Food Chemistry 217 (2017) 610–619

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Food Chemistry

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Analytical Methods

Natural variation in folate levels among tomato (Solanum lycopersicum)accessions

http://dx.doi.org/10.1016/j.foodchem.2016.09.0310308-8146/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail addresses: pravas43@gmail.com (P. Upadhyaya), tyagi.kamal6672@

gmail.com (K. Tyagi), supu.megha@gmail.com (S. Sarma), vajirchem@gmail.com(V. Tamboli), syellamaraju@gmail.com (Y. Sreelakshmi), rameshwar.sharma@gmail.com (R. Sharma).

1 Joint first authors.

Pallawi Upadhyaya 1, Kamal Tyagi 1, Supriya Sarma, Vajir Tamboli, Yellamaraju Sreelakshmi,Rameshwar Sharma ⇑Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

a r t i c l e i n f o

Article history:Received 19 August 2015Received in revised form 2 June 2016Accepted 5 September 2016Available online 7 September 2016

Keywords:FolateSolanum lycopersicumNatural accessionsSingle nucleotide polymorphismEcoTILLING

a b s t r a c t

Folate content was estimated in tomato (Solanum lycopersicum) accessions using microbiological assay(MA) and by LC-MS. The MA revealed that in red-ripe fruits folate levels ranged from 4 to 60 lg/100 gfresh weight. The LC-MS estimation of red-ripe fruits detected three folate forms, 5-CH3-THF, 5-CHO-THF, 5,10-CH+THF and folate levels ranged from 14 to 46 lg/100 g fresh weight. In mature green andred ripe fruit, 5-CH3-THF was the most abundant folate form. Comparison of LC-MS with MA revealed thatMA inaccurately estimates folate levels. The accumulation of folate forms and their distribution variedamong accessions. The single nucleotide polymorphism was examined in the key genes of the folate path-way to understand its linkage with folate levels. Despite the significant variation in folate levels amongtomato accessions, little polymorphism was found in folate biosynthesis genes. Our results indicate thatvariation in folate level is governed by a more complex regulation at cellular homeostasis level.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Folates, water soluble B9 vitamins, play very important role inthe prevention of some cardiovascular diseases, neural tube defects(NTDs), spina bifida and anencephaly in infants, megaloblastic ane-mia, and certain cancers in adults (Lucock, 2000). Humans and ani-mals lack the ability to synthesize folates, consequently solelydepend upon dietary sources to obtain folate (Rébeillé et al.,2006; Scott, Rébeillé, & Fletcher, 2000). In developing countriesdietary deficiency of folate increases the incidences of neural tubedefects in fetal development (Scott, Weir, & Kirke, 1995). The ade-quate daily dietary folate intake during the gestation period isessential to ensure normal growth and development of the fetus.Developed countries like Australia and USA have mandated theaddition of folic acid to wheat flour for bread-making. However,the developing countries do not have food fortification programbecause of the high cost of synthetic folic acid and absence of anindustrial food system.

Plant-based foods are the main dietary sources of folate forhumans and other animals. Among plant-based foods; fruits, nuts,

and vegetables provide about 30% requirement of folate in theAmerican diet (Kader, Perkins-Veazie, & Lester, 2004). The leafyvegetables such as spinach, lettuce, broccoli, asparagus, and fruitssuch as citrus are good source of dietary folate (Kader & Perkins-Veazie, 2004; Delchier, Herbig, Rychlik, & Renard, 2016). The natu-ral forms of folate are also better for intestinal absorption than thesynthetic form. Staple foods consumed in developing countriessuch as wheat, maize, and rice contain very low amount of folatewhich is insufficient to meet folate RDA of 400 lg/day. Consideringthis, there are concerted efforts to biofortify common cereal grainswith folate using transgenic approaches. Rice biofortification wassuccessfully achieved by simultaneous overexpression of two Ara-bidopsis genes involved in the pteridine and para-aminobenzoatebranches of the folate biosynthesis pathway (Storozhenko et al.,2007). The biofortified rice seeds have nearly 100 times higherfolate level than the parental plant and its level was sufficient forrequired RDA for folate (Storozhenko et al., 2007). Similar trans-genic enhancement in folate level was also achieved in tomatoby stimulating biosynthesis of folate in fruits (Díaz de La Garza,Gregory, & Hanson, 2007). However, transgenic tomato and riceare considered as genetically modified (GM) food, which faces con-siderable consumer resistance amid concerns for its safety.

Due to concerns over GM food, there have been efforts to iden-tify and exploit the natural variations of folate content in differentcrop plants. Moreover, linkage of natural variation in folate withgenes/QTLs can be used to increase the folate levels in crops by

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conventional plant breeding. A large scale screen of wheat (175genotypes) revealed that folate level varied from 364 to 774 ng/gdry weight in winter wheat and from 323 to 741 ng/g dry weightin spring wheat. Significantly the durum wheat genotypes showedhighest folate level indicating scope for using these lines for breed-ing wheat genotypes with enriched folate content (Piironen,Edelmann, Kariluoto, & Bed}o, 2008). In spinach, examination of67 accessions showed folate level range from 54 to 173 lg/100 gof fresh weight, identifying potential genotypes that can be usedfor breeding (Shohag et al., 2011).

Notwithstanding above variation in folate content in differentgenotypes, little information is available about the regulation offolate biosynthesis at the genetic level. The folate biosynthesispathway in plants is distributed in three subcellular compart-ments. Pteridine and p-aminobenzoic acid (pABA) moieties aresynthesized in the cytosol and plastids respectively and later con-densed and glutamylated in mitochondria to form tetrahydrofo-late. The key regulatory enzyme for pterin synthesis is GTPcyclohydrolase I (GCHI) (Basset et al., 2002) while pABA is synthe-sized from chorismate using two enzymatic steps catalysed byaminodeoxychorismate synthase (ADCS) (Basset et al., 2004a)and aminodeoxychorismate lyase (ADCL) (Basset et al., 2004b). Inaddition, folate is glutamylated by the action of folylpolyglutamatesynthase (FPGS) (Mehrshahi et al., 2010). It is believed that thelevel of folate is also regulated by removal of glutamate moietyby gamma-glutamyl hydrolases (GGH) (Akhtar et al., 2010;Orsomando et al., 2005). While most genes contributing to folatebiosynthesis in plants have been identified, the identity of genescontrolling folate turnover and transport is not known. These genescould also regulate the folate levels in tissue-specific and alsospecies-specific manners.

Tomato (Solanum lycopersicum) is a plant food which is widelyconsumed in all parts of the world. It is also considered an impor-tant functional food due to enriched levels of bioactive compoundssuch as lycopene and b-carotene. Currently, little information isavailable about natural variation in folate level in tomato. Exami-nation of folate in eleven cultivars of tomato showed levels rangingfrom 6.5 to 28.6 lg/100 g of fresh weight (Iniesta, Perez-Conesa,Garcia-Alonso, Ros, & Periago, 2009). Several folate vitamersaccount for the total folate in tomato, of which 5-methyltetra-hydrofolate (5-CH3-THF) is the main vitamer. The distributionand developmental regulation of different folate vitamers intomato fruit are not known.

In the present study, we analyzed folate level in 160 accessionsof tomato by microbiological assay and 125 accessions by LC-MSmethod. Simultaneously, key genes of the folate biosynthesis andturnover were screened for single nucleotide polymorphism (SNPs)in tomato accessions. In this study, we report that though thefolate level in tomato accessions varies considerably, such a broadrange of variations was not observed in SNPs in key genes regulat-ing the folate level.

2. Materials and methods

2.1. Plant material

Tomato (Solanum lycopersicum L.) accessions were obtainedfrom TGRC (Tomato Genetics Resource Center at University of Cal-ifornia, Davis) (www.tgrc.ucdavis.edu); IIVR (Indian Institute ofVegetable Research, Varanasi, India) (www.iivr.org.in); IIHR(Indian Institute of Horticultural Research, Bengaluru, India(www.iihr.res.in); NBPGR (National Bureau of Plant GeneticResources, New Delhi, India) (www.nbpgr.ernet.in) and Bejo Shee-tal (Bejo Sheetal Seeds Pvt. Ltd., Jalna, India) (www.bejoshee-talseeds.com) (Supplementary Table 1). The detailed information

about the accessions used and their characters can be accessed/searched from the respective website/search portals of TGRC,NBPGR, IIHR, and IIVR.

We grew a population of 391 different accessions from Octoberto February in the year 2011–12 and 2012–13. SupplementaryTable 2 shows the average temperature and humidity for aboveseasons. The fruits from plants grown in 2011–12 were used forthe microbiological assay (MA) and from plants grown in 2012–13 were used for LC-MS estimation of folate. While SNPs wereexamined in all of above accessions using DNA isolated from leaf,only 160 accessions yielded 3 or more red ripe fruits for folate esti-mation using MA. Likewise for LC-MS based folate estimation, 3 ormore replicates were obtained from only 125 accessions and 82accessions for red ripe fruits and mature green fruits respectively.

2.2. Plant growth

All accessions were grown under similar conditions in the openfield at University of Hyderabad, Hyderabad, India. Fifteen seedsfrom each accession after surface sterilization with 4% (v/v) sodiumhypochlorite for 10–12 min and rinsing with tap water were grownin germination trays containing coconut peat (Sri Balaji Agro Ser-vices, Madanapalle, AP, India). After 21 days, seedlings were trans-ferred to open field with drip irrigation. The first and secondflowers from first and second truss (preferably 1st truss) of theplants were tagged, and fruits at mature green (MG) and red ripe(RR) stages were harvested from at least three different plants ofeach accession. The attainment of mature-green stage variedamong the accessions. Fruits at the 28–35 days after pollinationwere harvested for MG stage. The transition from mature-greento red-ripe stages also varied among the accessions, requiring8–15 day duration to attain the red-ripe stage. The fruits after har-vesting were placed on ice in an ice bucket and transferred to thelab. Since the open field and the lab were at a distance of 100 m,only a minimal time (1–2 h) elapsed between harvesting and fruithomogenization. The fruits were homogenized in liquid nitrogenusing homogenizer (IKA, A11 basic, Germany) and the powderwas stored at �80 �C till further use.

2.3. Chemicals and folate standards

The folate standards 5-methyltetrahydrofolate (5-CH3-THF),tetrahydrofolate (THF), 5,10 methenyltetrahydrofolate (5,10-CH+THF), 5-formyltetrahydrofolate (5-CHO-THF) and 5,10-methylenetetrahydrofolate (5,10-CH2THF) were purchased fromSchirck’s Laboratory, Bauma, Switzerland (http://www.schircks.ch/). The purity of above folate standards as per data sheet pro-vided by Schirck’s Laboratory was in the range of 95–99%. Folicacid (FA), ascorbic acid, b-mercaptoethanol, LC-MS grade acetoni-trile and a-amylase (from Bacillus sp., A6814) were obtained fromSigma Aldrich Co. (St. Louis, USA). Milli-Q water (18.2X at 25 �C)was obtained from Millipore water system (Millipore, Bradford,USA). LC-MS grade formic acid (HCOOH) was obtained from FisherScientific (Loughborough, UK). Potassium dihydrogen phosphate,dipotassium hydrogen phosphate, folic acid casei medium(M-543-100G), Protease (from Streptomyces griseus, RM6186) andactivated charcoal were obtained from HiMedia (Mumbai, India).For microbiological assay, Lactobacillus rhamnosus (ATCC 7469)was obtained from MTCC (Microbial Type Culture Collection)Chandigarh (http://mtcc.imtech.res.in/) (ATCC 7469 = MTCC1408). Protease (P5147) and sodium ascorbate were obtainedfrom Sigma Aldrich Co. (St. Louis, USA). Rat plasma was obtainedfrom National Institute of Nutrition (NIN), Hyderabad, India(http://ninindia.org/).

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2.4. Folate standards: preparation and purity correction

Stock solutions of folate standards (1 mg/mL) were prepared in50 mM potassium phosphate solution, pH 4.5 containing 1% (w/v)of ascorbic acid and 0.5% (v/v) of b-mercaptoethanol except FA,which was dissolved in basic pH potassium phosphate buffer.The standard stock solutions were freshly diluted in the extractionsolution to prepare working solutions. The remaining stock solu-tions were flushed with nitrogen gas, and small aliquots werestored at �80 �C. The purity of the folate standards were calculatedusing respective molar absorption coefficients. For spectrophoto-metric measurements, standards were dissolved in 0.01 M phos-phate buffer (100 ng/lL), except 5,10-methenyl THF and folicacid which were dissolved in 0.01 N HCl and 1 N NaOH respec-tively. The molar absorption coefficients for above folate standardswere obtained from Zhang et al. (2003), except for 5,10-MethenylTHF which was from Moldt et al. (2009). The respective purity of5-Methyl THF, 5-Formyl THF, THF, 5,10-Methenyl THF, 5,10-Methylene THF and folic acid were 90, 65, 90, 80, 77, 78%respectively. The standard curve for individual folate vitamerswere plotted after correcting for the purity.

2.5. Enzyme preparation for folate extraction

Protease (2 mg/mL) and a-amylase (20 mg/mL) were dissolvedin Milli-Q water, and aliquots were stored at �20 �C. To removeendogenous folate from rat plasma and a-amylase, 100 mL of ratplasma and a-amylase were mixed with 5 g of activated charcoal.This mixture was incubated on ice for 1 h with intermittent stirringfollowed by centrifugation at 5000g (Sorvall Lynx 6000, ThermoScientific, USA) for 10 min at room temperature. The supernatantwas filtered through a 0.22 lm filter, divided into 1 mL aliquots,and stored at �20 �C. Protease was used without pre-treatmentand was stored in �20 �C.

2.6. Sample extraction procedure for LC-MS

Total folate was extracted following the procedures of Tyagiet al. (2015) from 125 accessions (Supplementary Table 1). Briefly,100 mg homogenized tissue was suspended in 650 lL of extractionsolution (50 mM potassium phosphate, 1% (w/v) ascorbic acid, 0.5%(v/v) b-mercaptoethanol, 1 mM calcium chloride, pH 4.5, flushedwith nitrogen) in a 2 mL Eppendorf tube. The homogenate wasboiled for 10 min and then cooled on ice. Thereafter, 10 lL of a-amylase (20 mg/mL) was added, and tubes were incubated at roomtemperature for 10 min. Following that 2.5 lL protease (2 mg/mL)was added and incubation was carried out at 37 �C for 1 h. The pro-tease activity was terminated by transferring the tubes to boilingwater bath for 5 min and cooling on ice. For deconjugation of folatepolyglutamates to monoglutamates, 100 lL of rat plasma wasadded to each sample and tubes were incubated at 37 �C for 2 h.Enzymatic activity was stopped by transferring the tubes to boilingwater bath for 5 min and cooling on ice followed by centrifugationfor 30 min (14,000g, 4 �C). The supernatant was filtered throughthe 0.22 lm filter (MDI Advanced Micro-devices) and the filtratewas ultra-filtered at 12,000g for 12 min using 10 kDa molecularweight cut-off membrane filter (Pall Corporation, USA) for samplecleanup before LC-MS analysis. The resulting filtrate was trans-ferred to an autosampler vial and 7.5 lL aliquot was directlyinjected on the column.

2.7. Liquid chromatography condition and mass spectrometry settings

For LC-MS, all the parameters used were essentially the same asdescribed earlier by Tyagi et al. (2015). The folate derivatives wereseparated on a reversed phase Luna C18 column (5 lm particle

size, 250 mm � 4.60 mm ID) (Phenomenex, USA) using WatersAcquityTMUPLC system (Milford, USA) running in HPLC mode, cou-pled to a binary pump, an autosampler, and controlled by Xcalibur3.0 software (Thermo Fisher Scientific, San Jose, USA). For massspectrometry, ExactiveTMPlus Orbitrap mass spectrometer (ThermoFisher Scientific, USA) was operated in alternating full scan and allion fragmentation (AIF) mode equipped with positive heatedelectrospray ionization (ESI).

2.8. Folate quantification and recovery analysis

External standards were used for folate quantification. The sen-sitivity was confirmed by evaluating the limit of detection (LOD;calculated as 3.3r/S, where r is the standard deviation and S isthe slope of calibration curve) and limit of quantification (LOQ; cal-culated as 10r/S). Least-square regression analysis was used fordata fitting. After confirmation of individual peak identity on thebasis of m/z and their fragmentation products, quantification wasdone according to the response of the mass detector to the folatestandard. The linearity of each folate standard was evaluated byplotting the peak area at different concentrations and sample con-centrations were calculated from the equation y =mx + c. R2 valuesfor the calibration curves were FA (0.999), THF (0.997), 5,10-CH+THF (0.984), 5-CH3-THF (0.998) and 5-CHO-THF (0.994). Thebuffer blanks were run before first sample run and after every20 sample run to remove any carryover. After 240 runs, theHPLC columns were also cleaned as per the manufacturer’srecommendation.

Endogenous residual folate of trienzyme (rat plasma +a-amylase + protease) was corrected by running blank samplesand subtracting the values from the sample extracts. The sum ofall the folate vitamers was expressed as microgram per 100 g offresh weight. To determine the accuracy of the method, a recoverytest was performed in four randomly chosen tomato accessions byspiking of tomato extract with known amount of individual folatevitamer standards and calculating their final content in the extract.The recovery (R) was calculated as (B � C/A) � 100, where A = peakarea of neat folate standards, B = peak area of spiked extract,C = peak area of extract.

2.9. Folate extraction and estimation through microbiological assay

Folate was estimated in a population of 160 accessions oftomato (Supplementary Table 1). The stock culture of L. casei wasmade in Lactobacillus broth and maintained on agar medium. Manyparameters like aeration, incubation time and inoculum dose wereoptimized to achieve steady bacterial growth. A modified methodof Wilson and Horne (1982) was used to prepare cryoprotectedcells for using as inoculum. The standard curve for bacterial growthwas made against increasing folic acid concentrations. All parame-ters related to the bacterial growth were standardized for 200 lLvolume of microtiter plate wells.

2.10. Extraction method

Folate was extracted from tomato fruit tissue using trienzymeextraction described by Goyer and Navarre (2007) with some mod-ifications. Extraction was carried out in 2 mL wells of a 96 wellplate. Briefly, 100 mg fresh or stored homogenized fruit tissuewas suspended in 1 mL extraction buffer (0.1 M potassiumphosphate pH 7.0, 1% (w/v) ascorbic acid and 0.1% (v/v)b-mercaptoethanol, flushed with nitrogen gas). Plates were trans-ferred to boiling water bath for 10 min and immediately cooledon ice. After addition of 10 lL protease (10 mg/mL), the samplewas incubated at 37 �C for 2 h followed by transferring the platesto boiling water bath for 5 min and immediately cooling on ice.

P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 613

Thereafter, 25 lL a-amylase (20 mg/mL) and 25 lL rat plasma con-jugase was added to the sample and incubated at 37 �C for 3 h afterwhich samples were transferred to boiling water bath for 5 min,immediately cooled on ice and centrifuged for 10 min at 3000g.The clear supernatant was transferred to fresh plates. All stepsafter the first boiling step were carried out in a sterile airflowbench to avoid the need for filtration of extracts. No significant dif-ference in folate content was observed between samples processedwith or without filtration. Since folates are light sensitive theextracts were protected from light to prevent the oxidation offolates during extraction and storage.

2.11. Inoculation and incubation of extracts with bacterial culture

To the sample wells of a microtiter plate containing 100 lLassay medium, 50 lL buffer (50 mM potassium phosphate buffer,0.15% sodium ascorbate (w/v), pH 6.1), 40 lL cryoprotected cells(25 times diluted in 0.9% (w/v) NaCl), 10 lL of plant extract (3.2times diluted) was added. The wells with buffer blank contained100 lL assay medium and 100 lL buffer, while wells with inocu-lum blank contained 100 lL assay medium, 60 lL buffer and40 lL cryoprotected cells (25 times diluted in 0.9% (w/v) NaCl).Plates were incubated static at 37 �C for 18 h and thereafter absor-bance was recorded at 540 nm in a microplate reader.

2.12. SNPs in key genes of folate pathway

Genomic DNA was isolated from tomato accessions followingthe procedures of Sreelakshmi et al. (2010). Five genes of folatebiosynthesis pathway were selected namely GTP cyclohydrolase I(GCH1), aminodeoxychorismate synthase (ADCS), aminodeoxycho-rismate lyase (ADCL1 and ADCL2), folylpolyglutamate synthase(FPGSp and FPGSm), and c-glutamyl hydrolase (GGH1, GGH2, andGGH3). The sequences of above genes and their isoforms wereobtained from SOL GENOMICS NETWORK (solgenomics.net). Aweb-based software tool Codons Optimized to Discover Deleteri-ous Lesions (blocks.fhcrc.org/proweb/coddle) was used to predicta region of the gene where mutation/change(s) would cause themost deleterious effect. The primers for the CODDLE predictedregion were designed using Primer3web version 4.0.0 (bioinfo.ut.ee/primer3). Genes and primer sequences used are listed in Sup-plementary Table 3. The SNP detection was carried out using Eco-TILLING protocol (Mohan et al., 2016) with few modifications. Thefirst step PCR was carried out using 3 pmol unlabeled primers cov-ering the flanking sequence of targeted genomic region in a volumeof 20 lL with DNA of different accessions mixed with that of ArkaVikas in a 1:1 ratio. The PCR reaction carried out in 20 lL volumeconsisted of 5 ng of template DNA, 1X PCR buffer (10 mM Tris,50 mM KCl, 1.5 mM MgCl2, 0.1% (w/v) gelatin, 0.005% (v/v)Tween-20, 0.005% (v/v) NP-40, pH 8.8), 2.5 mM each dNTPs,2.0 mM MgCl2, 0.18 lL Taq polymerase (in-house isolated) and3 pmol each of forward and reverse primers. The cycling conditionsfor amplification were 94 �C-4 min, 35 cycles of 94 �C-20 s, 55 �C-45 s, 72 �C-2 min, 72 �C-10 min and incubation at 12 �C. The firststep PCR product was used as a template for second step PCR reac-tion using a combination of 0.29 pmol (0.015 lM) of unlabeled for-ward primer, 0.42 pmol (0.02 lM) of IRD700 M13 forward primer,0.20 pmol (0.01 lM) of unlabeled reverse primer and 0.50 pmol(0.025 lM) of IRD800 M13 reverse primer. PCR cycling conditionsfor second step were-94 �C-4 min, 3 cycles of 94 �C-20 s, 60 �C-45 swith a decrement of 2.0 �C per cycle, 72 �C-1 min 30 s followed by30 cycles of 94 �C-20 s, 52 �C-45 s, 72 �C-1 min 30 s, 72 �C-10 min.The amplified PCR products were subjected to denaturation andcooling (re-annealing) to generate heteroduplexes between wildtype and natural accession if any. Heteroduplexing conditions were

as follows: 99 �C-10 min, 80 �C-20 s, 70 cycles of 80 �C-7 s with adecrement of 0.3 �C per cycle and held at 4 �C.

The presence of heteroduplex was detected by using amismatch specific endonuclease, CEL I enzyme that cleaves theheteroduplex DNA at the site of mismatch resulting in fragmentedDNA. The mismatch cleavage reaction was performed in a total vol-ume of 45 lL containing 20 lL PCR product, and 25 lL CEL I diges-tion mixture (1X CEL I digestion buffer = 10 mM HEPES buffer pH7.0, 10 mM KCl, 10 mM MgCl2, 0.002% (v/v) Triton X-100 and10 lg/mL BSA) and CEL I enzyme (in-house isolated) at 1:300 dilu-tion (1 lL/300 lL CEL I digestion buffer). The mixture was incu-bated at 45 �C for 15 min and cleavage reaction was stopped byadding 10 lL stop solution (2.5 M NaCl, 75 mM EDTA, pH 8.0 and0.5 mg/mL blue dextran). The DNA was precipitated by additionof 125 lL of cold absolute ethanol and a brief incubation in�80 �C followed by centrifugation at 4500 rpm in a SH-3000 rotorfor 30 min. The DNA pellet was washed with 70% (v/v) ethanol andafter drying at 80 �C, was suspended in 8 lL formamide loadingbuffer consisting of 37% (v/v) deionized formamide, 1 mM EDTAand 0.02% (w/v) bromophenol blue. The PCR products were dena-tured by heating at 94 �C for 2 min and then were incubated onice. The fragmented products were resolved on high resolutiondenaturing PAGE (polyacrylamide gel electrophoresis). About0.5 lL of the sample was electrophoresed in a denaturing 6.5%(w/v) polyacrylamide gel in TBE buffer (89 mM Tris, 89 mM boricacid, 2 mM EDTA, pH 8.3) at 1500 V, 40 mA and 40 V setting onLI-COR 4300 DNA analyzer. The two TIFF images of 700 and 800channels were analyzed in Adobe Photoshop software (AdobeSystems Inc.), and the gel was visually assessed for the presenceof SNPs.

After detection of SNPs in a given accession, genomic DNA fromthat accession was re-amplified and subjected to agarose gel basedmismatch detection assay to reconfirm the presence of SNPs(Sharma, Tyagi, Narasu, Sreelakshmi, & Sharma, 2011). The acces-sions showing identical fragment size on LI-COR gels were groupedas a single haplotype.

2.13. Statistical analysis

A minimum of three biological replicates (nP 3) were used foreach sample. The results are expressed as the mean of all biologicalreplicates in microgram per 100 g fresh weight (FW) tissue.Statistical analysis of data was performed using SigmaPlot 11.0.

3. Results

3.1. LC-MS determination of total folate levels

The folate levels in fruits of 125 tomato accessions(Fig. 1A and B) at red ripe stage ranged from 13.8 to 45.8 lg/100 g FW, whereas at mature green stage it ranged from 12.5 to70.9 lg/100 g FW (Fig. 1C; Supplementary Figs. 1 and 2 showslevels of individual vitamers). The median of total folate level atthe red ripe and the mature green stage was at 25.5 and35 lg/100 g of FW respectively. Based on folate levels, the acces-sions were classified into 4 groups at the red ripe stage and 6groups at the mature green stage. At the red ripe stage, 82 acces-sions were in the range of 20–30 lg/100 g FW followed by 26accessions in the range of 30–40 lg/100 g of FW. Together thesetwo groups consisted of nearly 86% of total accessions. While redripe fruits of 3 accessions were in 40–50 lg/100 g FW group, 14accessions showed less than 20 lg/100 g FW folate. The highestfolate content 45.8 lg/100 g FW was observed in accessionEC498372 at the red ripe stage.

Fig. 2. Distribution of different folate forms in tomato fruits at red ripe and maturegreen stage. The data represented are mean values of folate levels in 125 accessionsfor RR and 82 accessions for MG stage. 5-CH3-THF was the predominant folate formpresent at RR and MG stage�THF was observed only at the mature green stage butnot at the red ripe stage of fruits.

Fig. 1. Total folate composition in fruits of tomato accessions at red ripe (A and B,125 accessions) and mature green (82 accessions) (C) using LC-MS. The legend onleft of the graph represents folate levels in tomato fruits and legend on the right ofthe graph shows tomato accessions falling in different ranges (marked with dottedlines) of folate levels. Data presented are mean of a minimum 3 biological replicates(P3 ± SE).

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At mature green (MG) stage, we screened only 82 accessionsdue to non-availability of enough biological replicates of fruitsfor remaining 43 accessions. An interesting observation was thatthe mature green fruits possessed the higher level of folate thanthe red ripe fruits. At the MG stage, 16 accessions were in the rangeof 40–50 lg/100 g FW followed by 30 accessions in the range of30–40 lg/100 g of FW. Together these two groups constituted56% of the total accessions. Only one accession showed folatehigher than 70 lg/100 g FW while 6 accessions showed folatelevels in the range of 50–60 lg/100 g FW. Twenty three accessionshad folate levels ranging from 20 to 30 lg/100 g of FW. Six acces-sions showed folate level below 20 lg/100 g of FW.

Considering that the tomato accessions were grown in the openfield the effect of seasons and growth conditions on the folate levelwas examined for three tomato cultivars. Supplementary Table 4shows that except Ailsa Craig that had high folate levels inthe open field (39 lg/100 g FW), than in the green house

(27 lg/100 g FW), folate levels for Arka Vikas (14.6–17.3 lg/100 g FW) and Periakulum-1 (33.5–40.6 lg/100 g FW) didnot show drastic variations. The folate levels of Arka Vikas andPeriakulum-1 in different seasons varied within 20%.

The vitamers of folates are labile in nature particularly to oxida-tive degradation during extraction which is stimulated by oxygen,heat, and light. To delineate the extent of degradation and effect ofvarying accessions, we performed the recovery test for differentfolate vitamers. The recovery for reference cultivar Arka Vikaswas in the range of 79–115% for 5-CH3-THF, 5,10-CH+THF, and5-CHO-THF. For other three tomato accessions, a variable recoverywas observed ranging from 65% to 77% for 5-CH3-THF, 87–123% for5-CHO-THF and 42–79% for 5,10-CH+THF. Our results indicatedthat barring 5,10-CH+THF that was present in small amount, therecovery of 5-CH3-THF and 5-CHO-THF vitamers were satisfactory.The higher loss observed for 5,10-CH+THF may be due to highersusceptibility of this folate to degradation (SupplementaryTable 5).

3.2. Distribution of folate forms in tomato accessions

Folate forms present in plants differ in their abundance and sta-bility, and therefore levels of different folate forms were analyzedin fruits of tomato accessions. At MG and RR stage, 5-CH3-THFwas the most abundant folate followed by 5-CHO-THF and5,10-CH+THF (Fig. 2, Supplementary Figs. 1 and 2). The level of5-CH3-THF in accessions varied from 11.6 to 36.1 and 10.7 to63 lg/100 g of FW at the red ripe and mature green stage respec-tively. The relative proportion of 5-CH3-THF in total folatelevel declined from MG (87.9%) to RR (74.6%) stage, whereasrelative proportion of 5-CHO-THF increased from MG (8.8%) toRR (18.5%), stage. A similar trend was also observed for5,10-CH+THF, whose levels increased from MG (2.3%) to RR stage(6.9%). The levels of 5,10-CH+THF varied from 0.5 to3.8 lg/100 g FW and, from 0.2 to 2.2 lg/100 g FW at RR and MGstage respectively. Interestingly, THF was observed only in fewaccessions in MG fruit but not in RR fruit and its level ranged from0.006 to 1.6 lg/100 g FW.

3.3. Effect of ripening on folate content of tomato accessions

We examined relative change in folate levels in fruits of differ-ent tomato accessions during the transition from mature green to

Fig. 3. The relative change in folate levels in tomato fruit during the transition frommature green (MG) to red ripe (RR) stage (RR/MG). The majority of the accessionsshowed decrease in folate level at the red ripe stage.

Fig. 4. Total folate content in fruits of tomato accessions at red ripe stage (160accessions) using microbiological assay. Data are mean of a minimum 3 biologicalreplicates (P3 ± SE).

P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 615

the red ripe stage (RR/MG). In general, folate level was eithernearly similar to MG stage or declined at the RR stage, 63 acces-sions showed declining folate content with ripening; while 11accessions showed increased folate levels with ripening. Eightaccessions showed nearly no change (RR/MG = 0.95 to 1.05) infolate content on ripening. The maximum reduction was observedin accession EC7317 where the folate level in RR fruit was 28% ofMG stage. Contrary to this, three accessions showed more than50% increase in the folate content during the transition from MGto RR stage (Fig. 3).

3.4. Microbiological assay (MA) of total folate level

Total folate content of red ripe tissue of tomato naturalaccessions was estimated by microbiological assay using L. caseisubspecies rhamnosus (strain ATCC 7469) which is an auxotrophicstrain and needs folate for growth. The examination of bacterialgrowth in the presence of varying concentration of folic acidrevealed that the growth was linear in the range of 0.001–0.009 ng/200 lL folate concentration. The correlation analysisshowed a good correlation value (r2 = 0.98) between bacterialgrowth and folate levels. In view of this, the folate extractswere diluted to fall within this range before microbiological assay.During initial studies tomato fruit extracts were serially diluted todetermine the suitable dilution for this assay. Based on these stud-ies 1/3.2 fold dilution was selected for all final inoculations.

The estimation of folate in RR fruits of 160 accessionsusing above assay showed wide variation in levels ranging 4.5–59.9 lg/100 g FW. The folate content in reference cultivar ArkaVikas (AV) was 39.5 lg/100 g FW (Fig. 4A and B). Based on folatelevels, the accessions were classified into 3 groups. Fifty-fouraccessions had less than 20 lg/100 g FW folate whereas 102 acces-sions showed a folate range of 20–50 lg/100 g FW. Four accessionsshowed more than 50 lg/100 g FW folate.

3.5. Comparison of microbiological assay and LC-MS estimation offolate levels

In this study, we analyzed folate levels in tomato using both MAand LC-MS. Though the accessions used for MA and LC-MS werenot grown concurrently, the plants were grown in same seasonand in experimental plots with nearly identical conditions (Supple-mentary Table 2). Only 49 accessions were common in both

seasons for which both MA and LC-MS data were obtained. Thecorrelation in folate level in common accessions was analyzed atthe RR stage by LC-MS and MA (Fig. 5). However, only little corre-lation was apparent betweenMA and LC-MS assay. Out of 49 acces-sions, 26 accessions showed higher folate values and 23 accessionsshowed nearly similar or lower values when estimated by MA thanby LC-MC. The analysis of the same sample from four differentaccessions revealed that the folate value by MA was in the rangeof 84–95% of the values obtained by LC-MS for mature green fruits.However, for red ripe fruit, the estimation by MA was in a muchwider range of 59–149% of the values obtained by LC-MS (Table 1).These variations likely reflect the difference in matrices used, dif-ferences in extraction protocols and sensitivity of the microbialassay to different folate forms. Taken together, it is apparent thatMA likely over/under estimates the folate levels compared toLC-MS for red ripe fruits.

3.6. Single nucleotide polymorphism in folate biosynthesis pathwaygenes

The folate biosynthesis in higher plants is distributed over threecompartments viz. mitochondria, plastids, and cytosol involving atotal of 11 enzymatic steps (Hanson & Gregory, 2011). In addition,the vacuole presumably serves as a storage site of folates (Akhtaret al., 2010). The folate precursor pterin is synthesized in thecytosol by GTP cyclohydrolase I (Basset et al., 2002) and another

Fig. 5. The correlation of folate levels determined by LC-MS and MA in fruits at thered ripe stage in 49 tomato accessions.

Table 1Folate content by LC-MS and microbiological assay (MA) in different accessions oftomato at mature green (MG) and red ripe (RR) stage. Values are mean (±SE) of aminimum of three biological replica (nP 3). The relative difference in folate levelsassayed by MA and LC-MS is given as percent value below the folate levels estimatedby MA, the level of folate estimated by LC-MS was taken as 100% for the respectivesamples.

Fruit stage AV EC8372 BL1208 EC398405

Folate content by LC-MSMG 18.1 (1.9) 36.9 (1.8) 29.5 (2.2) 36.3 (1.0)RR 14.8 (1.2) 36.0 (2.4) 32.7 (1.4) 32.7 (2.9)

Folate content by microbial assayMG 15.7 (1.0)

[86.7%]34.9 (2.8)[94.6%]

24.7 (1.7)[83.7%]

34.5 (2.2)[95.0%]

RR 22.1 (0.9)[149.3%]

36.0 (1.9)[100.0%]

19.4 (1.6)[59.3%]

27.4 (3.3)[83.8%]

616 P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619

precursor p-aminobenzoic acid (pABA) is synthesized in plastids byaminodeoxychorismate synthase (Basset et al., 2004a) and amin-odeoxychorismate lyase (Basset et al., 2004b). These precursorsare transported to mitochondria where end product tetrahydrofo-late (THF) is synthesized and glutamylated. The polyglutamylationof THF is assisted by folylpolyglutamate synthase (FPGS) that isencoded by two genes in tomato; a mitochondrial form (FPGSm)and a plastidial form (FPGSp) (Waller et al., 2010). The polygluta-mate tail of folate molecules can be shortened or removed by theaction of c-glutamyl hydrolases (GGH) which are encoded by threegenes in tomato (GGH1, GGH2, and GGH3), their activity is mainlyrestricted to vacuoles (Orsomando et al., 2005).

The activity of these key enzymes can vary if the genes encod-ing them exhibit polymorphism among different accessions oftomato. Presence of single nucleotide polymorphisms (SNP) wasinvestigated in these accessions in comparison with the referencecultivar Arka Vikas. Since folate is a critical molecule essential forplant survival, only limited polymorphism was observed in abovegenes. Eleven accessions showed SNPs in GCHI gene while 12accessions showed SNPs in ADCS gene. Seven accessions eachshowed SNPs in ADCL1 and ADCL2 gene and were distributedamong three haplotypes. Maximum numbers of accessions showedSNPs in FPGSm gene followed by GGH3 gene, but many of theseaccessions shared a common haplogroup having similar SNP(s).Interestingly, all the SNPs of FPGS and GGH were located in theintrons and SNPs in the ADCS gene were located in exons, whileSNPs detected in GCHI were both intronic and exonic (Supplemen-tary Table 6).

4. Discussion

Tomato is enriched in several antioxidants particularly carote-noids. However, it has a moderate level of folate. In recent years,local varieties and germplasm accessions collected from diverselocations have been increasingly used as a resource to enrich thecommercial cultivars with desirable traits. Essentially thisapproach uses a rigorous analysis of metabolite diversity amongaccessions to identify natural genetic variants and introgressionof beneficial alleles into target cultivars. Examination of red ripefruits of 125 tomato accessions revealed folate content rangingfrom 13.7 to 45.8 lg/100 g FW showing 3.4 fold variations withinthe accessions. 5-CH3-THF was the major form present in bothred ripe and mature green fruits, though its level slightly declinedduring ripening (Fig. 2). Though 5-CH3-THF is reported to be themost prevalent folate form, our data point towards genotypic dif-ferences among accessions for the differential accumulation of aparticular folate form. The relative contribution of each folate formto the total folate pool significantly varied among the accessions.Our results are in agreement with earlier reports in tomato(Iniesta et al., 2009), pepper (Phillips, Ruggio, Ashraf-Khorassani,& Haytowitz, 2006) and spinach (Shohag et al., 2011) where similarvariations were reported.

While 5-CH3-THF, 5-CHO-THF, and 5,10-CH+THF were presentin both red ripe and mature green fruits, minor amounts of THFwas detected only at the mature green stage that too in few acces-sions. Fourteen accessions showed very low (<20 lg/100 g FW) andthree accessions showed high folate level (>40 lg/100 g FW) by LC-MS estimation. A commercially grown local tomato cultivar ArkaVikas selected as a reference variety showed 17.2 lg/100 g FWtotal folate level. The median folate levels in tomato fruits(25.5 lg/100 g FW) was lower than other commonly consumedfruits such as strawberries (47 lg/100 g FW, Strålsjö, Witthöft,Sjöholm, & Jägerstad, 2003) and papaya (67 lg/100 g FW, Ramos-Parra, García-Salinas, Hernández-Brenes, & Díaz de la Garza,2013). Iniesta et al. (2009) examined 11 tomato cultivars andreported similar variations in folate content. A similar study con-ducted in 67 spinach accessions using HPLC-based estimation offolate showed a range of 54–173 lg/100 g FW with 4 accessionswith folate content above 150 lg/100 g FW (Shohag et al., 2011).

For animals, 5-CH3-THF is reported to be the most bioavailableform (Scott et al., 2000) making it the preferred form for the forti-fication of food items (Scott et al., 2000). Therefore, accessions withhigher levels of 5-CH3-THF can be ideal parental lines for breedingbased biofortification approach. Tomato fruits are enriched in 5-CH3-THF with 69.0% contribution to total folate in red ripe fruits.In almost all accessions second highest folate vitamer was 5-CHO-THF. Another study conducted by Díaz de La Garza et al.(2007) in Microtom cultivar of tomato reported 5,10-CH+THF asthe second highest vitamer. The variance between our study andDíaz de La Garza et al. (2007) study indicates the influence of geno-typic differences on relative accumulation of folate species in dif-ferent accessions. A comprehensive analysis of accessions atmolecular and genetic level may identify the factors regulatingin vivo level of folate in tomato fruits.

Unlike the increase in sugar levels and accumulation of carote-noid level observed during tomato fruit ripening, the analysis offolate level in fruit at mature green and red ripe stage did not indi-cate a fixed trend. Nonetheless, most accessions showed thedecline in folate levels during transition from mature green tored ripe stage of ripening. While fruits of some accessions didnot show such a decline during ripening, a small number of acces-sions showed increase in folate levels at the red ripe stage (Fig. 3).Similarly, ripening process in papaya and strawberry fruits too wasnot directly or inversely related to total folate levels (Ramos-Parraet al., 2013; Strålsjö et al., 2003).

P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 617

Currently, little information is available about the influence ofseasonal variation on the folate levels in fruits. In strawberries har-vested over a period of three years, the folate levels in a givenaccession differed by ±20% (Strålsjö et al., 2003). Though the folatelevels in five tomato cultivars harvested over three years periodshowed wide variations, the relative differences in folate levelsamong the cultivars were nearly the same, irrespective of the sea-son (Iniesta et al., 2009). In our study, tomato accessions weregrown in the open field during the season where climatic conditionshowed only little variation. Barring Ailsa Craig which showed avariation of 30%, the folate levels of Arka Vikas and Periakulam-1cultivars in different season/growth conditions differed by only20%. Considering above and earlier studies, it can be assumed thatobserved wide variation in folate levels in tomato accessions aremainly due to differences in their genotypes.

Compared to the animal system, where relative differencesbetween microbiological estimation of folate and LC-MS estima-tion of folate have been examined in several studies, little compar-ative information is available for plant systems. MA estimates thefolate content by measuring the proportional increase in turbidityof culture in relation to exogenous folate levels. Though MA cannotdistinguish between different forms of folate, it does provide anapproximate estimation of total folate levels. A major limitationof this assay is that L. casei growth response differs to differentforms of folate leading to imprecise results (Freisleben,Schieberle, & Rychlik, 2002). This assay is also susceptible to extra-neous folate and contamination by other microbes (Quinlivan,Hanson, & Gregory, 2006). Notwithstanding above limitations,due to its inexpensive nature, MA is routinely used for estimationof total folate in food samples. Our results indicate that microbio-logical assay was inaccurate, and it either overestimated the folatelevel or underestimated the folate levels compared to LC-MS assay(Fig. 5).

Comparison of relative efficiency of MA and LC-MS for estima-tion in human serum showed good correspondence between bothassays (Fazili, Pfeiffer, & Zhang, 2007). On the other hand, in ready-to-eat breakfast cereals, this assay overestimated folic acid levelsby 10–67% than the LC-MS based estimation (Phillips et al.,2010). Given the wide range of differences observed between MAand LC-MS assay, the latter assay more precisely estimates folatelevels. Moreover, LC-MS method also distinguishes different folatespecies and is less susceptible to interference from other metabo-lites and/or inhibitors. Though MA gives an imprecise estimation,being cost-effective and easy to setup, it is more widely used forfolate estimations. It is desirable that MA estimation of folate levelsis also validated by LC-MS method for precise estimations.

Since metabolome of an organism is determined by its geno-type, it has been advocated that comparison of genetic variationamong accessions with metabolome variation can provide infor-mation about linkage between genetic polymorphism and metabo-lite variation in the accessions (Keurentjes et al., 2006). Though,metabolic pathways are regulated by multiple genes and showpolygenic inheritance, there are reports that variation in a singleallele may dramatically influence the level of a metabolite (Belóet al., 2008; Harjes et al., 2008). In maize, a leucine-to-threoninesubstitution in fatty acid desaturase 2 (fad2) gene at a conservedposition 71 negatively affected the activity of the FAD2 enzymeleading to accumulation of oleic acid in the maize kernel (Belóet al., 2008). In maize, considerable variation exists for carotenoidaccumulation in different accessions which is related to the poly-morphic variation in lycopene epsilon cyclase (lcyE) locus. The rel-ative flux between a-carotene versus b-carotene branches of thecarotenoid pathway was largely determined by four natural poly-morphisms in the lcyE gene (Harjes et al., 2008). Similar to carote-noids, the folate biosynthesis is also regulated by multiple genes.

The establishment of links between SNPs in causative genes andvariation in metabolite levels may uncover key alleles that mayinfluence the level of a given metabolite. A genome-wide studycarried out using 96 Arabidopsis accessions indicated that thegenetic variation is a major component that controls the metabo-lome variation (Chan, Rowe, Hansen, & Kliebenstein, 2010). In viewof this, polymorphism in selected folate pathway genes was exam-ined using EcoTILLING.

Genomic DNA of 391 accessions was screened to score SNPs innine genes viz. GCHI, ADCS, ADCL1, ADCL2, FPGSm, FPGSp, GGH1,GGH2, and GGH3 contributing to folate biosynthesis and removalof glutamate tails in tomato. Our analysis showed one to severalSNPs in selected regions of above genes. Based on the size of frag-ments, the accessions showing similar sized fragments weregrouped into respective haplotype. GCHI gene regulating pterinbiosynthesis showed polymorphism in 11 accessions groupedunder 8 haplotypes. Only 12 accessions harboured SNPs in ADCSgene and belonged to three different haplotypes. Seven accessionseach showed SNPs in the exonic region of ADCL1 and ADCL2 gene.The paucity of exonic SNPs in GCHI, ADCS, and ADCL genes may berelated to their critical role in the biosynthesis of folate. Since GCHI,ADCS and ADCL genes act at the very initial steps of the folatebiosynthesis pathway, the SNP(s) affecting their function may belethal; therefore these genes are least likely to harbour genicpolymorphism.

Similar to above genes, little polymorphism was observed inFPGS and GGH genes. Though 17 accessions showed SNPs in theplastidial form of FPGSp gene, these were present only in 3 haplo-types. Fifty-seven accessions showed SNPs in FPGSm gene andformed 5 haplotypes. Similarly, GGH1 and GGH2 genes showedtwo haplotypes while accessions harbouring SNPs in GGH3 geneformed 3 haplotypes. Interestingly, observed polymorphism inFPGS and GGH isoforms was restricted to intronic regions. Takentogether with the low frequency of the polymorphisms in GCHI,ADCS and ADCL genes contributing to folate biosynthesis andoccurrence of polymorphism in the intronic region of FPGS andGGH genes, it appears that the genes regulating folate biosynthesisare recalcitrant to polymorphic changes. Since folate is one of thecritical vitamins that is needed for several metabolic reactionsincluding nucleic acid synthesis, it is likely that polymorphism inthese genes is not tolerated and may affect the optimal functionof plants.

Above observations are consistent with the literature reportswhere no mutants have been reported in genes regulating folatebiosynthesis in plants, except for mutations in FPGS gene. In Ara-bidopsis FPGS is encoded by three genes. However, their functionsare redundant, and phenotypes can be observed only with doublemutants (Mehrshahi et al., 2010) except fpgs1 (FPGSp) mutant thathas short root (Srivastava et al., 2011). The mutation in fpgs1 genethat is located at splice junction releases chromatin silencing on agenome-wide scale as it is indirectly essential for DNA and histonemethylation (Zhou et al., 2013). The mutation in another fpgs1mutant (moots koom 2) located in C-terminal of protein leads toexhaustion of root apical meristem soon after germination indicat-ing that folate is needed for stem cell specification and continua-tion of indeterminacy of root apical meristem (Reyes-Hernándezet al., 2014). Since in both mutants the mutations affecting FPGS1function are located in the exonic region, it can be assumed thatintronic SNPs observed in our study may not affect the function.

Though most of the SNPs in folate biosynthesis genes werelocated in introns, emerging evidences indicate that even intronicSNPs can affect the gene function. Such an effect was reportedfor yellow flesh tomato where an intronic SNP disrupted biosyn-thesis of carotenoids (Kang et al., 2014). Even synonymous muta-tions have been reported to affect gene functions by regulating

618 P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619

mRNA splicing, stability and translation regulation where a pre-ferred synonymous codon is more efficiently translated(Shabalina, Spiridonov, & Kashina, 2013).

Though a direct linkage of folate with observed SNPs in abovegene(s) remains to be established, the present study provides valu-able information for the natural variation in the folate levels andgenes encoding above pathway in tomato accessions. Our studyhighlights that wide range of variation in folate levels amongtomato accessions is not similarly reflected in SNPs present infolate biosynthesis genes. In essence, our results indicate that thefolate level in fruits of tomato accessions are governed by a morecomplex regulation at cellular homeostasis level, which remainsto be deciphered.

5. Conclusion

The present study examined the variation in folate levels in redripe fruits of tomato accessions using two high-throughput meth-ods; microbiological assay (MA) and LC-MS. The MA over/under-estimated folate levels in tomato fruits compared to LC-MS. Thethreefold variations in folate levels in accessions indicated geno-type dependent regulation of folate levels. Accessions identifiedwith very high and very low folate levels were selected for futurebreeding efforts to enhance folate levels in tomato. The limitedpolymorphism in genes encoding folate biosynthesis pathway indi-cated that due to essential requirement of folate for one-carbonmetabolism, the genes were recalcitrant to polymorphic variation.In general our study provides valuable information for the naturalvariation in the folate levels in tomato.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the Department of Biotechnology(Grant No. BT/PR11671/PBD/16/828/2008 to R.S. and Y.S.), theCouncil of Scientific and Industrial Research (research fellowshipto KT), University Grants Commission (research fellowship to PUand SS).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2016.09.031.

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