Metabolomics of capsicum ripening.pdf

Postharvest Biology and Technology 89 (2014) 1931

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Postharvest Biology and Technology

jou rn al h om epage: www.elsev ier .com/ l

Metabolomics of capsicum ripening reveals modethylene related-pathway and carbon metabolism

Wan M. Aizata, Daniel A. Diasb, James C.R. Stangoulis c, Jason AUte Roes b,d a,

a School of Agri ond, Sb Metabolomicc School of Biold Australian Ce

a r t i c l

Article history:Received 6 September 2013Accepted 12 November 2013

Keywords:Capsicum annuFruitGCMSLCMSNon-climacterPepper

Capsicum (Capsicum annuum L. cv. Aries) is a non-climacteric bell-pepper fruit, exhibiting limited eth-ylene and respiration levels during ripening. In contrast to climacteric fruit, such as tomato which islargely dependent upon ethylene to ripen, the regulation of non-climacteric ripening is still inadequatelyunderstood. A metabolomics approach was used to identify differentially abundant compounds betweenripening stages with the aim of elucidating metabolic pathways involved in the regulation of non-

1. Introdu

Fruit arnon-climacing ripeninexhibit notbut can alsoGiovannonifruit such a

CorresponTel.: +61 8 831

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climacteric ripening. Metabolite proling using gas chromatographymass spectrometry (GCMS) wasinitially employed to screen potential metabolite differences among three ripening stages (Green, BreakerRed 1 and Light Red). Targeted analyses using liquid chromatographymass spectrometry (LCMS) orenzymatic assays were subsequently employed to characterise selected metabolites in more ripeningstages. Starch, sugars and their derivatives were signicantly modied during ripening which may affectthe abundance of some glycolysis intermediates and consequently other metabolic pathways involvingamino acids, colour and pungency precursors, and tricarboxylic acid (TCA) cycle intermediates. Further-more, metabolites closely related to ethylene production such as cysteine and methionine graduallyincreased between the ripening stages, whereas putrescine signicantly decreased during ripening, sug-gesting that some parts of the ethylene pathway may still be functional in this non-climacteric fruit.Thus, this study which utilised both proling and targeted metabolomics, has identied a wide range ofmetabolites which are involved in various biochemical pathways and highlights the overall metabolicshifts during non-climacteric capsicum ripening.

2013 Elsevier B.V. All rights reserved.

ction

e generally classied as being either climacteric orteric based on their ethylene and respiration levels dur-g. Climacteric fruit such as banana, apple and tomato

only ethylene and respiration bursts during ripening be stimulated strongly by ethylene to ripen (Cara and, 2008; Bapat et al., 2010). In contrast, non-climacterics strawberry, grape and capsicum, are characterised by

ding author at: PMB1 Glen Osmond, SA 5064, Australia.3 7245; fax: +61 8 8313 7109.ddresses: [email protected] (W.M. Aizat),b.edu.au (D.A. Dias),[email protected] (J.C.R. Stangoulis),elaide.edu.au (J.A. Able), [email protected] (U. Roessner),adelaide.edu.au (A.J. Able).

their limited production of ethylene and carbon dioxide (Paran andvan der Knaap, 2007; Pech et al., 2008). Although a lot is knownabout the regulation of climacteric fruit ripening, especially thechief role of ethylene, the control of ripening in non-climactericfruit as well as the absence of autocatalytic ethylene production isstill not well understood (Pech et al., 2008).

Capsicum has become an increasingly important fruit systemto study the non-climacteric fruit ripening process because itsgenome has conserved sequence with the model fruit, tomato(Livingstone et al., 1999), thus allowing better comparisons withclimacteric ripening. Another benet for using capsicum is thatit also has different ripening abilities when harvested at differentripening stages, with fruit harvested during the Green (G) stage notripening normally compared with those harvested at Breaker (B) orbeyond (Krajayklang et al., 2000; Pham, 2007). Unknown ripeningfactors may be differentially expressed during the different stagesto induce capsicum ripening. Therefore, further in-depth analysisis required to identify these factors, especially using comparison-based approaches.

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.postharvbio.2013.11.004sner , Amanda J. Ableculture, Food and Wine, The University of Adelaide, Waite Research Institute, Glen Osms Australia, School of Botany, The University of Melbourne, Victoria, Australiaogical Science, Flinders University, Bedford Park, SA 5042, Australiantre for Plant Functional Genomics, The University of Melbourne, Victoria, Australia

e i n f o a b s t r a c tocate /postharvbio

ication of the

. Ablea,

A 5064, Australia

20 W.M. Aizat et al. / Postharvest Biology and Technology 89 (2014) 1931

Several comparison-based approaches such as transcriptomics,proteomics and metabolomics have been used to study fruit ripen-ing, particularly climacteric tomato (Roessner-Tunali et al., 2003;Fei et al., 2004; Alba et al., 2005; Carrari et al., 2006; Rocco et al.,2006; FauroRohrmann fruit includ2008; Bianc2011) and Grimplet etIn capsicumregulation enzyme, wh2013). Furthments in thsignalling ting (Lee et athat non-cltory mechausing metain capsicum

Metabolcal system uapproachesor a produerful tool any given Several teching gas chchromatogrnetic resonBowne, 200metabolite mentary anand enzymthe metabo

Thereformetabolomences betwcv. Aries) r(LR)]. Severusing targeing stages [Differenceswere mainlglycolysis apathways. Tripening reripening.

2. Materia

2.1. Plant m

Capsicumstated in Aof capsicumFebruaryAing (G, BR1for GCMS of six ripenBR1, BR2, Lbiological reach particu(2013).

2.2. GCMS

Approximately 30 mg fresh weight (FW) of homogenisedcapsicum was weighed into pre-chilled 2 mL microfuge tubes.

nol (rd (1

mixmin.

at roarateure wntrif

tter ets (5o folved

idinem. Thimethtandadecaiaconh dectionque mplet quad ac

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g at 3ss sp0 scaevaluralwoed u/wwx-Pla/csbd-houing mretening rf inteed m. There foS1). Itabowith

MS

rgetalidat (paundsilute

wasbert et al., 2007; Moco et al., 2007; Kok et al., 2008;et al., 2011; Qin et al., 2012) and a few non-climactericing strawberry (Aharoni and OConnell, 2002; Fait et al.,o et al., 2009; Ponce-Valadez et al., 2009; Zhang et al.,grapes (Deytieux et al., 2007; Giribaldi et al., 2007;

al., 2007; Martnez-Esteso et al., 2011; Dai et al., 2013). ripening, a recent proteomics study has revealed an up-of 1-aminocyclopropane-1-carboxylate (ACC) oxidaseich is important for ethylene production (Aizat et al.,ermore, transcriptomic analysis has revealed other ele-e ethylene pathway including ethylene receptors andranscripts also being increased during capsicum ripen-l., 2010; Osorio et al., 2012). Although this may suggestimacteric capsicum ripening shares a common regula-nism with climacteric ripening, further investigationbolomics may reveal other potential factors involved

ripening.omics aims to measure all metabolites within a biologi-sing both comprehensive, qualitative and quantitative, and detects metabolites which can be a substratect of a biochemical pathway and hence is a pow-to gauge the functional and cellular regulation inspecies (Fiehn, 2002; Roessner and Bowne, 2009).niques have been employed in metabolomics includ-romatographymass spectrometry (GCMS), liquidaphymass spectrometry (LCMS) and nuclear mag-ance (NMR) spectroscopy (Fiehn, 2002; Roessner and9). A recent study on capsicum ripening has reportedproling using GCMS (Osorio et al., 2012) but comple-d more targeted approaches/techniques such as LCMSatic assays are required to better quantify and validatelites (Roessner and Bowne, 2009).e, we have initially employed a GCMS-basedics approach to screen potential metabolite differ-een three bell pepper capsicum (Capsicum annuum L.ipening stages [G, Breaker Red 1 (BR1) and Light Redal metabolites of interest were further investigatedted LCMS or enzymatic analyses in six different ripen-G, B, BR1, Breaker Red 2 (BR2), LR and Deep Red (DR)].

in metabolites between capsicum ripening stagesy associated with sugar modication, possibly affectingnd carbon metabolism, as well as ethylene-relatedhe implication of these metabolites in non-climactericgulation is discussed in comparison to climacteric

ls and methods

aterials

plants (cv. Aries) were grown and prepared asizat et al. (2013). There were two independent setss, which ripened during OctoberDecember 2010 andpril 2011. The rst set consisted of three stages of ripen-, LR) with ve biological replicates at each stage usedmetabolomics analysis and the second set, compriseding stages with three biological replicates each (G, B,R, DR), used for LCMS and enzymatic analyses. Eacheplicate was an individual fruit from one single plant atlar harvest and homogenised as detailed in Aizat et al.

Methastandasamplefor 15 10 minin sepnanopthen cethis laAliquoin vacuredissoin pyr500 rpbis-(trtime sn-nonahexatr

Eacto injetechniautosaAgilenadjustetris-(p30 m Vgra gutempeion souwas usysis ofprograa 7 C mheatin

Ma5060were (Spectidenti(http:/of Ma(http:/the inmatchof the Resultarea oanalys(2001)compaTable the meshape

2.3. LC

A tacross vinterescompousing dwater)250 L of 100%), in addition to 10 L of an internal3C6-sorbitol in water, 0.2 mg mL1) was added. Thetures were vortexed for 30 s and incubated at 70 C

The samples were then centrifuged at 13,000 rpm forom temperature (RT, 23 C). Supernatants were stored

2 mL tubes and the pellets were mixed with 250 Later for the second extraction (vortexed for 30 s anduged at 13,000 rpm for 10 min at RT. Supernatants fromxtraction were then combined with the rst extracts.0 L) from the combined sample extracts were driedr subsequent derivatisation. The dried samples were

in 10 L of 30 mg mL1 methoxyamine hydrochloride and derivatised at 37 C for 120 min with mixing ate samples were then treated for 30 min with 20 L N,O-ylsilyl)triuoroacetamide (BSTFA) and 2.5 L retentionrd mixture [0.029% (v/v) n-dodecane, n-pentadecane,ne, n-docosane, n-octacosane, n-dotriacontane, n-tane dissolved in pyridine] with mixing at 500 rpm.rivatised sample was allowed to rest for 60 min prior. Samples (1 L) were injected using a hot needleinto a GCMS system comprised of a Gerstel 2.5.2r, a 7890A Agilent gas chromatograph and a 5975Cdrupole MS (Agilent, Santa Clara, USA). The MS wascording to the manufacturers recommendations usingorobutyl)-amine (CF43). The GC was performed on aS column with 0.2 m lm thickness and a 10 m Inte-olumn (Varian, Inc., Victoria, Australia). The injectione was set at 250 C, the MS transfer line at 280 C, thedjusted to 250 C and the quadrupole at 150 C. Helium

the carrier gas at a ow rate of 0.8 mL min1. The anal-ples was performed under the following temperature; start at injection 70 C, a hold for 1 min, followed by1 oven temperature, ramp to 325 C and a nal 6 min25 C.ectra were recorded at 2 scans s1 with an m/znning range. Both chromatograms and mass spectraated using the AnalyzerPro Deconvolution Programrks, UK). Mass spectra of eluting compounds weresing the commercial mass spectra library NIST 05

w.nist.gov), the public domain mass spectra librarynck-Institute for Plant Physiology, Golm, Germanyb.mpimp-golm.mpg.de/csbdb/dbma/msri.html) andse Metabolomics Australia mass spectral library. Allass spectra were additionally veried by determinationtion time by analysis of authentic standard substances.elative response ratios (area of analyte divided byrnal sorbitol standard) per sample FW (mg) for eachetabolite were prepared as described in Roessner et al.

data were also normalised to the G stage in order told differences between ripening stages (Supplementaryf a specic metabolite had multiple TMS derivatives,lite with the greater detector response and better peakin the dynamic range of the instrument was selected.

ed LCMS based metabolomics approach was used tote and conrm the abundance of several compounds ofrticularly amino acids and amines) as well as additional

not detected in the GCMS. A preliminary LCMS studyd capsicum samples (four-fold dilution with nanopure

done but metabolites which had less than 50 g mg1

W.M. Aizat et al. / Postharvest Biology and Technology 89 (2014) 1931 21

were more difcult to detect (data not shown). Therefore, undilutedoriginal capsicum samples were used for the LCMS analysis.

Approximately 30 mg FW of ground capsicum samples wereresuspended in 250 L cold MeOH and immediately vortexed.Nanopure wdards in col2-aminoantdiately at 1transferredpared; (a) of 25 aminsulphur-coning glutathitris(2-carbotwo stock soware with wformate to 0.1, 0.5, 1, aliquot of eborate buff1 mM ascosolution wahydrosysucin 100% ace2011). The then centrifcontaining

An Agilespray Ionisaexperimentwere 0.52.Dynamic Mfor each anatages werestandard wused: sheatpressure 45

For the Rapid Resoa ow rate oating pressuin Boughtomobile phatonitrile 0.1was 1 and thtively, folloconditions ied aminoof the specibilities of thQuantitativthe levels o

2.4. Enzym

Metabolfructose, suusing spectrof the six assays. For ples were thfor 10 min transferred13,000 rpmfor malate were made

respectively. Samples for starch determination were prepared byadding 1 mL hydrochloric acid (HCl) and 4 mL dimethylsulphoxide(DMSO) in 50 mL tubes with 1.0 g ground samples. The mixtureswere vortexed for 5 s, incubated at 60 C for 30 min and immedi-

mmeand ). Ane wa

the stratiive cpared

accomou

Spelia) aoichitive press

etabo

me bae opae/wwl othted uordeolic p

atisti

ess oGensans ignianceplemn thiscriis (PC

ults

etaboces i

acid

MS wns)th 6nt amt anaon thg stariancere sion into eriv

wn) (ater (250 L) and a solution containing internal stan-d methanol (10 L, 1 mM, 13C615N-valine, 13C6-sorbitol,hracene) were added, vortexed and centrifuged imme-3,200 rpm for 10 min. The resulting supernatant was

to a new vial. Two stock solutions were also pre-an amino acid solution containing a standard mixo acids in nanopure water 0.1% formic acid, and (b)taining compound solution: A 2.5 mM stock contain-one and S-adenosylhomocysteine in water with 10 mMxyethyl)phosphine (TCEP) and 1 mM ascorbate. Thelutions were mixed and diluted using volumetric glass-ater containing 10 mM TCEP, 1 mM ascorbate and 0.1%produce the following series of combined standards:5, 10, 20, 50, 100 and 150 M. For derivatisation, anach standard or sample (10 L) was added to 70 L ofer (200 mM, pH 8.8 at 25 C) containing 10 mM TCEP,rbic acid and 50 M 2-aminobutyrate. The resultings vortexed before adding 20 L of 6-aminoquinolyl-N-cinimidyl carbamate (AQC) reagent [200 mM dissolvedtonitrile (ACN)] immediately vortexing (Boughton et al.,samples were heated with shaking at 55 C for 10 minuged (13,000 rpm at RT) and transferred to HPLC vialsinserts (Agilent, springless glass inserts 250 L).nt 1200 LC-system coupled to an Agilent 6420 Electro-tion-Triple Quadrupole-MS was used for quantications. Injection volumes used for the samples or standards0 L. Ions were monitored in the positive mode using aultiple Reaction Monitoring (DMRM) method optimisedlyte. The source, collision energies and fragmentor vol-

optimised for each analyte by infusing a derivatisedith LC eluent. The following source conditions wereh gas temperature 315 C, gas ow 10 L min1, nebuliser

psi and capillary voltage 3800 V.chromatography, an Agilent Zorbax Eclipse XDB-C18lution HT 2.1 50 mm, 1.8 m column was used withf 300 L min1, maintained at 30 C, resulting in oper-res below 400 bar with a 19 min run time as described

n et al. (2011). A gradient LC method was used withses comprised of (A) water 0.1% formic acid and (B) ace-% formic acid (such that at 0.0 and 2.0 min, the % of Ben increased to 15 and 30% at 9.0 and 14.0 min, respec-

wed by a reduction to 1% at 14.1 and 19.0 min). Theseprovided suitable chromatographic separation of mod-

acids and although co-elution was observed for somees, this could be overcome by the mass-selective capa-e mass spectrometer using MRM. Agilent MassHuntere Analysis Software, Version 4.0 was used to quantifyf amino acids.

atic analyses

ites of interest not detected in the LCMS (glucose,crose, starch, malate and citrate) were determinedophotometric enzyme-based analyses. Three replicatesstages of ripening (second set) were used in thesesugars, malate and citrate analyses, 1.0 g ground sam-awed at RT in 1.5 mL tubes, centrifuged at 13,000 rpm

at RT to obtain clear fruit juices. The supernatant was into a new 1.5 mL tube and another centrifugation at

was performed for 5 min at RT. Original juice was useddetermination whereas 1 in 100 and 1 in 20 dilutionsup with nanopure water for sugars and citrate analyses,

ately iadded (NaOHmixturple forconcena posit

Preassays(AnonyUV/VISAustrawas strespecand ex

2.5. M

TheeratedwebsitEncycl(http:/severageneraally remetab

2.6. St

Unlusing ent meleast sof variin Supbetweemain danalys

3. Res

3.1. Mdifferenorganic

GCunknoLR) widiffereponenbased ripenintion vaPC2) wexpressied sugar dunknorsed in ice cold water. Nanopure water (10 mL) wasthe pH adjusted to 45 with 5 M sodium hydroxideother 3.5 mL of nanopure water was added before thes ltered through cheesecloth to give the original sam-tarch assay. For each assay, a standard solution of knownon was prepared and run together with the samples asontrol.

samples above were used in respective enzymaticrding to the Methods of Enzymatic Food Analysiss, 1984). All absorbances were determined using actrophotometer SP 8001 (AdeLab Scientic, Thebarton,t a wavelength of 340 nm. As the absorbance changeometric to the metabolite levels, the concentration ofmetabolite can be measured using the given formulaed as g FW L1 (Anonymous, 1984).

lic pathways

tabolic pathways (Fig. 4) were manually gen-sed on available information from the SolCyc(http://solcyc.solgenomics.net/) and the Kyotodia of Genes and Genome (KEGG) websitew.genome.jp/kegg/), and then cross-checked wither cited published reviews/papers. Heatmaps weresing the open-source R program (Version 3.0), manu-

red according to ripening stages and inserted into theathways.

cal analysis

therwise stated, all statistical analyses were performedtat 14 (Hemel Hempstead, UK). Signicantly differ-between ripening stages were determined using thecant difference (LSD) at P < 0.05 of one-way analysis

(ANOVA). Normalised values for GCMS metabolitesentary Table S1 were compared using t-tests (P < 0.05)e G stage and the other two stages (BR1 and LR). Theminative data analysis used was principal componentA) (the open-source R program, Version 3.0).

lite proling using GCMS analysis identiedn metabolism of amino acids, amines, sugars ands

analysis identied 99 metabolites (including 38 in capsicum fruit at different ripening stages (G, BR1,4 of these metabolites being present in signicantlyounts in one or more stages (Table 1). Principal com-

lysis (PCA) revealed that the samples are differentiatedeir metabolite prole representing the three differentges (Fig. 1A; Supplementary Fig. S1A) and the correla-es explained by the two principal components (PC1 and34% and 16%, respectively. The patterns of metaboliteacross the ripening stages were not necessarily clas-their functional groups (amino acids, amines, sugars,atives, organic acids, sterols, fatty acids, unclassied orTable 1).


Table 1The GCMS analysis of three capsicum ripening stages (Green, G, Breaker red 1, BR1; Light red, LR) has identied several metabolites which can be classied into amino acidsand amines, sugar and sugar derivatives, organic acids, sterols and fatty acids as well as others including unknowns. Metabolites detected are expressed as relative responseratio per mg fresh weight of ve biological replicates. Signicantly different metabolites between the stages of ripening (P < 0.05) were calculated based on respective leastsignicant difference (LSD) for each metabolite and are denoted by different letters at each mean.

Metabolites ratio mg1

ites (r1)

Amino acids nd fat-Aminob opherAlanine sterol-Alanine steroAsparagin noate Aspartate ecanoEthanolam oate Glutamate canoaGlutamineGlycine -1,4-lIsoleucine ethyPhenylala horatePutrescine Pyroglutam wn 1 Serine ThreonineValine

Sugars and s3-Deoxy-a-TrehaloDigalactosErythronaFructose Fructose-6GalactinolGalactonaGalactosylGentobiosGluconateGlucose Glucose-6Glycerate Glycerate-Glycerol-3Glycolate Myo-inosiMyo-inosiRafnose Ribonate Sucrose Xylose

Organic acidCitrate DehydroaMalate Malonate Oxalate Quinate ShikimateSuccinate ThreonateThreonate

3.1.1. Metaderivatives

There wysis. The trebe groupedeither relatpyroglutamand remainmetabolitessignicantlacid (GABA)stage. Morestages whilcantly drop(relative response)

G BR1 LR LSD Metabolratio mg

and amines Sterols autyric acid (GABA) 435.683ab 470.127b 306.024a 147.6 -Toc

321.342b 116.666a 179.427a 113.1 -Sito8.225a 10.396a 26.051b 2.2 Campe

e 1168.465a 2510.533b 2875.141b 1102.9 Hepta247.215c 156.107b 38.821a 49.8 Hexad

ine 74.357b 40.758a 36.321a 19.7 Nonan 306.950b 199.338a 253.377ab 72.6 Octade

289.566ab 379.211b 197.221a 170.3 Others200.541b 113.025a 63.769a 69.8 Butyro

127.700a 113.662a 104.888a 41.0 Monomnine 23.470a 42.404a 88.549b 32.4 Phosp

375.841b 78.610a 12.654a 103.9 Uracilate 3838.107a 3052.015a 2453.935a 1425.5 Unkno1035.061ab 1270.077b 712.327a 435.2 Unknown 2 180.142a 264.664b 277.970b 67.0 Unknown 3

253.199a 313.936a 327.081a 244.2 Unknown 4 ugar derivatives Unknown 5 rabino-hexarate 85.253a 133.271b 158.411c 20.3 Unknown 6 se 15.506a 13.224a 14.486a 5.7 Unknown 7 ylglycerol 81.653a 54.960a 54.767a 30.2 Unknown 8 te 13.093b 9.482a 11.251ab 3.6 Unknown 9

7729.978a 9981.993ab 10,690.146b 2608.2 Unknown 10-phosphate 21.572a 24.548a 9.098a 24.8 Unknown 11

2537.050b 194.899a 173.141a 1054.5 Unknown 12te 26.501a 19.190a 46.144b 15.3 Unknown 13glycerol 465.316b 619.723c 250.771a 121.6 Unknown 14e 22.904a 36.645b 54.080c 13.5 Unknown 15

52.552a 40.610a 33.517a 34.0 Unknown 164592.985a 6740.437ab 8154.820b 2280.4 Unknown 17

-phosphate 12.188a 17.166b 16.339b 3.3 Unknown 1839.072b 34.243b 22.955a 6.3 Unknown 19

3-phosphate 21.627b 5.948a 3.269a 7.9 Unknown 20-phosphate 19.833a 24.394a 25.942a 9.4 Unknown 21

19.015a 19.508a 17.685a 3.9 Unknown 22tol 2337.751a 3870.643b 4126.038b 995.0 Unknown 23tol-2-phosphate 53.450b 51.644b 38.915a 10.6 Unknown 24

15.369a 28.907a 9.497a 41.5 Unknown 2513.340b 5.065a 12.385b 4.6 Unknown 2629.221a 272.804b 90.107a 69.7 Unknown 2740.229a 111.628b 120.956b 17.5 Unknown 28

s Unknown 291663.470a 5067.340b 8218.310c 1033.4 Unknown 30

scorbate dimer 57.612a 79.255a 653.248b 74.9 Unknown 31649.571b 163.733a 127.516a 309.9 Unknown 328.435a 3.299a 27.933a 28.2 Unknown 3338.566ab 32.645a 52.902b 18.5 Unknown 344201.731c 2921.085b 1638.808a 1265.8 Unknown 35

178.043b 230.004c 111.094a 29.0 Unknown 3620.464a 29.280b 37.763c 4.7 Unknown 37

987.478b 1489.346c 223.305a 355.3 Unknown 38-1,4-lactone 15.058b 32.839c 2.920a 5.4

bolism of amino acids, amines, sugars and sugar

ere 15 amino acids and one amine detected in the anal-nd of these metabolites across G, BR1 and LR stages can

into seven different classes. Some metabolites wereively constant between the stages (isoleucine, valine,ate) or increased signicantly from G to BR1 stagesed high at LR stage (asparagine, threonine). Additional

such as phenylalanine and -alanine only increasedy at the LR stage whereas glutamine, -aminobutyric

and serine peaked at BR1 before they dropped at the LRover, aspartate continually decreased across all ripeninge alanine, glycine, ethanolamine and putrescine signi-ped at BR1 and remained low at the LR stage. Glutamate

also decreathe LR stage

Out of 2seven comthree ripeerol, fructophosphate,and 3-deoxripening wthe BR1 stag(glycerate-3decreased dincreased aconsistent aelative response G BR1 LR LSD

ty acidsol 5.508a 9.595a 35.179b 8.2

22.817a 165.052a 13.831a 199.0l 12.221c 8.325b 5.286a 2.3

12.196a 10.431a 11.782a 3.2ate (palmitate) 201.949a 192.273a 195.651a 43.1

11.100a 9.596a 10.080a 2.8te (stearate) 204.223a 186.621a 192.892a 32.8

actam 26.020b 24.481b 18.600a 4.4lphosphate 21.036b 16.178ab 12.226a 7.6

5098.320a 5164.940a 7694.819b 1103.05.632a 5.234a 4.935a 2.923.879c 12.743b 1.086a 9.3

1561.584a 1509.460a 1662.800a 307.43.744a 2.550a 3.157a 1.6112.114a 109.196a 119.359a 23.197.808a 52.139a 183.020a 175.738.003a 36.100a 38.787a 7.471.903a 50.401a 50.379a 23.0137.013a 132.621a 143.055a 29.7220.489a 211.377a 424.001a 332.0

107.464a 97.574a 109.840a 19.6 118.676a 119.583a 132.038a 36.6 9.663a 9.252a 10.746a 1.5 121.214b 167.444c 1.763a 34.3 39.745a 38.822a 37.588a 16.8 38.313a 38.982a 45.993a 16.0 88.827a 80.742a 88.572a 18.5 2568.070ab 2293.337a 3248.806b 813.2 44.488b 42.148b 14.408a 10.9 337.392b 371.228b 17.076a 83.5

5.893a 4.698a 2.186a 6.0 28.507b 31.976b 15.120a 8.1 16.335a 18.278ab 19.971b 3.2 81.881a 114.659b 68.263a 19.7 9.217b 15.391c 1.728a 2.2 53.966a 97.880b 41.435a 16.6 5.500c 2.870b 0.075a 1.6 127.088a 142.626ab 156.602b 28.8 13.728b 15.943b 7.284a 3.2 28.657b 65.983c 6.931a 16.2

92.485a 112.194a 708.109b 88.4 484.417a 814.200b 1133.660c 101.1 330.032a 404.155a 507.779a 233.5 466.819a 601.308a 813.522b 161.1 13.300a 14.525a 17.795b 2.9 7.647a 8.079a 11.664b 2.0 155.723a 214.684b 298.095c 44.8 27.530a 55.118b 74.068c 12.6 134.002a 187.696a 177.779a 60.2

sed signicantly at BR1 but slightly increased again at.2 sugars and their derivatives identied by GCMS,pounds were not signicantly different among thening stages namely -trehalose, digalactosylglyc-se-6-phosphate (fructose-6-P), gluconate, glycerol-3-

glycolate and rafnose. Fructose, glucose, gentobiosey-arabino-hexarate consistently increased throughouthereas sucrose and galactosylglycerol peaked duringe only. Furthermore, galactinol, glycerate-3-phosphate-P), erythronate and ribonate contents signicantlyuring the BR1 stage but only the latter two metabolitesgain during the LR stage. Three other metabolites werecross the rst two ripening stages before they increased


Fig. 1. Principetry (GCMS) pspectrometry of Green (G, s(LR, reverse trexplained by tgeted analysesG, Breaker (B, dDeep Red (DRrespectively foreplicate.

(galactonatphosphate)(glucose-6-ing BR1 bef

3.1.2. MetaMetabol

throughoutSeveral com1,4-lactoneit increaseddecreased adroascorbatmalonate w

There wby GCMSdecreased cantly incrsignicant sitosterol) o

heptanoate, hexadecanoate (palmitate), nonanoate and octade-canoate (stearate)] detected.

Metabolites of interest for quantitative analysistabolntaryandpenictedg, wctropdrasted bylyceeta

andalysiyl-mpone

al., 2 the olitesc acid3.1.3. Me

plemethe G vest riundeteripeninor spemost followfold), gother mlactoneThe anadenosas com(Lin etdue tometaborganial component analysis (PCA) of gas chromatographymass spectrom-roling (A) and targeted analyses using liquid chromatographymass

and enzymatic analyses (B). (A) For GCMS, ve biological replicatesquare symbols), Breaker Red 1 (BR1, round symbols) and Light Rediangle symbols) stages were analysed and the correlation varianceshe PC1 and PC2 components are 34% and 16%, respectively. (B) For tar-, three biological replicates of six stages of ripening were analysed;iamond symbols), BR1, Breaker Red 2 (BR2, triangle symbols), LR and

, pentagon symbols) and the correlation variances are 45% and 19%,r PC1 and PC2 components. Each symbol represents a single biological

e) or decreased signicantly (glycerate, myo-inositol-2- during the LR stage, respectively. Glucose-6-phosphateP), myo-inositol and xylose increased signicantly dur-ore remaining constant thereafter.

bolism of organic acids, sterols and fatty acidsites such as citrate and succinate continually increased

ripening whereas quinate consistently decreased.pounds including shikimate, threonate and threonate-

peaked during BR1 while oxalate dropped at BR1 before again at the LR stage. Meanwhile, malate signicantlyt BR1 and remained constant thereafter. The dehy-e dimer only increased signicantly during LR whereasas not signicantly different throughout ripening.ere three sterols and ve fatty acids identied. Sterols such as campesterol was consistentlyduring ripening and -tocopherol was only signi-eased at the LR stage compared to G and BR1. Nochanges were observed in the other sterol (-r fatty acids [9,12-(Z,Z)-octadecadienoate (linoleate),

3.2. Targete

Six staggated usingLCMS andidentied 3non-standaEnzymatic aand citrate)(Fig. 3B) dulites, 26 wripening. Eferentiationmiddle of DR) (Fig. 1explained b

3.2.1. Aminusing LCM

Out of 2detected usAsparagineing. Glutam(B and/or BRthe DR stagestage beforeacids werethey increaing the lateincreased inthen remaionly signiwhile glyciDR stage. Olysine, phedifferent in

The levealso differeproline wea slight inin the lateing but onites that had more than a two-fold difference (Sup- Table S1) and were statistically signicant between

BR1 stages (to explain the differences in posthar-ng behaviour of the two stages) (Table 1) or were

in all stages but known to be important in fruitere chosen for targeted analysis using either LCMShotometric enzyme-based assays. Galactinol had theic change between G and BR1 stages (12.9-fold),

sucrose (9.3-fold), putrescine (4.8-fold), malate (4.0-rate-3-P (3.6-fold) and citrate (3.1-fold). Meanwhile,bolites such as xylose, alanine, ribonate, threonate-1,4-

unknown 29 had differences between 2.2 and 2.8-fold.s did not detect cysteine, methionine, spermidine and S-ethionine (SAM) which have been previously describednts of the polyamine and ethylene synthesis pathways009; Mattoo et al., 2010; Pech et al., 2012). However,limitation of available methods to conrm all of these

of interest, only the amino acids, amines, sugars ands were measured in the subsequent analyses.

d metabolomics for metabolites of interest

es of ripening (G, B, BR1, BR2, LR, DR) were investi- targeted and quantitative metabolomics employing

spectrophotometric-based enzymatic analyses. LCMS3 metabolites that can be classied as amino acids,rd amino acids, polyamines as well as others (Fig. 2).nalysis was also used to measure organic acids (malate

(Fig. 3A), starch and sugars (glucose, fructose, sucrose)ring capsicum ripening. Out of these detected metabo-ere signicantly different in one or more stages ofxamination using PCA also revealed that there is dif-

according to ripening stages especially among G, B,ripening (BR1 and BR2) and full red stages (LR andB, Supplementary Fig. S1B). The correlation variancesy the PC1 and PC2 were 45% and 19%, respectively.

o acids, polyamines and other metabolites prolingS0 known standard amino acids, 19 were successfullying LCMS (only tryptophan was undetected) (Fig. 2A)., histidine and threonine increased throughout ripen-ine and serine peaked at the middle stages of ripening) although the latter compound increased again during. Moreover, alanine decreased signicantly during the B

it consistently increased thereafter. Three other amino consistent in the rst few stages of ripening beforesed (glutamate and proline) or decreased (leucine) dur-r stages (LR and/or DR). Both cysteine and methionine

the rst few ripening stages up to the BR2 stage andned constant thereafter. Furthermore, isoleucine wascantly higher at B compared to the LR and DR stagesne was only signicantly lower at B compared to thether identied amino acids such as arginine, aspartate,nylalanine, tyrosine and valine were not signicantly

any of the six analysed ripening stages.ls of a few detectable non-standard amino acids werential during ripening (Fig. 2B). -Alanine and hydroxy-re signicantly lower during the B stage, after whichcrease was detected during BR1 before decreasingr stages of ripening. GABA uctuated during ripen-ly LR stage was signicantly lower compared to

24

W.M

. A

izat et

al. /

Postharvest Biology

and Technology

89 (2014)

1931

Fig. 2. Liquid chromatographymass spectrometry (LCMS) analysis of six capsicum ripening stages (G, Green; B, Breaker; BR1, Breaker red 1; BR2, Breaker red 2; LR, Light red; DR, Deep red) identied several metaboliteswhich can be categorised into amino acids (A), non-standard amino acids (B) and polyamines (C) as well as other compounds (D). Bar graphs are coloured with increasing intensity to indicate ripening progress. Metabolites areexpressed as g mg1 fresh weight of three biological replicates (SE) except for -alanine which have only two replicates at both LR and DR stages. Signicantly different metabolites across ripening stages were determinedusing respective least signicant difference (P < 0.05) for each metabolite (Supplementary Table S2) as indicated by different letters on each bar. nd, not detected. (For interpretation of the references to colour in this gure legend,the reader is referred to the web version of this article.)


Fig. 3. SpectroBreaker red 2;of three biologmetabolite (Suto the web ver

the G and in the rsLR and DRand homosand B.

Four polBoth putresduring the Bout ripeninDR. Meanwthe early stB stages fowere reducrespectively

There wacetyl-5-OHripening stnot detecteand serotostages, respin taurine, tryptamineand DR.photometric analysis of selected organic acids (A), starch and sugars (B) using six stages LR, Light red; DR, Deep red). Bar graphs are coloured with increasing intensity to indicaical replicates (SE). Signicantly different metabolites across ripening stages were detepplementary Table S2) as indicated by different letters on each bar. (For interpretation osion of this article.)

BR2 stages. Meanwhile, citrulline remained constantt few stages before signicantly decreasing during. No signicant changes were observed in ornithineerine levels but homoserine was undetectable in G

yamines were detected in the LCMS analysis (Fig. 2C).cine and spermidine content was signicantly reduced-BR stages but only putrescine remained low through-g whereas spermidine increased again during LR andhile, agmantine and cadaverine were not detectable inages of ripening (G stage for agmantine and both G andr cadaverine) although the levels of both metabolitesed from B or BR1 towards the later stages of ripening,.ere four other metabolites detected (taurine, N--tryptamine, serotonin and tryptamine) across severalages (Fig. 2D). However, all four metabolites wered during the G stage, while N-acetyl-5-OH-tryptaminenin were also not detected during the BR2 and Bectively. Overall, there were no signicant changesserotonin and tryptamine. However, N-acetyl-5-OH-

was signicantly higher in B and BR1 compared to LR

3.2.2. EnzymCitrate s

signicantlthereafter (and fructosonly signiacross the fexplain thesignicantlthroughout

4. Discussi

Capsicumnormally obut not whPham, 2007to screen mdifferentialtargeted anto quantifystages (G, Bthese two a of capsicum ripening (G, Green; B, Breaker; BR1, Breaker red 1; BR2,te ripening progress. Metabolites are expressed as g L1 fresh weightrmined using respective least signicant difference (P < 0.05) for eachf the references to colour in this gure legend, the reader is referred

atic analyses of organic acids, sugars and starchignicantly increased during ripening whereas malatey decreased during the B stage before remaining lowFig. 3A). Meanwhile, monosaccharides such as glucosee gradually increased during ripening whereas sucrosecantly increased during G to B and remained constantollowing stages (Fig. 3B). Starch was also measured to

increase in these simple sugars and was found to bey reduced during B before remaining consistently low

the rest of the ripening process.

on

has a unique ripening behaviour where it only ripensff the plant when harvested at B and onward stagesen harvested at the G stage (Krajayklang et al., 2000;). Metabolite proling (GCMS) was initially employedetabolites from G, BR1 and LR stages in order to identifyly expressed molecules during ripening. The followingalyses (LCMS and enzymatic analysis) were then used

several classes of metabolites in additional ripening, BR1, BR2, LR, DR). The patterns of several metabolites innalyses including monosaccharides (glucose, fructose),


organic acids (malate, citrate), an amine (putrescine) and aminoacids (glutamine, isoleucine, serine, valine) were consistent amongG, BR1 and LR stages which highlight the reproducibility and consis-tency between these analyses and fruit harvesting. For the purposeof this studgeted metalatter analyand quantit

The usepresent stubroader covpared to thmethod (Os81 unique cof which 65pathways (or more ripalso revealediscriminating consideand the mementary Figstudy: the mthe changes

4.1. Sugarscould inuecapsicum rip

Glycolyserate precuenergy comthat glycolybased on tand their d(approximathat starching. Starch cultivars dulel with redet al., 2012cose, fructoincrease, paof translocaOther sugaring capsicu1992; Nava2008; Florelar ripeningand an incrfruit such a2006; Gaut2008; ZahaLycaon et al(Fortes et al2004; Fait cation is fruit, regardfurther invetration affesucrose and(Dumville aby inuencmechanism

Anothermetabolism

analysis, several metabolites closely related to its metabolismexhibited some level of alteration. Galactose can be degradedto galactosylglycerol and galactonate which were increased byapproximately 1.8 and 1.3-fold when comparing G with BR1 and

pectvia Us suc

maystach

ripeen G alactecursed bting ose pere l) espetabing aue etg aleakdetab

peni moirectays. Te-6-Ples ed gcosestarcose-ne aneninantl

is fur stach aaldeh

Calvet al

to ao mte, warr hen

ted and hrkers

redsionte (Polysolitessynthce, Pds ahaewsisedages ive cays. Hs tyrbetwy, where there are results from both proling and tar-bolomics, unless otherwise stated, the results from thesis are discussed due to having denser sampling stagesative results.

of both proling and targeted metabolomics in thisdy was able to generate a more comprehensive anderage of metabolites during capsicum ripening com-e previous report which has only used the GCMSorio et al., 2012). Indeed, our study has identied at leastompounds (excluding unknowns) from both analyses

of them can be mapped into several major biochemicalFig. 4; only statistically signicant metabolites in oneening stages are represented by heatmaps). The PCAd that the metabolites detected in both analyses wereed into different clusters according to ripeness, indicat-rable differentiation of metabolites at different stagestabolic shifts during capsicum ripening (Fig. 1, Supple-. S1). Two main observations can be described from thisodication of sugars and downstream compounds, and

of metabolites related to the ethylene pathway.

and their derivatives exhibit dramatic changes thatnce glycolysis and carbon metabolism duringening

is is a process of breaking down carbohydrates to gen-rsors for various metabolic pathways while producingpounds such as ATP and NADH. Our results suggestedsis is altered dramatically during capsicum ripening

he change in several identied carbohydrates/sugarserivatives. For example, starch decreased signicantlytely 1.8-fold) during G to later stages (Fig. 3B), implying

degradation has taken place during capsicum ripen-degradation has also been observed in other capsicumring ripening (Hubbard and Pharr, 1992), in paral-uced expression of starch biosynthetic genes (Osorio). Concomitantly, simple sugars such as sucrose, glu-se and gentiobiose were increased (Fig. 4). The sugarrticularly sucrose, in capsicum fruit might be as a resulttion from photosynthesising tissues (Yamaki, 2010).s, particularly glucose and fructose, also increased dur-m ripening (Nielsen et al., 1991; Hubbard and Pharr,rro et al., 2006; Matsufuji et al., 2007; Bernardo et al.,s et al., 2009; Serrano et al., 2010), indicating a simi-

event across different cultivars. A reduction in starchease in sugars has also been observed in climacterics tomato (Roessner-Tunali et al., 2003; Carrari et al.,ier et al., 2008; Osorio et al., 2012), mango (Simo et al.,rah et al., 2013) and banana (Agopian et al., 2011; Fils-., 2011), as well as non-climacteric fruit including grape., 2011; Dai et al., 2013) and strawberry (Souleyre et al.,et al., 2008; Zhang et al., 2011). Carbohydrate modi-therefore a general consequence of ripening in mostless of climacteric or non-climacteric nature. However,stigation is required to conrm if altering sugar concen-cts ripening, even though sugar application, especially

gentiobiose, on strawberry (Jia et al., 2011) and tomatond Fry, 2003) respectively, promoted ripening possiblying the induction of phytohormones and/or signallings (Field, 2009).

potential carbohydrate modication is galactose. Although galactose was not detected in our

LR, restose is such aearlierto the duringbetweUDP-gas a princreassuggesstachyelsewhinositotwo msignallties (Xripeninwall brthese mwith ri

Thelites dpathwglucosmolecuincreasple, gluby the in gluchistidithe ripsignicwhichin lateucts suGlycering the(Farr knownand twribonaway (Fstage wassocia2010) bioma

Theconverpyruvain glycmetabas bioinstanalkaloivon ScsyntheBR1 stble actpathwsuch aferent ively (Table 1). Another metabolic pathway for galac-DP-galactose which can then be synthesised into sugarsrose and stachyose. The increase in sucrose as discussed

be contributed by this metabolic pathway as the routeyose biosynthesis through galactinol may not be activening. Galactinol was signicantly reduced by 14-foldand later stages (BR1 and LR) which may preserve moreose for sucrose synthesis. Myo-inositol which is usedor for UDP-galactose to galactinol conversion was alsoy 2-fold at the same period of ripening (Table 1), furtherthat the myo-inositol may not have been utilised in theathway. An almost similar pattern was also observed

in capsicum for both metabolites (galactinol and myo-ecially at the G to BR period (Osorio et al., 2012). These

olites and their derivatives have been associated withnd stress-related events due to their antioxidant abili-

al., 2007; Valluru and Van den Ende, 2011). Given thatso involves oxidation processes, especially during cellown and textural softening (Fry et al., 2001; Fry, 2003),olites may well coordinate the stress signals associated

ng and hence, merit further research.dication of carbohydrates can also affect metabo-ly involved in glycolysis and hence other associatedhree glycolysis compounds were detected by GCMS;, fructose-6-P and glycerate-3-P (Table 1) and theseexhibited considerable changes that could suggestlycolytic activity during capsicum ripening. For exam--6-P increased in later stages which can be contributedh breakdown and sugar accumulation. Such an increase6-P may also inuence the level of its products such asd fructose-6-P. Histidine gradually accumulated acrossg period (Fig. 2A). Meanwhile, fructose-6-P was noty different between ripening stages and glycerate-3-Prther downstream in the pathway (Fig. 4) was reducedges (BR1 and LR), indicating that intermediate prod-s glyceraldehyde-3-P may be used in other pathways.yde-3-P is a key metabolite in various pathways includ-in cycle and monoterpene and carotenoid biosynthesis., 2010; Taniguchi and Miyake, 2012). Carotenoids areccumulate during capsicum ripening (Ha et al., 2007)etabolites measured in our analysis, -tocopherol andhich are generated in parallel with the carotenoid path-et al., 2010) were also increased signicantly at the LR

compared to the BR1 stage. Carotenoids are mainlywith colour development (Ha et al., 2007; Farr et al.,ence can be further analysed for their use as potential

of fruit ripening.uction of glycerate-3-P may also be caused by further

towards other downstream metabolites. PhosphoenolEP), pyruvate and acetyl-CoA are the key metabolitesis that follow after glycerate-3-P, sequentially. These

can be utilised in various metabolic pathways suchesis of secondary metabolites and amino acids. ForEP is a precursor for the biosynthesis of terpenoids,nd phenylpropanoids (Casati et al., 1999; Kruger anden, 2003; Taiz and Zeiger, 2010). Shikimate which is

early in these pathways increased 1.3-fold from G tobefore decreasing at the LR stage, which implies possi-onversion of PEP towards shikimate and correspondingowever, amino acids generated along these pathways,osine and phenylalanine, were not signicantly dif-een ripening stages (Fig. 2A). This could indicate that


Fig. 4. A meta(LCMS) and edetailed in Maof ripening (Gronly signicanonly signicanMore than oneaccumulation a high metabobold. GABA, glyceraldehydmyo-inositol-2article.)

these aminophenylalanimportant f2005; Sutohquinate (Stebolic map for non-climacteric capsicum ripening. Data from gas chromatographymassnzymatic spectrophotometry analyses were represented by heatmaps corresponding toterials and Methods). Three stages of ripening (Green, Breaker Red 1 and Light Red) were ueen, Breaker, Breaker Red 1, Breaker Red 2, Light Red and Deep Red) were used for LCMtly different metabolites in one or more ripening stages were shown. Where there are ret metabolites from the latter two analyses were included. Any heatmaps for metabolites

arrow in a pathway represents multiple biochemical reactions to produce a subsequenfor respective analyses (1 to 1 scale for GCMS and 2 to 2 scale for LCMS and spectrophlite level during capsicum ripening. Major biochemical pathways/cycles were enclosed in-aminobutyric acid; glucose-6-P, glucose-6-phosphate; fructose-6-P, fructose-6-phosphe-3-phosphate; glycerate-1,3-diP, glycerate-1,3-diphosphate; glycerate-3-P, glycerate-3--phosphate; SAM, S-adenosyl-methionine. (For interpretation of the references to colour

acids are used in other pathways as well. For instance,ine can be used for caffeoyl-CoA synthesis which isor capsaicinoid and capsinoid generation (Stewart et al.,

et al., 2006). One of the precursors in this pathway,wart et al., 2005), was signicantly reduced by roughly

2-fold betwstudies havduring capsand its relaripening (Si spectrometry (GCMS), liquid chromatographymass spectrometry their mean levels during capsicum ripening stages (constructed assed in GCMS analysis (n = 5 biological replicates) whereas six stages

S and enzymatic analyses (n = 3 biological replicates). Heatmaps frompeat metabolites from GCMS, LCMS as well as enzymatic analyses,

which were undetectable in any ripening stages were also removed.t metabolite. The heatmap scale represents the degree of metaboliteotometic analyses). Red indicates a low level whereas green indicates

squares with dotted lines and major functional groups are shown inate; fructose-1,6-biP, fructose-1,6-biphosphate; glyceraldehyde-3-P,phosphate; glycerate-2-P, glycerate-2-phosphate; myo-inositol-2-P,

in this gure legend, the reader is referred to the web version of this

een G and later stages (BR1 and LR). Indeed, somee documented the progressive increase in capsaicinoidsicum ripening (Barrera et al., 2008; Siddiqui et al., 2013)tionship with the development of pungency duringddiqui et al., 2013).


The last two metabolites in the glycolysis pathway are pyru-vate and acetyl-CoA which can be supplied to several biosyntheticpathways. Pyruvate, for instance is a precursor for alanine biosyn-thesis which can then be synthesised to valine and leucine. The uxof the pathalanine to vall six ripenduring G tothe same pbranches ofTCA cycle aZeiger, 2010heptanoatedifferent bacetyl-CoA eral detectechanged thactivated dsuccinate aincreased gthere was uCitrate has 2012) as wOsorio et aity in plantreduced froA proteomimalate dehetate, was suggesting contributedthe TCA cycet al., 2006;similarly deserved ripewith the reet al., 2012starch and min various pand conseqaccumulati2009) as wproperties accumulateend of ripeble effect oet al., 2011stand the roredox regulhelp to expof ethylene

Acetyl-Ccan be furthduring ripewhich are mtate can alswhich is im

4.2. Metabowere differe

Several methionineing ripeninB-BR stagesine which a

increase in serine and subsequently cysteine can be contributed bythe glycerate-3-P metabolism during glycolysis as discussed ear-lier and again signifying glycolysis role during capsicum ripening.The increase in cysteine may inuence the downstream methio-

enera pat

et a thate speine siblewherhioniweremat010)eningen tone primacn. Me

may ino

en sh0122012rison

remred tethioyleneine

ne pted tecreg1

d stacine

synts therest incr

beforly beosynMSnvolve an

funce waf its dropgrad

indics folso dyamincludattoo

(La and2010haveh ovecarb

polyway might be favouring alanine to leucine rather thanaline as valine was not statistically different betweening stages, whereas alanine was signicantly reduced

B stages concomitantly with the increase of leucine ateriod. Meanwhile, acetyl-CoA can be used for different

biochemical pathways such as fatty acid metabolism,s well as ethylene and polyamine pathways (Taiz and). Fatty acids detected in the GCMS analysis (linoleate,, palmitate, nonanoate, stearate) were not signicantlyetween ripening which possibly indicates that mostwas supplied to the other two pathways. Indeed, sev-d metabolites in these pathways were substantially

roughout ripening. For instance, the TCA cycle may beuring capsicum ripening based on the levels of citrate,nd malate (Table 1 and Fig. 3A). Citrate and succinateradually throughout ripening, particularly citrate wherep to 14-fold difference between the G and DR stages.been shown to accumulate in capsicum (Osorio et al.,ell as tomato ripening (Roessner-Tunali et al., 2003;l., 2012) and has been linked to high glycolysis activ-

cells (Fait et al., 2008). On the other hand, malate wasm G to later ripening stages by about 2-fold (Fig. 3A).c analysis on capsicum ripening stages revealed thatydrogenase (MDH), which catalyses malate to oxaloac-up-regulated during the BR1 stage (Aizat et al., 2013)that the malate decrease during capsicum ripening was

by its metabolism to the subsequent intermediate inle. Malate levels in other fruit such as tomato (Carrari

Osorio et al., 2012) and grape (Dai et al., 2013) were alsocreased during ripening, potentially indicating a con-ning event. Malate decrease was positively correlatedduced expression of starch biosynthetic genes (Osorio) which is in agreement with the reduction in bothalate presented here. Malate is known to be involved

lant processes which include regulating redox activityuently mediating cellular metabolism, especially starchon (Tiessen et al., 2002; Scheibe, 2004; Sweetman et al.,ell as inuencing some postharvest and/or ripening(Centeno et al., 2011). Tomato mdh knockout mutantsd more malate and higher lycopene content by thening, compared to wild-type fruit, suggesting a possi-n ripening when malate content was altered (Centeno). However, additional research is required to under-le of malate in capsicum ripening but the possibility ofation in a non-climacteric fruit is intriguing as this maylain the physiological changes that occur independent

regulation.oA can also be used as a precursor for aspartate whicher converted to other amino acids. Aspartate decreasedning (Table 1). Concurrently, asparagine and threonineetabolised from aspartate also increased (Fig. 4). Aspar-o be utilised in a pathway that generates methionineportant for ethylene and polyamine biosyntheses.

lites related methionine and polyamine metabolismntial during capsicum ripening

metabolites detected in our analyses indicated that and polyamine metabolism might be activated dur-g. For example, methionine was up-regulated during

of capsicum ripening. Cysteine is synthesised from ser-lso signicantly increased from G to B-BR stages. The

nine gsimilar(Osoriomeansof thestathionrespon2006) in metcripts both toet al., 2ing ripbetweethylenon-clductiowhichdant amhas beet al., 2et al., compaysis tocompaonly m

Ethpolyamethyleconverually d17 g mfull reputresmidinesuggesing. Intdid nostagespossibsine biand LCways iargininnot beas therlevel ocantly also a whichpatternwere a

Poltions iand Mtomato(MalikSingh, 2012) ThrougSAM dtent oftion as well. Interestingly, methionine displayed veryterns of peaking at the B-BR stages in other capsicuml., 2012) and climacteric tomato (Katz et al., 2006). This

the regulation of methionine may be conserved in bothcies, despite having different ripening behaviour. Cys-gamma synthase (CGS) is the rst committed enzyme

for cysteine conversion towards methionine (Katz et al.,eas methionine sulphoxide reductase (MSR) is involvedne recycling (Alba et al., 2005). Both CGS and MSR trans-

found to be accumulated particularly at the B stage ofo (Alba et al., 2005; Katz et al., 2006) and capsicum (Lee, and this may underlie the increase in methionine dur-. Although methionine may be regulated quite similarlymato and capsicum ripening, its level and the followingecursors such as SAM and ACC may be limiting in theteric capsicum which causes the lack of ethylene pro-thionine was not detected during our GCMS analysisindicate its low level and it is also one of the lower abun-

acids detected using LCMS (Fig. 2A). Furthermore, ACCown to be lower in capsicum (Pretel et al., 1995; Tan

) compared to tomato (Bulens et al., 2011; Van de Poel) during respective ripening. However, a more direct

between these two species is needed in a single anal-ove any experimental variation. Osorio et al. (2012) hashe metabolites of both capsicum and tomato, howevernine in capsicum was reported and not tomato.

precursors such as SAM can also be used in thepathway which may further deprive them from theathway. SAM is rstly decarboxylated before beingo spermidine by utilising putrescine. Putrescine grad-ased from approximately 100 g mg1 in G to aroundin BR1 before further reduction to 4 g mg1 in theges (LR and DR) (Fig. 3 C). The dramatic decline inwas also accompanied by the up-regulation of sper-thase during the BR1 stage (Aizat et al., 2013) whiche activation of this polyamine pathway during ripen-tingly, spermidine which is the product of this reactionease as expected, but instead declined during the B-BRe increasing again during later stages. Spermidine could

almost immediately used up for spermine and hypu-thesis but both compounds were not detected in GCMS

analyses. Putrescine can also be recycled through path-ing -alanine, glutamate, proline, ornithine, citrulline,d agmantine. Interestingly, this recycling pathway maytional especially during late ripening stages (BR2-DR)s no increase in putrescine during this period and theprecursors, ornithine and agmantine, did not signi-

to indicate this was the case. Furthermore, there wasual increase in proline and glutamate during BR2-DRates they were not utilised to make ornithine. Similarr proline and glutamate during late capsicum ripeningocumented elsewhere (Osorio et al., 2012).nes are known to have multiple roles in cellular func-ing stress tolerance, senescence and ripening (Handa, 2010). External applications of amines on fruit such asw et al., 1991), peach (Torrigiani et al., 2012), mango

Singh, 2006), plum (Serrano et al., 2003; Khan and), apple (Kramer et al., 1991) and kiwi (Jhalegar et al.,

been shown to prolong ripening and/or fruit shelf life.erexpression of key enzymes in the pathway such asoxylase and spermidine synthase, the endogenous con-amines was increased in tomato and shown to extend


the ripening period and reduce postharvest symptoms, respectively(Mehta et al., 2002; Nambeesan et al., 2010). In capsicum ripen-ing, as shown here, the reduction of polyamines especially at theG to B-BR stages as well as an inability to recycle putrescine atlater ripeniregulation. sicum culti2001) and iincreased (2006), indipound in dihas been ptowards thea small amonon-climacin various cet al., 1986;1999; Phamthe B-BR stsensitivity tene applica(Fox et al., were no sigKrajayklangB stage rip(Krajayklandifferences stages. Diffeof polyaminneeds to betor gene, Ninduced in p2012). Howdown-reguet al., 2007)receptor levels, as well treatments

5. Conclus

The use oof sugars, aprimary mestate metabux througity assays acorrelate thripening stto identify further expars, malate non-climacon determiof exogenowell as meene receptoripening.

Acknowled

We wousicum seedScholarshipplatforms A

Infrastructure Strategy (NCRIS) with co-investment from the Vic-torian State Government and The University of Melbourne.

dix A. Supplementary data

plemonli1.00

nces

, R.G.D., Cordges inarch t, A., Orecep.M., Aicum r cand

Paytoannontiple p954ous, 1.A., Trieshy f07.

J.A., H. Physur Amo, A., Mn of ts) in ., Lop009. Sning a., Walng ma480.n, B.A

McCotitati

I., Van. Protenzym

Giovalopm., Baxforovagrateds thatetabo., DrinP-ma, D.C.,hausarrari

abolisharve., Loevealy (Vitix, C., Geome e, J.C., . PlantHanh

Aharoorks , Sanace on rt, M.,007. lopmTang, annonmics ., 2002. Biol. ng stages may have a major consequence for ripeningPutrescine was also signicantly reduced in other cap-vars during ripening (Serrano et al., 1995; Yahia et al.,n contrast, the level of putrescine in tomato graduallyYahia et al., 2001; Carrari et al., 2006; Tassoni et al.,cating possible differences in regulation of this com-fferent ripening behaviour. The decline in polyaminesroposed to increase the sensitivity of plant tissues

ethylene hormone (Yahia et al., 2001) such that onlyunt of ethylene may be needed to induce ripening in

teric capsicum. A minute level of ethylene productionapsicum has been documented during ripening (Gross

Biles et al., 1993; Pretel et al., 1995; Villavicencio et al.,, 2007) which suggests ethylene may signicantly affectages where the polyamines would be limited and theowards ethylene is increased. Indeed, exogenous ethyl-tion towards B-BR fruit in capsicum quickens ripening2005) and yet when applied during the G stage, therenicant effects on ripening (Knavel and Kemp, 1973;

et al., 2000). Given that capsicum harvested at theens normally but not when harvested at the G stageg et al., 2000; Pham, 2007), we suggest that there arein sensitivity towards ethylene in different ripeningrent sensitivities may be caused by the different levelses or different ethylene receptor levels or both which

conrmed in future experiments. An ethylene recep-r and three EIL1-like ethylene signalling genes wereepper during the B stage (Lee et al., 2010; Osorio et al.,ever, ethylene receptors such as Nr are also known to belated despite the increase in Nr transcript levels (Kevany. Further studies to isolate and characterise the ethyleneels in capsicum, both at the transcript and protein lev-as to determine capsicum sensitivity towards ethyleneare needed to verify this notion.

ion

f metabolomics in this study has revealed modicationmino acids, organic acids, polyamines and some othertabolites during capsicum ripening. Although steady-olomics alone cannot directly determine the actual

h biochemical pathways, which requires enzyme activ-nd isotope labelling experiments, we were still able toe changes of several corresponding metabolites acrossages. This suggests the applicability of metabolomicsspecic pathways and/or metabolites of interest forerimentation. In this case, the signicant change of sug-and putrescine between the G and B/BR stages duringteric ripening is of interest. Future work will also focusning the sensitivity of capsicum using different levelsus ethylene and 1-methylcyclopropene treatments asasuring the abundance of ethylene precursors, ethyl-rs and ethylene signalling transcripts during capsicum

gements

ld like to thank Monsanto Australia for providing cap-s. WMA was supported by an Adelaide Graduate Fee

while Metabolomics Australia is funded through Bio-ustralia Pty Ltd., a National Collaborative Research

Appen

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Metabolomics of capsicum ripening reveals modification of the ethylene related-pathway and carbon metabolism1 Introduction2 Materials and methods2.1 Plant materials2.2 GCMS2.3 LCMS2.4 Enzymatic analyses2.5 Metabolic pathways2.6 Statistical analysis

3 Results3.1 Metabolite profiling using GCMS analysis identified differences in metabolism of amino acids, amines, sugars and orga...3.1.1 Metabolism of amino acids, amines, sugars and sugar derivatives3.1.2 Metabolism of organic acids, sterols and fatty acids3.1.3 Metabolites of interest for quantitative analysis

3.2 Targeted metabolomics for metabolites of interest3.2.1 Amino acids, polyamines and other metabolites profiling using LCMS3.2.2 Enzymatic analyses of organic acids, sugars and starch

4 Discussion4.1 Sugars and their derivatives exhibit dramatic changes that could influence glycolysis and carbon metabolism during cap...4.2 Metabolites related methionine and polyamine metabolism were differential during capsicum ripening

5 ConclusionAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data

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