Endowooza ez’enjawulo ez’okukuuma ebimera ziwa obulagirizi obukulu obw’enzikiriziganya okunnyonnyola enkola z’enkyukakyuka y’ebimera mu ngeri ey’enjawulo, naye okulagula kwazo okukulu kukyalina okugezesebwa. Wano, twakozesa okwekenneenya okutaliimu kyekubiira kwa tandem mass spectrometry (MS/MS) okunoonyereza mu nkola enkola ya metabolome ya taaba attenuated strains okuva ku bimera ssekinnoomu okutuuka ku bungi n’ebika ebikwatagana ennyo, era ne tukola ku muwendo omunene ogw’endowooza z’ebifaananyi eby’amass spectrometric nga tusinziira ku compound spectra mu mawulire. Enkola okugezesa okulagula okukulu okw’endowooza z’okwekuuma okulungi (OD) n’ekigendererwa ekigenda (MT). Ekitundu ky’amawulire ekya metabolomics y’ebimera kikwatagana n’endowooza ya OD, naye kikontana n’okuteebereza okukulu okw’endowooza ya MT ku nkyukakyuka ya metabolomics ereetebwa ebisolo ebirya ebimera. Okuva ku micro okutuuka ku macro evolutionary scale, akabonero ka jasmonate kazuulibwa ng’ekikulu ekisalawo OD, ate akabonero ka ethylene kaawa okulongoosa okulungi okw’okuddamu okw’enjawulo eri ebisolo ebirya ebimera okwawandiikibwako omukutu gwa molekyu ogwa MS/MS.
Ebirungo eby’enjawulo ebikyusa ebiriisa ebirina ensengekera ez’enjawulo bye bisinga okwetaba mu kukwatagana kw’ebimera n’obutonde naddala mu kulwanirira abalabe (1). Enjawulo eyeewuunyisa ey’enkyukakyuka ey’enjawulo esangibwa mu bimera evuddeko okunoonyereza okw’obwegendereza okumala emyaka mingi ku bintu byayo bingi eby’emirimu gy’obutonde, era ekoze olukalala oluwanvu olw’endowooza z’okukuuma ebimera, nga zino ze nkulaakulana n’enkulaakulana y’obutonde bw’ensi ey’enkolagana y’ebimera n’ebiwuka. Okunoonyereza okukoleddwa mu ngeri ey’okugezesa (empirical research) kuwa obulagirizi obukulu (2). Naye, endowooza zino ez’okukuuma ebimera tezaagoberera kkubo lya normative erya hypothetical deductive reasoning, nga mu kino okulagula okukulu kwali ku mutendera gwe gumu ogw’okwekenneenya (3) era ne kugezesebwa mu ngeri y’okugezesa okutumbula enzirukanya eddako ey’enkulaakulana y’enzikiriziganya (4). Ebikoma mu by’ekikugu bikoma ku kukungaanya amawulire ku biti ebitongole eby’enkyukakyuka mu mubiri era ne biggyako okwekenneenya okujjuvu okw’ebirungo eby’enjawulo ebikyukakyuka, bwe kityo ne kiziyiza okugeraageranya wakati w’ebiti ebyetaagisa mu nkulaakulana y’enzikiriziganya (5). Obutabeera na data ya metabolomics enzijuvu n’ensimbi ez’awamu okugeraageranya enkola y’emirimu gy’okulongoosa ekifo ky’enkyukakyuka wakati w’ebibinja by’ebimera eby’enjawulo kiremesa okukula kwa ssaayansi kw’ennimiro.
Enkulaakulana ezisembyeyo mu kisaawe kya tandem mass spectrometry (MS/MS) metabolomics zisobola okulaga mu bujjuvu enkyukakyuka z’enkyukakyuka mu mubiri munda ne wakati w’ebika by’ensengekera y’ensengekera eweereddwa, era zisobola okugattibwa n’enkola z’okubalirira okubala okufaanagana kw’enzimba wakati w’ensengekera zino enzibu. Okumanya nga tonnaba ku kemiko (5). Okugatta tekinologiya ow’omulembe mu kwekenneenya n’okukozesa kompyuta kiwa omusingi ogwetaagisa ogw’okugezesa okumala ebbanga eddene okw’okuteebereza kungi okukoleddwa endowooza z’obutonde n’enkulaakulana ez’enjawulo mu nkyukakyuka y’emmere. Shannon (6) yaleeta endowooza y’amawulire omulundi gwe ogwasooka mu kiwandiiko kye eky’omusingi mu 1948, n’ateekawo omusingi gw’okwekenneenya amawulire mu kubala, ekibadde kikozesebwa mu bintu bingi okuggyako okugakozesa mu kusooka. Mu genomics, endowooza y’amawulire ebadde ekozesebwa bulungi okugera obungi bw’amawulire agakuuma ensengekera (7). Mu kunoonyereza ku transcriptomics, endowooza y’amawulire yeekenneenya enkyukakyuka okutwalira awamu mu transcriptome (8). Mu kunoonyereza okwasooka, twakozesa enkola y’ebibalo ey’endowooza y’amawulire ku metabolomics okunnyonnyola obukugu mu nkyukakyuka y’emmere ey’omutendera gw’ebitundu mu bimera (9). Wano, tugatta enkola y’emirimu eyesigamiziddwa ku MS/MS n’enkola y’emitindo ey’endowooza y’amawulire, emanyiddwa olw’enjawulo y’enkyukakyuka y’emmere mu ssente eza bulijjo, okugeraageranya okulagula okukulu okw’endowooza y’okukuuma ebimera eya metabolome ereetebwa ebisolo ebirya ebimera.
Enkola z’enzikiriziganya z’okukuuma ebimera zitera okuba nga zirimu buli omu era zisobola okwawulwamu ebika bibiri: ezo ezigezaako okunnyonnyola ensaasaanya y’ebirungo ebikyusa ebimera ebitongole nga byesigamiziddwa ku mirimu gy’okwekuuma, gamba ng’okuziyiza okulungi (OD) (10), ekigendererwa ekigenda (MT) (11) ) N’endowooza (12) n’endabika (12) endowooza, ate endala zinoonya ennyonyola ez’ebyuma ku ngeri enkyukakyuka mu kubeerawo kw’eby’obugagga gye zikwata ku bimera okukula n’okukuŋŋaanyizibwa kw’ebirungo eby’enjawulo ebikyusa ebiriisa, nga kaboni: endowooza y’okutebenkeza ebiriisa (13), endowooza y’omutindo gw’okukula (14 ), n’endowooza y’emyenkanonkano y’okukula n’enjawulo (15). Ensengekera z’endowooza zombi ziri ku mitendera egy’enjawulo egy’okwekenneenya (4). Naye, endowooza bbiri ezirimu emirimu gy’okwekuuma ku ddaala ly’emirimu zifuga emboozi ku kuziyiza ebimera ebikola n’okuleetebwa: endowooza ya OD, ekitwala nti ebimera biteeka ssente mu kuziyiza kwabyo okw’eddagala okw’ebbeeyi nga kyetaagisa, okugeza, nga biyingidde Ensolo y’omuddo bw’erumbye, n’olwekyo, okusinziira ku busobozi bw’okulumba mu biseera eby’omu maaso, ekibangirizi ekirina omulimu ogw’okwekuuma kiweebwa (10); endowooza ya MT etegeeza nti tewali axis ya nkyukakyuka ya directional metabolite, naye metabolite ekyuka randomly, bwe kityo ne kitondekawo okusobola Okulemesa metabolic “motion target” of attacking herbivores. Mu ngeri endala, endowooza zino ebbiri zikola okulagula okw’enjawulo ku kuddamu okukola kw’enkyukakyuka y’emmere okubaawo oluvannyuma lw’okulumba ebisolo ebirya ebimera: enkolagana wakati w’okukuŋŋaanyizibwa kw’ebirungo ebikyusa ebiriisa mu ngeri emu n’omulimu ogw’okwekuuma (OD) n’enkyukakyuka mu nkyukakyuka mu mubiri ezitali za bulagirizi (MT) (11 ).
Endowooza za OD ne MT tezizingiramu nkyukakyuka ezireetebwa mu metabolome zokka, naye n’ebiva mu butonde n’enkulaakulana olw’okukuŋŋaanyizibwa kw’ebirungo bino ebikyukakyuka, gamba ng’ebisale by’okukyusakyusa n’emigaso gy’enkyukakyuka zino ez’enkyukakyuka mu mbeera y’obutonde (16). Newankubadde endowooza zombi zimanyi omulimu gw’okwekuuma ogw’ebirungo eby’enjawulo ebikyusa ebiriisa, ebiyinza oba ebitayinza kuba bya bbeeyi, okulagula okukulu okwawula endowooza za OD ne MT kuli mu ndagiriro y’enkyukakyuka z’enkyukakyuka ezireetebwa. Okuteebereza kw’endowooza ya OD kwe kusinga okufaayo mu kugezesa okutuusa kati. Ebigezo bino mulimu okunoonyereza ku mirimu gy’okwekuuma obutereevu oba obutatereevu obw’ebitundu eby’enjawulo eby’ebirungo ebitongole mu girinaawuzi n’embeera z’obutonde, awamu n’enkyukakyuka mu mutendera gw’enkula y’ebimera (17-19). Naye, okutuusa kati, olw’obutaba na nkola ya mirimu n’enkola y’emitindo ey’okwekenneenya okujjuvu mu nsi yonna okw’enjawulo y’enkyukakyuka y’emmere y’ekiramu kyonna, okulagula kw’enjawulo enkulu wakati w’endowooza zombi (kwe kugamba, obulagirizi bw’enkyukakyuka mu nkyukakyuka mu mubiri) kukyalina okugezesebwa. Wano, tuwaayo okwekenneenya ng’okwo.
Ekimu ku bisinga okulaga ebirungo ebikyukakyuka mu bimera (plan-specific metabolites) kwe kukyukakyuka kwabyo okuyitiridde mu nsengeka ku mitendera gyonna okuva ku bimera ebimu, ebika okutuuka ku bika ebifaanagana ( 20 ). Enkyukakyuka nnyingi mu bungi mu metabolites ez’enjawulo zisobola okulabibwa ku mutendera gw’omuwendo gw’abantu, ate enjawulo ez’amaanyi ez’omutindo zitera okukuumibwa ku mutendera gw’ebika ( 20 ). Nolwekyo, enjawulo mu nkyukakyuka y’ebimera y’ensonga enkulu ey’enjawulo y’emirimu, eraga okukyusakyusa mu bifo eby’enjawulo naddala ebifo ebyo ebirina obusobozi obw’enjawulo obw’okulumba ebiwuka eby’enjawulo n’ebirya ebimera ebya bulijjo ( 21 ). Okuva ekiwandiiko kya Fraenkel (22) ekyamenyawo ensonga eziviirako okubaawo kw’ebirungo ebikyusa ebimera ebitongole, enkolagana n’ebiwuka eby’enjawulo ebadde etwalibwa ng’okunyigirizibwa okukulu mu kulonda, era enkolagana zino kirowoozebwa nti zaabumba ebimera mu kiseera ky’enkulaakulana. Ekkubo ly’enkyukakyuka mu mubiri (23). Enjawulo wakati w’ebika mu njawulo y’ebirungo ebikyukakyuka eby’enjawulo era eyinza okulaga enzikiriziganya y’omubiri ekwatagana n’okuziyiza ebimera ebikola n’okuleetebwa okuva ku bukodyo bw’okulya omuddo, kubanga ebika bino byombi bitera okukwatagana obubi ne bannaabwe ( 24 ). Wadde nga kiyinza okuba eky’omugaso okukuuma okwekuuma okulungi ekiseera kyonna, enkyukakyuka mu budde mu nkyukakyuka y’emmere ezikwatagana n’okwekuuma ziwa emigaso egy’olwatu mu kusobozesa ebimera okugabanya eby’obugagga eby’omuwendo mu nsimbi endala ez’omubiri (19, 24), n’okwewala obwetaavu bw’okubeera awamu. Okwonoonebwa okw’omusingo (25). Okugatta ku ekyo, okuddamu okutegeka kuno okw’ebirungo eby’enjawulo ebikyukakyuka okuva ku biwuka ebirya omuddo kuyinza okuvaako okusaasaana okuzikiriza mu kibinja (26), era kuyinza okulaga okusoma obutereevu enkyukakyuka ez’obutonde ennene mu kabonero ka asidi wa jasmonic (JA), akayinza okukuumibwa mu kibinja. Obubonero bwa JA obwa waggulu n’obwa wansi busuubulagana wakati w’okwekuuma ebisolo ebirya ebimera n’okuvuganya n’ebika ebitongole ( 27 ). Okugatta ku ekyo, amakubo ag’enjawulo aga metabolite biosynthetic gajja kuyita mu kufiirwa okw’amangu n’okukyuka mu kiseera ky’enkulaakulana, ekivaamu okusaasaana kw’enkyukakyuka mu bitundutundu mu bika ebikwatagana ennyo ( 28 ). Enkyukakyuka zino ez’enjawulo zisobola okuteekebwawo amangu nga ziddamu enkyukakyuka mu nkola z’ebisolo ebirya ebimera (29), ekitegeeza nti enkyukakyuka y’ebitundu by’ebisolo ebirya ebimera nsonga nkulu evuga obutafaanagana mu nkyukakyuka y’emmere.
Wano, mu ngeri ey’enjawulo twagonjoola ebizibu bino wammanga. (I) Ekiwuka ekirya omuddo kiddamu kitya okusengeka metabolome y’ekimera? (Ii) Bitundu ki ebikulu eby’amawulire eby’obuveera bw’enkyukakyuka mu mubiri ebiyinza okupimibwa mu bungi okugezesa okulagula kw’endowooza y’okwekuuma okw’ekiseera ekiwanvu? (Iii) Oba okuddamu okukola pulogulaamu y’enkyukakyuka y’ebimera mu ngeri ey’enjawulo eri oyo alumba, bwe kiba bwe kityo, kifo ki obusimu bw’ekimera kye bukola mu kutunga eky’okuddamu eky’enjawulo eky’enkyukakyuka mu mubiri, era biki ebikyusakyusa ebiyamba mu ngeri ey’enjawulo ey’okwekuuma? (Iv) Okuva okulagula okukolebwa endowooza nnyingi ez’okwekuuma bwe kuyinza okugaziwa mu mitendera gyonna egy’ebitundu by’ebiramu, twabuuza engeri okuddamu kw’enkyukakyuka y’emmere ereetebwa gye kukwataganamu okuva mu kugeraageranya okw’omunda okutuuka ku kugeraageranya wakati w’ebika? Ku nsonga eno, tusomye mu nkola entegeke enkyukakyuka y’amakoola ga taaba nicotine, ekimera eky’ekyokulabirako eky’obutonde nga kirimu enkyukakyuka ey’enjawulo ennungi, era kikola bulungi ku nkwaso z’ebisolo bibiri eby’enjawulo ebirya omuddo, Lepidoptera Datura (Ms) ( Very aggressive, mainly eaten) Ku Solanaceae ne Spodoptera litoralis (Sl), ensowera z’amakoola ga ppamba kika kya “ekika”, n’ebimera ebikyaza Solanaceae n’ebimera ebirala ebikyaza ebika ebirala n’amaka Emmere y’ebimera. Twasengejja MS/MS metabolomics spectrum ne tuggyamu ebinyonyola by’emitindo gy’endowooza y’amawulire okugeraageranya endowooza za OD ne MT. Tonda maapu z’obutonde (specificity maps) okulaga enjawulo y’ebirungo ebikulu ebikyusa ebiriisa. Okwekenenya kuno kwagaziyizibwa ku bantu enzaalwa za N. nasi n’ebika bya taaba ebikwatagana ennyo okwongera okwekenneenya enjawulo wakati w’obubonero bw’obusimu bw’ebimera n’okuyingiza OD.
Okusobola okukwata maapu okutwalira awamu ku buveera n’ensengekera y’amakoola metabolome ya taaba alya omuddo, twakozesa enkola y’emirimu eyakolebwa emabegako okwekenneenya n’okubalirira okukung’aanya mu bujjuvu n’okuggyawo data ey’obulungi obw’amaanyi eyeetongodde MS / MS spectra okuva mu bimera ebiggiddwamu ( 9 ). Enkola eno etali ya njawulo (eyitibwa MS/MS) esobola okuzimba spektira z’ebirungo ezitali za muwendo, oluvannyuma eziyinza okukozesebwa mu kwekenneenya kwonna okw’omutendera gw’ekirungo okunnyonnyoddwa wano. Ebirungo bino ebikyukakyuka mu bimera ebitali bifuuse bya bika bya njawulo, nga birimu ebikumi n’ebikumi by’ebirungo ebikyukakyuka (nga 500-1000-s/MS/MS wano). Wano, tulowooza ku buveera bw’enkyukakyuka mu mubiri mu nkola y’endowooza y’amawulire, era ne tugera obungi bw’enjawulo n’obukugu bwa metabolome nga tusinziira ku Shannon entropy y’ensaasaanya y’emirundi gy’enkyukakyuka y’emmere. Nga tukozesa ensengekera eyateekebwa mu nkola emabegako (8), twabala ekibinja ky’ebiraga ebiyinza okukozesebwa okugera obungi bw’enjawulo ya metabolome (Hj indicator), metabolic profile specialization (δj indicator) ne metabolic specificity of a single metabolite (Si indicator) . Okugatta ku ekyo, twakozesa Relative Distance Plasticity Index (RDPI) okugera obungi bwa metabolome inducibility y’ebisolo ebirya ebimera (Figure 1A) (30). Mu nkola eno ey’ebibalo, tukwata MS/MS spectrum nga ekitundu ky’amawulire ekikulu, era ne tukola ku bungi obw’enjawulo obwa MS/MS mu maapu y’okusaasaanya frequency, n’oluvannyuma ne tukozesa Shannon entropy okubalirira enjawulo ya metabolome okuva mu yo. Okukuguka kwa metabolome kupimibwa n’obutuufu bwa wakati obwa MS/MS spectrum emu. N’olwekyo, okweyongera mu bungi bw’ebibinja ebimu ebya MS/MS oluvannyuma lw’okuyingiza ebimera ebirya ebimera kukyusibwa ne bifuuka spectral inducibility, RDPI n’okukuguka, kwe kugamba, okweyongera mu δj index, kubanga ebirungo ebikyusa ebiriisa eby’enjawulo ebisingawo bikolebwa era omuwendo gwa Si ogw’amaanyi ne gukolebwa. Okukka kw’omuwendo gw’enjawulo ya Hj kulaga nti oba omuwendo gwa MS/MS ezikoleddwa gukendedde, oba engabanya ya frequency ya profile ekyuka mu kkubo eritali lya kimu, ate nga kikendeeza ku butakakasa bwayo okutwalira awamu. Okuyita mu kubala omuwendo gwa Si, kisoboka okulaga MS/MS ki ezireetebwa ebisolo ebimu ebirya ebimera, okwawukana ku ekyo, MS/MS ki ezitaddamu kuyingizibwa, nga kino kye kiraga ekikulu okwawula okuteebereza kwa MT ne OD.
(A) Ebinnyonnyola ebibalo ebikozesebwa ku bimera ebirya ebimera (H1 okutuuka ku Hx) MS/MS data-inducibility (RDPI), enjawulo (Hj index), okukuguka (δj index) n’obutonde obw’enjawulo (Si index). Okweyongera mu ddaala ly’okukuguka (δj) kiraga nti, ku kigero, ebirungo ebikyusa ebiriisa ebitongole ebisingawo ebirya ebimera bijja kukolebwa, ate okukendeera mu njawulo (Hj) kulaga okukendeera mu kukola ebirungo ebikyusa ebiriisa oba okusaasaana okutali kwa bwenkanya okw’ebirungo ebikyusa ebiriisa mu maapu y’okusaasaanya . Omuwendo gwa Si gwekenneenya oba ekirungo ekikyukakyuka (metabolite) kyetongodde ku mbeera eweereddwa (wano, ekirya omuddo) oba mu ngeri endala kikuumibwa ku ddaala lye limu. (B) Ekifaananyi ky’endowooza eky’okuteebereza endowooza y’okwekuuma nga tukozesa ekisiki ky’endowooza y’amawulire. Endowooza ya OD eragula nti okulumba kw’ebisolo ebirya ebimera kujja kwongera ku bikozesebwa mu kwekuuma, bwe kityo ne kyongera δj. Mu kiseera kye kimu, Hj ekendeera kubanga profile eddaamu okutegekebwa okutuuka ku butakakasa obukendedde obw’amawulire agakwata ku nkyukakyuka y’emmere. Endowooza ya MT eragula nti okulumba kw’ebisolo ebirya ebimera kujja kuleeta enkyukakyuka ezitali za ndagiriro mu metabolome, bwe kityo ne kyongera Hj ng’ekiraga okweyongera kw’obutali bukakafu bw’amawulire agakwata ku nkyukakyuka y’emmere era ne kireetawo okusaasaana kwa Si mu ngeri ey’ekifuulannenge. Twateesa n’ekyokulabirako ekitabuddwa, MT esinga obulungi, nga mu bino ebirungo ebimu ebikyusa ebiriisa ebirina emiwendo egy’okwekuuma egy’oku ntikko bijja kwongerwako naddala (omuwendo gwa Si ogw’amaanyi), ate ebirala biraga okuddamu okw’ekifuulannenge (omuwendo gwa Si ogwa wansi).
Nga tukozesa ennyonyola z’endowooza y’amawulire, tutaputa endowooza ya OD okulagula nti enkyukakyuka ez’enjawulo ezireetebwa ebimera ebirya ebimera mu mbeera etandikibwawo ejja kuleeta (i) okweyongera mu metabolic specificity (Si index) okuvuga metabolic specificity (δj index) Okweyongera kwa) ebibinja ebimu eby’enjawulo metabolite ebirina omuwendo gw’okwekuuma ogwa waggulu, ne (ii) okukendeera kw’enjawulo ya metabolome (Hj index) olw’enkyukakyuka y’ensaasaanya y’emirundi gy’enkyukakyuka mu mubiri okudda mu kugabanya kw’omubiri gwa leptin okusingawo. Ku ddaala lya metabolite emu, ensaasaanya ya Si etegekeddwa esuubirwa, nga metabolite ejja kwongera ku muwendo gwa Si okusinziira ku muwendo gwakyo ogw’okwekuuma (Figure 1B). Ku layini eno, tunnyonnyola endowooza ya MT okulagula nti okusikirizibwa kujja kuleeta (i) enkyukakyuka ezitali za ndagiriro mu metabolites ekivaamu okukendeera mu δj index, ne (ii) okweyongera mu Hj index olw’okweyongera mu metabolic uncertainty. Oba randomness, eyinza okupimibwa mu bungi ne Shannon entropy mu ngeri ya generalized diversity. Ku ky’obutonde bw’enkyukakyuka mu mubiri, endowooza ya MT ejja kulagula ensaasaanya ya Si mu ngeri ey’ekifuulannenge. Nga tussa mu nkola nti ebirungo ebimu ebikyusa ebiriisa biri wansi w’embeera ezenjawulo wansi w’embeera ezenjawulo, era embeera endala teziri wansi wa mbeera ntongole, era omuwendo gwazo ogw’okwekuuma gusinziira ku butonde, era twateesa ku nkola y’okwekuuma okutabuliddwa, nga δj ne Hj zisaasaanyizibwa mu bibiri okumpi ne Si Increase mu njuyi zonna, ebibinja by’ebirungo ebimu byokka, ebirina emiwendo egy’okwekuuma egy’oku ntikko, bye bijja kwongera naddala Si, ate ebirala bijja kuba n’eky’okwekuuma ekitali kigenderere okusaasaana (Ekifaananyi 1B).
Okusobola okugezesa okulagula kw’endowooza y’okwekuuma okuddamu okunnyonnyolwa ku kisenge ky’ennyonnyola y’endowooza y’amawulire, twakuza enkwaso z’ebimera ebirya ebimera eby’ekikugu (Ms) oba eby’enjawulo (Sl) ku bikoola bya Nepenthes pallens (Ekifaananyi 2A). Nga tukozesa okwekenneenya kwa MS/MS, twaggya 599 non-redundant MS/MS spectra (data file S1) okuva mu bikuta bya methanol eby’ebitundu by’amakoola ebyakung’aanyizibwa oluvannyuma lw’okuliisa enkwale. Okukozesa RDPI, Hj, ne δj indexes okulaba okuddamu okusengeka ebirimu amawulire mu fayiro z’okusengeka MS/MS kiraga enkola ezisikiriza (Ekifaananyi 2B). Omuze okutwalira awamu guli nti, nga bwe kinyonyoddwa omunnyonnyola w’amawulire, ng’enkwale zeeyongera okulya ebikoola, diguli y’okuddamu okutegeka kwonna okw’enkyukakyuka y’emmere yeeyongera okumala ekiseera: essaawa 72 oluvannyuma lw’ekisolo ekirya omuddo okulya, RDPI yeeyongera nnyo. Bw’ogeraageranya n’ekifuga ekitali kwonooneka, Hj yakendeera nnyo, ekyava ku ddaala eryeyongedde ery’okukuguka mu nkyukakyuka y’emmere, eyapimibwa mu bungi n’omuwendo gwa δj. Omuze guno ogulabika gukwatagana n’okuteebereza kw’endowooza ya OD, naye tegukwatagana n’okuteebereza okukulu okw’endowooza ya MT, ekkiriza nti enkyukakyuka ezitali za bulijjo (ezitali za ndagiriro) mu mitendera gy’ebirungo ebikyusa ebiriisa zikozesebwa ng’okukweka okw’okwekuuma (Ekifaananyi 1B). Newankubadde nga ebirimu oral secretion (OS) elicitor n’enneeyisa y’emmere y’ebisolo bino ebibiri ebirya omuddo bya njawulo, okuliisa kwabyo obutereevu kwavaamu enkyukakyuka ezifaanagana mu ndagiriro za Hj ne δj mu biseera by’okukungula eby’essaawa 24 n’essaawa 72. Enjawulo yokka yabaddewo ku ssaawa 72 eza RDPI. Bw’ogeraageranya n’ekyo ekyava ku Ms feeding, okutwalira awamu metabolism eyaleetebwa Sl feeding yali waggulu.
(A) Enteekateeka y’okugezesa: embizzi eza bulijjo (S1) oba abakugu (Ms) ezirya ebimera ziliisibwa ebikoola by’ebimera by’ensuwa ebitaliimu munnyo, ate ku kulya omuddo okukoppa, OS ya Ms (W + OSMs) ekozesebwa okukwata okuboola ebiwundu mu bifo by’amakoola ebituufu. S1 (W + OSSl) enkwaso oba amazzi (W + W). Ekifuga (C) kibala ekitayonoonebwa. (B) Inducibility (RDPI bw’ogeraageranya n’ekipande ekifuga), enjawulo (Hj index) n’okukuguka (δj index) index ebaliriddwa ku maapu ey’enjawulo ey’ebirungo ebikyusa ebiriisa (599 MS/MS; data file S1). Emmunyeenye ziraga enjawulo ennene wakati w’okuliisa ebisolo ebirya ebimera obutereevu n’ekibinja ekifuga (Student’s t-test with paired t-test, *P<0.05 and ***P<0.001). ns, si kikulu. (C) Omuwendo gw’okusalawo kw’obudde obw’ebikulu (ekibokisi kya bbululu, amino asidi, asidi ow’obutonde ne ssukaali; fayiro ya data S2) n’ensengekera y’ebirungo ebikyusa ebiriisa eby’enjawulo (ekibokisi ekimyufu 443 MS/MS; fayiro ya data S1) oluvannyuma lw’okujjanjaba ebimera ebikoppa. Bandi ya langi kitegeeza ekiseera ky’obwesige ekya 95%. Emmunyeenye eraga enjawulo ennene wakati w’obujjanjabi n’okufuga [quadratic analysis of variance (ANOVA), n’eddirirwa Tukey’s honestly significant difference (HSD) for post hoc multiple comparisons, *P<0.05, **P<0.01 And *** P <0.001]. (D) Okukuguka kwa puloti z’okusaasaana n’ebifaananyi eby’enjawulo eby’ebirungo ebikyusa ebiriisa (sampuli eziddiŋŋanwa nga zirina enzijanjaba ez’enjawulo).
Okunoonyereza oba okuddamu okukola okuva mu bimera ebirya ebimera ku ddaala lya metabolome kweyolekera mu nkyukakyuka mu ddaala ly’ebirungo ebikyusa ebiriisa ssekinnoomu, twasooka kussa essira ku bikozesebwa mu kukyusa ebiriisa ebyasomesebwa emabegako mu bikoola bya Nepenthes pallens nga bikakasiddwa nti bigumira ebimera ebirya ebimera. Phenolic amides ze hydroxycinnamamide-polyamine conjugates ezikuŋŋaanyizibwa mu nkola y’okulya ebiwuka era nga zimanyiddwa okukendeeza ku nkola y’ebiwuka ( 32 ). Twanoonya ebisookerwako ebya MS/MS ebikwatagana ne tukola pulaani ya cumulative kinetic curves zaabwe (Figure S1). Tekyewuunyisa nti ebiva mu phenol ebitakwatibwako butereevu mu kuziyiza ebisolo ebirya omuddo, nga chlorogenic acid (CGA) ne rutin, biba bitereezebwa wansi oluvannyuma lw’okulya omuddo. Okwawukana ku ekyo, ebisolo ebirya ebimera bisobola okufuula phenol amides ez’amaanyi ennyo. Okuliisa okutambula obutasalako kw’ebisolo ebibiri ebirya omuddo kwavaamu kumpi ensengekera y’okusikirizibwa y’emu eya phenolamides, era enkola eno yali yeeyoleka nnyo naddala ku de novo synthesis ya phenolamides. Ekintu kye kimu kijja kulabibwa nga tunoonyereza ku kkubo lya 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), erikola omuwendo omunene ogwa acyclic diterpenes ezirina emirimu emirungi egy’okulwanyisa omuddo (33), nga ku zino Ms Feeding with Sl yasitula enkola y’okwolesebwa efaananako (Figure S1)).
Ekizibu ekiyinza okubaawo mu kugezesa okuliisa ebimera ebirya ebimera obutereevu y’enjawulo mu kigero ky’okulya ebikoola n’obudde bw’okuliisa ebisolo ebirya omuddo, ekizibuwalira okumalawo ebikosa ebisolo ebirya omuddo ebitongole ebiva ku biwundu n’ebisolo ebirya omuddo. Okusobola okugonjoola obulungi ebika by’ebimera ebirya ebimera eby’enjawulo eby’okuddamu kw’enkyukakyuka y’amakoola okuleetebwa, twakoppa okuliisa enkwaso za Ms ne Sl nga tusiiga amangu ddala OS eyaakakung’aanyizibwa (OSM ne OSS1) ku mutindo gw’okuboola W ogw’ebifo by’amakoola ebikwatagana. Enkola eno eyitibwa obujjanjabi bwa W + OS, era essa omutindo ku induction nga eteeka obudde obutuufu okutandika kw’okuddamu okuleetebwa ekisolo ekirya omuddo awatali kuleeta bikolwa bitabula eby’enjawulo mu kigero oba obungi bw’okufiirwa kw’ebitundu (Figure 2A) (34). Nga tukozesa payipu y’okwekenneenya n’okubalirira eya MS/MS, twaggyayo ebifaananyi bya MS/MS 443 (fayiro ya data S1), ebyakwatagana ne spektira ezaakuŋŋaanyizibwa emabegako okuva mu kugezesa emmere ey’obutereevu. Okwekenenya endowooza y’amawulire mu kibiina kino ekya MS/MS data set kwalaga nti okuddamu okukola pulogulaamu ya metabolomes ez’enjawulo ez’amakoola nga tukoppa ebisolo ebirya omuddo kwalaga ebisikiriza ebikwata ku OS (Figure 2C). Okusingira ddala, bw’ogeraageranya n’obujjanjabi bwa OSS1, OSM yaleeta okunywezebwa kw’okukuguka kwa metabolome ku ssaawa 4. Kikulu okumanya nti bw’ogeraageranya n’ensengeka y’ebikwata ku kugezesa okuliisa ebimera ebirya ebimera obutereevu, entambula y’enkyukakyuka y’emmere eyalabibwa mu kifo eky’ebitundu bibiri nga tukozesa Hj ne δj nga enkwatagana n’obulagirizi bw’okukuguka kw’ebimera mu kuddamu obujjanjabi bw’ebisolo ebirya ebimera ebikoppa okumala ekiseera Okwongera obutakyukakyuka (Figure 2D). Mu kiseera kye kimu, twagera obungi bw’ebirimu amino asidi, asidi ez’obutonde ne ssukaali (data file S2) okunoonyereza oba kino ebigendererwamu okweyongera mu bukugu mu metabolome kiva ku kuddamu okusengeka enkyukakyuka ya kaboni ey’omu makkati mu kuddamu eri ebisolo ebirya ebimera ebikoppa ( Ekifaananyi S2). Okusobola okunnyonnyola obulungi enkola eno, twayongera okulondoola enkola y’okukuŋŋaanyizibwa kw’enkyukakyuka y’emmere (metabolic accumulation kinetics) ey’amakubo ga phenolamide ne 17-HGL-DTG agaayogerwako emabegako. Enkola ya OS-specific induction y’ebisolo ebirya ebimera ekyusibwa mu nkola ey’okuddamu okusengeka ey’enjawulo munda mu nkyukakyuka ya phenolamide (Figure S3). Phenolic amides ezirimu ebitundu bya coumarin ne caffeoyl zisinga kuleetebwa OSS1, ate OSMs zisitula okuyingiza okwetongodde okwa ferulyl conjugates. Ku kkubo lya 17-HGL-DTG, differential OS induction nga ekolebwa wansi wa malonylation ne dimalonylation products yazuulibwa (Figure S3).
Ekiddako, twasoma obuveera bwa OS-induced transcriptome nga tukozesa time-course microarray data set, ekoppa enkozesa ya OSMs okujjanjaba ebikoola by’ebikoola by’ekimera kya rosette mu bimera ebirya ebimera. Enkola y’okutwala sampuli okusinga ekwatagana ne kinetics ekozesebwa mu kunoonyereza kuno okwa metabolomics ( 35 ). Okugerageranya n’okuddamu okusengeka metabolome nga mu kino obuveera bw’enkyukakyuka bweyongera naddala mu biseera, tulaba okubutuka kw’okuwandiika okw’ekiseera mu bikoola okuleetebwa Ms, nga transcriptome inducibility (RDPI) n’okukuguka (δj) biri ku 1 Waaliwo okweyongera okw’amaanyi mu ssaawa, n’enjawulo (Hj) mu kiseera kino, okwolesebwa kwa BMP1 kwakendeera nnyo, ne kugobererwa okuwummulamu wa transcriptome okukuguka (Ekifaananyi S4). Amaka g’obuzaale bw’enkyukakyuka (nga P450, glycosyltransferase, ne BAHD acyltransferase) geetaba mu nkola y’okukuŋŋaanya ebirungo eby’enjawulo ebikyukakyuka okuva mu yuniti z’enzimba ezisibuka mu nkyukakyuka y’ebirungo ebisookerwako, nga bagoberera enkola eyogeddwako waggulu ey’okukuguka okw’amaanyi okwasooka. Nga okunoonyereza ku mbeera, ekkubo lya phenylalanine lyakeberebwa. Okwekenenya kukakasa nti obuzaale obukulu mu nkyukakyuka ya phenolamide buleetebwa nnyo OS mu bimera ebirya ebimera bw’ogeraageranya n’ebimera ebitasikirizibwa, era bikwatagana nnyo mu nkola zaabyo ez’okwolesebwa. Ensonga y’okuwandiika MYB8 n’obuzaale bw’enzimba PAL1, PAL2, C4H ne 4CL mu waggulu w’ekkubo lino byalaga okutandika amangu okuwandiika. Acyltransferases ezikola omulimu mu kukungaanya okusembayo okwa phenolamide, nga AT1, DH29, ne CV86, ziraga enkola y’okulinnyisa amaanyi okumala ebbanga eddene (Ekifaananyi S4). Ebintu ebyo waggulu biraga nti okutandika amangu okukuguka mu transcriptome n’oluvannyuma okutumbula okukuguka mu metabolomics ngeri ya kugatta, ekiyinza okuba nga kiva ku nkola y’okulungamya ey’okukwatagana (synchronous regulatory system) etandikawo okuddamu okw’amaanyi okw’okwekuuma.
Okuddamu okusengeka mu bubonero bw’obusimu bw’ebimera kukola nga oluwuzi olulung’amya olugatta amawulire agalya omuddo okuddamu okukola pulogulaamu y’omubiri gw’ebimera. Oluvannyuma lw’okukoppa ebisolo ebirya ebimera, twapima enkyukakyuka y’okukuŋŋaanyizibwa kw’ebika by’obusimu bw’ebimera ebikulu era ne tulaba okulaba awamu okwolesebwa okw’ekiseera wakati waabwe [Pearson correlation coefficient (PCC)> 0.4] (Ekifaananyi 3A). Nga bwe kisuubirwa, obusimu bw’ebimera obukwatagana n’okusengejja ebiramu bukwatagana mu mutimbagano gw’okulaga obusimu bw’ebimera awamu. Okugatta ku ekyo, metabolic specificity (Si index) eteekebwa ku maapu ku mutimbagano guno okulaga obusimu bw’ebimera obuleetebwa obujjanjabi obw’enjawulo. Ebitundu bibiri ebikulu eby’okuddamu okwetongodde kw’ebimera ebirya ebimera bikubiddwa: ekimu kiri mu kibinja kya JA, nga JA (engeri yaayo ekola mu biramu JA-Ile) n’ebiva mu JA ebirala biraga obubonero bwa Si obusinga obunene; ekirala ye ethylene (ET). Gibberellin yalaga okweyongera okw’ekigero kwokka mu butonde bw’ebisolo ebirya ebimera, ate obusimu obulala obw’ebimera, nga cytokinin, auxin, ne abscisic acid, bwalina obutonde obutono obw’okuyingiza ebimera. Bw’ogeraageranya n’okukozesa W + W yokka, okugaziya kw’omuwendo gw’entikko ogw’ebiva mu JA okuyita mu nkola ya OS (W + OS) okusinga kuyinza okukyusibwa okufuuka ekiraga ekitongole eky’amaanyi ekya JAs. Mu ngeri etasuubirwa, OSM ne OSS1 ezirina elicitor ez’enjawulo zimanyiddwa okuleeta okukuŋŋaanyizibwa okufaanagana okwa JA ne JA-Ile. Okwawukana ku OSS1, OSM ekwatibwako mu ngeri ey’enjawulo era ey’amaanyi OSMs, ate OSS1 tegaziya kuddamu kwa biwundu bya basal (Figure 3B).
(A) Okwekenenya omukutu gw’okwolesebwa awamu nga kwesigamiziddwa ku kubala kwa PCC okw’okusiiga enkola y’okukuŋŋaanyizibwa kw’obusimu bw’ebimera obuleetebwa ebisolo ebirya ebimera. Ensigo ekiikirira obusimu bw’ekimera bumu, ate obunene bw’ennyingo bukiikirira omuwendo gwa Si ogw’enjawulo ku busimu bw’ekimera wakati w’obujjanjabi. (B) Okukuŋŋaanyizibwa kwa JA, JA-Ile ne ET mu bikoola okuva ku nkola ez’enjawulo eziragiddwa langi ez’enjawulo: apricot, W + OSM; bbululu, W + OSSl; omuddugavu, W + W; enzirugavu, C (okufuga) . Emmunyeenye ziraga enjawulo ennene wakati w’obujjanjabi n’okufuga (two-way ANOVA egobererwa Tukey HSD post hoc multiple comparison, *** P <0.001). Okwekenenya endowooza y’amawulire (C)697 MS/MS (data file S1) mu JA biosynthesis ne impaired perception spectrum (irAOC ne irCOI1) ne (D)585 MS/MS (data file S1) mu ETR1 nga zirina obuzibu bwa ET signal Enzijanjaba bbiri ez’ebimera ebirya ebimera ezikoppa zatandika layini z’ebimera n’ebimera ebifuga mmotoka ekyerere (EV). Emmunyeenye ziraga enjawulo ennene wakati w’obujjanjabi bwa W+OS n’okufuga okutali kwonooneka (ANOVA ey’engeri bbiri ng’eddirirwa Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 ne ***P<0.001). (E) Grafu ezisaasaanyiziddwa ez’okuwakanya okusaasaana kw’okukuguka. Langi zino zikiikirira ebika eby’enjawulo ebikyusiddwa mu buzaale; obubonero bukiikirira enkola ez’enjawulo ez’obujjanjabi: enjuyi essatu, W + OSS1; enjuyi ennya, W + OSM; enzirugavu C
Ekiddako, tukozesa ekika kya Nepenthes ekikyusiddwa mu buzaale (irCOI1 ne sETR1) mu mitendera emikulu egy’okusengejja obulamu bwa JA ne ET (irAOC ne irACO) n’okutegeera (irCOI1 ne sETR1) okwekenneenya enkyukakyuka y’obusimu buno obubiri obw’ebimera ku bimera ebirya ebimera The relative contribution of reprogramming. Okukwatagana n’okugezesa okwasooka, twakakasa okuyingiza herbivore-OS mu bimera empty carrier (EV) (Ekifaananyi 3, C okutuuka ku D) n’okukendeera okutwalira awamu mu Hj index evudde ku OSM, ate δj index yeeyongera. Okuddamu kweyolekera nnyo okusinga okuddamu okuleetebwa OSS1. Grafu ya layini bbiri nga ekozesa Hj ne δj nga coordinates eraga deregulation eyetongodde (Figure 3E). Omuze ogusinga okweyoleka kwe kuba nti mu bika ebitaliimu kabonero ka JA, enkyukakyuka za metabolome n’okukuguka ezireetebwa ebisolo ebirya ebimera kumpi biweddewo ddala (Figure 3C). Okwawukana ku ekyo, endowooza ya ET esirifu mu bimera bya sETR1, wadde ng’okutwalira awamu ekikolwa ku nkyukakyuka mu nkyukakyuka y’ebimera ebirya omuddo kiri wansi nnyo okusinga eky’obubonero bwa JA, kikendeeza ku njawulo mu miwendo gya Hj ne δj wakati w’okusikirizibwa kwa OSM ne OSS1 (Ekifaananyi 3D n’Ekifaananyi S5). . Kino kiraga nti ng’oggyeeko omulimu omukulu ogw’okukyusa obubonero bwa JA, okukyusa obubonero bwa ET nakyo kikola ng’okulongoosa obulungi enkola y’okukyusakyusa emmere ey’ebika (species-specific metabolic response) ey’ebisolo ebirya ebimera. Okukwatagana n’omulimu guno ogw’okulongoosa obulungi, tewaaliwo nkyukakyuka mu kuyingiza metabolome okutwalira awamu mu bimera bya sETR1. Ku luuyi olulala, bw’ogeraageranya n’ebimera bya sETR1, ebimera bya irACO byaleeta amplitudes okutwalira awamu ezifaanagana ez’enkyukakyuka mu metabolic ezireetebwa ebimera ebirya ebimera, naye byalaga obubonero bwa Hj ne δj obw’enjawulo ennyo wakati w’okusoomoozebwa kwa OSM ne OSS1 (Figure S5).
Okusobola okuzuula ebirungo ebikyukakyuka eby’enjawulo ebirina ebikulu ebiyamba mu kuddamu kw’ebika by’ebimera ebirya ebimera n’okulongoosa obulungi okukola kwabyo okuyita mu bubonero bwa ET, twakozesa enkola ya structural MS/MS eyakolebwa emabegako. Enkola eno yeesigamye ku nkola ya bi-clustering okuddamu okuteebereza famire y’enkyukakyuka mu mubiri okuva mu bitundutundu bya MS/MS [normalized dot product (NDP)] n’obubonero bw’okufaanagana okusinziira ku neutral loss (NL). Ekibiina kya MS/MS data set ekyazimbibwa okuyita mu kwekenneenya layini za ET transgenic kyavaamu 585 MS/MS (data file S1), ekyagonjoolwa nga bazikuŋŋaanyiziddwa mu modulo musanvu enkulu eza MS/MS (M) (Figure 4A) . Ebimu ku modulo zino bijjudde nnyo ebirungo eby’enjawulo ebimanyiddwa edda: okugeza, M1, M2, M3, M4 ne M7 birimu ebirungo eby’enjawulo ebiva mu phenol (M1), flavonoid glycosides (M2), ssukaali wa acyl (M3 Ne M4), ne 17-HGL-DTG (M7). Okugatta ku ekyo, amawulire agakwata ku nkyukakyuka y’emmere (Si index) ag’ekirungo ekimu mu buli modulo gabalibwa, era ensaasaanya yaayo eya Si esobola okulabibwa mu ngeri ey’okutegeera. Mu bufunze, MS/MS spectra eziraga herbivory ne genotype specificity enkulu zimanyiddwa olw’emiwendo gya Si egy’amaanyi, era ebibalo bya kurtosis biraga ensaasaanya y’ebyoya ku nsonda y’omukira ogwa ddyo. Ekimu ku kusaasaana kwa koloyidi okugonvu ng’okwo kwazuulibwa mu M1, nga mu kino phenol amide yalaga ekitundu kya Si ekisinga obunene (Ekifaananyi 4B). Eddagala eryayogeddwako erya herbivorous inducible 17-HGL-DTG mu M7 yalaga obubonero bwa Si obw’ekigero, ekiraga nti waliwo ekigero eky’ekigero eky’okulungamya enjawulo wakati w’ebika bya OS ebibiri. Okwawukana ku ekyo, ebisinga okukolebwa mu ngeri ya constitutively specialized metabolites, nga rutin, CGA, ne acyl sugars, bye bimu ku bubonero bwa Si obusinga wansi. Okusobola okunoonyereza obulungi ku buzibu bw’enzimba n’ensaasaanya ya Si wakati w’ebirungo eby’enjawulo ebikyukakyuka, omukutu gwa molekyu gwazimbibwa ku buli modulo (Ekifaananyi 4B). Okulagula okukulu okw’endowooza ya OD (okufunzibwa mu kifaananyi 1B) kwe kuba nti okuddamu okutegeka ebirungo eby’enjawulo ebikyukakyuka oluvannyuma lw’okulya omuddo kulina okuvaako enkyukakyuka ez’engeri emu mu bikyukakyuka ebirina omuwendo ogw’okwekuuma ogw’amaanyi, naddala nga kwongera ku butonde bwabyo (okuwukana ku kusaasaana okw’ekifuulannenge) Mode) Ebirungo ebikyukakyuka eby’okwekuuma ebyalagulwa endowooza ya MT. Ebisinga ku biva mu phenol ebikung’aanyiziddwa mu M1 bikwatagana mu nkola n’okukendeera kw’emirimu gy’ebiwuka ( 32 ). Bwe tugeraageranya emiwendo gya Si mu bikozesebwa mu M1 wakati w’ebikoola ebisikirizibwa n’ebikoola ebikola ebimera ebifuga EV ku ssaawa 24, twalaba nti enkyukakyuka y’ebirungo bingi ebikyukakyuka oluvannyuma lw’ebiwuka ebirya omuddo erina omuze ogw’okweyongera ogw’amaanyi (Figure 4C). Okweyongera okwetongodde mu muwendo gwa Si kwazuulibwa mu phenolamides ezikuuma zokka, naye tewali kweyongera mu muwendo gwa Si kwazuulibwa mu phenols endala ne metabolites ezitamanyiddwa ezibeera awamu mu modulo eno. Eno nkola ya njawulo, ekwatagana n’endowooza ya OD. Okulagula okukulu okw’enkyukakyuka mu nkyukakyuka mu mubiri ezireetebwa ebisolo ebirya ebimera bikwatagana. Okusobola okugezesa oba kino particularity ya phenolamide spectrum yaleetebwa OS-specific ET, twa plotted metabolite Si index era ne kireeta differential expression value wakati wa OSM ne OSS1 mu EV ne sETR1 genotypes (Figure 4D ). Mu sETR1, enjawulo ereetebwa phenamide wakati wa OSM ne OSS1 yakendeera nnyo. Enkola ya bi-clustering era yakozesebwa ku MS/MS data eyakunganyizibwa mu bika ebitalina JA emala okuteebereza modulo enkulu eza MS/MS ezikwatagana ne JA-regulated metabolic specialization (Figure S6).
(A) Ebivudde mu kugatta 585 MS/MS nga byesigamiziddwa ku kitundu ekigabanyizibwa (NDP similarity) n’okugabana okufiirwa okutaliimu (NL okufaanagana) bivaamu modulo (M) okubeera nga ekwatagana n’amaka g’ebirungo agamanyiddwa, oba olw’obutonde bwa Metabolite obutamanyiddwa oba obutakyukakyuka bulungi. Okumpi ne buli modulo, ensaasaanya ya metabolite (MS/MS) specific (Si) eragiddwa. (B) Omukutu gwa molekyu ogwa modulo: Ennyiriri zikiikirira MS/MS n’empenda, obubonero bwa NDP (emmyufu) ne NL (bbulu) MS/MS (okusalako,> 0.6). Omuwendo gwa metabolite specificity index (Si) ogwa graded metabolite specificity index gwa langi okusinziira ku modulo (ku kkono) era ne guteekebwa ku maapu ku mutimbagano gwa molekyu (ku ddyo). (C) Module M1 y’ekimera kya EV mu mbeera ya constitutive (control) ne induced (simulated herbivore) ku ssaawa 24: molekyu network diagram (Si value is the node size, defensive phenolamide is highlighted in blue). (D) Ekifaananyi kya M1 molekyu network diagram ya spectrum line sETR1 n’okukosebwa EV ne ET okutegeera: phenolic compound ekiikirira green circle node, n’enjawulo enkulu (P value) wakati W + OSM ne W + OSS1 obujjanjabi nga Node size. CP, N-kafeyoyili-tayirosiyini; CS, N-kafeyoyili-spermidini, nga zino za; FP, asidi wa N-ferulic ester-uric asidi; FS, N-ferulili-spermidini, nga bano; CoP, N’, N “-Kumaroli-tyrosine;DCS, N’, N”-dikaffeoyili-spermidine; CFS, N’, N”-caffeoyl, feruloyl-spermidine, Lycium barbarum mu mutabani w’omusege, Nick.O-AS, ssukaali wa O-acyl.
Twayongera okugaziya okwekenneenya okuva ku genotype emu eya Nepenthes attenuated okutuuka ku populations ez’obutonde, nga enkyukakyuka ez’amaanyi intraspecific mu herbivorous JA levels ne specific metabolite levels zaayogerwako emabegako mu natural populations ( 26 ). Kozesa data set eno okubikka ku germplasms 43. Ensigo zino zirimu ebika by’ebimera 123 okuva mu N. pallens. Ebimera bino byaggyibwa mu nsigo ezaakuŋŋaanyizibwa mu bifo eby’enjawulo eby’enjawulo enzaalwa mu Utah, Nevada, Arizona, ne California (Figure S7), twabala enjawulo ya metabolome (wano eyitibwa population level) β diversity) n’okukuguka okwaleetebwa OSM. Okukwatagana n’okunoonyereza okwakolebwa emabegako, twalaba enkyukakyuka nnyingi mu nkyukakyuka y’emmere ku bikondo bya Hj ne δj, ekiraga nti obuwuka obuyitibwa germplasms bulina enjawulo nnene mu buveera bw’engeri gye ziddamu mu nkyukakyuka y’emmere eri ebisolo ebirya ebimera (Figure S7). Ekibiina kino kijjukiza ebyo bye twalaba emabegako ku nkyukakyuka za JAs ezikyukakyuka ezireetebwa ebisolo ebirya ebimera, era ekuumye omuwendo gwa waggulu nnyo mu kibinja kimu ( 26 , 36 ). Nga tukozesa JA ne JA-Ile okugezesa enkolagana y’omutendera okutwalira awamu wakati wa Hj ne δj, twakizuula nti waliwo enkolagana ennungi enkulu wakati wa JA ne metabolome β diversity and specialization index (Figure S7). Kino kiraga nti obutafaanagana obuleetebwa ebisolo ebirya omuddo mu kuyingiza JAs obuzuuliddwa ku mutendera gw’omuwendo gw’abantu buyinza okuba nga buva ku nkyukakyuka enkulu ez’enkyukakyuka mu mubiri ezireetebwa okusunsulwa okuva mu biwuka ebirya omuddo.
Okunoonyereza okwakolebwa emabegako kwalaga nti ebika bya taaba byawukana nnyo mu kika n’okwesigamira okw’enjawulo ku induced and constitutive metabolic defenses. Kiteeberezebwa nti enkyukakyuka zino mu kukyusa obubonero obulwanyisa ebimera n’obusobozi bw’okwekuuma zifugibwa puleesa y’omuwendo gw’ebiwuka, enzirukanya y’obulamu bw’ebimera, n’ensimbi ezisaasaanyizibwa mu kukola eby’okwerinda mu kifo ekika ekimu we kikula. Twasoma obutakyukakyuka bw’okuddamu okukola mu bitundu by’amakoola (leaf metabolome remodeling) okuleetebwa ebisolo ebirya ebimera eby’ebika mukaaga ebya Nicotiana ebizaalibwa mu North America ne South America. Ebika bino bikwatagana nnyo ne Nepenthes North America, kwe kugamba Nicolas Bociflo. La, N. ebiwuka ebiyitibwa nicotinis, Nicotiana n. omuddo ogukendedde, Nicotiana tabacum, taaba ow’ennyiriri, taaba (Nicotiana spegazzinii) ne taaba ow’amakoola ga taaba (Nicotiana obtusifolia) (Ekifaananyi 5A) (37). Mukaaga ku bika bino, omuli n’ekika ekimanyiddwa obulungi ekya N. please, bimera ebibeerawo buli mwaka eby’ekika kya petunia, ate obtusifolia N. bimera ebiwangaala eby’ekika kya mwannyinaffe Trigonophyllae (38). Oluvannyuma, W + W, W + OSM ne W + OSS1 induction zakolebwa ku bika bino omusanvu okunoonyereza ku nsengeka y’ebika ku ddaala ly’enkyukakyuka y’emmere y’emmere y’ebiwuka.
(A) Omuti gwa bootstrap phylogenetic nga gwesigamiziddwa ku maximum likelihood [for nuclear glutamine synthesis (38)] n’ensaasaanya y’ebika bya Nicotiana musanvu ebikwatagana ennyo mu bitundu (langi ez’enjawulo) (37). (B) Ploti y’okusaasaana ey’enjawulo ey’enjawulo ku bifaananyi by’enkyukakyuka y’emmere y’ebika bya Nicotiana musanvu (939 MS/MS; fayiro ya data S1). Ku mutendera gw’ebika, enjawulo ya metabolome ekwatagana bubi n’eddaala ly’okukuguka. Okwekenenya enkolagana y’omutindo gw’ebika wakati w’enjawulo y’enkyukakyuka y’emmere n’okukuguka n’okukuŋŋaanyizibwa kwa JA kulagibwa mu kifaananyi 2. S9. Langi, ebika eby’enjawulo; enjuyi essatu, W + OSS1; enjuyi ennya, W + OSM; (C) Nicotiana JA ne JA-Ile dynamics zisengekeddwa okusinziira ku OS excitation amplitude (two-way ANOVA ne Tukey HSD oluvannyuma lw’okugeraageranya emirundi mingi, * P <0.05, ** P <0.01 ne * ** Okugeraageranya W + OS ne W + W, P <0.001). Box plot ya (D) enjawulo ne (E) okukuguka kwa buli kika oluvannyuma lw’okukoppa herbivorous ne methyl JA (MeJA). Emmunyeenye eraga enjawulo ennene wakati wa W + OS ne W + W oba lanolin plus W (Lan + W) oba Lan plus MeJA (Lan + MeJa) ne Lan control (okwekenneenya enjawulo mu ngeri bbiri, nga kugobererwa Tukey’s HSD post hoc multiple comparison , *P<0.05, **P<0.01 ne ***P<0.001).
Nga tukozesa enkola ya dual cluster, twazuula modulo 9 eza 939 MS/MS (data file S1). Ebitonde bya MS/MS ebizzeemu okutegekebwa enzijanjaba ez’enjawulo byawukana nnyo mu modulo ez’enjawulo wakati w’ebika (Ekifaananyi S8). Okulaba mu birowoozo Hj (eyogerwako wano nga γ-enjawulo ku ddaala ly’ebika) ne δj kiraga nti ebika eby’enjawulo bikuŋŋaanyizibwa mu bibinja eby’enjawulo ennyo mu kifo ky’enkyukakyuka y’emmere, ng’okugabanyaamu ku ddaala ly’ebika kutera okweyoleka okusinga okusikirizibwa. Ng’oggyeeko N. linear ne N. obliquus, ziraga enkyukakyuka nnyingi ez’ebikolwa eby’okuyingiza (Figure 5B). Okwawukana ku ekyo, ebika nga N. purpurea ne N. obtusifolia birina okuddamu kw’enkyukakyuka y’emmere okutali kweyoleka nnyo mu bujjanjabi, naye enkyukakyuka y’ebimera ya njawulo nnyo. Ensaasaanya y’ebika ebitongole ey’okuddamu kw’enkyukakyuka y’emmere okuleetebwa yavaamu enkolagana embi ey’amaanyi wakati w’okukuguka n’enjawulo ya gamma (PCC = -0.46, P = 4.9×10-8). Enkyukakyuka ezireetebwa OS mu mitendera gya JAs zikwatagana bulungi n’okukuguka mu metabolome, era zikwatagana bubi n’enjawulo ya metabolic gamma eyolesebwa buli kika (Figure 5B and Figure S9). Kinajjukirwa nti ekika kino mu njogera ekiyitibwa “signal response” species mu kifaananyi 5C, nga Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, ne Nepenthes attenuated, kyaleeta obubonero obw’amaanyi ku ddakiika 30. Okubutuka kwa JA ne JA-Ile okukwata ku OS gye buvuddeko, ate nga bakitiriya endala eziyitibwa “signal unresponsive”, nga ebyuma bya Nepenthes, Nepenthes powdery ne N. obtusifolia byokka biraga JA-Ile Edge induction yokka awatali OS specificity yonna (Figure 5C). Ku ddaala ly’enkyukakyuka y’emmere, nga bwe kyayogeddwa waggulu, ku Nepenthes ezikendedde, ebintu ebiddamu obubonero byalaga OS specificity era ne byongera nnyo δj, ate nga bikendeeza Hj. Kino OS-specific priming effect teyazuulibwa mu bika ebisengekeddwa nga signal non-reactive species (Figure 5, D ne E). OS-specific metabolites zigabanyizibwa nnyo wakati w’ebika ebiddamu obubonero, era ebibinja bino eby’obubonero bikuŋŋaana n’ebika ebirina okuddamu kw’obubonero obunafu, ate ebika ebirina okuddamu kw’obubonero obunafu biraga okwesigamizibwa okutono (Figure S8 ). Ekivaamu kino kiraga nti okuyingiza JAs mu OS-specific n’okuddamu okusengeka OS-specific eya metabolome eya wansi biyungibwa ku ddaala ly’ebika.
Ekiddako, twakozesa ekikuta kya lanolin ekirimu methyl JA (MeJA) okulongoosa ebimera okunoonyereza oba engeri zino ez’okuyunga zikugirwa olw’okubaawo kwa JA essiddwako exogenous JA, ejja okuba mu cytoplasm y’ebimera. Okukendeeza ester mu bwangu ye JA. Twazuula omuze gwe gumu ogw’enkyukakyuka mpolampola okuva ku bika ebiddamu obubonero okudda ku bika ebitaddamu bubonero ebiva ku kugabibwa kwa JA okutambula obutasalako (Ekifaananyi 5, D ne E). Mu bufunze, obujjanjabi bwa MeJA bwaddamu nnyo pulogulaamu ya metabolomes za linear nematodes, N. obliquus, N. aquaticus, N. pallens, ne N. mikimotoi, ekyavaamu okweyongera okw’amaanyi mu δj n’okukendeera kwa Hj. N. purpurea yalaga okweyongera kwa δj kwokka, naye si Hj. N. obtusifolia, emabegako eyalagibwa okukung’aanya emiwendo gya JAs egya wansi ennyo, nayo eddamu bubi ku bujjanjabi bwa MeJA mu nsonga z’okuddamu okusengeka metabolome. Ebivuddemu bino biraga nti okukola JA oba okukyusa obubonero kuziyizibwa mu mubiri mu bika ebitaddamu bubonero. Okugezesa endowooza eno, twasoma ebika bina (N. pallens, N. mills, N. pink ne N. microphylla) ebireetebwa W + W, W + OSMs ne W + OSS1 Transcriptome ( 39 ). Okukwatagana n’enkola y’okuddamu okukola metabolome, ebika byawuddwamu bulungi mu kifo kya transcriptome, nga mu bino N. attenuated yalaga RDPI esinga obunene eya OS-induced, ate N. gracilis yalina esinga wansi (Figure 6A). Wabula kyazuulibwa nti enjawulo ya transcriptome eyaleetebwa N. oblonga ye yali esinga wansi mu bika bina, okwawukana ku njawulo ya metabolic esinga obunene eya N. oblonga eyalagibwa emabegako mu bika musanvu. Okunoonyereza okwakolebwa emabegako kwalaga nti ekibinja ky’obuzaale obukwatagana n’obubonero bw’okwekuuma obusooka, omuli n’obubonero bwa JA, bunnyonnyola obutonde bw’okuddamu okw’okwekuuma okwasooka okuleetebwa elicitors ezikwatagana n’ebisolo ebirya ebimera mu bika bya Nicotiana ( 39 ). Okugeraageranya amakubo g’obubonero bwa JA wakati w’ebika bino ebina kyalaga enkola eyeesigika (Figure 6B). Ensengekera z’obuzaale ezisinga obungi mu kkubo lino, nga AOC, OPR3, ACX ne COI1, zalaga emitendera egy’okuyingiza egy’amaanyi ennyo mu bika bino ebina. Naye, ensengekera y’obutonde enkulu, JAR4, ekyusa JA mu ngeri yaayo ekola mu biramu ey’ebiwandiiko ebikung’aanyiziddwa mu JA-Ile, era omutindo gwayo ogw’okuwandiika guli wansi nnyo naddala mu byuma bya N., Nepenthes pieris ne N. microphylla. Okugatta ku ekyo, ebiwandiiko byokka eby’obuzaale obulala AOS tebyazuulibwa mu N. bifidum. Enkyukakyuka zino mu kwolesebwa kw’obuzaale ziyinza okuba nga ze zivunaanyizibwa ku nkula ezisukkiridde ezireetebwa okukola kwa JA okutono mu bika bya signal anergic n’okuleetebwa kwa N. gracilis.
(A) Okwekenenya endowooza y’amawulire ku kuddamu okukola pulogulaamu y’okuddamu kw’okuwandiika okwasooka okw’ebika bya taaba bina ebikwatagana ennyo ebya sampuli oluvannyuma lw’eddakiika 30 oluvannyuma lw’okuyingiza omuddo. RDPI ebalwa nga tugeraageranya ebikoola ebiva mu OS ekika kya herbivore n’ekirungo ekifuga ebiwundu. Langi ziraga ebika eby’enjawulo, ate obubonero bulaga enkola ez’enjawulo ez’obujjanjabi. (B) Okwekenenya okwolesebwa kw’obuzaale mu makubo g’obubonero bwa JA mu bika bina. Ekkubo lya JA eryanguyiziddwa liragibwa okumpi ne puloti y’akabokisi. Langi ez’enjawulo ziraga enkola ez’enjawulo ez’okulongoosa. Emmunyeenye eraga nti waliwo enjawulo nnene wakati w’obujjanjabi bwa W + OS n’okufuga W + W (ku Student’s t-test for pairwise differences, *P<0.05, **P<0.01 ne ***P<0.001). OPDA, asidi wa 12-oxophytodienoic; OPC-8: 0,3-okso-2 (2′ (Z)-penteni)-sayikilopentani-1-oktanoyiki asidi.
Mu kitundu ekisembayo, twasoma engeri okuddamu okukola mu bika by’ebiwuka ebikwata ku metabolome y’ebika by’ebimera eby’enjawulo gye kuyinza okugumira ebisolo ebirya ebimera. Okunoonyereza okwakolebwa emabegako kwassa essira ku kika kya Nicotiana. Obuziyiza bwazo eri Ms ne larvae bwawukana nnyo (40). Wano, twasoma akakwate akaliwo wakati w’ekyokulabirako kino n’obuveera bwazo obw’enkyukakyuka y’emmere. Nga tukozesa ebika bya taaba ebina waggulu, n’okugezesa enkolagana wakati w’enjawulo n’okukuguka kwa metabolome ereetebwa ebisolo ebirya omuddo n’okuziyiza kw’ebimera eri Ms ne Sl, twazudde nti okuziyiza, enjawulo n’okukuguka eri generalist Sl Byonna bikwatagana bulungi, so ng’ate enkolagana wakati w’okuziyiza abakyala abakugu n’okukuguka nnafu, era enkolagana n’enjawulo si nnene (Figure S10). Ku bikwata ku kuziyiza S1, byombi N. chinensis ne N. gracilis ebikendedde, ebyalagibwa emabegako okulaga emitendera gyombi egy’okukyusa obubonero bwa JA n’obuveera bwa metabolome, byalina eby’okuddamu eby’enjawulo ennyo ku kuyingizibwa kw’ebisolo ebirya ebimera, era nabyo byalaga okuziyiza okw’amaanyi okufaananako. Okwegatta.
Mu myaka nkaaga egiyise, endowooza y’okukuuma ebimera ewadde enkola y’enzikiriziganya, abanoonyereza kwe basinziira okulagula omuwendo omunene ogw’enkulaakulana n’emirimu gy’ebirungo eby’enjawulo ebikyukakyuka mu bimera. Endowooza zino ezisinga tezigoberera nkola ya bulijjo ey’okuteebereza okw’amaanyi (41). Bateesa ku kuteebereza okukulu (3) ku mutendera gwe gumu ogw’okwekenneenya. Okugezesa okulagula okukulu bwe kukkiriza endowooza ezenjawulo okwekenneenya, kino kijja kufuula Ennimiro egenda mu maaso. Beera kuwagirwa, naye mugaane abalala (42). Wabula, endowooza empya ekola okulagula ku mitendera egy’enjawulo egy’okwekenneenya era n’eyongerako oluwuzi olupya olw’okulowooza okunnyonnyola (42). Naye, endowooza ebbiri eziteeseddwa ku ddaala ly’emirimu, MT ne OD, zisobola bulungi okunnyonnyolwa ng’okuteebereza okukulu okw’enkyukakyuka ez’enjawulo ez’enkyukakyuka mu mubiri ezireetebwa ebisolo ebirya omuddo: endowooza ya OD ekkiriza nti enkyukakyuka mu “ekifo” eky’enjawulo eky’enkyukakyuka y’emmere zirina obulagirizi bungi . Endowooza ya MT ekkiriza nti enkyukakyuka zino zijja kuba tezikwata ku ndagiriro era nga zisangibwa mu ngeri ya kimpowooze mu kifo ky’enkyukakyuka y’emmere, era zitera okuba n’ebirungo ebikyusa ebiriisa eby’omuwendo gw’okwekuuma ogw’amaanyi. Okukebera okwasooka okw’okuteebereza kwa OD ne MT kugezesebwa nga tukozesa ekibinja ekifunda eky’ebirungo “eby’okwekuuma” ebya a priori. Ebigezo bino ebikwata ku metabolite biziyiza obusobozi okwekenneenya obunene n’enkola y’okuddamu okusengeka metabolome mu kiseera ky’okulya omuddo, era tebikkiriza kugezesebwa mu nkola y’ebibalo ekwatagana okwetaagisa okulagula okukulu okuyinza okulowoozebwako okutwaliza awamu Okubala enkyukakyuka mu metabolome y’ebimera. Wano, twakozesa tekinologiya omuyiiya mu metabolomics nga tusinziira ku computational MS era ne tukola deconvolution MS analysis mu ssente eza bulijjo ez’ebinyonyola endowooza y’amawulire okugezesa enjawulo wakati w’ebibiri ebiteeseddwa ku mutendera gwa metabolomics ogw’ensi yonna. Okulagula okukulu okw’endowooza eno. Endowooza y’amawulire ekozesebwa mu bintu bingi naddala mu mbeera y’okunoonyereza ku bitonde eby’enjawulo n’okutambula kw’ebiriisa (43). Naye nga bwe tumanyi, eno y’enkozesa esoose okukozesebwa okunnyonnyola ekifo ky’amawulire agakwata ku nkyukakyuka y’emmere y’ebimera n’okugonjoola ebizibu by’obutonde ebikwatagana n’enkyukakyuka mu nkyukakyuka y’emmere ey’ekiseera nga tuddamu ebiraga obutonde. Okusingira ddala, obusobozi bw’enkola eno buli mu busobozi bwayo okugeraageranya enkola munda ne wakati w’ebika by’ebimera okwekenneenya engeri ebisolo ebirya omuddo gye bikyuse okuva mu bika eby’enjawulo okudda mu nkola z’enkulaakulana ennene wakati w’ebika ku mitendera egy’enjawulo egy’enkulaakulana. Enkyukakyuka y’ebirungo.
Okwekenenya ebitundu ebikulu (PCA) kukyusa ekibiina kya data ekikyukakyuka ennyo mu kifo eky’okukendeeza ku bipimo (dimensionality reduction space) olwo omuze omukulu ogwa data gusobole okunnyonnyolwa, kale kitera okukozesebwa ng’enkola ey’okunoonyereza okusengejja ekibiina kya data, gamba nga deconvolution metabolome. Naye, okukendeeza ku bipimo kijja kufiirwa ekitundu ku mawulire agali mu kibiina ky’amawulire, era PCA tesobola kuwa mawulire ga bungi ku mpisa ezikwatagana naddala n’endowooza y’obutonde, gamba nga: engeri ebisolo ebirya ebimera gye biddamu okusengeka enjawulo mu nnimiro ez’enjawulo (okugeza, obugagga, okusaasaana) N’obungi) ebirungo ebikyusa ebiriisa? Biki ebikyusa ebiriisa ebiragula embeera ereeteddwa ey’ekisolo ekirya omuddo ekiweereddwa? Okusinziira ku ndowooza y’obutonde, enjawulo n’okuleetebwa, amawulire agali mu nsengeka y’ebirungo ebikyukakyuka mu bikoola (leaf-specific metabolite profile) gavunda, era kizuuliddwa nti okulya ebisolo ebirya ebimera bisobola okukola enkyukakyuka ey’enjawulo. Nga tetusuubira, twalaba nti, nga bwe kyayogerwako mu bipimo by’endowooza y’amawulire ebyassibwa mu nkola, embeera y’enkyukakyuka y’emmere evuddemu erina okukwatagana okunene oluvannyuma lw’okulumba kw’ebisolo ebibiri ebirya ebimera (the night-fed generalist Sl) n’omukugu mu Solanaceae Ms. Wadde nga enneeyisa yaabwe ey’okulya n’okusengejja kwabwe kwa njawulo nnyo. Omutandisi wa asidi w’amasavu-amino asidi (FAC) mu OS (31). Nga bakozesa herbivore OS okujjanjaba ebiwundu ebifumita ebituufu, obujjanjabi bw’ebimera ebirya ebimera ebikoppa nabwo bwalaga omuze ogufaananako. Enkola eno etuukiridde ey’okukoppa engeri ebimera gye biddamu okulumbibwa ebimera ebirya omuddo emalawo ensonga ezitabula ezireetebwa enkyukakyuka mu nneeyisa y’okulya kw’ebisolo ebirya ebimera, ebivaako okwonooneka okw’enjawulo mu biseera eby’enjawulo ( 34 ). FAC, emanyiddwa okuba nga y’esinga okuvaako OSM, ekendeeza ku JAS n’okuddamu kw’obusimu bw’ebimera obulala mu OSS1, ate OSS1 ekendeeza emirundi ebikumi n’ebikumi ( 31 ). Wabula OSS1 yaleeta emitendera egy’enjawulo egy’okukuŋŋaanyizibwa kwa JA bw’ogeraageranya ne OSM. Emabegako kyalagibwa nti okuddamu kwa JA mu Nepenthes ezikendedde kukwata nnyo ku OSM, FAC gy’esobola okukuuma emirimu gyayo ne bwe kiba nga kifukiddwa 1:1000 n’amazzi ( 44 ). N’olwekyo, bw’ogeraageranya ne OSM, wadde nga FAC mu OSS1 ntono nnyo, emala okuleeta okubutuka kwa JA okumala. Okunoonyereza okwakolebwa emabegako kwalaga nti puloteyina eziringa porin ( 45 ) ne oligosaccharides ( 46 ) zisobola okukozesebwa ng’obubonero bwa molekyu okuleeta okuddamu kw’okwekuuma kw’ebimera mu OSS1. Wabula tekinnategeerekeka oba elicitors zino mu OSS1 ze zivunaanyizibwa ku kukungaanya JA etunuuliddwa mu kunoonyereza okuliwo kati.
Newankubadde nga waliwo okunoonyereza okutono okunnyonnyola engalo ez’enjawulo ez’enkyukakyuka mu mubiri ezireetebwa okukozesa ebisolo eby’enjawulo ebirya omuddo oba exogenous JA oba SA (salicylic acid) ( 47 ), tewali muntu yenna ataataaganya okutaataaganyizibwa kw’ebika by’ebimera ebirya ebimera mu mutimbagano gw’omuddo gw’ebimera n’ebikosa ebitongole The overall impact of metabolism is systematically studied. ebikwata ku muntu. Okwekenenya kuno kwayongera okukakasa nti okuyungibwa kw’omukutu gw’obusimu ogw’omunda n’obusimu obulala obw’ebimera okuggyako JAs kukola obutonde bw’okuddamu okutegekebwa kw’enkyukakyuka y’emmere okuleetebwa ebisolo ebirya ebimera. Okusingira ddala, twakizuula nti ET ereetebwa OSM yali nnene nnyo okusinga eyo ereetebwa OSS1. Mode eno ekwatagana n’ebirimu FAC ebisingawo mu OSM, nga eno embeera eyeetaagisa era emala okutandika okubutuka kwa ET ( 48 ). Mu mbeera y’enkolagana wakati w’ebimera n’ebirya ebimera, omulimu gw’okulaga obubonero ogwa ET ku nkyukakyuka y’ebirungo ebikyukakyuka mu bimera (plan-specific metabolite dynamics) gukyaliwo era gutunuulidde ekibinja ky’ebirungo kimu kyokka. Okugatta ku ekyo, okunoonyereza okusinga kukozesezza okukozesa ET oba ebigisooka oba ebiziyiza eby’enjawulo okunoonyereza ku kulungamya ET, nga mu bino okukozesa eddagala lino ery’ebweru kujja kuleeta ebizibu bingi ebitali bya njawulo. Okusinziira ku kye tumanyi, okunoonyereza kuno kukiikirira okwekenneenya okusooka okw’enteekateeka okw’amaanyi okw’omulimu gwa ET mu kukozesa ET okufulumya n’okutegeera ebimera ebikyusiddwa obuzaale ebikoseddwa okukwasaganya enkyukakyuka y’ebimera mu metabolome. Herbivore-specific ET induction ku nkomerero esobola okukyusakyusa mu kuddamu kwa metabolome. Ekisinga obukulu kwe kukyusa obuzaale mu buzaale bwa ET biosynthesis (ACO) n’okutegeera (ETR1) okwalaga okukuŋŋaanyizibwa kw’ebimera ebirya ebimera (herbivore-specific de novo accumulation of phenolamides). Emabegako kyalagibwa nti ET esobola okulongoosa obulungi okukuŋŋaanyizibwa kwa nicotine okuleetebwa JA ng’etereeza putrescine N-methyltransferase ( 49 ). Naye okusinziira ku ndaba y’ebyuma, tekitegeerekeka bulungi ngeri ET gy’elongoosaamu enkola ya phenamide. Ng’oggyeeko omulimu gw’okukyusa obubonero (signal transduction function) ogwa ET, ekisengejjo ky’enkyukakyuka (metabolic flux) nakyo kisobola okukyusibwa okudda ku S-adenosyl-1-methionine okulungamya okuteeka ssente mu polyaminophenol amides. S-adenosyl-1-methionine ye ET era Common intermediate of polyamine biosynthetic ekkubo. Enkola ET signal gy’etereeza omutindo gwa phenolamide yeetaaga okwongera okunoonyereza.
Okumala ebbanga ddene, olw’omuwendo omunene ogw’ebirungo eby’enjawulo ebikyukakyuka eby’ensengekera etamanyiddwa, okufaayo ennyo ku biti by’enkyukakyuka ebitongole tekusobodde kwekenneenya nnyo nkyukakyuka ez’ekiseera ez’enjawulo y’enkyukakyuka mu mubiri oluvannyuma lw’enkolagana y’ebiramu. Mu kiseera kino, okusinziira ku kwekenneenya endowooza y’amawulire, ekikulu ekiva mu kufuna spectrum ya MS/MS nga kwesigamiziddwa ku biwuka ebikyusa ebiriisa ebitaliiko kyekubiira kwe kuba nti ebisolo ebirya omuddo ebirya oba okukoppa ebirya ebimera bikyagenda mu maaso n’okukendeeza ku njawulo y’enkyukakyuka y’ebimera okutwalira awamu eya metabolome y’amakoola ate nga byongera ku ddaala lyayo ery’okukuguka. Okweyongera kuno okw’ekiseera mu butonde bwa metabolome obuleetebwa ebisolo ebirya ebimera kukwatagana n’okweyongera kw’okukwatagana mu butonde bwa transcriptome. Ekintu ekisinga okuyamba mu kukuguka kuno okunene okwa metabolome (okulina omuwendo gwa Si ogw’oku ntikko) ye metabolite ey’enjawulo erimu omulimu gw’okulya omuddo ogwamanyibwa emabegako. Omuze guno gukwatagana n’okuteebereza kwa OD theory, naye okulagula kwa MT okukwatagana n’obutabeera bwa randomness bwa metabolome reprogramming tekukwatagana. Naye, data eno era ekwatagana n’okuteebereza kw’ekyokulabirako ekitabuddwa (MT esinga obulungi; Ekifaananyi 1B), kubanga ebirungo ebirala ebitamanyiddwa nga metabolites ebirina emirimu gy’okwekuuma egitamanyiddwa biyinza okuba nga bikyagoberera ensaasaanya ya Si eya random.
Omuze ogweyoleka ogweyongedde okukwatibwa okunoonyereza kuno kwe kuba nti okuva ku ddaala ly’enkulaakulana entonotono (ekimera kimu ne taaba omuwendo) okutuuka ku mutendera omunene ogw’enkulaakulana (ebika bya taaba ebikwatagana ennyo), emitendera egy’enjawulo egy’enteekateeka y’enkulaakulana giri mu “kwekuuma okusinga obulungi” Waliwo enjawulo ennene mu busobozi bw’ebisolo ebirya ebimera. Moore ne banne. (20) ne Kessler ne Kalske (1) mu bwetwaze baateesa okukyusa emitendera esatu egy’emirimu egy’ebitonde eby’enjawulo egyasooka okwawulwa Whittaker (50) mu nkyukakyuka ez’ekiseera ezikola n’ezireetebwa ez’enjawulo y’eddagala; abawandiisi bano wadde mu bufunze Enkola z’okukung’aanya ebikwata ku metabolome ku mutendera omunene nazo tezirambika ngeri ya kubalirira njawulo ya metabolic okuva mu data zino. Mu kunoonyereza kuno, ennongoosereza entonotono mu kugabanya emirimu gya Whittaker zijja kulowooza ku njawulo ya α-metabolic ng’enjawulo ya MS/MS spectra mu kimera ekiweereddwa, n’enjawulo ya β-metabolic nga basic intraspecific metabolism y’ekibinja ky’abantu Space, ne γ-metabolic diversity ejja kuba kugaziya ku kwekenneenya ebika ebifaanagana.
Akabonero ka JA keetaagibwa nnyo mu ngeri nnyingi ez’okuddamu kw’ebisolo ebirya ebimera. Naye, waliwo obutaba na kugezesebwa kwa bungi okukakali okw’omugabo gw’okulungamya mu ngeri ey’enjawulo (intraspecific regulation of JA biosynthesis) mu njawulo ya metabolome, era oba akabonero ka JA kifo kya bulijjo eky’enjawulo y’enkyukakyuka y’emmere ereetebwa situleesi ku mutendera ogw’enkulaakulana ogw’amaanyi (higher macroevolutionary scale) kikyali kizuuliddwa. Twalaba nti obutonde bw’okulya omuddo obwa Nepenthes herbivorous buleeta okukuguka mu metabolome n’enkyukakyuka y’okukuguka mu metabolome mu bungi bw’ebika bya Nicotiana ne mu bika bya Nicotiana ebikwatagana ennyo ekwatagana bulungi mu nkola n’obubonero bwa JA. Okugatta ku ekyo, akabonero ka JA bwe kakosebwa, obutonde bw’enkyukakyuka mu mubiri obuleetebwa ekisolo ekimu eky’omuddo eky’ekika kya genotype bujja kusazibwamu (Ekifaananyi 3, C ne E). Okuva enkyukakyuka mu nsengekera y’enkyukakyuka y’emmere ey’ebika bya Nepenthes ebikendeezeddwa mu butonde bwe zisinga okuba ez’omuwendo, enkyukakyuka mu njawulo ya β y’enkyukakyuka mu mubiri n’obutonde mu kwekenneenya kuno ziyinza okusinga okuva ku kusikirizibwa okw’amaanyi okw’ebika by’ebirungo ebirimu ebirungo ebikyusa ebiriisa. Ebika bino eby’ebirungo bifuga ekitundu ky’ensengekera ya metabolome era bivaako enkolagana ennungi n’obubonero bwa JA.
Olw’okuba enkola z’ebiramu ez’ekika kya taaba ekikwatagana ennyo nayo za njawulo nnyo, ebirungo ebikyukakyuka (metabolites) bizuulibwa mu ngeri ey’enjawulo mu nsonga y’omutindo, kale esinga kwekenneenya. Enkola y’endowooza y’amawulire ku nkola y’enkyukakyuka y’emmere ekwatibwa eraga nti okuyingiza ebimera ebirya ebimera kyongera okusuubulagana wakati w’enjawulo y’enkyukakyuka ya gamma n’okukuguka. Siginini ya JA ekola kinene mu kusuubulagana kuno. Okweyongera mu kukuguka mu metabolome kukwatagana n’okuteebereza kwa OD okukulu era kukwatagana bulungi n’akabonero ka JA, ate akabonero ka JA kakwatagana bubi n’enjawulo ya metabolic gamma. Ebikozesebwa bino biraga nti obusobozi bwa OD bw’ebimera businga kusalibwawo obuveera bwa JA, oba ku mutendera gwa microevolutionary oba ku mutendera omunene ogw’enkulaakulana. Okugezesa okukozesa JA okuva ebweru okwetooloola obulema mu JA biosynthesis kyongera okulaga nti ebika bya taaba ebikwatagana ennyo bisobola okwawulwamu ebika ebiddamu obubonero n’ebitaddamu bubonero, nga bwe kiri ku ngeri yaabwe eya JA n’obuveera bwa metabolome obuleetebwa ebisolo ebirya ebimera. Ebika ebitaddamu ku bubonero tebisobola kuddamu olw’obutasobola kukola JA ey’omunda era n’olwekyo biba n’obuzibu mu mubiri. Kino kiyinza okuva ku nkyukakyuka mu buzaale obumu obukulu mu kkubo ly’obubonero bwa JA (AOS ne JAR4 mu N. crescens) of. Ekivaamu kino kiraga nti enkola zino ez’enkulaakulana ennene wakati w’ebika ziyinza okusinga okuvugibwa enkyukakyuka mu kutegeera n’okuddamu kw’obusimu obw’omunda.
Ng’oggyeeko enkolagana wakati w’ebimera n’ebirya ebimera, okunoonyereza ku njawulo mu nkyukakyuka y’emmere kukwatagana n’enkulaakulana yonna enkulu mu ndowooza mu kunoonyereza ku kukwatagana kw’ebiramu n’obutonde n’enkulaakulana y’engeri enzibu ez’enkula. Olw’okweyongera kw’obungi bw’amawulire agafunibwa ebikozesebwa eby’omulembe ebya MS, okugezesa endowooza ku njawulo mu nkyukakyuka y’emmere kati kusobola okusukkuluma ku njawulo z’ebirungo ebikyusa ebiriisa eby’omuntu kinnoomu/ekika n’okukola okwekenneenya okw’ensi yonna okulaga enkola ezitasuubirwa. Mu nkola y’okwekenneenya okw’amaanyi, olugero olukulu kwe ndowooza y’okulowooza ku maapu ez’amakulu eziyinza okukozesebwa okunoonyereza ku biwandiiko. N’olwekyo, ekikulu ekivudde mu kugatta okuliwo kati okwa MS/MS metabolomics okutaliimu kyekubiira n’endowooza y’amawulire kwe kuba nti kiwa metric ennyangu eyinza okukozesebwa okuzimba maapu okulambula enjawulo y’enkyukakyuka ku minzaani ez’enjawulo ez’ensengeka. Kye kyetaagisa ekikulu eky’enkola eno. Okunoonyereza ku nkulaakulana ya micro/macro n’obutonde bw’ensi mu kitundu.
Ku mutendera gwa macro-evolutionary, omusingi gw’endowooza y’enkulaakulana y’ebimera n’ebiwuka eya Ehrlich ne Raven (51) kwe kuteebereza nti enkyukakyuka y’enjawulo y’enkyukakyuka y’ebimera wakati w’ebika y’ensonga y’enjawulo y’ennyiriri z’ebimera. Naye mu myaka ataano bukya omulimu guno ogw’ensigo gufulumizibwa, endowooza eno tetera kugezesebwa (52). Kino kisinga kuva ku mpisa z’ensengekera z’ensengekera z’ebimera ezigeraageranyizibwa mu nnyiriri z’ebimera eby’ewala. Rarity esobola okukozesebwa okunyweza enkola z’okwekenneenya ebigendererwa. Enkola y’emirimu gya MS/MS eriwo kati ekolebwa endowooza y’amawulire egera obungi bw’okufaanagana kw’enzimba ya MS/MS okw’ebirungo ebikyukakyuka ebitali bimanyiddwa (nga tewali kulonda bikozesebwa mu kusooka) era n’ekyusa MS/MS zino mu kibinja kya MS/MS, bwe kityo mu nkyukakyuka y’emmere ey’ekikugu Ebikozesebwa bino eby’enkulaakulana ennene (macro-evolutionary models) bigeraageranyizibwa mu minzaani y’okugabanya. Ebiraga ebibalo ebyangu. Enkola eno efaananako n’okwekenneenya ensengekera y’obuzaale (phylogenetic analysis), eyinza okukozesa okukwatagana kw’ensengekera (sequence alignment) okugera obungi bw’omutindo gw’enjawulo oba enkyukakyuka y’obubonero awatali kuteebereza nga tekunnabaawo.
Ku mutendera gw’ebiramu, endowooza y’okukebera eya Firn ne Jones (53) eraga nti enjawulo mu nkyukakyuka y’emmere ekuumibwa ku mitendera egy’enjawulo okusobola okuwa ebigimusa okukola emirimu gy’ebiramu egy’ebirungo ebikyukakyuka eby’edda ebitali bikwatagana oba ebikyusiddwa. Enkola z’endowooza y’amawulire ziwa enkola nga enkyukakyuka zino ez’enkulaakulana ezikwata ku metabolite ezibeerawo mu kiseera ky’okukuguka mu metabolite zisobola okupimibwa mu bungi ng’ekitundu ky’enkola y’okukebera enkulaakulana eteeseddwa: okukyusakyusa mu biramu okuva ku specificity entono okutuuka ku specificity enkulu Ebirungo ebiziyiza eby’obutonde obuweereddwa.
Byonna awamu, mu nnaku ezasooka ez’obulamu bwa molekyu, endowooza enkulu ez’okukuuma ebimera zakolebwa, era enkola ezikulemberwa endowooza (deductive hypothesis-driven methods) zitwalibwa nnyo ng’engeri yokka ey’okukulaakulana mu bya ssaayansi. Kino kisinga kuva ku buzibu bw’eby’ekikugu mu kupima metabolome yonna. Newankubadde enkola ezikulemberwa endowooza (hypothesis-driven methods) za mugaso nnyo mu kulonda enkola endala ezireeta, obusobozi bwazo okutumbula okutegeera kwaffe ku mikutu gy’ebiramu (biochemical networks) bukoma nnyo okusinga enkola z’okubalirira eziriwo mu kiseera kino mu sayansi ow’omulembe guno akozesa amawulire amangi. N’olwekyo, endowooza ezitasobola kuteebereza zisukka wala obuwanvu bwa data eziriwo, kale hypothetical formula/test cycle of progress in the research field teyinza kuggyibwawo (4). Tulaba nti enkola y’emirimu gy’okubalirira eya metabolomics eyanjuddwa wano esobola okuddamu okusitula obwagazi mu nsonga ezisembyeyo (engeri) n’ezisembayo (lwaki) ez’enjawulo mu nkyukakyuka y’emmere, n’okuyamba ku mulembe omupya ogwa ssaayansi wa data alungamizibwa mu ndowooza. Omulembe gwaddamu okwekenneenya endowooza enkulu ezaaluŋŋamya emirembe egyayita.
Okuliisa obutereevu ebimera ebirya omuddo kukolebwa nga tukunda enkwaso ey’omutendera ogw’okubiri oba enkwaso ya Sl ku kikoola kimu eky’ekimera ky’ensuwa ekya langi enzirugavu eky’ekimera kimu ekifuumuuka ebimuli bya rose, nga buli kimera kirimu ebikoppi by’ebimera 10. Enkwaso z’ebiwuka zaasibirwa n’ebikwaso, era ebitundu by’amakoola ebyasigalawo ne bikuŋŋaanyizibwa oluvannyuma lw’essaawa 24 ne 72 nga bifunye obulwadde ne bifukibwa mangu, era ebirungo ebikyusa ebiriisa ne biggyibwamu.
Koppa obujjanjabi obw’okulya omuddo mu ngeri ekwatagana ennyo. Enkola eno kwe kukozesa nnamuziga z’omusono gw’olugoye okuboola ennyiriri ssatu ez’amaggwa ku buli ludda lw’omugongo ogw’omu makkati g’ebikoola by’ekimera ebisatu ebigaziyiziddwa mu bujjuvu mu kiseera ky’okukula kw’ekimuli ky’olugoye, n’osiiga amangu ddala 1:5 Diluted Ms. Oba kozesa engalo eziriko ggalavu okuyingiza S1 OS mu kiwundu eky’okufumita. Kungula era olongoose ekikoola nga bwe kyayogeddwa waggulu. Kozesa enkola eyayogerwako emabegako okuggyamu ebirungo ebisookerwako ebikyusa ebiriisa n’obusimu bw’ebimera (54).
Ku nkola ya JA ey’ebweru, ebikoola by’ebikoola ebisatu ebya buli kimu ku bimera omukaaga ebibuuka ebimuli bya rose ebya buli kika bijjanjabwa ne 20μl za lanolin paste ezirimu 150μg MeJA (Lan + MeJA), ne 20μl za lanolin nga kwogasse n’okujjanjaba ebiwundu (Lan + W), oba kozesa 20μl za lanolin omulongoofu ng’ekifuga. Ebikoola byakungula oluvannyuma lw’essaawa 72 nga bimaze okulongoosebwa, ne bifukibwa mu bbugumu mu nayitrojeni ow’amazzi, ne biterekebwa ku -80°C okutuusa lwe byakozesebwa.
Layini nnya eza JA ne ET transgenic, okuli irAOC (36), irCOI1 (55), irACO ne sETR1 (48), zizuuliddwa mu kibinja kyaffe eky’okunoonyereza. irAOC yalaga nnyo okukendeera mu miwendo gya JA ne JA-Ile, ate irCOI1 teyali sensitive ku JAs. Bw’ogeraageranya ne EV, okukuŋŋaanyizibwa kwa JA-Ile kweyongera. Mu ngeri y’emu, irACO ejja kukendeeza ku kukola ET, era bw’ogeraageranya ne EV, sETR1, etali ya ET, ejja kwongera ku kukola ET.
Ekyuma ekipima ekitangaala (photoacoustic laser spectrometer) (Sensor Sense ETD-300 real-time ET sensor) kikozesebwa okukola okupima ET mu ngeri etali ya kuyingirira. Amangu ddala nga bamaze okulongoosebwa, ekitundu ky’ebikoola byasalibwa ne bitwalibwa mu kidomola ky’endabirwamu ekya 4-ml, era ekifo ky’omutwe ne kikkirizibwa okukuŋŋaanyizibwa mu ssaawa 5. Mu kiseera ky’okupima, buli kibbo kyafukibwa n’omugga gwa liita 2/essaawa ey’empewo ennongoofu okumala eddakiika 8, nga emabegako yali eyise mu kiziyiza ekyaweebwa Sensor Sense okuggyawo CO2 n’amazzi.
Ebikwata ku microarray byasooka kufulumizibwa mu (35) era ne biterekebwa mu National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (ennamba y’okuyingira GSE30287). Ebiwandiiko ebikwatagana n’ebikoola ebiva ku bujjanjabi bwa W + OSMs n’okufuga okutayonooneka byaggyibwamu okukola okunoonyereza kuno. Amaanyi amabisi gali log2. Nga tetunnaba kwekenneenya bibalo, omusingi gwakyusibwa ne gufuulibwa ogwa bulijjo okutuuka ku kitundu kyagwo ekya 75 ku buli kikumi nga tukozesa pulogulaamu ya R.
Ebikwata ku RNA sequencing (RNA-seq) ebyasooka eby’ebika bya Nicotiana byaggyibwa mu NCBI Short Reading Archives (SRA), ennamba ya pulojekiti ye PRJNA301787, eyaloopebwa Zhou et al. (39) era ogende mu maaso nga bwe kinyonyoddwa mu (56). Ebiwandiiko ebibisi ebyakolebwako W + W, W + OSM ne W + OSS1 ebikwatagana n’ebika bya Nicotiana byalondebwa okwekenneenya mu kunoonyereza kuno, era ne bikolebwa mu ngeri eno wammanga: Okusooka, ebisomeddwa ebibisi ebya RNA-seq byakyusibwa ne bifuulibwa enkola ya FASTQ. HISAT2 ekyusa FASTQ okudda mu SAM, ate SAMtools ekyusa fayiro za SAM mu fayiro za BAM ezisunsuddwa. StringTie ekozesebwa okubala okulaga kw’ensengekera y’obutonde (gene expression), era enkola yaayo ey’okwolesebwa eri nti waliwo ebitundutundu buli lukumi mu bitundutundu by’omusingi buli bukadde bw’ebitundutundu by’okuwandiika ebisengekeddwa.
Empagi ya Acclaim chromatographic (mm 150 x mm 2.1; obunene bw’obutundutundu 2.2μm) ekozesebwa mu kwekenneenya ate empagi y’omukuumi eya mm 4 x mm 4 zirimu ekintu kye kimu. Enkyukakyuka eno wammanga ey’ebibiri ekozesebwa mu nkola ya Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): eddakiika 0 okutuuka ku 0.5, isocratic 90% A [amazzi agataliimu ayoni, 0.1% (v/v) acetonitrile ne 0.05% formic acid], 10% B (Acetonitrile ne 0.05% formic acid); eddakiika 0.5 okutuuka ku 23.5, omutendera gw’okusereba guli 10% A ne 90% B, mu kulondako; eddakiika 23.5 okutuuka ku 25, isocratic 10% A ne 90% B. Omuwendo gw’okukulukuta guli 400μl/min. Ku kwekenneenya kwonna okwa MS, ssaako ekirungo ekirongoosa eky’empagi mu kyuma ekikebera ebyuma ebina n’ekiseera ky’okubuuka (qTOF) ekirimu ensibuko y’amasannyalaze ekola mu mbeera ya ionization ennungi (capillary voltage, 4500 V; capillary outlet 130 V ; Drying temperature 200°C; drying airflow 10 liters/min).
Kola okwekenneenya ebitundu bya MS / MS (wano okuyitibwa MS / MS) okutali kwa mugaso oba okutayawulwa ku data okufuna amawulire ag’enzimba ku nsengeka y’enkyukakyuka y’emmere okutwalira awamu ezuulibwa. Endowooza y’enkola ya MS/MS etali ya kusosola yeesigamye ku nsonga nti quadrupole erina eddirisa eddene ennyo ery’okwawula obuzito [n’olwekyo, lowooza ku bubonero bwonna obw’omugerageranyo gw’obuzito n’omusannyalazo (m/z) ng’ebitundutundu]. Olw’ensonga eno, olw’okuba ekintu kya Impact II tekyasobola kutondawo CE tilt, okwekenneenya okwetongodde okuwerako kwakolebwa nga tukozesa emiwendo gy’amasoboza ag’okutomeragana agava mu kutomeragana (CE) egy’okwongera. Mu bufunze, sooka okwekenneenya sampuli nga okozesa UHPLC-electrospray ionization/qTOF-MS ng’okozesa enkola ya single mass spectrometry mode (embeera z’okukutula okutono ezikolebwa okukutukakutuka mu nsibuko), okusika okuva ku m/z 50 okutuuka ku 1500 ku frequency y’okuddiŋŋana eya 5 Hz. Kozesa nayitrojeni nga ggaasi y’okutomeragana mu kwekenneenya kwa MS/MS, era okole ebipimo ebyetongodde ku vvulovumenti zino wammanga ennya ez’enjawulo ez’okukutula ezireetebwa okutomeragana: 20, 30, 40, ne 50 eV. Mu nkola yonna ey’okupima, quadrupole erina eddirisa erisinga obunene ery’okwawula obuzito, okuva ku m/z 50 okutuuka ku 1500. Okugezesa kw’omubiri gw’omu maaso m/z n’obugazi bw’okwawula bwe kuteekebwa ku 200, ekipimo ky’obuzito kikolebwa mu ngeri ey’otoma software ekola ku kikozesebwa ne 0 Da. Sikaani ku bitundutundu by’amasasi nga mu mbeera y’obuzito obumu. Kozesa sodium formate (50 ml isopropanol, 200 μl formic acid ne 1 ml 1M NaOH aqueous solution) okupima obuzito. Nga tukozesa enkola ya Bruker ey’okupima okw’obutuufu obw’amaanyi, fayiro ya data epimibwa oluvannyuma lw’okuddukanya spektrum eya wakati mu kiseera ekigere. Kozesa omulimu gw’okufulumya ebweru ogwa pulogulaamu ya Data Analysis v4.0 (Brook Dalton, Bremen, Germany) okukyusa fayiro za data embisi mu nkola ya NetCDF. Ekiwandiiko kya MS/MS kiterekeddwa mu kifo ekiggule ekya metabolomics database MetaboLights (www.ebi.ac.uk) nga kiriko ennamba y’okuyingira. MTBLS1471.
Okukuŋŋaanya kwa MS/MS kuyinza okutuukirira okuyita mu kwekenneenya enkolagana wakati wa MS1 ne MS/MS omutindo gw’obubonero ku maanyi g’okutomeragana amatono n’amangi n’amateeka amapya agateekeddwa mu nkola. Ekiwandiiko kya R kikozesebwa okutegeera okwekenneenya enkolagana y’ensaasaanya y’ekisookerwako eri ekintu, ate ekiwandiiko kya C # (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) kikozesebwa okussa mu nkola amateeka.
Okusobola okukendeeza ku nsobi ez’obulimba ennungi ezireetebwa amaloboozi ag’emabega n’enkolagana ey’obulimba ereetebwa okuzuula ebifaananyi ebimu ebya m/z mu sampuli ntono zokka, tukozesa omulimu gwa “filled peak” ogwa R package XCMS (for background noise correction) Should be used to replace the “NA” (undetected peak) intensity. Omulimu gw’entikko y’okujjuza bwe gukozesebwa, wakyaliwo emiwendo mingi egy’amaanyi “0′′ mu kibiina kya data egijja okukosa okubala kw’enkolagana.Awo, tugeraageranya ebivudde mu kukola ku data ebifunibwa ng’omulimu gw’entikko ogujjuziddwa gukozesebwa era nga omulimu gw’entikko ogujjuziddwa tegukozesebwa, ne tubala omuwendo gw’amaloboozi g’emabega okusinziira ku muwendo ogubalirirwamu ogwa wakati ogutereezeddwa, n’oluvannyuma ne tukyusa emiwendo gino egy’amaanyi 0 ne tussaamu omuwendo gw’amaanyi ogw’emabega ogwabalirirwa yatwalanga ebifaananyi byokka amaanyi gaabyo agasukka emirundi esatu omuwendo ogw’emabega era n’abitwala nga “entikko entuufu.” Ku kubalirira kwa PCC, obubonero bwa m/z bwokka obw’ekisookerwako eky’ekyokulabirako (MS1) n’ebitundutundu by’ebiwandiiko ebirina waakiri entikko munaana entuufu bye bitunuulirwa.
Singa amaanyi g’ekifaananyi ky’omutindo gw’ekisookerwako mu sampuli yonna gakwatagana nnyo n’amaanyi agakendedde ag’ekifaananyi ky’omutindo kye kimu ekikolebwako amasoboza ag’okutomeragana amatono oba amangi, era ekifaananyi ekyo tekiwandiikibwako ntikko ya isotopu nga CAMERA, kiyinza okwongera okunnyonnyolwa. Olwo, nga tubalirira ebibiri byonna ebisoboka ebisookerwako-ebivaamu mu s 3 (eddirisa ly’obudde bw’okusigala obubalirirwamu okusigala ku ntikko), okwekenneenya kw’enkolagana kukolebwa. Omuwendo gwa m/z bwe guba wansi okusinga omuwendo gw’ekisookerwako era ng’okukutukakutuka kwa MS/MS kubaawo mu kifo kye kimu eky’ekyokulabirako mu kibiina kya data n’ekisookerwako mwe kiva, kye kitwalibwa ng’ekitundutundu.
Okusinziira ku mateeka gano abiri amangu, tuggyako ebitundutundu ebiragiddwa nga emiwendo gya m/z gisinga m/z egy’ekisookerwako ekizuuliddwa, era nga tusinziira ku kifo kya sampuli ekisookerwako we kirabika n’ekitundu ekiragiddwa. Era kisoboka okulonda ebifaananyi by’omutindo ebikolebwa ebitundutundu bingi ebiri mu nsibuko ebikolebwa mu mbeera ya MS1 ng’ebisookerwako ebivuganya, bwe kityo ne bivaamu ebirungo bya MS/MS ebitaliimu. Okusobola okukendeeza ku data redundancy eno, singa NDP similarity ya spectra esukka 0.6, era nga za chromatogram “pcgroup” annotated by CAMERA, tujja kuzigatta. Mu kusembayo, tugatta ebivudde mu CE byonna ebina ebikwatagana n’ekisookerwako n’ebitundutundu mu spectrum esembayo eya deconvoluted composite spectrum nga tulonda entikko y’amaanyi esinga obunene mu ntikko zonna ezigenda okwesimbawo nga zirina omuwendo gwa m/z gwe gumu ku masoboza ag’okutomeragana ag’enjawulo. Emitendera gy’okulongoosa egiddako gyesigamiziddwa ku ndowooza ya spektrum y’ekirungo ekigatta era gitwala mu nkola embeera za CE ez’enjawulo ezeetaagisa okutumbula emikisa gy’okukutukakutuka, kubanga ebitundu ebimu bisobola okuzuulibwa wansi w’amasoboza ag’okutomeragana ag’enjawulo gokka.
RDPI (30) yakozesebwa okubala inducibility ya metabolic profile. Enjawulo y’ensengekera y’enkyukakyuka y’emmere (Hj index) eggibwa mu bungi bw’ebisookerwako ebya MS/MS nga tukozesa entropi ya Shannon ey’okugabanya kwa frequency ya MS/MS nga tukozesa ensengekera eno wammanga eyayogerwako Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) nga Pij ekwatagana n’emirundi egy’enjawulo egya MS/MS ey’omulundi ogwa i mu sampuli ey’omulundi ogwa j (j = 1, 2,..., m) (i = 1, 2, ..., m) t).
Metabolic specificity (Si index) etegeezebwa nga expression identity ya MS/MS eweereddwa mu bikwatagana n’emirundi wakati wa sampuli ezitunuuliddwa. MS/MS specificity ebalwa nga Si = 1t (∑j = 1tPijPilog2PijPi) .
Kozesa ensengekera eno wammanga okupima omuwendo gwa δj ogukwata ku metabolome ogwa buli sampuli ya j, ne average ya MS/MS specificity δj = ∑i = 1mPijSi
MS/MS spectra zisengekeddwa mu babiri babiri, era okufaanagana kubalibwa okusinziira ku bubonero bubiri. Okusooka, nga okozesa NDP eya mutindo (era emanyiddwa nga enkola ya cosine correlation), kozesa ensengekera eno wammanga okuteeba okufaanagana kw’ekitundu wakati wa spektira NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 nga S1 ne S2 Mu ngeri ekwatagana, ku spektrum 1 ne spektrum 2, awamu ne WS1, i ne WS2, i ekiikirira obuzito okusinziira ku maanyi g’entikko nti enjawulo y’entikko ey’awamu ey’omulundi ogwa i wakati wa spektira zombi eri wansi wa 0.01 Da. Obuzito bubalibwa bwe buti: W = [amaanyi ag’oku ntikko] m [omutindo] n, m = 0.5, n = 2, nga bwe kyateesebwa MassBank.
Enkola eyokubiri ey’okugaba obubonero yassibwa mu nkola, nga muno mwe muli okwekenneenya NL egabanyizibwa wakati wa MS/MS. Ku nkomerero eno, twakozesa enkalala za NL 52 ezitera okusangibwa mu nkola y’okukutulakutula kwa MS mu tandem, ne NL esinga okwetongodde (fayiro ya data S1) eyali emaze okuwandiikibwako obugambo ku MS/MS spectrum y’ebirungo eby’okubiri ebikyusa ebiriisa eby’ekika kya Nepenthes ekinafuye ( 9, 26). Tonda vekita ya binary eya 1 ne 0 ku buli MS/MS, ekwatagana n’akaseera kano n’akatali ka NL ezimu. Okusinziira ku kufaanagana kw’ebanga lya Euclidean, obubonero bw’okufaanagana kwa NL bubalirirwa ku buli pair ya binary NL vectors.
Okukola okukuŋŋaanya emirundi ebiri, twakozesa ekintu kya R ekya DiffCoEx, nga kino kyesigamiziddwa ku kugaziya kwa Weighted Gene Co-expression Analysis (WGCNA). Nga tukozesa NDP ne NL scoring matrices za MS/MS spectra, twakozesa DiffCoEx okubala matrix y’enkolagana ey’okugeraageranya. Okukuŋŋaanya ebibiri kukolebwa nga oteeka “cutreeDynamic” parameter ku nkola = “hybrid”, cutHeight = 0.9999, deepSplit = T, ne minClusterSize = 10. Ensibuko ya R eya DiffCoEx yawanulibwa okuva mu fayiro endala 1 nga Tesson et al. (57) era (57); Enkola ya pulogulaamu ya R WGCNA eyeetaagisa esobola okusangibwa mu https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Okusobola okukola okwekenneenya omukutu gwa molekyu ogwa MS/MS, twabalirira okuyungibwa kwa spectral okugatta nga tusinziira ku bika by’okufaanagana kwa NDP ne NL, era ne tukozesa pulogulaamu ya Cytoscape okulaba mu birowoozo topology y’omukutu nga tukozesa ensengeka y’obutonde mu nkola y’okugaziya enkola ya CyFilescape yFiles layout algorithm.
Kozesa R version 3.0.1 okukola okwekenneenya kw’ebibalo ku data. Obukulu bw’ebibalo bwakeberebwa nga tukozesa okwekenneenya enjawulo okw’engeri bbiri (ANOVA), ne kugobererwa okugezesebwa kwa Tukey okw’enjawulo okw’amazima okw’amazima (HSD) okw’oluvannyuma. Okusobola okwekenneenya enjawulo wakati w’obujjanjabi obw’okulya omuddo n’okufuga, ensaasaanya y’emikira ebiri egy’ebibinja bibiri ebya sampuli ebirina enjawulo y’emu yeekenneenyezebwa nga tukozesa ekigezo kya Student’s t.
Okumanya ebikozesebwa eby’okugatta ku kiwandiiko kino, laba http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
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Endowooza y’amawulire egaba ssente ey’ensi yonna ey’okugeraageranya metabolome ez’enjawulo n’okuteebereza endowooza z’okuziyiza okugezesebwa.
Endowooza y’amawulire egaba ssente ey’ensi yonna ey’okugeraageranya metabolome ez’enjawulo n’okuteebereza endowooza z’okuziyiza okugezesebwa.
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