Favonoid ak repons fito-oksilipin nan fèy grenad (Punica Granatum L.) Pati 1

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Résumé:Methyl jasmonate (MeJA) pwodwi nan plant yo ka medyatè repons yo nan estrès anviwònman an. Yo te montre aplikasyon ekzojèn MeJA pou aktive chemen siyal ak pwovoke akimilasyon fitoaleksin nan anpil espès plant. Pou konprann ki jan plant grenad yo reyaji byochimik a estrès anviwònman an, yo te fè analiz metabolit nan fèy grenad ki sibi aplikasyon MeJA ak revele chanjman inik nan tanen idroliz, flavonoid, ak Phyto-oxylipins. Anplis de sa, transcriptome ak analiz qPCR an tan reyèl nan fèy grenad mock ak MeJA-trete idantifye diferan eksprime jèn metabolik ak faktè transkripsyon ki kapab patisipe nan kontwòl la.hydrolyzabletanen, flavonoid, ak chemen Phyto-oxylipin. Molekilè, byochimik, ak byoenfòmatik karakterizasyon nan sèlmanlipoksijenazak soutni, ekspresyon MeJA-induit te montre ke li se kapab nan oksidasyon asid gra poliensature, menm si li pa sitiye nan lòj la subselilè kote ki pa jasmonate (ki pa JA) Phyto-oxylipins yo te pwodwi. Rezilta sa yo kolektivman sijere ke pandan ke gwo repwesyon nan flavonoid ak antosyanin yo omwen pasyèlman kontwole nan nivo transkripsyon an, byosentèz la pwovoke nan ki pa JA.Phyto-oxylipinsgen anpil chans pa reglemante transcriptionally. An jeneral, yon pi bon konpreyansyon sou ki jan fèy grenad reponn a estrès anviwònman an pa pral sèlman ankouraje sante plant ak pwodiktivite, men tou gen yon enpak sou sante moun kòm fwi ki pwodui pa plant grenad se yon sous rich nan konpoze nitrisyonèl.

Mo kle:Methyl jasmonate.Flavonoid·Anthocyanin· Asid gra.Phyto-oxylipin·Lipoxygenase

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Entwodiksyon

Lè yo blese oswa atake pa èbivò ak patojèn, plant yo pwodui ak emèt methyl jasmonate (MeJA), ki se konnen pa tisi plant ki pa blese ak plant vwazen yo aktive repons defans (Cheong and Choi 2003). Anplis de sa, yo te montre aplikasyon ekzojèn MeJA nan yon plant yo pwovoke chemen siyal osi byen ke pwodiksyon an nan pwoteyin ki gen rapò ak patojèn ak pwodwi chimik defans ke yo rekonèt kòm phytoalexins. Phenolic phytoalexins, eg flavonoids and anthocyanins, te ekspoze ogmante akimilasyon an repons a tretman MeJA nan diferan plant, tankou Arabidopsis thaliana, rezen (Vitis vinifera), bannann (Musa acuminate), pòm (Malus Domestica), ak Franbwaz wouj (Rubus idaeus). )(Pandey et al.2016; Portu et al.2015; De Geyteret al.2012; Shafiq et al.2011; Flo-res and Ruiz del Castillo 2014). Biosentèz flavonoid ak anthocyanins kòmanse ak fòmasyon nan naringenin chalcone soti nan koumaroyl CoA ak twa molekil malonyl CoA katalize pa chalcone sentaz (CHS) ak izomerizasyon ki vin apre nan naringenin chalcone nan naringenin pa chalcone isomerase (CHILCON). Lè sa a, Naringenin yo itilize pou jenere eskèlèt nwayo flavonoid ak anthocyanin, ki plis modifye ak gwoup fonksyonèl glycosyl, methyl, hydroxyl, ak prenyl pou bay divès estrikti ak fonksyon (Tian et al. 2008).

Cistanche ka amelyore iminite

In addition to phenolic phytoalexins, the production of Phyto-oxylipins upon MeJA induction has also been reported in a few plant species (Deboever et al.2020). Phyto-oxylipins are oxygenated fatty acids and derivatives that play a role in plant growth, development, stress response, and innate immunity(Wasternack and Feussner 2018). The initial step of Phyto-oxylipin biosynthesis involves oxidation of polyunsaturated fatty acids(PUFAs) to fatty acid hydroperoxides (HPOs)by lipoxygenases(LOXs)(Andreou and Feussner 2009). Plant LOXs are grouped into two subfamilies according to their protein sequences; type ILOXs are highly homologous(>75 pousan resanblans) epi yo pa genyen yon peptide siyal, tandiske LOXs tip II posede yon resanblans sekans ki ba an jeneral (<35%)but all="" contain="" a="" chloroplast="" target="" peptide="" (feussner="" and="" wasternack="" 2002).="" plant="" loxs="" can="" also="" be="" classified="" based="" on="" enzymatic="" activities;9-loxs="" and="" 13-loxs="" target="" the="" c-9="" and="" c-13="" position="" of="" the="" fatty="" acid="" substrate,="" respectively="" (feussner="" and="" wasternack="" 2002).="" hpos="" generated="" by="" loxs="" can="" be="" further="" transformed="" to="" various="" phyto-oxylipins,="" such="" as="" hydroxy="" fatty="" acids="" by="" reductases,="" keto="" fatty="" acids="" by="" loxs,="" epoxy="" fatty="" acids="" by="" per-oxygenases(pxgs),="" and="" dihydroxy="" fatty="" acids="" by="" loxs="" or="" α-dioxygenase="" α-doxs).="" notably,13-hydroperoxy-linolenic="" acid(an="" hpo)produced="" from="" linolenic="" acid="" by="" 13-lox="" can="" initiate="" a="" series="" of="" reactions="" to="" form="" jasmonic="" acid(ja),="" meja,="" and="" the="" bioactive="" ja-isoleucine="">

Grenad (Punica granatum L.) se yon rekòt ortikultur espesyalize ki gen valè pou konpoze fenolik abondan nan fwi li yo, tankou flavonoid, antosyanin, ak tanen idrolizable (HTs) ki sòti nan yon entèmedyè nan chemen chikimat la (Ono et al.2016). ). Anpil etid te byen lwen tèlman konsantre sou wòl nan fenolik grenad nan soulaje estrès ak maladi nan imen (Wu and Tian 2017). Kontrèman, yo konnen ti kras sou fonksyon an nan fenolik ak lòt fitochimik nan defann grenad kont faktè abyotik ak byotik (egzanp). blesi, ajan patojèn, endiksyon MeJA) nan fèy ak fwi. De dènye rapò evalye tretman MeJA pre-rekòlte sou bon jan kalite a apre rekòt nan fwi grenad (Koushesh Saba and Zarei 2019; Garcia-Pastor et al.2020). Antosyanin total, flavonoid, ak fenolik fwi trete MeJA, men pa fèy, yo te analize kolektivman lè l sèvi avèk yon espektrofotomèt. Youn nan etid yo te analize tou anthocyanins endividyèl lè l sèvi avèk espektrometri mas (Garcia-Pastor et al.2020). Sepandan, li rete klè ki jan tisi plant grenad, swa fèy oswa fwi, reponn a estrès anviwònman an anvan yo mete fwi a.

Pou kòmanse diseksyon entèraksyon ant plant grenad ak faktè anviwònman an, nou te envestige repons metabolik fèy grenad nan aplikasyon MeJA ekzojèn lè l sèvi avèk segondè-pèfòmans likid chromatography (HPLC) ak likid chromatografi-electrospray ionizasyon tandem espektrometri mas (LC-ESI-MS/MS) . Chanjman inik nan HTs, flavonoid, ak anthocyanins osi byen ke chanjman nan lipid, asid gra, ak Phyto-oxylipins yo te obsève nan fèy grenad trete ak MeJA. Analiz transcriptome konparatif, valide pa analiz qPCR an tan reyèl, revele ke jèn estriktirèl ak / oswa regilasyon ki enplike nan HT, flavonoid, anthocyanin, ak metabolis Phyto-oxylipin yo te eksprime diferan nan fèy grenad mock- ak MeJA-trete. Sèl jèn LOX ki te montre yon ekspresyon soutni regilasyon nan fèy trete MeJA te sibi plis karakterizasyon molekilè, byochimik, ak filogenetik.

Materyèl ak metòd

Yo te achte estanda glikoz -glucogallin ak pentagalloyl nan Shanghai Yuanye Bio-Technology Co Ltd (Shanghai, Lachin). Pwodwi chimik yo itilize nan tès LOX yo te jwenn nan machann sa yo: 3-(dimethylamino)benzoic acid (DMAB)(Adamas Reagent, Co., Ltd., Shanghai, China), asid linoleik (Sigma-Aldrich, St. Louis, MO, USA), 3-methyl-2-benzothiazolinone (MBTH) ak emoglobin (Sangon Biotech Co., Ltd., Shanghai, Lachin).

Materyèl plant Grenad fwi ak grenn (cv. Wonderful) te bay san gad dèyè pa Akademi Panzhihua nan Syans Agrikòl ak Forest ak idantifye pa Dr Binjie Ge nan Shanghai Chenshan Botanical Garden. Yo te depoze yon echantiyon bon (No CSHO173966) nan èrbyòm Shanghai Chenshan Botanical Garden, Shanghai, Lachin. Grenad plant yo te grandi nan yon chanm kwasans kontwole tanperati pou 6 semèn nan 25 degre degre ak 16 h limyè / 8 h fè nwa. Konsantrasyon MeJA pou aplikasyon espre nan tisi plant yo rapòte varye ant 100 ak 250 μM（Ku et al.2014; Hickman et al.2017). Diferan konsantrasyon MeJA te okòmansman aplike nan fèy grenad, ki 200μM MeJA te mennen nan yon repons metabolik disernanb nan analiz preliminè a epi yo te itilize pou metabolit ak analyse ekspresyon jèn yo dekri nan etid sa a. Anvan tretman MeJA a, mwatye nan plant grenad yo te deplase nan yon lòt chanm kwasans ak kondisyon menm jan an. Pandan ke plant nan yon chanm kwasans yo te flite ak 200 μM MeJA, sa yo ki nan lòt chanm nan kwasans yo te flite ak dlo (sa vle di kontwòl mock). h,12-h,24-h, 30-h,36-h,48-h, ak72-h apre tretman an, kite soti nan3 a 5 plant mock- oswa MeJA-trete yo te pisin, ki konstitye yon sèl replike byolojik. Twa replike byolojik yo te kolekte pou mock- ak MeJA-tretman eksperyans yo; chak replike byolojik te divize an aliquot pou pwofil metabolit ak analiz ekspresyon jèn.

Analiz pwofil metabolit

Fèy grenad yo te lyofilize, peze, epi mouye nan yon poud amann lè l sèvi avèk pèl zirkonya nan yon batè pèl (Mixer Mill MM 400, Retsch GmbH, Haan, Almay) pou 90s yo nan 30 Hz. Pou analiz HPLC, echantiyon fèy la te ekstrè nan 70 pousan metanol pou 60 min anba sonication ak santrifuje nan 13, 000 rpm pou 10 min. Supernatant la te transfere nan yon flakon HPLC, nan ki 30μL yo te enjekte sou yon faz ranvèse HPLC (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) ak analize jan sa dekri deja (Wil-son et al.2019). Metabolit yo te detekte pa absòpsyon UV nan 254 nm, 280 nm, 320 nm, ak 360 nm. Yo te konstwi koub kalibrasyon estanda nan -glucogallin ak pentagalloyl glikoz; yo te itilize pou konvèti zòn yo nan pik ki matche ak tan yo retansyon ak spectre absòpsyon nan -glucogallin ak pentagalloyl glikoz nan konsantrasyon respektif yo.

For LC-ESI-MS/MS analysis, the homogenized leaf sample(100 mg) was extracted in 1 mL of 70% methanol at 4℃Covernight.On the following day, the methanolic extract was centrifuged at 10,000×g for 10 min and the supernatant was passed through a CNWBOND Carbon-GCB SPE cartridge（ANPEL, Shanghai, China） and a 0.22-μm syringe filter(ANPEL) prior to metabolite analysis. The extract （2 μL） was analyzed using LC-ESI-MS/MS (Shim-pack UFLC, Shimadzu, Kyoto, Japan; QTRAP6500, Applied Biosystems, Foster City, CA, USA)and a reverse phase C1s column(ACQUITY UPLC HSS T3,1.8 βm, 2.1 mm×100 mm, Waters, Milford, MA, USA).Metabolites were eluted using solvents(A)water containing 0.04%acetic acid,and(B)acetonitrile containing0.04% acetic acid at a gradient of 0-11 min,95-5%A;11-12 min,5% A;12-12.1 min,5-95%A;12.1-15min,95%A.The flow rate was maintained at 0.4 mL min-1. Linear ion trap (LIT) and triple quadrupole(QQQ)MS scans were acquired in positive-and negative-ion modes. The turbo spray ion source was operated at 500℃C with an ionization voltage of 5500 V. The ion source gas I, gas I, and curtain gas were set at 55 psi,60 psi, and 25 psi, respectively. The collision gas (nitro-gen) was set at 5 psi. For the MRM analysis, declustering potential (DP)and collision energy(CE) were optimized for each precursor-product ion transition. For metabolite identification, the LC-ESI-MS/MS data were compared with an MS2T library of commercial standards and previously identified compounds published in mass spectral databases (when commercial standards are not available)(Chen et al.2013). Pomegranate metabolites were annotated based on the retention times, accumulate m/z values, and fragmentation patterns that match the MS2T library entries(Chen et al.2013). Metabolite quantification was performed using the MRM method as described by Dresen et al. (Dresen et al.2010). The biological replicates of each treatment (mock or MeJA)were averaged for comparative metabolite analysis. The Variable Importance in Projection(VIP) value was obtained from the Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)model. Metabolites with ILog, FCl>1, and VIP>=1 yo te konsidere siyifikativman chanje.

Analiz transcriptome

Total RNA te extrait nan fèy grenad lè l sèvi avèk TRIzol reyaktif (Invitrogen, Carlsbad, CA, USA) ak quantifye lè l sèvi avèk Nanodrop2000 (ThermoFisher Scientific, Waltham, MA, USA). Yo te verifye entegrite echantiyon RNA yo atravè separasyon sou yon jèl agaroz (pa gen okenn degradasyon vizib) ak detèminasyon rapò OD20/28o (ant 1.8 ak 2.2) lè l sèvi avèk Nanodrop2000. Anrichisman mRNA de total de RNA te pote soti lè l sèvi avèk oligo (dT) pèl mayetik (Invitrogen). Bibliyotèk mRNA-Seq yo te konstwi lè l sèvi avèk twous preparasyon bibliyotèk Truseq RNA (Illumina, San Diego, CA, USA). Analiz transcriptome yo te fèt sou Illumina HiSeq4000 epi yo te jwenn 55-60 milyon 150-bp lekti fen pè (PE150) pou chak bibliyotèk echantiyon. Done sekans anvan tout koreksyon yo te trete lè yo retire sekans adaptè yo ak kout (<50 bp),="" low="" quality=""><30), and="" poly(="">10%)reads using SeqPrep (https://github.com/jstjohn/seqprep)and Sickle (HTTPS: GitHub. com/Joshi/sickle). Over 95% of the cleaned reads were uniquely mapped to the reference pomegranate genome for each sample library using HISAT2(Kim et al.2015) and the mapped reads were assembled using StringTie(Pertea et al.2015). The assembled transcriptome sequences were annotated using NCBI NR(ftp://ftp.ncbi.nlm.nih.gov/blast/db/). Transcript abundance was determined by the RNA-Seq by Expectation-Maximization(RSEM) method and expressed as Transcripts Per Kilobase Million(TPM)(Li and Dewey 2011). Differential gene expression analysis was performed using DESeq2 (Love et al.2014), with a thresh-old of the log, FCl>1, ak ajiste valè P<>

An tan reyèl analiz qPCR

Total RNA te extrait de mock- ak MeJA-trete fèy grenad itilize RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, Lachin). Transkripsyon ranvèse (RT) yo te fèt lè l sèvi avèk RNA total ak PrimeScriptTM RT Reagent Kit la (Takara Bio Inc., Kusatsu, Japon). Yo te pote PCR (qPCR) kantite lè l sèvi avèk TB GreenTM Premix Ex Taq TM (Tli RNaseH Plus) twous ( Takara) ak yon sistèm PCR an tan reyèl StepOnePlus (Ther-moFisher Scientific). Yo te fè analiz koub k ap fonn epi li te montre yon sèl pwodwi anplifikasyon pou chak pè Jadendanfan. Pou analiz RT-qPCR la, yo te egzamine twa replike byolojik ak chak ak twa replika teknik pou echantiyon trete mock ak MeJA. Ekspresyon jèn yo te analize lè l sèvi avèk metòd konparatif C,(△AC) (Livak and Schmittgen 2001), ak nivo siyifikasyon yo te detèmine lè l sèvi avèk yon tès t Student de-ke. Tablo S1 montre sekans primè pou analiz qPCR an tan reyèl ak efikasite anplifikasyon pè primè yo.

Ekspresyon ak pirifikasyon nan pwoteyin recombinant ak tès anzim

Ankadreman lekti ouvè Pgr025417 (kode yon LOX putatif) te sentèz pou itilizasyon kodon pi bon nan E. coli (Genewiz, Suzhou, Lachin) epi li te klonaj nan pET28a. Yo te transfòme plasmid recombinant nan E. coli BL21 (DE3) selil yo. Yon kilti 5-mL Luria Bertani (LB) ak 50ug mL-Ikanamycin te kòmanse nan yon sèl koloni ak enkubasyon lannwit lan ak souke nan 37C. Kilti lannwit lan te itilize pou vaksinen yon mwayen LB 100-mL ak 50ug mL-'kanamycin epi pèmèt yo grandi nan yon OD600 de 0.5. Isopropyl - - D-thiogalactoside (IPTG) te ajoute nan yon konsantrasyon final 0.1 mM pou endiksyon ekspresyon pwoteyin. Apre enkubasyon ak souke nan 16 degre degre pou 18 èdtan, selil yo te rekòlte pa santrifujasyon. Granules selil yo te resuspend nan tanpon lysis la (50 mM NaH, PO, pH 7.4,300 mM NaCl, 10 mM imidazol) ak omojenize lè l sèvi avèk yon disruptor selil (Constant Systems Ltd, Northants, UK). Pwoteyin ki make li yo te pirifye lè l sèvi avèk. Ni-NTA pèl (ThermoFisher Scientific) ak tanpon lave a (50 mM NaH, PO, pH 7.4,300 mM NaCl, 25 mM imidazol) ak tanpon elisyon an (50 mM NaH, PO, pH 7.4,300 mM NaCl, 500 mM). imidazol). Pwoteyin pirifye yo te separe sou yon jèl SDS-PAGE 10 pousan pou vizyalizasyon pite pwoteyin. Konsantrasyon pwoteyin ki pirifye yo te detèmine lè l sèvi avèk tès Bradford (Bradford 1976).

Pou tès LOX la, yo te itilize asid linoleik kòm substra nan yon metòd kolorimetrik de etap ak ti modifikasyon （Anthon and Barrett 2008）. Melanj reyaksyon 500-μL, ki gen ladan 50 mM Na-fosfat, pH 6, 10 mM DMAB, 0.5 mM asid linoleik, ak divès kantite pwoteyin recombinant pirifye (1.5 mg mL). -), yo te enkube nan 25 degre pou 10 min.Yon dezyèm solisyon (500 μL) ki gen 0.2 mM MBTH ak 0.1 mg mL-'emoglobin te ajoute nan melanj reyaksyon an, ki te enkube pou yon lòt 5min. Reyaksyon an te sispann lè yo ajoute 500μL 1 pousan (w/v) sodyòm lauryl sulfat. Limyè absòpsyon nan 598 nm te detèmine. Lokalizasyon subselilè ak analiz filogenetik Yon rechèch nan jenom grenad anote (Qin et al. 2017) te idantifye 1l LOXs plen longè potatif (786 aa a 970 aa), ki gen ladan Pgr025413, Pgr020032, Pgr, Pgr0125413, Pgr025413, Pgr0254132, Pgr0254188250488040254188080402541880180180180180180180180001 -length sekans nan GenBank XP _031395793), Pgr009839, Pgr008562, Pgr025678, ak PgrO13780. Lokalizasyon subselilè ak sit klivaj nan peptides siyal pou LOXs grenad yo te prevwa lè l sèvi avèk TargetP 2.0 (http://www.cbs.dtu.dk/servi ces/TargetP/)(Almagro Armenteros et al.2019).

Analiz sit TF-obligatwa

Pou predi sit obligatwa TF yo, yo te jwenn 1000 bp en nan kodon kòmanse ATG nan jèn sib yo nan GenBank epi yo te fouye kont Eucalyptus Grandis TF yo nan PlantRegMap (vèsyon 5) (Tian et al.2020). Valè papòt la pou obligatwa. idantifikasyon sit te fikse nan P Mwens pase oswa egal a le-4. Analiz estatistik pou quantification metabolit, transcriptome, ak done qPCR an tan reyèl yo dekri nan seksyon respektif yo.

Rezilta yo

MeJA modile siyal selilè ak wout metabolik nan fèy grenad

Pou konprann repons transkripsyon grenad la nan tout genòm nan elicitation MeJA, transcriptom fèy grenad nan 2-h,6-h,24-h, ak 72-h apre MeJA oswa mock tretman (yo chak ak twa replike byolojik) yo te analize (figi S1). Apeprè 55 milyon lekti sekans anvan tout koreksyon (2 × 150 bp pè-fen) yo te jwenn pou chak transcriptome ak GC-kontni alantou 52 pousan ak valè Q30 sòti nan 91.6 a 95 pousan (Tablo S2). Pou tout transkriptòm yo, plis pase 96 pousan nan lekti sekans netwaye yo te trase nan jenom grenad referans (Qin et al.2017) (Table S3). 2000 bp (32.9 pousan), oswa 2000 bp—3000 bp (18.1 pousan) (Tablo S4).

To identify pathways that are significantly enriched with differentially expressed genes (DEGs)at the above-mentioned time points, genes that show significantly different expression(log, FCl>1, ajiste P<0.05)between meja-="" and="" mock-treated="" leaves="" were="" compared="" to="" the="" kyoto="" encyclopedia="" of="" genes="" and="" genomes(kegg)database="" (fig.s1).="" application="" of="" meja="" modulated="" the="" expression="" of="" genes="" in="" plant="" hormone="" and="" mitogen-activated="" protein="" kinase="" (mapk)signaling="" pathways="" as="" well="" as="" fatty="" acid="" metabolism="" at="" all="" time="" points,="" with="" the="" only="" exception="" of="" those="" in="" plant="" hormone="" pathways="" at="" 24-h="" (fig.="" s1).="" while="" changes="" in="" aromatic="" amino="" acid="" metabolic="" (including="" the="" shikimate="" pathway)genes="" became="" evident="" at="" 6-h="" after="" meja="" treatment="" (fig.s1b),="" a="" surge="" of="" modified="" expression="" of="" flavonoid="" metabolic="" genes="" was="" observed="" for="" the="" 24-h="" and="" 72-h="" post-meja="" treatment="" leaves="" (figs.="" slc="" and="" s1d).="" shikimate="" and="" ht="" pathway="" genes="" and="" ht="" metabolites="" were="" induced="" in="" meja-treated="" pomegranate="" leave="" as="" revealed="" in="" the="" transcriptome="" and="" kegg="" pathway="" enrichment="" analysis,="" three="" shikimate="" biosynthetic="" path-way="" genes="" showed="" upregulated="" expression="" in="" meja-treated="" leaves="" relative="" to="" mock="" controls="" at="" 6-h,="" including="" 3-deoxy-d-arabinose-heptulosonate-7-phosphate="" synthase="" (dahps),="" 3-dehydrogenate="" synthase(dhs),="" and="" the="" bifunctional="" 3-dehydrogenate="" dehydratase/shikimate="" dehydrogenase="" (dhq/sdh;="" abbreviated="" as="" sdh)(figs.s1b="" and="" la).in="" particular,="" three="" isoforms="" of="" pomegranate="" sdhs="" were="" identified="" and="" showed="" differential="" expression="" in="" the="" transcriptome="" analysis,="" pgr020271,="" pgr019030,="" and="">

Pou detèmine si chanjman nan kantite transkripsyon jèn shikimate ak HT biosentetik ka afekte nivo metabolit ki sòti nan chemen sa yo, yo te retire metabolit fenolik nan fèy rekòlte nan 24-h, 30-h. 36-h,48-h, ak 72-h apre MeJA oswa mock aplikasyon epi analize pa HPLC (Fig.2). Li ta dwe remake ke pwen tan sa yo te chwazi pou konte pou tan ki nesesè pou sentèz pwoteyin ak pwodiksyon metabolit ak akimilasyon apre chanjman ekspresyon yo obsève nan shikimate ak HT byosentetik jèn nan 6-h. Tan yo retansyon ak spectre absòpsyon de metabolit eluye nan 4.57 min (pik 1) ak 24.98 min (pik 2) matche ak sa yo ki nan entèmedyè yo chemen HT -glucogallin ak Penta alyaj-glikoz, respektivman (figi la ak 2a). Tou de pik yo te montre chanjman enpòtan nan zòn pik entegre nan plizyè pwen tan (Fig.2b). Espesyalman, pik 1 ogmante nan fèy trete ak MeJA parapò ak kontwòl mok nan 30-h, 36-h, ak 48-h (Fig.2b). Enteresan, pik 2 nan fèy trete MeJA te okòmansman diminye nan 24-h, men answit te ogmante nan 30-h ak 36-h anvan yo te retounen nan yon nivo ki sanble ak kontwòl mok nan 48- h ak 72-h (Fig.2b). Rediksyon nan pifò flavonoid ak anthocyanins, osi byen ke ogmante flavon methylated ak flavonol, te aparan nan fèy grenad trete MeJA.

To investigate whether exogenous application of MeJA may trigger broad-scale metabolic changes in pomegranate, metabolite profiling analysis was conducted on leaves collected at 72-h after mock- or MeJA treatment using LC-ESI-MS/MS. Metabolite annotation and quantification were performed using an MS/MS spectral tag (MS2T)library and multiple reaction monitoring(MRM), respectively. Of the 658 metabolites that were detected,29 showed increased and 73 exhibited decreased accumulation in MeJA-treated leaves compared to the mock controls (ILog, FCl>1; Tablo S5 ak S6). Pou metabolit yo akimile diferansye, te gen yon anrichisman jeneral nan metabolit ki enplike nan metabolis segondè / espesyalize plant (67 nan 102), patikilyèman konpoze fenolik (63 nan 102) (Tablo S6).

Pami fenolik, yon rediksyon konsèté nan yon pakèt flavonoid ak antosyanin (42 nan 73 konpoze yo diminye) te aparan nan fèy trete MeJA (Fig. 3; Tablo S6). Entrigan, twa flavon mono- oswa di-O-methylated ak flavonol, ki gen ladan di-O-methyl quercetin, chrysoberyl O-hexosyl-O-hexoside, ak vann 5-O-hexoside, te ogmante nan fèy trete MeJA. (Fig. 3; Tablo S6). Plizyè entèmedyè nan wout flavonoid ak anthocyanin, ki gen ladan luteolin, chrysoberyl, dihydrokaempferol, dihydroquercetin, dihydromyricetin, epicatechin, delphinidin, ak pelargonidin, te detekte men yo pa t montre chanjman enpòtan nan fèy trete MeJA (Fig.3; Tablo S5). Hydroxycin-namoyl dérivés, isoflavones, ak coumarins te pami lòt phenolics ki te montre redwi akimilasyon sou endiksyon MeJA (Tablo S6). Kontrèman, de asid fenolik, 2,3-asid dihydroxybenzoic ak asid protocatechuic (3,4-asid dihydroxybenzoic), ak yon koumarin, 6-methyl coumarin, te ogmante nan fèy trete MeJA. (Tablo S6).

Konfòm ak flavonoid yo lajman redwi ak anthocyanins nan fèy grenad trete MeJA, relve nòt yo nan de anzim kle pou flavonoid ak anthocyanin byosentèz. CHS(Pgr005566)ak CHI (Pgr025966), yo te siyifikativman diminye nan 6-h ak 24-h apre aplikasyon MeJA dapre analiz transcriptome a (figi 4). Yo te fè analiz qPCR an tan reyèl pou egzamine ekspresyon CHS ak CHI ak pwen tan adisyonèl, tankou 2-h, 3-h, 6-h,12-h,{{ 11}}h,48-h, ak 72-h (Fig.4). Transkripsyon CHS te tonbe nan fèy trete ak MeJA nan 3-h epi yo te rete siyifikativman pi ba pase sa ki nan mok. kontwole jiska 72-h, ak pi gwo diminisyon nan 12-h. Kontrèman, rediksyon nan ekspresyon CHI te sèlman enpòtan nan 24-h, 48-h, ak 72-h apre tretman MeJA (figi 4).

Atik sa a soti nan Planta (2021) 254:89 https://doi.org/10.1007/s00425-021-03735-9