Postharvest Biology and Technology 89 (2014) 1931

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Postharvest Biology and Technology

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Metabolomics of capsicum ripening reveals modication of the ethylene related-pathway and carbon metabolism
Wan M. Aizat a , Daniel A. Dias b , James C.R. Stangoulis c , Jason A. Able a , Ute Roessner b,d , Amanda J. Able a,
a

School of Agriculture, Food and Wine, The University of Adelaide, Waite Research Institute, Glen Osmond, SA 5064, Australia Metabolomics Australia, School of Botany, The University of Melbourne, Victoria, Australia School of Biological Science, Flinders University, Bedford Park, SA 5042, Australia d Australian Centre for Plant Functional Genomics, The University of Melbourne, Victoria, Australia
b c

a r t i c l e

i n f o

a b s t r a c t
Capsicum (Capsicum annuum L. cv. Aries) is a non-climacteric bell-pepper fruit, exhibiting limited ethylene and respiration levels during ripening. In contrast to climacteric fruit, such as tomato which is largely dependent upon ethylene to ripen, the regulation of non-climacteric ripening is still inadequately understood. A metabolomics approach was used to identify differentially abundant compounds between ripening stages with the aim of elucidating metabolic pathways involved in the regulation of nonclimacteric ripening. Metabolite proling using gas chromatographymass spectrometry (GCMS) was initially employed to screen potential metabolite differences among three ripening stages (Green, Breaker Red 1 and Light Red). Targeted analyses using liquid chromatographymass spectrometry (LCMS) or enzymatic assays were subsequently employed to characterise selected metabolites in more ripening stages. Starch, sugars and their derivatives were signicantly modied during ripening which may affect the abundance of some glycolysis intermediates and consequently other metabolic pathways involving amino acids, colour and pungency precursors, and tricarboxylic acid (TCA) cycle intermediates. Furthermore, metabolites closely related to ethylene production such as cysteine and methionine gradually increased between the ripening stages, whereas putrescine signicantly decreased during ripening, suggesting 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 of metabolites which are involved in various biochemical pathways and highlights the overall metabolic shifts during non-climacteric capsicum ripening. 2013 Elsevier B.V. All rights reserved.

Article history: Received 6 September 2013 Accepted 12 November 2013 Keywords: Capsicum annuum Fruit GCMS LCMS Non-climacteric Pepper

1. Introduction Fruit are generally classied as being either climacteric or non-climacteric based on their ethylene and respiration levels during ripening. Climacteric fruit such as banana, apple and tomato exhibit not only ethylene and respiration bursts during ripening but can also be stimulated strongly by ethylene to ripen (Cara and Giovannoni, 2008; Bapat et al., 2010). In contrast, non-climacteric fruit such as strawberry, grape and capsicum, are characterised by

Corresponding author at: PMB1 Glen Osmond, SA 5064, Australia. Tel.: +61 8 8313 7245; fax: +61 8 8313 7109. E-mail addresses: wan.wankamaruddin@adelaide.edu.au (W.M. Aizat), ddias@unimelb.edu.au (D.A. Dias), james.stangoulis@inders.edu.au (J.C.R. Stangoulis), jason.able@adelaide.edu.au (J.A. Able), u.roessner@unimelb.edu.au (U. Roessner), amanda.able@adelaide.edu.au (A.J. Able). 0925-5214/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.11.004

their limited production of ethylene and carbon dioxide (Paran and van der Knaap, 2007; Pech et al., 2008). Although a lot is known about the regulation of climacteric fruit ripening, especially the chief role of ethylene, the control of ripening in non-climacteric fruit as well as the absence of autocatalytic ethylene production is still not well understood (Pech et al., 2008). Capsicum has become an increasingly important fruit system to study the non-climacteric fruit ripening process because its genome has conserved sequence with the model fruit, tomato (Livingstone et al., 1999), thus allowing better comparisons with climacteric ripening. Another benet for using capsicum is that it also has different ripening abilities when harvested at different ripening stages, with fruit harvested during the Green (G) stage not ripening normally compared with those harvested at Breaker (B) or beyond (Krajayklang et al., 2000; Pham, 2007). Unknown ripening factors may be differentially expressed during the different stages to induce capsicum ripening. Therefore, further in-depth analysis is required to identify these factors, especially using comparisonbased approaches.

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Several comparison-based approaches such as transcriptomics, proteomics and metabolomics have been used to study fruit ripening, particularly climacteric tomato (Roessner-Tunali et al., 2003; Fei et al., 2004; Alba et al., 2005; Carrari et al., 2006; Rocco et al., 2006; Faurobert et al., 2007; Moco et al., 2007; Kok et al., 2008; Rohrmann et al., 2011; Qin et al., 2012) and a few non-climacteric fruit including strawberry (Aharoni and OConnell, 2002; Fait et al., 2008; Bianco et al., 2009; Ponce-Valadez et al., 2009; Zhang et al., 2011) and grapes (Deytieux et al., 2007; Giribaldi et al., 2007; Grimplet et al., 2007; Martnez-Esteso et al., 2011; Dai et al., 2013). In capsicum ripening, a recent proteomics study has revealed an upregulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase enzyme, which is important for ethylene production (Aizat et al., 2013). Furthermore, transcriptomic analysis has revealed other elements in the ethylene pathway including ethylene receptors and signalling transcripts also being increased during capsicum ripening (Lee et al., 2010; Osorio et al., 2012). Although this may suggest that non-climacteric capsicum ripening shares a common regulatory mechanism with climacteric ripening, further investigation using metabolomics may reveal other potential factors involved in capsicum ripening. Metabolomics aims to measure all metabolites within a biological system using both comprehensive, qualitative and quantitative approaches, and detects metabolites which can be a substrate or a product of a biochemical pathway and hence is a powerful tool to gauge the functional and cellular regulation in any given species (Fiehn, 2002; Roessner and Bowne, 2009). Several techniques have been employed in metabolomics including gas chromatographymass spectrometry (GCMS), liquid chromatographymass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy (Fiehn, 2002; Roessner and Bowne, 2009). A recent study on capsicum ripening has reported metabolite proling using GCMS (Osorio et al., 2012) but complementary and more targeted approaches/techniques such as LCMS and enzymatic assays are required to better quantify and validate the metabolites (Roessner and Bowne, 2009). Therefore, we have initially employed a GCMS-based metabolomics approach to screen potential metabolite differences between three bell pepper capsicum (Capsicum annuum L. cv. Aries) ripening stages [G, Breaker Red 1 (BR1) and Light Red (LR)]. Several metabolites of interest were further investigated using targeted LCMS or enzymatic analyses in six different ripening stages [G, B, BR1, Breaker Red 2 (BR2), LR and Deep Red (DR)]. Differences in metabolites between capsicum ripening stages were mainly associated with sugar modication, possibly affecting glycolysis and carbon metabolism, as well as ethylene-related pathways. The implication of these metabolites in non-climacteric ripening regulation is discussed in comparison to climacteric ripening.

2.2. GCMS Approximately 30 mg fresh weight (FW) of homogenised capsicum was weighed into pre-chilled 2 mL microfuge tubes. Methanol (250 L of 100%), in addition to 10 L of an internal standard (13 C6 -sorbitol in water, 0.2 mg mL1 ) was added. The sample mixtures were vortexed for 30 s and incubated at 70 C for 15 min. The samples were then centrifuged at 13,000 rpm for 10 min at room temperature (RT, 23 C). Supernatants were stored in separate 2 mL tubes and the pellets were mixed with 250 L nanopure water for the second extraction (vortexed for 30 s and then centrifuged at 13,000 rpm for 10 min at RT. Supernatants from this latter extraction were then combined with the rst extracts. Aliquots (50 L) from the combined sample extracts were dried in vacuo for subsequent derivatisation. The dried samples were redissolved in 10 L of 30 mg mL1 methoxyamine hydrochloride in pyridine and derivatised at 37 C for 120 min with mixing at 500 rpm. The samples were then treated for 30 min with 20 L N,Obis-(trimethylsilyl)triuoroacetamide (BSTFA) and 2.5 L retention time standard mixture [0.029% (v/v) n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotriacontane, nhexatriacontane dissolved in pyridine] with mixing at 500 rpm. Each derivatised sample was allowed to rest for 60 min prior to injection. Samples (1 L) were injected using a hot needle technique into a GCMS system comprised of a Gerstel 2.5.2 autosampler, a 7890A Agilent gas chromatograph and a 5975C Agilent quadrupole MS (Agilent, Santa Clara, USA). The MS was adjusted according to the manufacturers recommendations using tris-(peruorobutyl)-amine (CF43). The GC was performed on a 30 m VF-5MS column with 0.2 m lm thickness and a 10 m Integra guard column (Varian, Inc., Victoria, Australia). The injection temperature was set at 250 C, the MS transfer line at 280 C, the ion source adjusted to 250 C and the quadrupole at 150 C. Helium was used as the carrier gas at a ow rate of 0.8 mL min1 . The analysis of samples was performed under the following temperature programme; start at injection 70 C, a hold for 1 min, followed by a 7 C min1 oven temperature, ramp to 325 C and a nal 6 min heating at 325 C. Mass spectra were recorded at 2 scans s1 with an m/z 50600 scanning range. Both chromatograms and mass spectra were evaluated using the AnalyzerPro Deconvolution Program (Spectralworks, UK). Mass spectra of eluting compounds were identied using the commercial mass spectra library NIST 05 (http://www.nist.gov), the public domain mass spectra library of Max-Planck-Institute for Plant Physiology, Golm, Germany (http://csbdb.mpimp-golm.mpg.de/csbdb/dbma/msri.html) and the in-house Metabolomics Australia mass spectral library. All matching mass spectra were additionally veried by determination of the retention time by analysis of authentic standard substances. Resulting relative response ratios (area of analyte divided by area of internal sorbitol standard) per sample FW (mg) for each analysed metabolite were prepared as described in Roessner et al. (2001). The data were also normalised to the G stage in order to compare fold differences between ripening stages (Supplementary Table S1). If a specic metabolite had multiple TMS derivatives, the metabolite with the greater detector response and better peak shape within the dynamic range of the instrument was selected. 2.3. LCMS A targeted LCMS based metabolomics approach was used to cross validate and conrm the abundance of several compounds of interest (particularly amino acids and amines) as well as additional compounds not detected in the GCMS. A preliminary LCMS study using diluted capsicum samples (four-fold dilution with nanopure water) was done but metabolites which had less than 50 g mg1

2. Materials and methods 2.1. Plant materials Capsicum plants (cv. Aries) were grown and prepared as stated in Aizat et al. (2013). There were two independent sets of capsicums, which ripened during OctoberDecember 2010 and FebruaryApril 2011. The rst set consisted of three stages of ripening (G, BR1, LR) with ve biological replicates at each stage used for GCMS metabolomics analysis and the second set, comprised of six ripening stages with three biological replicates each (G, B, BR1, BR2, LR, DR), used for LCMS and enzymatic analyses. Each biological replicate was an individual fruit from one single plant at each particular harvest and homogenised as detailed in Aizat et al. (2013).

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were more difcult to detect (data not shown). Therefore, undiluted original capsicum samples were used for the LCMS analysis. Approximately 30 mg FW of ground capsicum samples were resuspended in 250 L cold MeOH and immediately vortexed. Nanopure water (250 L) and a solution containing internal standards in cold methanol (10 L, 1 mM, 13 C6 15 N-valine, 13 C6 -sorbitol, 2-aminoanthracene) were added, vortexed and centrifuged immediately at 13,200 rpm for 10 min. The resulting supernatant was transferred to a new vial. Two stock solutions were also prepared; (a) an amino acid solution containing a standard mix of 25 amino acids in nanopure water 0.1% formic acid, and (b) sulphur-containing compound solution: A 2.5 mM stock containing glutathione and S-adenosylhomocysteine in water with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and 1 mM ascorbate. The two stock solutions were mixed and diluted using volumetric glassware with water containing 10 mM TCEP, 1 mM ascorbate and 0.1% formate to produce the following series of combined standards: 0.1, 0.5, 1, 5, 10, 20, 50, 100 and 150 M. For derivatisation, an aliquot of each standard or sample (10 L) was added to 70 L of borate buffer (200 mM, pH 8.8 at 25 C) containing 10 mM TCEP, 1 mM ascorbic acid and 50 M 2-aminobutyrate. The resulting solution was vortexed before adding 20 L of 6-aminoquinolyl-Nhydrosysuccinimidyl carbamate (AQC) reagent [200 mM dissolved in 100% acetonitrile (ACN)] immediately vortexing (Boughton et al., 2011). The samples were heated with shaking at 55 C for 10 min then centrifuged (13,000 rpm at RT) and transferred to HPLC vials containing inserts (Agilent, springless glass inserts 250 L). An Agilent 1200 LC-system coupled to an Agilent 6420 Electrospray Ionisation-Triple Quadrupole-MS was used for quantication experiments. Injection volumes used for the samples or standards were 0.52.0 L. Ions were monitored in the positive mode using a Dynamic Multiple Reaction Monitoring (DMRM) method optimised for each analyte. The source, collision energies and fragmentor voltages were optimised for each analyte by infusing a derivatised standard with LC eluent. The following source conditions were used: sheath gas temperature 315 C, gas ow 10 L min1 , nebuliser pressure 45 psi and capillary voltage 3800 V. For the chromatography, an Agilent Zorbax Eclipse XDB-C18 Rapid Resolution HT 2.1 50 mm, 1.8 m column was used with a ow rate of 300 L min1 , maintained at 30 C, resulting in operating pressures below 400 bar with a 19 min run time as described in Boughton et al. (2011). A gradient LC method was used with mobile phases comprised of (A) water 0.1% formic acid and (B) acetonitrile 0.1% formic acid (such that at 0.0 and 2.0 min, the % of B was 1 and then increased to 15 and 30% at 9.0 and 14.0 min, respectively, followed by a reduction to 1% at 14.1 and 19.0 min). These conditions provided suitable chromatographic separation of modied amino acids and although co-elution was observed for some of the species, this could be overcome by the mass-selective capabilities of the mass spectrometer using MRM. Agilent MassHunter Quantitative Analysis Software, Version 4.0 was used to quantify the levels of amino acids. 2.4. Enzymatic analyses Metabolites of interest not detected in the LCMS (glucose, fructose, sucrose, starch, malate and citrate) were determined using spectrophotometric enzyme-based analyses. Three replicates of the six stages of ripening (second set) were used in these assays. For sugars, malate and citrate analyses, 1.0 g ground samples were thawed at RT in 1.5 mL tubes, centrifuged at 13,000 rpm for 10 min at RT to obtain clear fruit juices. The supernatant was transferred into a new 1.5 mL tube and another centrifugation at 13,000 rpm was performed for 5 min at RT. Original juice was used for malate determination whereas 1 in 100 and 1 in 20 dilutions were made up with nanopure water for sugars and citrate analyses,

respectively. Samples for starch determination were prepared by adding 1 mL hydrochloric acid (HCl) and 4 mL dimethylsulphoxide (DMSO) in 50 mL tubes with 1.0 g ground samples. The mixtures were vortexed for 5 s, incubated at 60 C for 30 min and immediately immersed in ice cold water. Nanopure water (10 mL) was added and the pH adjusted to 45 with 5 M sodium hydroxide (NaOH). Another 3.5 mL of nanopure water was added before the mixture was ltered through cheesecloth to give the original sample for the starch assay. For each assay, a standard solution of known concentration was prepared and run together with the samples as a positive control. Prepared samples above were used in respective enzymatic assays according to the Methods of Enzymatic Food Analysis (Anonymous, 1984). All absorbances were determined using a UV/VIS Spectrophotometer SP 8001 (AdeLab Scientic, Thebarton, Australia) at a wavelength of 340 nm. As the absorbance change was stoichiometric to the metabolite levels, the concentration of respective metabolite can be measured using the given formula and expressed as g FW L1 (Anonymous, 1984).

2.5. Metabolic pathways The metabolic pathways (Fig. 4) were manually generated based on available information from the SolCyc website (http://solcyc.solgenomics.net/) and the Kyoto Encyclopaedia of Genes and Genome (KEGG) website (http://www.genome.jp/kegg/), and then cross-checked with several other cited published reviews/papers. Heatmaps were generated using the open-source R program (Version 3.0), manually reordered according to ripening stages and inserted into the metabolic pathways.

2.6. Statistical analysis Unless otherwise stated, all statistical analyses were performed using Genstat 14 (Hemel Hempstead, UK). Signicantly different means between ripening stages were determined using the least signicant difference (LSD) at P < 0.05 of one-way analysis of variance (ANOVA). Normalised values for GCMS metabolites in Supplementary Table S1 were compared using t-tests (P < 0.05) between the G stage and the other two stages (BR1 and LR). The main discriminative data analysis used was principal component analysis (PCA) (the open-source R program, Version 3.0).

3. Results 3.1. Metabolite proling using GCMS analysis identied differences in metabolism of amino acids, amines, sugars and organic acids GCMS analysis identied 99 metabolites (including 38 unknowns) in capsicum fruit at different ripening stages (G, BR1, LR) with 64 of these metabolites being present in signicantly different amounts in one or more stages (Table 1). Principal component analysis (PCA) revealed that the samples are differentiated based on their metabolite prole representing the three different ripening stages (Fig. 1A; Supplementary Fig. S1A) and the correlation variances explained by the two principal components (PC1 and PC2) were 34% and 16%, respectively. The patterns of metabolite expression across the ripening stages were not necessarily classied into their functional groups (amino acids, amines, sugars, sugar derivatives, organic acids, sterols, fatty acids, unclassied or unknown) (Table 1).

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Table 1 The 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 acids and amines, sugar and sugar derivatives, organic acids, sterols and fatty acids as well as others including unknowns. Metabolites detected are expressed as relative response ratio per mg fresh weight of ve biological replicates. Signicantly different metabolites between the stages of ripening (P < 0.05) were calculated based on respective least signicant difference (LSD) for each metabolite and are denoted by different letters at each mean. Metabolites (relative response ratio mg1 ) Amino acids and amines -Aminobutyric acid (GABA) Alanine -Alanine Asparagine Aspartate Ethanolamine Glutamate Glutamine Glycine Isoleucine Phenylalanine Putrescine Pyroglutamate Serine Threonine Valine Sugars and sugar derivatives 3-Deoxy-arabino-hexarate -Trehalose Digalactosylglycerol Erythronate Fructose Fructose-6-phosphate Galactinol Galactonate Galactosylglycerol Gentobiose Gluconate Glucose Glucose-6-phosphate Glycerate Glycerate-3-phosphate Glycerol-3-phosphate Glycolate Myo-inositol Myo-inositol-2-phosphate Rafnose Ribonate Sucrose Xylose Organic acids Citrate Dehydroascorbate dimer Malate Malonate Oxalate Quinate Shikimate Succinate Threonate Threonate-1,4-lactone G BR1 LR LSD Metabolites (relative response ratio mg1 ) Sterols and fatty acids -Tocopherol -Sitosterol Campesterol Heptanoate Hexadecanoate (palmitate) Nonanoate Octadecanoate (stearate) Others Butyro-1,4-lactam Monomethylphosphate Phosphorate Uracil Unknown 1 Unknown 2 Unknown 3 Unknown 4 Unknown 5 Unknown 6 Unknown 7 Unknown 8 Unknown 9 Unknown 10 Unknown 11 Unknown 12 Unknown 13 Unknown 14 Unknown 15 Unknown 16 Unknown 17 Unknown 18 Unknown 19 Unknown 20 Unknown 21 Unknown 22 Unknown 23 Unknown 24 Unknown 25 Unknown 26 Unknown 27 Unknown 28 Unknown 29 Unknown 30 Unknown 31 Unknown 32 Unknown 33 Unknown 34 Unknown 35 Unknown 36 Unknown 37 Unknown 38 G BR1 LR LSD

435.683ab 321.342b 8.225a 1168.465a 247.215c 74.357b 306.950b 289.566ab 200.541b 127.700a 23.470a 375.841b 3838.107a 1035.061ab 180.142a 253.199a 85.253a 15.506a 81.653a 13.093b 7729.978a 21.572a 2537.050b 26.501a 465.316b 22.904a 52.552a 4592.985a 12.188a 39.072b 21.627b 19.833a 19.015a 2337.751a 53.450b 15.369a 13.340b 29.221a 40.229a 1663.470a 57.612a 649.571b 8.435a 38.566ab 4201.731c 178.043b 20.464a 987.478b 15.058b

470.127b 116.666a 10.396a 2510.533b 156.107b 40.758a 199.338a 379.211b 113.025a 113.662a 42.404a 78.610a 3052.015a 1270.077b 264.664b 313.936a 133.271b 13.224a 54.960a 9.482a 9981.993ab 24.548a 194.899a 19.190a 619.723c 36.645b 40.610a 6740.437ab 17.166b 34.243b 5.948a 24.394a 19.508a 3870.643b 51.644b 28.907a 5.065a 272.804b 111.628b 5067.340b 79.255a 163.733a 3.299a 32.645a 2921.085b 230.004c 29.280b 1489.346c 32.839c

306.024a 179.427a 26.051b 2875.141b 38.821a 36.321a 253.377ab 197.221a 63.769a 104.888a 88.549b 12.654a 2453.935a 712.327a 277.970b 327.081a 158.411c 14.486a 54.767a 11.251ab 10,690.146b 9.098a 173.141a 46.144b 250.771a 54.080c 33.517a 8154.820b 16.339b 22.955a 3.269a 25.942a 17.685a 4126.038b 38.915a 9.497a 12.385b 90.107a 120.956b 8218.310c 653.248b 127.516a 27.933a 52.902b 1638.808a 111.094a 37.763c 223.305a 2.920a

147.6 113.1 2.2 1102.9 49.8 19.7 72.6 170.3 69.8 41.0 32.4 103.9 1425.5 435.2 67.0 244.2 20.3 5.7 30.2 3.6 2608.2 24.8 1054.5 15.3 121.6 13.5 34.0 2280.4 3.3 6.3 7.9 9.4 3.9 995.0 10.6 41.5 4.6 69.7 17.5 1033.4 74.9 309.9 28.2 18.5 1265.8 29.0 4.7 355.3 5.4

5.508a 22.817a 12.221c 12.196a 201.949a 11.100a 204.223a 26.020b 21.036b 5098.320a 5.632a 23.879c 1561.584a 3.744a 112.114a 97.808a 38.003a 71.903a 137.013a 220.489a 107.464a 118.676a 9.663a 121.214b 39.745a 38.313a 88.827a 2568.070ab 44.488b 337.392b 5.893a 28.507b 16.335a 81.881a 9.217b 53.966a 5.500c 127.088a 13.728b 28.657b 92.485a 484.417a 330.032a 466.819a 13.300a 7.647a 155.723a 27.530a 134.002a

9.595a 165.052a 8.325b 10.431a 192.273a 9.596a 186.621a 24.481b 16.178ab 5164.940a 5.234a 12.743b 1509.460a 2.550a 109.196a 52.139a 36.100a 50.401a 132.621a 211.377a 97.574a 119.583a 9.252a 167.444c 38.822a 38.982a 80.742a 2293.337a 42.148b 371.228b 4.698a 31.976b 18.278ab 114.659b 15.391c 97.880b 2.870b 142.626ab 15.943b 65.983c 112.194a 814.200b 404.155a 601.308a 14.525a 8.079a 214.684b 55.118b 187.696a

35.179b 13.831a 5.286a 11.782a 195.651a 10.080a 192.892a 18.600a 12.226a 7694.819b 4.935a 1.086a 1662.800a 3.157a 119.359a 183.020a 38.787a 50.379a 143.055a 424.001a 109.840a 132.038a 10.746a 1.763a 37.588a 45.993a 88.572a 3248.806b 14.408a 17.076a 2.186a 15.120a 19.971b 68.263a 1.728a 41.435a 0.075a 156.602b 7.284a 6.931a 708.109b 1133.660c 507.779a 813.522b 17.795b 11.664b 298.095c 74.068c 177.779a

8.2 199.0 2.3 3.2 43.1 2.8 32.8 4.4 7.6 1103.0 2.9 9.3 307.4 1.6 23.1 175.7 7.4 23.0 29.7 332.0 19.6 36.6 1.5 34.3 16.8 16.0 18.5 813.2 10.9 83.5 6.0 8.1 3.2 19.7 2.2 16.6 1.6 28.8 3.2 16.2 88.4 101.1 233.5 161.1 2.9 2.0 44.8 12.6 60.2

3.1.1. Metabolism of amino acids, amines, sugars and sugar derivatives There were 15 amino acids and one amine detected in the analysis. The trend of these metabolites across G, BR1 and LR stages can be grouped into seven different classes. Some metabolites were either relatively constant between the stages (isoleucine, valine, pyroglutamate) or increased signicantly from G to BR1 stages and remained high at LR stage (asparagine, threonine). Additional metabolites such as phenylalanine and -alanine only increased signicantly at the LR stage whereas glutamine, -aminobutyric acid (GABA) and serine peaked at BR1 before they dropped at the LR stage. Moreover, aspartate continually decreased across all ripening stages while alanine, glycine, ethanolamine and putrescine signicantly dropped at BR1 and remained low at the LR stage. Glutamate

also decreased signicantly at BR1 but slightly increased again at the LR stage. Out of 22 sugars and their derivatives identied by GCMS, seven compounds were not signicantly different among the three ripening stages namely -trehalose, digalactosylglycerol, fructose-6-phosphate (fructose-6-P), gluconate, glycerol-3phosphate, glycolate and rafnose. Fructose, glucose, gentobiose and 3-deoxy-arabino-hexarate consistently increased throughout ripening whereas sucrose and galactosylglycerol peaked during the BR1 stage only. Furthermore, galactinol, glycerate-3-phosphate (glycerate-3-P), erythronate and ribonate contents signicantly decreased during the BR1 stage but only the latter two metabolites increased again during the LR stage. Three other metabolites were consistent across the rst two ripening stages before they increased

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heptanoate, hexadecanoate (palmitate), nonanoate and octadecanoate (stearate)] detected. 3.1.3. Metabolites of interest for quantitative analysis Metabolites that had more than a two-fold difference (Supplementary Table S1) and were statistically signicant between the G and BR1 stages (to explain the differences in postharvest ripening behaviour of the two stages) (Table 1) or were undetected in all stages but known to be important in fruit ripening, were chosen for targeted analysis using either LCMS or spectrophotometric enzyme-based assays. Galactinol had the most drastic change between G and BR1 stages (12.9-fold), followed by sucrose (9.3-fold), putrescine (4.8-fold), malate (4.0fold), glycerate-3-P (3.6-fold) and citrate (3.1-fold). Meanwhile, other metabolites such as xylose, alanine, ribonate, threonate-1,4lactone and unknown 29 had differences between 2.2 and 2.8-fold. The analysis did not detect cysteine, methionine, spermidine and Sadenosyl-methionine (SAM) which have been previously described as components of the polyamine and ethylene synthesis pathways (Lin et al., 2009; Mattoo et al., 2010; Pech et al., 2012). However, due to the limitation of available methods to conrm all of these metabolites of interest, only the amino acids, amines, sugars and organic acids were measured in the subsequent analyses. 3.2. Targeted metabolomics for metabolites of interest Six stages of ripening (G, B, BR1, BR2, LR, DR) were investigated using targeted and quantitative metabolomics employing LCMS and spectrophotometric-based enzymatic analyses. LCMS identied 33 metabolites that can be classied as amino acids, non-standard amino acids, polyamines as well as others (Fig. 2). Enzymatic analysis was also used to measure organic acids (malate and citrate) (Fig. 3A), starch and sugars (glucose, fructose, sucrose) (Fig. 3B) during capsicum ripening. Out of these detected metabolites, 26 were signicantly different in one or more stages of ripening. Examination using PCA also revealed that there is differentiation according to ripening stages especially among G, B, middle of ripening (BR1 and BR2) and full red stages (LR and DR) (Fig. 1B, Supplementary Fig. S1B). The correlation variances explained by the PC1 and PC2 were 45% and 19%, respectively. 3.2.1. Amino acids, polyamines and other metabolites proling using LCMS Out of 20 known standard amino acids, 19 were successfully detected using LCMS (only tryptophan was undetected) (Fig. 2A). Asparagine, histidine and threonine increased throughout ripening. Glutamine and serine peaked at the middle stages of ripening (B and/or BR) although the latter compound increased again during the DR stage. Moreover, alanine decreased signicantly during the B stage before it consistently increased thereafter. Three other amino acids were consistent in the rst few stages of ripening before they increased (glutamate and proline) or decreased (leucine) during the later stages (LR and/or DR). Both cysteine and methionine increased in the rst few ripening stages up to the BR2 stage and then remained constant thereafter. Furthermore, isoleucine was only signicantly higher at B compared to the LR and DR stages while glycine was only signicantly lower at B compared to the DR stage. Other identied amino acids such as arginine, aspartate, lysine, phenylalanine, tyrosine and valine were not signicantly different in any of the six analysed ripening stages. The levels of a few detectable non-standard amino acids were also differential during ripening (Fig. 2B). -Alanine and hydroxyproline were signicantly lower during the B stage, after which a slight increase was detected during BR1 before decreasing in the later stages of ripening. GABA uctuated during ripening but only LR stage was signicantly lower compared to

Fig. 1. Principal component analysis (PCA) of gas chromatographymass spectrometry (GCMS) proling (A) and targeted analyses using liquid chromatographymass spectrometry and enzymatic analyses (B). (A) For GCMS, ve biological replicates of Green (G, square symbols), Breaker Red 1 (BR1, round symbols) and Light Red (LR, reverse triangle symbols) stages were analysed and the correlation variances explained by the PC1 and PC2 components are 34% and 16%, respectively. (B) For targeted analyses, three biological replicates of six stages of ripening were analysed; G, Breaker (B, diamond symbols), BR1, Breaker Red 2 (BR2, triangle symbols), LR and Deep Red (DR, pentagon symbols) and the correlation variances are 45% and 19%, respectively for PC1 and PC2 components. Each symbol represents a single biological replicate.

(galactonate) or decreased signicantly (glycerate, myo-inositol-2phosphate) during the LR stage, respectively. Glucose-6-phosphate (glucose-6-P), myo-inositol and xylose increased signicantly during BR1 before remaining constant thereafter.

3.1.2. Metabolism of organic acids, sterols and fatty acids Metabolites such as citrate and succinate continually increased throughout ripening whereas quinate consistently decreased. Several compounds including shikimate, threonate and threonate1,4-lactone peaked during BR1 while oxalate dropped at BR1 before it increased again at the LR stage. Meanwhile, malate signicantly decreased at BR1 and remained constant thereafter. The dehydroascorbate dimer only increased signicantly during LR whereas malonate was not signicantly different throughout ripening. There were three sterols and ve fatty acids identied by GCMS. Sterols such as campesterol was consistently decreased during ripening and -tocopherol was only signicantly increased at the LR stage compared to G and BR1. No signicant changes were observed in the other sterol (sitosterol) or fatty acids [9,12-(Z,Z)-octadecadienoate (linoleate),

24 W.M. Aizat 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 metabolites which 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 are expressed 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 determined using 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.)

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Fig. 3. Spectrophotometric analysis of selected organic acids (A), starch and sugars (B) using six stages of capsicum ripening (G, Green; B, Breaker; BR1, Breaker red 1; BR2, Breaker red 2; LR, Light red; DR, Deep red). Bar graphs are coloured with increasing intensity to indicate ripening progress. Metabolites are expressed as g L1 fresh weight of three biological replicates (SE). Signicantly different metabolites across ripening stages were determined using respective least signicant difference (P < 0.05) for each metabolite (Supplementary Table S2) as indicated by different letters on each bar. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

the G and BR2 stages. Meanwhile, citrulline remained constant in the rst few stages before signicantly decreasing during LR and DR. No signicant changes were observed in ornithine and homoserine levels but homoserine was undetectable in G and B. Four polyamines were detected in the LCMS analysis (Fig. 2C). Both putrescine and spermidine content was signicantly reduced during the B-BR stages but only putrescine remained low throughout ripening whereas spermidine increased again during LR and DR. Meanwhile, agmantine and cadaverine were not detectable in the early stages of ripening (G stage for agmantine and both G and B stages for cadaverine) although the levels of both metabolites were reduced from B or BR1 towards the later stages of ripening, respectively. There were four other metabolites detected (taurine, Nacetyl-5-OH-tryptamine, serotonin and tryptamine) across several ripening stages (Fig. 2D). However, all four metabolites were not detected during the G stage, while N-acetyl-5-OH-tryptamine and serotonin were also not detected during the BR2 and B stages, respectively. Overall, there were no signicant changes in taurine, serotonin and tryptamine. However, N-acetyl-5-OHtryptamine was signicantly higher in B and BR1 compared to LR and DR.

3.2.2. Enzymatic analyses of organic acids, sugars and starch Citrate signicantly increased during ripening whereas malate signicantly decreased during the B stage before remaining low thereafter (Fig. 3A). Meanwhile, monosaccharides such as glucose and fructose gradually increased during ripening whereas sucrose only signicantly increased during G to B and remained constant across the following stages (Fig. 3B). Starch was also measured to explain the increase in these simple sugars and was found to be signicantly reduced during B before remaining consistently low throughout the rest of the ripening process. 4. Discussion Capsicum has a unique ripening behaviour where it only ripens normally off the plant when harvested at B and onward stages but not when harvested at the G stage (Krajayklang et al., 2000; Pham, 2007). Metabolite proling (GCMS) was initially employed to screen metabolites from G, BR1 and LR stages in order to identify differentially expressed molecules during ripening. The following targeted analyses (LCMS and enzymatic analysis) were then used to quantify several classes of metabolites in additional ripening stages (G, B, BR1, BR2, LR, DR). The patterns of several metabolites in these two analyses including monosaccharides (glucose, fructose),

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organic acids (malate, citrate), an amine (putrescine) and amino acids (glutamine, isoleucine, serine, valine) were consistent among G, BR1 and LR stages which highlight the reproducibility and consistency between these analyses and fruit harvesting. For the purpose of this study, where there are results from both proling and targeted metabolomics, unless otherwise stated, the results from the latter analysis are discussed due to having denser sampling stages and quantitative results. The use of both proling and targeted metabolomics in this present study was able to generate a more comprehensive and broader coverage of metabolites during capsicum ripening compared to the previous report which has only used the GCMS method (Osorio et al., 2012). Indeed, our study has identied at least 81 unique compounds (excluding unknowns) from both analyses of which 65 of them can be mapped into several major biochemical pathways (Fig. 4; only statistically signicant metabolites in one or more ripening stages are represented by heatmaps). The PCA also revealed that the metabolites detected in both analyses were discriminated into different clusters according to ripeness, indicating considerable differentiation of metabolites at different stages and the metabolic shifts during capsicum ripening (Fig. 1, Supplementary Fig. S1). Two main observations can be described from this study: the modication of sugars and downstream compounds, and the changes of metabolites related to the ethylene pathway. 4.1. Sugars and their derivatives exhibit dramatic changes that could inuence glycolysis and carbon metabolism during capsicum ripening Glycolysis is a process of breaking down carbohydrates to generate precursors for various metabolic pathways while producing energy compounds such as ATP and NADH. Our results suggested that glycolysis is altered dramatically during capsicum ripening based on the change in several identied carbohydrates/sugars and their derivatives. For example, starch decreased signicantly (approximately 1.8-fold) during G to later stages (Fig. 3B), implying that starch degradation has taken place during capsicum ripening. Starch degradation has also been observed in other capsicum cultivars during ripening (Hubbard and Pharr, 1992), in parallel with reduced expression of starch biosynthetic genes (Osorio et al., 2012). Concomitantly, simple sugars such as sucrose, glucose, fructose and gentiobiose were increased (Fig. 4). The sugar increase, particularly sucrose, in capsicum fruit might be as a result of translocation from photosynthesising tissues (Yamaki, 2010). Other sugars, particularly glucose and fructose, also increased during capsicum ripening (Nielsen et al., 1991; Hubbard and Pharr, 1992; Navarro et al., 2006; Matsufuji et al., 2007; Bernardo et al., 2008; Flores et al., 2009; Serrano et al., 2010), indicating a similar ripening event across different cultivars. A reduction in starch and an increase in sugars has also been observed in climacteric fruit such as tomato (Roessner-Tunali et al., 2003; Carrari et al., 2006; Gautier et al., 2008; Osorio et al., 2012), mango (Simo et al., 2008; Zaharah et al., 2013) and banana (Agopian et al., 2011; FilsLycaon et al., 2011), as well as non-climacteric fruit including grape (Fortes et al., 2011; Dai et al., 2013) and strawberry (Souleyre et al., 2004; Fait et al., 2008; Zhang et al., 2011). Carbohydrate modication is therefore a general consequence of ripening in most fruit, regardless of climacteric or non-climacteric nature. However, further investigation is required to conrm if altering sugar concentration affects ripening, even though sugar application, especially sucrose and gentiobiose, on strawberry (Jia et al., 2011) and tomato (Dumville and Fry, 2003) respectively, promoted ripening possibly by inuencing the induction of phytohormones and/or signalling mechanisms (Field, 2009). Another potential carbohydrate modication is galactose metabolism. Although galactose was not detected in our

analysis, several metabolites closely related to its metabolism exhibited some level of alteration. Galactose can be degraded to galactosylglycerol and galactonate which were increased by approximately 1.8 and 1.3-fold when comparing G with BR1 and LR, respectively (Table 1). Another metabolic pathway for galactose is via UDP-galactose which can then be synthesised into sugars such as sucrose and stachyose. The increase in sucrose as discussed earlier may be contributed by this metabolic pathway as the route to the stachyose biosynthesis through galactinol may not be active during ripening. Galactinol was signicantly reduced by 14-fold between G and later stages (BR1 and LR) which may preserve more UDP-galactose for sucrose synthesis. Myo-inositol which is used as a precursor for UDP-galactose to galactinol conversion was also increased by 2-fold at the same period of ripening (Table 1), further suggesting that the myo-inositol may not have been utilised in the stachyose pathway. An almost similar pattern was also observed elsewhere in capsicum for both metabolites (galactinol and myoinositol) especially at the G to BR period (Osorio et al., 2012). These two metabolites and their derivatives have been associated with signalling and stress-related events due to their antioxidant abilities (Xue et al., 2007; Valluru and Van den Ende, 2011). Given that ripening also involves oxidation processes, especially during cell wall breakdown and textural softening (Fry et al., 2001; Fry, 2003), these metabolites may well coordinate the stress signals associated with ripening and hence, merit further research. The modication of carbohydrates can also affect metabolites directly involved in glycolysis and hence other associated pathways. Three glycolysis compounds were detected by GCMS; glucose-6-P, fructose-6-P and glycerate-3-P (Table 1) and these molecules exhibited considerable changes that could suggest increased glycolytic activity during capsicum ripening. For example, glucose-6-P increased in later stages which can be contributed by the starch breakdown and sugar accumulation. Such an increase in glucose-6-P may also inuence the level of its products such as histidine and fructose-6-P. Histidine gradually accumulated across the ripening period (Fig. 2A). Meanwhile, fructose-6-P was not signicantly different between ripening stages and glycerate-3-P which is further downstream in the pathway (Fig. 4) was reduced in later stages (BR1 and LR), indicating that intermediate products such as glyceraldehyde-3-P may be used in other pathways. Glyceraldehyde-3-P is a key metabolite in various pathways including the Calvin cycle and monoterpene and carotenoid biosynthesis (Farr et al., 2010; Taniguchi and Miyake, 2012). Carotenoids are known to accumulate during capsicum ripening (Ha et al., 2007) and two metabolites measured in our analysis, -tocopherol and ribonate, which are generated in parallel with the carotenoid pathway (Farr et al., 2010) were also increased signicantly at the LR stage when compared to the BR1 stage. Carotenoids are mainly associated with colour development (Ha et al., 2007; Farr et al., 2010) and hence can be further analysed for their use as potential biomarkers of fruit ripening. The reduction of glycerate-3-P may also be caused by further conversion towards other downstream metabolites. Phosphoenol pyruvate (PEP), pyruvate and acetyl-CoA are the key metabolites in glycolysis that follow after glycerate-3-P, sequentially. These metabolites can be utilised in various metabolic pathways such as biosynthesis of secondary metabolites and amino acids. For instance, PEP is a precursor for the biosynthesis of terpenoids, alkaloids and phenylpropanoids (Casati et al., 1999; Kruger and von Schaewen, 2003; Taiz and Zeiger, 2010). Shikimate which is synthesised early in these pathways increased 1.3-fold from G to BR1 stages before decreasing at the LR stage, which implies possible active conversion of PEP towards shikimate and corresponding pathways. However, amino acids generated along these pathways, such as tyrosine and phenylalanine, were not signicantly different between ripening stages (Fig. 2A). This could indicate that

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Fig. 4. A metabolic map for non-climacteric capsicum ripening. Data from gas chromatographymass spectrometry (GCMS), liquid chromatographymass spectrometry (LCMS) and enzymatic spectrophotometry analyses were represented by heatmaps corresponding to their mean levels during capsicum ripening stages (constructed as detailed in Materials and Methods). Three stages of ripening (Green, Breaker Red 1 and Light Red) were used in GCMS analysis (n = 5 biological replicates) whereas six stages of ripening (Green, Breaker, Breaker Red 1, Breaker Red 2, Light Red and Deep Red) were used for LCMS and enzymatic analyses (n = 3 biological replicates). Heatmaps from only signicantly different metabolites in one or more ripening stages were shown. Where there are repeat metabolites from GCMS, LCMS as well as enzymatic analyses, only signicant metabolites from the latter two analyses were included. Any heatmaps for metabolites which were undetectable in any ripening stages were also removed. More than one arrow in a pathway represents multiple biochemical reactions to produce a subsequent metabolite. The heatmap scale represents the degree of metabolite accumulation for respective analyses (1 to 1 scale for GCMS and 2 to 2 scale for LCMS and spectrophotometic analyses). Red indicates a low level whereas green indicates a high metabolite level during capsicum ripening. Major biochemical pathways/cycles were enclosed in squares with dotted lines and major functional groups are shown in bold. GABA, -aminobutyric acid; glucose-6-P, glucose-6-phosphate; fructose-6-P, fructose-6-phosphate; fructose-1,6-biP, fructose-1,6-biphosphate; glyceraldehyde-3-P, glyceraldehyde-3-phosphate; glycerate-1,3-diP, glycerate-1,3-diphosphate; glycerate-3-P, glycerate-3-phosphate; glycerate-2-P, glycerate-2-phosphate; myo-inositol-2-P, myo-inositol-2-phosphate; SAM, S-adenosyl-methionine. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

these amino acids are used in other pathways as well. For instance, phenylalanine can be used for caffeoyl-CoA synthesis which is important for capsaicinoid and capsinoid generation (Stewart et al., 2005; Sutoh et al., 2006). One of the precursors in this pathway, quinate (Stewart et al., 2005), was signicantly reduced by roughly

2-fold between G and later stages (BR1 and LR). Indeed, some studies have documented the progressive increase in capsaicinoids during capsicum ripening (Barrera et al., 2008; Siddiqui et al., 2013) and its relationship with the development of pungency during ripening (Siddiqui et al., 2013).

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The last two metabolites in the glycolysis pathway are pyruvate and acetyl-CoA which can be supplied to several biosynthetic pathways. Pyruvate, for instance is a precursor for alanine biosynthesis which can then be synthesised to valine and leucine. The ux of the pathway might be favouring alanine to leucine rather than alanine to valine as valine was not statistically different between all six ripening stages, whereas alanine was signicantly reduced during G to B stages concomitantly with the increase of leucine at the same period. Meanwhile, acetyl-CoA can be used for different branches of biochemical pathways such as fatty acid metabolism, TCA cycle as well as ethylene and polyamine pathways (Taiz and Zeiger, 2010). Fatty acids detected in the GCMS analysis (linoleate, heptanoate, palmitate, nonanoate, stearate) were not signicantly different between ripening which possibly indicates that most acetyl-CoA was supplied to the other two pathways. Indeed, several detected metabolites in these pathways were substantially changed throughout ripening. For instance, the TCA cycle may be activated during capsicum ripening based on the levels of citrate, succinate and malate (Table 1 and Fig. 3A). Citrate and succinate increased gradually throughout ripening, particularly citrate where there was up to 14-fold difference between the G and DR stages. Citrate has been shown to accumulate in capsicum (Osorio et al., 2012) as well as tomato ripening (Roessner-Tunali et al., 2003; Osorio et al., 2012) and has been linked to high glycolysis activity in plant cells (Fait et al., 2008). On the other hand, malate was reduced from G to later ripening stages by about 2-fold (Fig. 3A). A proteomic analysis on capsicum ripening stages revealed that malate dehydrogenase (MDH), which catalyses malate to oxaloacetate, was up-regulated during the BR1 stage (Aizat et al., 2013) suggesting that the malate decrease during capsicum ripening was contributed by its metabolism to the subsequent intermediate in the TCA cycle. Malate levels in other fruit such as tomato (Carrari et al., 2006; Osorio et al., 2012) and grape (Dai et al., 2013) were also similarly decreased during ripening, potentially indicating a conserved ripening event. Malate decrease was positively correlated with the reduced expression of starch biosynthetic genes (Osorio et al., 2012) which is in agreement with the reduction in both starch and malate presented here. Malate is known to be involved in various plant processes which include regulating redox activity and consequently mediating cellular metabolism, especially starch accumulation (Tiessen et al., 2002; Scheibe, 2004; Sweetman et al., 2009) as well as inuencing some postharvest and/or ripening properties (Centeno et al., 2011). Tomato mdh knockout mutants accumulated more malate and higher lycopene content by the end of ripening, compared to wild-type fruit, suggesting a possible effect on ripening when malate content was altered (Centeno et al., 2011). However, additional research is required to understand the role of malate in capsicum ripening but the possibility of redox regulation in a non-climacteric fruit is intriguing as this may help to explain the physiological changes that occur independent of ethylene regulation. Acetyl-CoA can also be used as a precursor for aspartate which can be further converted to other amino acids. Aspartate decreased during ripening (Table 1). Concurrently, asparagine and threonine which are metabolised from aspartate also increased (Fig. 4). Aspartate can also be utilised in a pathway that generates methionine which is important for ethylene and polyamine biosyntheses. 4.2. Metabolites related methionine and polyamine metabolism were differential during capsicum ripening Several metabolites detected in our analyses indicated that methionine and polyamine metabolism might be activated during ripening. For example, methionine was up-regulated during B-BR stages of capsicum ripening. Cysteine is synthesised from serine which also signicantly increased from G to B-BR stages. The

increase in serine and subsequently cysteine can be contributed by the glycerate-3-P metabolism during glycolysis as discussed earlier and again signifying glycolysis role during capsicum ripening. The increase in cysteine may inuence the downstream methionine generation as well. Interestingly, methionine displayed very similar patterns of peaking at the B-BR stages in other capsicum (Osorio et al., 2012) and climacteric tomato (Katz et al., 2006). This means that the regulation of methionine may be conserved in both of these species, despite having different ripening behaviour. Cystathionine gamma synthase (CGS) is the rst committed enzyme responsible for cysteine conversion towards methionine (Katz et al., 2006) whereas methionine sulphoxide reductase (MSR) is involved in methionine recycling (Alba et al., 2005). Both CGS and MSR transcripts were found to be accumulated particularly at the B stage of both tomato (Alba et al., 2005; Katz et al., 2006) and capsicum (Lee et al., 2010), and this may underlie the increase in methionine during ripening. Although methionine may be regulated quite similarly between tomato and capsicum ripening, its level and the following ethylene precursors such as SAM and ACC may be limiting in the non-climacteric capsicum which causes the lack of ethylene production. Methionine was not detected during our GCMS analysis which may indicate its low level and it is also one of the lower abundant amino acids detected using LCMS (Fig. 2A). Furthermore, ACC has been shown to be lower in capsicum (Pretel et al., 1995; Tan et al., 2012) compared to tomato (Bulens et al., 2011; Van de Poel et al., 2012) during respective ripening. However, a more direct comparison between these two species is needed in a single analysis to remove any experimental variation. Osorio et al. (2012) has compared the metabolites of both capsicum and tomato, however only methionine in capsicum was reported and not tomato. Ethylene precursors such as SAM can also be used in the polyamine pathway which may further deprive them from the ethylene pathway. SAM is rstly decarboxylated before being converted to spermidine by utilising putrescine. Putrescine gradually decreased from approximately 100 g mg1 in G to around 17 g mg1 in BR1 before further reduction to 4 g mg1 in the full red stages (LR and DR) (Fig. 3 C). The dramatic decline in putrescine was also accompanied by the up-regulation of spermidine synthase during the BR1 stage (Aizat et al., 2013) which suggests the activation of this polyamine pathway during ripening. Interestingly, spermidine which is the product of this reaction did not increase as expected, but instead declined during the B-BR stages before increasing again during later stages. Spermidine could possibly be almost immediately used up for spermine and hypusine biosynthesis but both compounds were not detected in GCMS and LCMS analyses. Putrescine can also be recycled through pathways involving -alanine, glutamate, proline, ornithine, citrulline, arginine and agmantine. Interestingly, this recycling pathway may not be functional especially during late ripening stages (BR2-DR) as there was no increase in putrescine during this period and the level of its precursors, ornithine and agmantine, did not signicantly drop to indicate this was the case. Furthermore, there was also a gradual increase in proline and glutamate during BR2-DR which indicates they were not utilised to make ornithine. Similar patterns for proline and glutamate during late capsicum ripening were also documented elsewhere (Osorio et al., 2012). Polyamines are known to have multiple roles in cellular functions including stress tolerance, senescence and ripening (Handa and Mattoo, 2010). External applications of amines on fruit such as tomato (Law et al., 1991), peach (Torrigiani et al., 2012), mango (Malik and Singh, 2006), plum (Serrano et al., 2003; Khan and Singh, 2010), apple (Kramer et al., 1991) and kiwi (Jhalegar et al., 2012) have been shown to prolong ripening and/or fruit shelf life. Through overexpression of key enzymes in the pathway such as SAM decarboxylase and spermidine synthase, the endogenous content of polyamines was increased in tomato and shown to extend

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the ripening period and reduce postharvest symptoms, respectively (Mehta et al., 2002; Nambeesan et al., 2010). In capsicum ripening, as shown here, the reduction of polyamines especially at the G to B-BR stages as well as an inability to recycle putrescine at later ripening stages may have a major consequence for ripening regulation. Putrescine was also signicantly reduced in other capsicum cultivars during ripening (Serrano et al., 1995; Yahia et al., 2001) and in contrast, the level of putrescine in tomato gradually increased (Yahia et al., 2001; Carrari et al., 2006; Tassoni et al., 2006), indicating possible differences in regulation of this compound in different ripening behaviour. The decline in polyamines has been proposed to increase the sensitivity of plant tissues towards the ethylene hormone (Yahia et al., 2001) such that only a small amount of ethylene may be needed to induce ripening in non-climacteric capsicum. A minute level of ethylene production in various capsicum has been documented during ripening (Gross et al., 1986; Biles et al., 1993; Pretel et al., 1995; Villavicencio et al., 1999; Pham, 2007) which suggests ethylene may signicantly affect the B-BR stages where the polyamines would be limited and the sensitivity towards ethylene is increased. Indeed, exogenous ethylene application towards B-BR fruit in capsicum quickens ripening (Fox et al., 2005) and yet when applied during the G stage, there were no signicant effects on ripening (Knavel and Kemp, 1973; Krajayklang et al., 2000). Given that capsicum harvested at the B stage ripens normally but not when harvested at the G stage (Krajayklang et al., 2000; Pham, 2007), we suggest that there are differences in sensitivity towards ethylene in different ripening stages. Different sensitivities may be caused by the different levels of polyamines or different ethylene receptor levels or both which needs to be conrmed in future experiments. An ethylene receptor gene, Nr and three EIL1-like ethylene signalling genes were induced in pepper during the B stage (Lee et al., 2010; Osorio et al., 2012). However, ethylene receptors such as Nr are also known to be down-regulated despite the increase in Nr transcript levels (Kevany et al., 2007). Further studies to isolate and characterise the ethylene receptor levels in capsicum, both at the transcript and protein levels, as well as to determine capsicum sensitivity towards ethylene treatments are needed to verify this notion. 5. Conclusion The use of metabolomics in this study has revealed modication of sugars, amino acids, organic acids, polyamines and some other primary metabolites during capsicum ripening. Although steadystate metabolomics alone cannot directly determine the actual ux through biochemical pathways, which requires enzyme activity assays and isotope labelling experiments, we were still able to correlate the changes of several corresponding metabolites across ripening stages. This suggests the applicability of metabolomics to identify specic pathways and/or metabolites of interest for further experimentation. In this case, the signicant change of sugars, malate and putrescine between the G and B/BR stages during non-climacteric ripening is of interest. Future work will also focus on determining the sensitivity of capsicum using different levels of exogenous ethylene and 1-methylcyclopropene treatments as well as measuring the abundance of ethylene precursors, ethylene receptors and ethylene signalling transcripts during capsicum ripening. Acknowledgements We would like to thank Monsanto Australia for providing capsicum seeds. WMA was supported by an Adelaide Graduate Fee Scholarship while Metabolomics Australia is funded through Bioplatforms Australia Pty Ltd., a National Collaborative Research

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

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.postharvbio. 2013.11.004.

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