8). OS-induced changes in JAs levels are positively correlated with metabolome specialization, and negatively correlated with metabolic gamma diversity exhibited by species each species (Figure 5B and Figure S9). Ber ni ngeyo nia lei ma julwongo ni "signal response" in cal mir 5C, calu Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, man Nepenthes attenuated, ukelo lanyuth ma pire tek i dakika 30. OS-specific JA man JA-Ile ma kawoni, ma bacteria mange ma julwongo ni "signal unresponsive", calu Nepenthes mills, Nepenthes powdery man N. obtusifolia nyutho kende JA-Ile Edge induction manangu OS specificity ope (Figure 5C). I rwom mi metabolic, calu ma juweco pire malu, pi Nepenthes ma judwoko piny, piny ma dwoko wang lembe unyutho OS specificity man umedire δj, man udwoko piny Hj. Adwogi pa OS-specific priming eni junwango ngo i lei ma jupoko ni lei ma timo ngo tic (lanyut 5, D man E). OS-specific metabolites jupoko kare ku kare ikind lei ma dwoko wang lembe, man dul pa lanyuth eni gicokiri ku lei ma dwoko wang lembe ma nok, man lei ma dwoko wang lembe ma goro nyuthu bedo ku winjiri manok (Figure S8 ). Adwogi eni nyutho nia OS-specific induction pa JAs man OS-specific reconfiguration pa downstream metabolome gi rwate i rwom pa lei.
I ngeye, watiyo ku lanolin paste ma tye ku methyl JA (MeJA) pi thiedho yen ni neno ka nyo kit mi rwate eni jukwero ku bedo tiye pa JA ma jutiyo kudu ku JA ma woko, ma bibedo i cytoplasm pa yen. Rapid deesterification ubedo JA. Wanwango kit ma rom pa aloka loka ma nok nok kud i lei ma dwoko wang lembe ni lei ma dwoko ngo lembe ma nyayu rwom pa JA (lanyut 5, D man E). I lembe ma nok, yath pa MeJA uketho metabolomes pa linear nematodes, N. obliquus, N. aquaticus, N. pallens, man N. mikimotoi udoko tek, ma ukelo dongo madit pa δj man dwoko piny pa Hj. N. purpurea unyutho kende kende mediri pa δj, ento ungo Hj. N. obtusifolia, ma jular junyutho nya ecoko JAs ma nok akeca, de dwoko wang marac ikum yath pa MeJA i yore mi dwoko metabolome. Adwogi eni nyutho nia JA production nyo signal transduction utiye piny ma juketho i kum lei ma dwoko ngo signal. Mi temo paru eni, waponjo kit lei angwen (N. pallens, N. mills, N. pink man N. microphylla) ma wok kud i W + W, W + OSMs man W + OSS1 Transcriptome (39). Malube ku kit ma metabolome timo kudu, lei ne gipokere maber i transcriptome space, ikindgi N. attenuated unyutho RDPI ma malu uloyu, man N. gracilis unyutho RDPI ma piny uloyu (lanyut 6A). Ento, junwango ni lembe ma jugoro ma N. oblonga ukelo ubedo piny uloyu ikind lei angwen, ma ukoc ku apoka poka madit pa N. oblonga ma jular junyutho i kind lei abiro. Somo mukadho unyutho nia dul pa genes ma rwate ku lanyuth mi gwoko kwo, ikinde utiye lanyuth pa JA, gonyo kit ma gwoko kwo ma kwong ma jukelo ku herbimore-related elicitors in Nicotiana species (39). Ka juporo JA signaling pathways ikind lei angwen eni unyutho kit ma ber (lanyut 6B). Pol pa genes i yore eni, calu AOC, OPR3, ACX man COI1, unyutho rwom ma malu pa induction ikind lei angwen eni. Ento, gene moko ma pire tek, JAR4, loko JA ni kit ma tiyo ku JA-Ile, man rwom pare utiye piny akeca, asaga in N. mills, Nepenthes pieris man N. microphylla. Mi medo, kende kende lembe ma jugoro pa gene mange AOS re ma junwange ngo i N. bifidum. Aloka loka eni i nyuthu pa gene copo bedu the lembe ma nyayu aloka loka madit ma wok ikum JA ma nok in lei ma timo signal anergic man kelo pa N. gracilis.
(A) Lembe ma jukoro ikum dwoko wang lembe ma jugoro con pa kit taba angwen ma rwate ma jucaku i ngei dakika 30 ingey camu lum. RDPI ju kwanu ku poro lum ma wok kud i herbivore OS ku wound control. Rangi nyuthu kit lei matung tung, man lanyuth nyuthu yore mi yath matung tung. (B) Nyamu lembe pa gene in JA signaling pathways ikind lei angwen. Yore mi JA ma juyubu yot nen inget box plot. Rangi matung tung nyutho yore matung tung mi tic. Asterisk nyutho nya tung tung madit utiye ikind yath pa W + OS ku yath pa W + W (pi Student's t-test pi tung tung pa ario, *P<0.05, **P<0.01 man ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
I thenge mir ajiki, wabeponjo kite ma loko kwo pa lei makwiny ma tung tung copo bedu tek ni lei ma camu lum. Lembe mukadho uketho cwiny ikum kit pa Nicotiana. Teko migi ikum Ms man larvae tung tung (40). Kae, wabeponjo dul pa lanyuth eni ku kit ma gi tiyo kudu. Ni tiyo ku kit taba angwen ma malu, man temo rwate pa tung tung man rwom pa metabolome ma wok kud i lei ma camu lum man kero pa yen ni Ms man Sl, wanwango ni rwom, tung tung man rwom ma malu ni generalist Sl All utiye ku winjiri maber, ento rwom pa rwom pa mon ma ngec migi tek man rwom ma malu utiye nyanok, man rwom ma tung tung ku Sl epe (Figure 10). Ku lembe pa S1 resistance, N. chinensis man N. gracilis ma judwoko piny, ma jular junyutho ni nyutho JA signal transduction levels man metabolome plasticity, gibedo ku dwokowang ma tung tung ni herbimore induction, man gin de ginyutho resistance ma malu marom. Butu.
I kind oro pyer abusiel mukadho, theori mi gwok pa yen umiyu yub, ma ju ma timo sayusac giporo wel madit pa dongo man tic pa yen. Pol pa paru eni lubu ngo yore ma rwinyo pa lembe ma tek (41). Gi ketho lembe ma pire tek (3) i rwom marom mi nyang ikum lembe. Ka temo pa lembe ma pire tek ma jular jukoro uketho juporo theories moko, eni biweko The field ube medere anyim. Bed ku kony, ento kwer jumange (42). Kakare, paru manyen timo lembe ma jular jukoro i rwom matung tung mi nyang ikum lembe man umedu rwom manyen mi lembe ma jukoro (42). Ento, paru ario ma juyero i thenge mi tic, MT ku OD, jucopo gonyo gi yot calu lembe ma pire tek ma jular jukoro ikum aloka loka ma jutimo ikum lei ma camu lum: paru pa OD yiyu niya aloka loka ikum “kabedo” pa metabolism utiye ku yore madit. The MT theory yiyu niya aloka loka eni bibedo ma nen ungo man bibedo iwi kabedo mi metabolic, man bibedo ku metabolites ma pire tek. Lembe mukadho ma jutimo ikum OD ku MT jutemo ku tic ku dul ma nok pa a priori "defense" compounds. Temo eni ma mako metabolite nyayu kero mi ngiyo kadhiri man kite ma metabolome loko kudu i kare mi camu lum, man eweko ngo temo i kind lembe ma rwate ma copo kwayu lembe ma pire tek ma juromo kwanu ni piny acel acel. Kae, watiyo ku diro manyen mi metabolomics ma ucungo iwi computational MS man watimo deconvolution MS analysis i rwom ma malu pa lembe mi ngec pi temo tung tung ma nuti kind lembe aryo ma juyero i rwom mi metabolomics mi ng'om zoo. Lembe ma pire tek ma jukoro i theori eni. Theory mi ngec jutiyo kudu i kabedo madwong, asaga in lembe mi kwo pa lei man rwom pa cam (43). Ento, kite ma wangeyo kudu, eni ubedo tic mir acel ma jutiyo kudu pi nyuthu kabedo mi ngec pa yen man cobo peko mi kwo ma mako aloka loka pa kwo pa yen. I andha, kero pa yore eni utiye i kero pare mi poro kit ma piny nen kudu i kind kit yen man ikind kit yen ni neno kite ma lei ma camu lum gicaku kudu kud i kit yen matung tung ni kit ma gikwo kudu i kind kit yen matung tung i rwom matung tung mi dongo. Metabolism.
Principal component analysis (PCA) loko dul pa lembe matung tung ni kabedu mi dwoko piny ma dit kara lembe ma pire tek utimiri, pieno pol kare jutiyo kudu calu yore mi ngiyo lembe, calu deconvolution metabolome. Ento, dwoko piny pa dimensionality bi rwenyo dul pa lembe ma nwang i data set, man PCA copo mio ngo lembe ma jukwanu ikum kit ma pire tek ni ecological theory, calu: kite ma lei ma camu lum loko kit ma tung tung i kabedo matung tung (lapor, lonyo, poko) man dwong) metabolites? Metabolites makani ma copo nyutho kite ma lei moko ma camu lum bibedo kudu? Ku neno pa lembe ma pire tek, tung tung man nyayu lembe, lembe ma nwang i kum kit ma ith yen moko loko kudu, man junwangu niya camu lei ma camu lum copo ketho tic pa piny ma tung tung. Ma dhanu ung'eyo ongo, waneno nia, calu ma jukoro i theori mi ngec ma jutiyo kudu, lembe ma wok kud i iye utiye ku rwom ma malu ingey lwiny pa lei ario ma camu lum (ju ma camu dyewor Sl) man Solanaceae expert Ms. Kadi bende kit cam migi man kero migi mi cam opokiri. Fatty acid-amino acid conjugate (FAC) initiator in OS (31). Ni tiyo ku herbimore OS pi timo yath pa ret ma juyiko, yath pa herbimore de unyutho kit marom. Lembe maeni ma juyiko pi lubo tok lembe ma yen timo ikum lwiny pa lei ma camu lum, jwigo lembe ma nyayu lworo ma wok ikum aloka loka mi kura pa lei ma camu lum, ma kelo peko matung tung i kare matung tung (34). FAC, ma ungeyere ni etiye the lembe madit ma nyayu OSM, dwoko piny JAS man adwogi pa hormone pa yen i OSS1, man OSS1 dwoko piny wang mapol (31). Ento, OSS1 ukelo rwom marom pa JA ma juporo ku OSM. Jular junyutho nia dwokowang' penji pa JA i Nepenthes ma judwoko piny ubedo ku kero madit ni OSM, kama FAC copo gwoko tic pare kadi nangu juyike 1:1000 ku pii (44). Pieno, ka juporo ku OSM, kadi bende FAC in OSS1 utiye piny akeca, eromo ni kelo two JA. Somo mukadho unyutho nia porin-like proteins (45) man oligosaccharides (46) jucopo tiyo kudu calu lanyuth pa molecule pi nyayu adwogi pa yen i OSS1. Ento, podi ungeyere ngo ka elicitors eni i OSS1 gitiye ku twero ikum coko pa JA ma juneno i somo ma kawoni.
Kadok nwango lembe manok kende re ma nyutho tung tung pa tic pa lei ma camu lum ma tung tung nyo JA nyo SA (salicylic acid) (47), ngati moko mbe ma nyayu aloka loka pa lei ma camu lum i lum pa lum man adwogi pare ikum piny ma tung tung. lembe peri giri. Lapor eni umedire ni ketho cing ikum dul pa hormone ma iye ku hormone pa yen mukende ma epe ku JAs loko kite ma metabolic reorganization ma lei ma camu lum kelo. I andha, wanwango ni ET ma OSM ukelo ubedo malac uloyu ma OSS1 ukelo. Kit maeni rwate ku FAC madwong in OSM, ma en e piny ma pire tek man ma romo pi nyayu ET burst (48). I lembe mi wec ikind yen ku lei ma camu lum, tic pa ET ikum kit ma yen moko lokore kudu podi utiye nya yot man etiye kende kende ikum dul acel kende. Mi medo, pol pa somo otiyu ku exogenous application pa ET nyo precursors pare nyo inhibitors matung tung pi ponjo regulation pa ET, ikindgi tic pa exogenous chemical maeni bi kelo adwogi marac mapol. Ni ngec mwa, somu eni nyutho tic ma dit mir acel ma jutimo ikum tic pa ET i tic ku ET pi yubu man nyang ikum yen ma juloko kugi ma rac pi yubu kit ma yen tiyo kudu. ET induction pa lei ma camu lum copo loko dwokowang pa metabolome. Ma pire tek uloyu utiye tic pa ET biosynthesis (ACO) man perception (ETR1) genes ma unyutho herbimore-specific de novo accumulation pa phenolamides. Jular junyutho nia ET copo dwoko rwom pa nicotine ma JA-ukelo ni rwom pa putrescine N-methyltransferase (49). Ento, kud i wang tic, epe kamaleng kite ma ET timo kudu tic pa phenamide. Mi medo ikum tic mi ET, tic pa metabolic flux copo bedu S-adenosyl-1-methionine pi jwigo wil i polyaminophenol amides. S-adenosyl-1-methionine ubedo ET man piny ma rwinyo polyamine biosynthetic pathway. Kite ma ET signal tiyo kudu pi jwigo kadhiri pa phenolamide mitu somo mukende.
Pi kare malac, pi wel madit pa metabolites ma pire tek ma jung'eyo ngo kite ma gikwo kudu, bedo moth madit ikum kit metabolic moko ubedu tek ni ngiyo aloka loka pa metabolic diversity ingey tic pa piny makwo. I kare eni, malubiri ku lembe ma jukoro, adwogi madit pa MS/MS spectrum acquisition ma lubere ku unbiased metabolites utiye nia lei ma camu lum ma camu nyo ma lubo lei ma camu lum mediri ni dwoko piny kit ma cam ma tung tung pa ithe gi medire kudu. Medu pa rwom pa metabolome ma wok kud i lei ma camu lum utiye ku rwom pa rwom pa transcriptome. Piny ma konyo lee i metabolome madit eni (ma utiye ku Si value ma malu) utiye metabolite ma pire tek ku tic mi camu lum. Model eni urombo ku paru pa OD theory, ento paru pa MT ma mako randomness pa metabolome reprogramming urombo ngo. Ento, lembe eni de urombo ku lembe ma jular jukoro ikum mixed model (MT maber loyo; cal mir 1B), kum metabolites mange ma jung'eyo ngo ku tic mi gwoko kum ma jung'eyo ngo copo lubo apoka poka pa Si.
Lembe ma pire tek ma jutimo i lembe maeni utiye nia nicaku i micro-evolution level (yen acel man taba) nitundu i evolutionary scale (kit taba ma rwate), rwom matung tung mi evolution organization utiye i "best defense" Utie tung tung madit i kero pa lei ma camu lum. Moore et al. (20) man Kessler ku Kalske (1) giparu giri ni loko rwom adek mi tic pa kwo ma tung tung ma Whittaker (50) unyutho ni aloka loka ma pire tek man ma kelo aloka loka pa kwo ma tung tung; jugor eni de gi goru ngo. Lembe ma jutimo pi coko lembe madwong ikum metabolome de unyutho ngo kite mi kwanu metabolic diversity kud i lembe eni. I lembe maeni, aloka loka manok ikum poko pa tic pa Whittaker bi ketho α-metabolic diversity calu tung tung pa MS/MS spectra in yen moko, man β-metabolic diversity calu basic intraspecific metabolism pa dul pa dhanu Space, man γ-metabolic diversity bibedo medo pa lembe ma rom.
JA signal piretek pi dwokowang lembe pa lei ma camu lum. Ento, epe ku rwom ma malu mi pimo kony pa intraspecific regulation pa JA biosynthesis ni metabolome diversity, man ka JA signal utiye kabedo ma pire tek pi stress-induced metabolic diversification i rwom ma malu mi macroevolutionary scale podi ungeyere ngo. Waneno nia kit pa Nepenthes ma camu lum nyayu tic pa metabolome man aloka loka pa metabolome ikind lei pa Nicotiana man ikind lei pa Nicotiana ma ceng ikumgi utiye ku winjiri maber ku JA signaling. Mi medo, ka JA signal ubedo marac, metabolic specificity ma jukelo ku genotype acel pa lei ma camu lum bibedo mbe (lanyut 3, C ku E). Calu ma aloka loka mi dul pa Nepenthes ma judwokogi piny ubedo wel, aloka aloka mi metabolic β diversity man specificity i lembe maeni copo bedu ni piny ma wok kud i rwom ma malu pa metabolite-rich compound categories. Juruot maeni gibedo ku twero iwi thenge mi metabolome profile man gikelo winjiri maber ku JA signals.
Pien kit ma kwo pa kodi taba ma rwate kude utiye tung tung, metabolites ungiyure i yore mi rwom, ci etiye lembe ma jutimo i iye sayusac. Theory mi lembe ma jukoro ikum kit ma piny ma jucaku kudu nyutho nia herbivorous induction medo rwom pa tic pa gamma man tic ma pire tek. JA signal timo tic ma pire tek i wil eni. Medu pa tic pa metabolome urombo ku OD ma pire tek man utiye ku winjiri maber ku JA signal, man JA signal utiye ku winjiri marac ku metabolic gamma diversity. Models eni nyutho nia OD capacity pa yen ucungo iwi plasticity pa JA, kadi i microevolutionary scale nyo i larger evolutionary scale. Expogenous JA application experiments ma circumvent JA biosynthesis defects medo nyutho nia kit taba ma ceng ceng jucopo poko gi ikind kit ma gi dwoko wang lembe man kit ma gi dwoko ngo wang lembe, calu kit migi mi JA man metabolome plasticity ma wok kud i lei ma camu lum. Lei ma timo ngo lembe maber copo ngo ni dwoko lembe pi bedo mbe migi mi kelo JA man kumeno gibedo ku peko mi kum. Eni copo bedu pi aloka loka pa genes moko ma pire tek in JA signaling pathway (AOS man JAR4 in N. crescens) pa. Adwogi eni nyutho nia kit kwo pa lei ma tung tung copo bedu pi aloka loka mi nen pa hormone man dwokowang lembe.
Mi medo ikum wec ma nuti kind yen ku lei ma camu lum, sayu lembe ikum tung tung pa piny ma kwo utiye ku winjiri ku lembe ma pire tek ma jutimo i sayu lembe ikum kwo pa lei man dongo pa kit ma kwo pa dhanu. Ku medu pa wel mi lembe ma ju nwango ku jam tic mi MS ma kawoni, pimo paru ikum tung tung pa metabolic dong copo kadho tung tung pa metabolite pa ngati acel acel/dul man timo sayusac mi ng'om zoo ni nyuthu kit ma jukuro ngo. Ikind timo sayusac madit, lanyuth ma pire tek utiye paru mi yubu map ma pire tek ma juromo tiyo kudu pi sayu lembe. Pieno, adwogi ma pire tek pa rwom ma kawoni pa MS/MS metabolomics man theori mi ngec utiye nya emiyo metric mayot ma juromo tiyo kudu pi yubu map pi neno tung tung pa metabolic i rwom matung tung. Etiye piny ma pire tek ma mitere pi yore eni. Somo pa micro/macro evolution man kwo pa dhanu.
I thenge mi macro-evolution, the lembe ma pire tek pa theory pa Ehrlich ku Raven (51) utiye ni nyutho nia aloka loka pa dul pa lei makwiny utiye the lembe ma nyayu apoka poka pa thek pa lei. Ento, ikind oro pyer abic nicaku i kare ma juwodho tic ma pire tek eni, paru eni jutemo ngo nyanok (52). Eni utiye pi kit pa phylogenetic pa kit pa metabolic ma rom ikind thek pa yen mabor. Rarity ecopu tiyo kudu pi ketho yore mi ngiyo lembe. Tic mi MS/MS ma kawoni ma jutimo ku theori mi ngec kwanu MS/MS marom pa metabolites ma jungeyo ngo (labongo yero metabolite macon) man loko MS/MSs eni ni dul pa MS/MS, kumeno i metabolism pa jurutic Models macro-evolutionary maeni juporo gi i rwom mi poko. Lembe ma yot ma nyutho wel. Lembe ne urombo ku phylogenetic analysis, ma copo tiyo ku sequence alignment pi pimo kadhiri pa tung tung nyo kit pa dhanu manangu epe ku paru moku cii.
I thenge mi biochemical, paru pa Firn ku Jones (53) nyutho nia metabolic diversity jugwoko i rwom matung tung mi mio piny ma jutiyo kudu pi timo tic pa metabolites ma con nyo ma juloko. Yore mi theori mi ngec miyo yore ma loko loko pa metabolite ma timere i saa mi metabolite ma jutimo kudu tic copo bedu acel ikin yub mi ngiyo evolution: biologically active adaptation kud i low specificity ni high specificity Inhibited metabolites of a given environment.
I lembe zo, i acaki pa molecular biology, lembe ma pire tek ikum gwoko yen juyubu, man yore mi deductive hypothesis-driven jubedu ka neno ni ubedu yore acel kende mi dongo ngec mi ngec. Eni utiye pi peko mi pimo metabolome zo. Kadi bende yore ma juporo ku lembe ma juparu pire tek i yero yore mukende ma kelo peko, kero migi mi medo ngec mwa ikum biochemical networks utiye nok uloyu yore mi kwan ma kawoni nuti i ngec ma kawoni ma tiyo ku lembe madwong. Pieno, paru ma jucopo ngo ni nyang ikume utiye mabor ku lembe ma nwangre, ci formula/test cycle mi dongo i tic mi sayu lembe copo bedu ngo (4). Waneno nia tic mi kwan pa metabolomics ma jukelo keni copo dwoko miti i kum lembe manyen (kite) man ma tok cen (pirango) mi metabolic diversity, man ekonyo i kare manyen mi theoretically guided data science. I kare ne udwoko wang lembe ma pire tek ma uketho dhanu macon ubedo ku miti.
Cam pa lei ma camu lum jutimo ku gwoko larva mir arionde nyo Sl larva iwi lum acel ma rangi pa rose blooming, ku lum 10 acel acel. Larvae pa lei eca ju mako ku clamps, man dul pa lum ma udong ju kwalo ingey sawa 24 ku 72 ingey nwangu two man ju ketho gi i friji pio pio, man ju kwanyo piny ma jucwalo.
Tim yath pa herbivorous i yore maber. Yore utiye ni tiyo ku tyend bongo mi goyo thwon adek i thenge acel acel mi theng lum adek ma dongo i kare ma bongo ubedo ka dongo, man ndhundhu ni tiyo ku 1:5 Diluted Ms. Nyo ti ku cing ma juketho i gloves pi ketho S1 OS i wang ret. Kwany man itim tic ku ith moko calu ma jukoro malu. Ti ku yore ma jukoro con ni kwanyu metabolites man hormones pa yen (54).
Pi tic pa JA ma woko, ith adek pa lum acel acel pa lum abusiel ma dongo pa lum acel acel juyiko ku 20μl pa lanolin paste ma tye ku 150μg MeJA (Lan + MeJA), man 20μl pa lanolin man yath pa ret (Lan + W), nyo jutiyo ku 20μl pa lanolin calu lanyuth. Ith ne ju kwalo ingey sawa 72 ingey yath, ju ketho gi i nitrogen ma pii, man ju gwoko gi i -80°C nitundu jutiyu kudu.
JA man ET angwen ma juloko, ma julwongo irAOC (36), irCOI1 (55), irACO man sETR1 (48), junwango gi i dul mwa mi sayu lembe. irAOC unyutho rwom ma piny pa JA man JA-Ile, ma irCOI1 ubedu ungo ku kero ikum JAs. Ka juporo ku EV, JA-Ile umedire. Kumeno bende, irACO bi dwoko piny ET, man ka juporo ku EV, sETR1, ma epe ku ET, bi medo ET.
Laser spectrometer (Sensor Sense ETD-300 real-time ET sensor) jutiyo kudu pi pimo ET ma epe ku peko. I ngei yath, nusu pa ith ne jupoko man jucwalo gi i 4-ml ma jugwoko maber, man juweko kabedo mi wiye upong ikind sawa abic. I kare mi pimo, vial acel acel juyike ku pii ma romo lita 2/sawa pi dakika 8, ma ukadhu kud i catalyst ma Sensor Sense umio pi kwanyu CO2 ku pii.
Lembe pa microarray jular juwodho i (35) man jugwoko i National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (accession number GSE30287). Lembe ma rwate ku ith ma wok kud i W + OSMs man yath ma unyothere juwodho pi somo eni. Teko ma rwinyo ubedo log2. Mapodi jutimo sayusac ikum wel, judwoko rwom pa piny man juyike ni 75th percentile ku tic ku R software package.
Lembe macon mi RNA sequencing (RNA-seq) pa kit Nicotiana juwodho kud i NCBI Short Reading Archives (SRA), namba pa tic ubedo PRJNA301787, ma jukoro ni Zhou et al. (39) man mediri calu ma jukoro i (56). Lembe ma jutiyo kudu ku W + W, W + OSM man W + OSS1 ma rwate ku kit Nicotiana juyeru pi nyang i somo eni, man jutiyo kudu i yo ma e: Mir acel, kwanu pa RNA-seq ma jutiyo kudu juloko ni FASTQ format. HISAT2 loko FASTQ ni SAM, man SAMtools loko SAM file ni BAM ma jupoko. StringTie jutiyo kudu pi kwanu gene expression, man yore pare mi nyuthu utiye nya etiye ku fragments pi thousand base fragments pi million sequenced transcription fragments.
Acclaim chromatographic column (150 mm x 2.1 mm; particle size 2.2μm) ma jutiyo kudu i sayu lembe man 4 mm x 4 mm guard column utiye ku piny marom. Gradient ma e piny eni jutiyo kudu i Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system: 0 ku 0.5 dakika, isocratic 90% A [deionized water, 0.1% (v/v) acetonitrile and 0.05% formic acid] B, 10% Acetonitrile and Acetonitrile 0.05% formic acid); 0.5 ku 23.5 dakika, rwom pa rwom ubedo 10% A man 90% B, kubang wadi; 23.5 ku 25 dakika, isocratic 10% A man 90% B. Kite ma pii mol kudu ubedo 400μl/min. Pi MS analyses zo, med column eluent i quadrupole man time-of-flight (qTOF) analyzer ma juketho ku electrospray source ma tiyo i positive ionization mode (capillary voltage, 4500 V; capillary outlet 130 V ; Drying temperature 200°C; drying airflow 10 liters/min).
Tim MS / MS fragment analysis (ma kawoni julwongo ni MS / MS) ma epe ku tic nyo ma copo poko ngo kud i lembe ma jugwoku pi nwangu lembe ma pire tek ikum metabolic profile. Lembe pa yore mi MS/MS ma rwinyore ucungo iwi lembe ma nia quadrupole utiye ku wang ma dit mi poko piny [pi meno, keth lembe mi pek-ku-charge (m/z) calu fragments]. Pi the lembe eni, pilembe jam tic mi Impact II ubedu ku copo mi cwio CE tilt, jutimo sayusac mapol ma jutiyo kudu ku rwom ma malu pa collision-induced dissociation collision energy (CE). I lembe ma nok, mir acel nen lanyuth ku UHPLC-electrospray ionization/qTOF-MS ma tiyo ku single mass spectrometry mode (low fragmentation conditions generated by in-source fragmentation), scanning from m/z 50 to 1500 at a repetition frequency of 5 Hz. Ti ku nitrogen calu gas mi turo pi MS/MS analysis, man itim pima peri giri i voltage angwen matung tung ma turo: 20, 30, 40, man 50 eV. Ikind kare mi pimo, quadrupole utiye ku wang dirisa madit mi poko pek, nicaku m/z 50 nituc i 1500. Ka m/z mi kore man lac mi poko piny juketho i 200, pek pa piny eca juyike kende kende ku software mi tic pa jam tic man 0 Da. Nen pi dul ma nok calu i kit pek acel. Ti ku sodium formate (50 ml isopropanol, 200 μl formic acid man 1 ml 1M NaOH aqueous solution) pi pimo pek pa piny. Ni tiyo ku Bruker's high-precision calibration algorithm, data file ju calibrate ingey timo spectrum ma rwom pare lapiny i kare moko. Ti ku tic mi cwalo piny woko pa Data Analysis v4.0 software (Brook Dalton, Bremen, Germany) mi loko lembe ma jukwanu i NetCDF. Lembe pa MS/MS jugwoko i metabolomics database MetaboLights (www.ebi.ac.uk) ku namba mi donjo. MTBLS1471.
MS/MS assembly copu bedu ni kadhu kud i nyang ikum MS1 man MS/MS quality signals pi kero ma nok man ma malu man cik manyen maju ketho. R script jutiyu kudu pi timo nyang ikum poko pa piny ma jucwalo, man C# script (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) jutiyo kudu pi ketho cik.
Pi ni jwigo lembe ma rac ma wok kud i dwand mi ngeye man lembe ma rac ma wok kud i nwangu m/z moko i lanyuth manok kende, watiyo ku "filled peak" pa R package XCMS (pi dwoko dwand mi ngeye) umaku jutii kudu pi loko "NA" (undetected peak) intensity. Ka jutiyo ku fill peak function, podi “0′′ intensity values ​​mapol nuti i data set ma bi loko wel mi correlation. I ngeye, waporo adwogi mi tic ku data ma wanwango ka jutiyo ku filled peak function man ka filled peak function jutiyu kudu ngo, man wa kwanu background noise value malube ki average corrected corrected, man wa dwoko ku 0 intensity values ​​eni wel pa piny ma ju kwanu. Wan bende waparu ikum piny ma tek migi ukadhu wang adek man waneno gi ni "peaks mandha." Pi kwan pa PCC, m/z signals pa sample precursor (MS1) man fragment data sets ku at least eight true peaks kende re ma jukwanu.
Ka kero pa kit ma ber pa precursor i lanyuth zoo utiye ku winjiri ku tek pa kit ma ber marom ma juketho i the tego pa collision ma nok nyo ma malu, man kit ne jukwanu ngo ni isotope peak ku CAMERA, ecopu bedu ma juloko. I ngeye, ku kwanu piny ceke ma copo bedu i kind 3 s (kare ma juporo pi gwoko piny ma malu), jutimo sayusac mi rwate. Ka wel pa m/z utiye piny uloyu wel pa precursor man MS/MS fragmentation timere i kabedo acel mi sample i data set calu precursor ma juwodho kud i iye, jubi kwanu ni fragment.
Malube ku cik aryo ma yot eni, waweko dul ma jukwanu ku m/z ma dit uloyu m/z pa piny ma jular juyero, man malubiri ku kabedo pa lanyuth kama piny ma kwong eca nen i iye man dul ma juyiko. Ecopu bedu bende ni yero kit maber ma juyubu ku dul mapol ma juyubu i MS1 mode calu precursors, ma kumeno juyubu redundant MS/MS compounds. Pi ni jwigo rwom pa lembe eni, ka NDP marom pa spectra ukadhu 0.6, man gitiye i chromatogram "pcgroup" ma CAMERA ukyewu, wabiketho gi karacelu. I ajiki, wa ketho adwogi angwen pa CE ma rwate ku precursor man fragments i final deconvoluted composite spectrum ku yero peak ma tek uloyu ikind peak ceke ma copo bedu ku m/z marom i collision energies matung tung. Thenge mi tic ma lubo ucungo iwi paru pa composite spectrum man uketho i iye lembe matung tung mi CE ma mitere pi medo kero mi poko, kum poko moko copo nwangire kende kende i the tego pa collision moko.
RDPI (30) jutiyo kudu pi kwanu inducibility pa metabolic profile. Kit ma metabolic spectrum diversity (Hj index) wok kudu kud i wel pa MS/MS precursors ma jutiyo kudu ku Shannon entropy pa MS/MS frequency distribution ma jutiyo kudu ku equation ma e ma Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) kama Pij rwate ku frequency pa i-th MS/MS in j-th sample (j = 1, 2,..., m) (i = 1, 2, …, m) t).
Metabolic specificity (Si index) gonyo ni rwom pa MS/MS ma jumiyo nimakere ku frequency ikind samples ma jubeparu. MS/MS specificity jukwanu ni Si = 1t (∑j = 1tPijPilog2PijPi)
Ti ku formula ma e ni pimo metabolome-specific δj index pa j acel acel, man wel pa MS/MS specificity δj = ∑i = 1mPijSi
MS/MS spectra juporo gi ario, man rom pa gi juporo malube ku wel aryo. Mir acel, nitio ku NDP (ma julwongo bende ni cosine correlation method), ti ku equation ma e ni pimo rwom pa dul ma rom ikind spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 kama S1 ku S2 Ma rom, pi spectrum 1 ku 2, man iWS2, 2 nyuthu pek pa rwom ma malo ma tung tung pa i-th common peak ikind spectra aryo utiye piny pa 0.01 Da. Pek ne ju kwanu kudu: W = [peak intensity] m [quality] n, m = 0.5, n = 2, calu ma MassBank uwacu.
Yore mir aryo mi poko wel jutiyo kudu, ma uketho i iye nyang ikum NL ma jupoko ikind MS/MS. Pi timo eni, watiyo ku 52 NL lists ma nwangre pol kare i kare mi MS fragmentation process i tandem, man NL ma pire tek (data file S1) ma jular jugoro pi MS/MS spectrum pa secondary metabolites pa wekened Nepenthes species ( 9, 26). Cwec binary vector pa 1 ku 0 pi MS/MS acel acel, ma rwate ku NL moko ma kawoni man ma epe. Malube ku rom pa bor pa Euclidean, NL marom pa wel ju kwanu pi dul acel acel pa NL vectors.
Mi timo dual clustering, watiyo ku R package DiffCoEx, ma ucungo iwi medo pa Weighted Gene Co-expression Analysis (WGCNA). Ni tiyo ku NDP man NL scoring matrices pa MS/MS spectra, watiyo ku DiffCoEx pi kwanu comparative correlation matrix. Binary clustering jutimo ku ketho "cutreeDynamic" parameter ni method = "hybrid", cutHeight = 0.9999, deepSplit = T, man minClusterSize = 10. R source code pa DiffCoEx juwodho kud i file 1 pa Tesson et al. (57); R WGCNA software package ma mitere i https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Pi timo MS/MS molecular network analysis, wa kwanu paired spectral connectivity malubiri ku NDP ku NL similarity types, man watiyo ku Cytoscape software mi neno network topology ni tiyo ku organic layout in CyFilescape yFiles layout algorithm extension application.
Ti ku R version 3.0.1 pi timo kwan pa lembe ikum lembe. Lembe ma pire tek ikum wel jupimu ku tic ku two-way analysis of variance (ANOVA), man ulubu ku Tukey's honestly significant difference (HSD) post-hoc test. Pi ni nyang tung tung mi yath ma camu lum ku yath ma jucamu ngo, poko pa dul aryo pa lanyuth ma tung tung marom juporo ku tic pa Student's t test.
Pi lembe mukende ma medo ikum lembe maeni, nen http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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