1. Analytical Methods
Natural variation in folate levels among tomato (Solanum lycopersicum)
accessions
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 2015
Received in revised form 2 June 2016
Accepted 5 September 2016
Available online 7 September 2016
Keywords:
Folate
Solanum lycopersicum
Natural accessions
Single nucleotide polymorphism
EcoTILLING
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 g
fresh 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 and
red ripe fruit, 5-CH3-THF was the most abundant folate form. Comparison of LC-MS with MA revealed that
MA inaccurately estimates folate levels. The accumulation of folate forms and their distribution varied
among 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 among
tomato accessions, little polymorphism was found in folate biosynthesis genes. Our results indicate that
variation in folate level is governed by a more complex regulation at cellular homeostasis level.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Folates, water soluble B9 vitamins, play very important role in
the 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 solely
depend upon dietary sources to obtain folate (Rébeillé et al.,
2006; Scott, Rébeillé, & Fletcher, 2000). In developing countries
dietary deficiency of folate increases the incidences of neural tube
defects in fetal development (Scott, Weir, & Kirke, 1995). The ade-
quate daily dietary folate intake during the gestation period is
essential to ensure normal growth and development of the fetus.
Developed countries like Australia and USA have mandated the
addition of folic acid to wheat flour for bread-making. However,
the developing countries do not have food fortification program
because of the high cost of synthetic folic acid and absence of an
industrial food system.
Plant-based foods are the main dietary sources of folate for
humans and other animals. Among plant-based foods; fruits, nuts,
and vegetables provide about 30% requirement of folate in the
American diet (Kader, Perkins-Veazie, & Lester, 2004). The leafy
vegetables such as spinach, lettuce, broccoli, asparagus, and fruits
such 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 the
synthetic form. Staple foods consumed in developing countries
such as wheat, maize, and rice contain very low amount of folate
which is insufficient to meet folate RDA of 400 lg/day. Considering
this, there are concerted efforts to biofortify common cereal grains
with folate using transgenic approaches. Rice biofortification was
successfully achieved by simultaneous overexpression of two Ara-
bidopsis genes involved in the pteridine and para-aminobenzoate
branches of the folate biosynthesis pathway (Storozhenko et al.,
2007). The biofortified rice seeds have nearly 100 times higher
folate level than the parental plant and its level was sufficient for
required RDA for folate (Storozhenko et al., 2007). Similar trans-
genic enhancement in folate level was also achieved in tomato
by stimulating biosynthesis of folate in fruits (Díaz de La Garza,
Gregory, & Hanson, 2007). However, transgenic tomato and rice
are 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 different
crop plants. Moreover, linkage of natural variation in folate with
genes/QTLs can be used to increase the folate levels in crops by
http://dx.doi.org/10.1016/j.foodchem.2016.09.031
0308-8146/Ó 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.
Food Chemistry 217 (2017) 610–619
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

2. conventional plant breeding. A large scale screen of wheat (175
genotypes) revealed that folate level varied from 364 to 774 ng/g
dry weight in winter wheat and from 323 to 741 ng/g dry weight
in spring wheat. Significantly the durum wheat genotypes showed
highest 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 of
67 accessions showed folate level range from 54 to 173 lg/100 g
of fresh weight, identifying potential genotypes that can be used
for breeding (Shohag et al., 2011).
Notwithstanding above variation in folate content in different
genotypes, little information is available about the regulation of
folate biosynthesis at the genetic level. The folate biosynthesis
pathway in plants is distributed in three subcellular compart-
ments. Pteridine and p-aminobenzoic acid (pABA) moieties are
synthesized 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 GTP
cyclohydrolase I (GCHI) (Basset et al., 2002) while pABA is synthe-
sized from chorismate using two enzymatic steps catalysed by
aminodeoxychorismate synthase (ADCS) (Basset et al., 2004a)
and aminodeoxychorismate lyase (ADCL) (Basset et al., 2004b). In
addition, folate is glutamylated by the action of folylpolyglutamate
synthase (FPGS) (Mehrshahi et al., 2010). It is believed that the
level of folate is also regulated by removal of glutamate moiety
by gamma-glutamyl hydrolases (GGH) (Akhtar et al., 2010;
Orsomando et al., 2005). While most genes contributing to folate
biosynthesis in plants have been identified, the identity of genes
controlling folate turnover and transport is not known. These genes
could also regulate the folate levels in tissue-specific and also
species-specific manners.
Tomato (Solanum lycopersicum) is a plant food which is widely
consumed in all parts of the world. It is also considered an impor-
tant functional food due to enriched levels of bioactive compounds
such as lycopene and b-carotene. Currently, little information is
available about natural variation in folate level in tomato. Exami-
nation of folate in eleven cultivars of tomato showed levels ranging
from 6.5 to 28.6 lg/100 g of fresh weight (Iniesta, Perez-Conesa,
Garcia-Alonso, Ros, & Periago, 2009). Several folate vitamers
account for the total folate in tomato, of which 5-methyltetra-
hydrofolate (5-CH3-THF) is the main vitamer. The distribution
and developmental regulation of different folate vitamers in
tomato fruit are not known.
In the present study, we analyzed folate level in 160 accessions
of tomato by microbiological assay and 125 accessions by LC-MS
method. Simultaneously, key genes of the folate biosynthesis and
turnover were screened for single nucleotide polymorphism (SNPs)
in tomato accessions. In this study, we report that though the
folate level in tomato accessions varies considerably, such a broad
range 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 obtained
from TGRC (Tomato Genetics Resource Center at University of Cal-
ifornia, Davis) (www.tgrc.ucdavis.edu); IIVR (Indian Institute of
Vegetable Research, Varanasi, India) (www.iivr.org.in); IIHR
(Indian Institute of Horticultural Research, Bengaluru, India
(www.iihr.res.in); NBPGR (National Bureau of Plant Genetic
Resources, 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 October
to February in the year 2011–12 and 2012–13. Supplementary
Table 2 shows the average temperature and humidity for above
seasons. The fruits from plants grown in 2011–12 were used for
the microbiological assay (MA) and from plants grown in 2012–
13 were used for LC-MS estimation of folate. While SNPs were
examined 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 or
more replicates were obtained from only 125 accessions and 82
accessions for red ripe fruits and mature green fruits respectively.
2.2. Plant growth
All accessions were grown under similar conditions in the open
field at University of Hyderabad, Hyderabad, India. Fifteen seeds
from each accession after surface sterilization with 4% (v/v) sodium
hypochlorite for 10–12 min and rinsing with tap water were grown
in 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 second
flowers from first and second truss (preferably 1st truss) of the
plants were tagged, and fruits at mature green (MG) and red ripe
(RR) stages were harvested from at least three different plants of
each accession. The attainment of mature-green stage varied
among the accessions. Fruits at the 28–35 days after pollination
were harvested for MG stage. The transition from mature-green
to red-ripe stages also varied among the accessions, requiring
8–15 day duration to attain the red-ripe stage. The fruits after har-
vesting were placed on ice in an ice bucket and transferred to the
lab. 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 fruit
homogenization. The fruits were homogenized in liquid nitrogen
using homogenizer (IKA, A11 basic, Germany) and the powder
was stored at À80 °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 from
Schirck’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%. Folic
acid (FA), ascorbic acid, b-mercaptoethanol, LC-MS grade acetoni-
trile and a-amylase (from Bacillus sp., A6814) were obtained from
Sigma Aldrich Co. (St. Louis, USA). Milli-Q water (18.2 X at 25 °C)
was obtained from Millipore water system (Millipore, Bradford,
USA). LC-MS grade formic acid (HCOOH) was obtained from Fisher
Scientific (Loughborough, UK). Potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, folic acid casei medium
(M-543-100G), Protease (from Streptomyces griseus, RM6186) and
activated 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 = MTCC
1408). Protease (P5147) and sodium ascorbate were obtained
from Sigma Aldrich Co. (St. Louis, USA). Rat plasma was obtained
from National Institute of Nutrition (NIN), Hyderabad, India
(http://ninindia.org/).
P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 611

3. 2.4. Folate standards: preparation and purity correction
Stock solutions of folate standards (1 mg/mL) were prepared in
50 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 extraction
solution to prepare working solutions. The remaining stock solu-
tions were flushed with nitrogen gas, and small aliquots were
stored at À80 °C. The purity of the folate standards were calculated
using 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 folic
acid which were dissolved in 0.01 N HCl and 1 N NaOH respec-
tively. The molar absorption coefficients for above folate standards
were obtained from Zhang et al. (2003), except for 5,10-Methenyl
THF which was from Moldt et al. (2009). The respective purity of
5-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 vitamers
were plotted after correcting for the purity.
2.5. Enzyme preparation for folate extraction
Protease (2 mg/mL) and a-amylase (20 mg/mL) were dissolved
in Milli-Q water, and aliquots were stored at À20 °C. To remove
endogenous folate from rat plasma and a-amylase, 100 mL of rat
plasma and a-amylase were mixed with 5 g of activated charcoal.
This mixture was incubated on ice for 1 h with intermittent stirring
followed by centrifugation at 5000g (Sorvall Lynx 6000, Thermo
Scientific, USA) for 10 min at room temperature. The supernatant
was filtered through a 0.22 lm filter, divided into 1 mL aliquots,
and stored at À20 °C. Protease was used without pre-treatment
and was stored in À20 °C.
2.6. Sample extraction procedure for LC-MS
Total folate was extracted following the procedures of Tyagi
et al. (2015) from 125 accessions (Supplementary Table 1). Briefly,
100 mg homogenized tissue was suspended in 650 lL of extraction
solution (50 mM potassium phosphate, 1% (w/v) ascorbic acid, 0.5%
(v/v) b-mercaptoethanol, 1 mM calcium chloride, pH 4.5, flushed
with nitrogen) in a 2 mL Eppendorf tube. The homogenate was
boiled for 10 min and then cooled on ice. Thereafter, 10 lL of a-
amylase (20 mg/mL) was added, and tubes were incubated at room
temperature 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 boiling
water bath for 5 min and cooling on ice. For deconjugation of folate
polyglutamates to monoglutamates, 100 lL of rat plasma was
added to each sample and tubes were incubated at 37 °C for 2 h.
Enzymatic activity was stopped by transferring the tubes to boiling
water bath for 5 min and cooling on ice followed by centrifugation
for 30 min (14,000g, 4 °C). The supernatant was filtered through
the 0.22 lm filter (MDI Advanced Micro-devices) and the filtrate
was ultra-filtered at 12,000g for 12 min using 10 kDa molecular
weight cut-off membrane filter (Pall Corporation, USA) for sample
cleanup before LC-MS analysis. The resulting filtrate was trans-
ferred to an autosampler vial and 7.5 lL aliquot was directly
injected on the column.
2.7. Liquid chromatography condition and mass spectrometry settings
For LC-MS, all the parameters used were essentially the same as
described earlier by Tyagi et al. (2015). The folate derivatives were
separated on a reversed phase Luna C18 column (5 lm particle
size, 250 mm  4.60 mm ID) (Phenomenex, USA) using Waters
AcquityTM
UPLC system (Milford, USA) running in HPLC mode, cou-
pled to a binary pump, an autosampler, and controlled by Xcalibur
3.0 software (Thermo Fisher Scientific, San Jose, USA). For mass
spectrometry, ExactiveTM
Plus Orbitrap mass spectrometer (Thermo
Fisher Scientific, USA) was operated in alternating full scan and all
ion fragmentation (AIF) mode equipped with positive heated
electrospray 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 is
the slope of calibration curve) and limit of quantification (LOQ; cal-
culated as 10r/S). Least-square regression analysis was used for
data fitting. After confirmation of individual peak identity on the
basis of m/z and their fragmentation products, quantification was
done according to the response of the mass detector to the folate
standard. The linearity of each folate standard was evaluated by
plotting the peak area at different concentrations and sample con-
centrations were calculated from the equation y = mx + c. R2
values
for 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). The
buffer blanks were run before first sample run and after every
20 sample run to remove any carryover. After 240 runs, the
HPLC columns were also cleaned as per the manufacturer’s
recommendation.
Endogenous residual folate of trienzyme (rat plasma +
a-amylase + protease) was corrected by running blank samples
and subtracting the values from the sample extracts. The sum of
all the folate vitamers was expressed as microgram per 100 g of
fresh weight. To determine the accuracy of the method, a recovery
test was performed in four randomly chosen tomato accessions by
spiking of tomato extract with known amount of individual folate
vitamer standards and calculating their final content in the extract.
The recovery (R) was calculated as (B À C/A) Â 100, where A = peak
area 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 of
tomato (Supplementary Table 1). The stock culture of L. casei was
made in Lactobacillus broth and maintained on agar medium. Many
parameters like aeration, incubation time and inoculum dose were
optimized to achieve steady bacterial growth. A modified method
of Wilson and Horne (1982) was used to prepare cryoprotected
cells for using as inoculum. The standard curve for bacterial growth
was made against increasing folic acid concentrations. All parame-
ters related to the bacterial growth were standardized for 200 lL
volume of microtiter plate wells.
2.10. Extraction method
Folate was extracted from tomato fruit tissue using trienzyme
extraction described by Goyer and Navarre (2007) with some mod-
ifications. Extraction was carried out in 2 mL wells of a 96 well
plate. Briefly, 100 mg fresh or stored homogenized fruit tissue
was suspended in 1 mL extraction buffer (0.1 M potassium
phosphate 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 cooled
on ice. After addition of 10 lL protease (10 mg/mL), the sample
was incubated at 37 °C for 2 h followed by transferring the plates
to boiling water bath for 5 min and immediately cooling on ice.
612 P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619

4. 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 after
which 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 steps
after the first boiling step were carried out in a sterile airflow
bench to avoid the need for filtration of extracts. No significant dif-
ference in folate content was observed between samples processed
with or without filtration. Since folates are light sensitive the
extracts were protected from light to prevent the oxidation of
folates during extraction and storage.
2.11. Inoculation and incubation of extracts with bacterial culture
To the sample wells of a microtiter plate containing 100 lL
assay 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 (3.2
times diluted) was added. The wells with buffer blank contained
100 lL assay medium and 100 lL buffer, while wells with inocu-
lum blank contained 100 lL assay medium, 60 lL buffer and
40 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 following
the procedures of Sreelakshmi et al. (2010). Five genes of folate
biosynthesis 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, and
GGH3). The sequences of above genes and their isoforms were
obtained from SOL GENOMICS NETWORK (solgenomics.net). A
web-based software tool Codons Optimized to Discover Deleteri-
ous Lesions (blocks.fhcrc.org/proweb/coddle) was used to predict
a region of the gene where mutation/change(s) would cause the
most deleterious effect. The primers for the CODDLE predicted
region 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. The
first step PCR was carried out using 3 pmol unlabeled primers cov-
ering the flanking sequence of targeted genomic region in a volume
of 20 lL with DNA of different accessions mixed with that of Arka
Vikas in a 1:1 ratio. The PCR reaction carried out in 20 lL volume
consisted 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) and
3 pmol each of forward and reverse primers. The cycling conditions
for 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 first
step 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 conditions
for second step were-94 °C-4 min, 3 cycles of 94 °C-20 s, 60 °C-45 s
with a decrement of 2.0 °C per cycle, 72 °C-1 min 30 s followed by
30 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 and
cooling (re-annealing) to generate heteroduplexes between wild
type 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 a
decrement of 0.3 °C per cycle and held at 4 °C.
The presence of heteroduplex was detected by using a
mismatch specific endonuclease, CEL I enzyme that cleaves the
heteroduplex DNA at the site of mismatch resulting in fragmented
DNA. 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 pH
7.0, 10 mM KCl, 10 mM MgCl2, 0.002% (v/v) Triton X-100 and
10 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 by
adding 10 lL stop solution (2.5 M NaCl, 75 mM EDTA, pH 8.0 and
0.5 mg/mL blue dextran). The DNA was precipitated by addition
of 125 lL of cold absolute ethanol and a brief incubation in
À80 °C followed by centrifugation at 4500 rpm in a SH-3000 rotor
for 30 min. The DNA pellet was washed with 70% (v/v) ethanol and
after drying at 80 °C, was suspended in 8 lL formamide loading
buffer consisting of 37% (v/v) deionized formamide, 1 mM EDTA
and 0.02% (w/v) bromophenol blue. The PCR products were dena-
tured by heating at 94 °C for 2 min and then were incubated on
ice. The fragmented products were resolved on high resolution
denaturing PAGE (polyacrylamide gel electrophoresis). About
0.5 lL of the sample was electrophoresed in a denaturing 6.5%
(w/v) polyacrylamide gel in TBE buffer (89 mM Tris, 89 mM boric
acid, 2 mM EDTA, pH 8.3) at 1500 V, 40 mA and 40 V setting on
LI-COR 4300 DNA analyzer. The two TIFF images of 700 and 800
channels were analyzed in Adobe Photoshop software (Adobe
Systems Inc.), and the gel was visually assessed for the presence
of SNPs.
After detection of SNPs in a given accession, genomic DNA from
that accession was re-amplified and subjected to agarose gel based
mismatch 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 grouped
as a single haplotype.
2.13. Statistical analysis
A minimum of three biological replicates (n P 3) were used for
each sample. The results are expressed as the mean of all biological
replicates 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 to
70.9 lg/100 g FW (Fig. 1C; Supplementary Figs. 1 and 2 shows
levels of individual vitamers). The median of total folate level at
the red ripe and the mature green stage was at 25.5 and
35 lg/100 g of FW respectively. Based on folate levels, the acces-
sions were classified into 4 groups at the red ripe stage and 6
groups 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 26
accessions in the range of 30–40 lg/100 g of FW. Together these
two groups consisted of nearly 86% of total accessions. While red
ripe fruits of 3 accessions were in 40–50 lg/100 g FW group, 14
accessions showed less than 20 lg/100 g FW folate. The highest
folate content 45.8 lg/100 g FW was observed in accession
EC498372 at the red ripe stage.
P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 613

5. At mature green (MG) stage, we screened only 82 accessions
due to non-availability of enough biological replicates of fruits
for remaining 43 accessions. An interesting observation was that
the mature green fruits possessed the higher level of folate than
the red ripe fruits. At the MG stage, 16 accessions were in the range
of 40–50 lg/100 g FW followed by 30 accessions in the range of
30–40 lg/100 g of FW. Together these two groups constituted
56% of the total accessions. Only one accession showed folate
higher than 70 lg/100 g FW while 6 accessions showed folate
levels in the range of 50–60 lg/100 g FW. Twenty three accessions
had 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 open
field the effect of seasons and growth conditions on the folate level
was examined for three tomato cultivars. Supplementary Table 4
shows that except Ailsa Craig that had high folate levels in
the 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) did
not show drastic variations. The folate levels of Arka Vikas and
Periakulum-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 of
varying accessions, we performed the recovery test for different
folate vitamers. The recovery for reference cultivar Arka Vikas
was in the range of 79–115% for 5-CH3-THF, 5,10-CH+
THF, and
5-CHO-THF. For other three tomato accessions, a variable recovery
was observed ranging from 65% to 77% for 5-CH3-THF, 87–123% for
5-CHO-THF and 42–79% for 5,10-CH+
THF. Our results indicated
that barring 5,10-CH+
THF that was present in small amount, the
recovery of 5-CH3-THF and 5-CHO-THF vitamers were satisfactory.
The higher loss observed for 5,10-CH+
THF may be due to higher
susceptibility of this folate to degradation (Supplementary
Table 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 analyzed
in fruits of tomato accessions. At MG and RR stage, 5-CH3-THF
was the most abundant folate followed by 5-CHO-THF and
5,10-CH+
THF (Fig. 2, Supplementary Figs. 1 and 2). The level of
5-CH3-THF in accessions varied from 11.6 to 36.1 and 10.7 to
63 lg/100 g of FW at the red ripe and mature green stage respec-
tively. The relative proportion of 5-CH3-THF in total folate
level declined from MG (87.9%) to RR (74.6%) stage, whereas
relative proportion of 5-CHO-THF increased from MG (8.8%) to
RR (18.5%), stage. A similar trend was also observed for
5,10-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 to
3.8 lg/100 g FW and, from 0.2 to 2.2 lg/100 g FW at RR and MG
stage respectively. Interestingly, THF was observed only in few
accessions in MG fruit but not in RR fruit and its level ranged from
0.006 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. 2. Distribution of different folate forms in tomato fruits at red ripe and mature
green stage. The data represented are mean values of folate levels in 125 accessions
for RR and 82 accessions for MG stage. 5-CH3-THF was the predominant folate form
present at RR and MG stageÁTHF was observed only at the mature green stage but
not 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 on
left of the graph represents folate levels in tomato fruits and legend on the right of
the graph shows tomato accessions falling in different ranges (marked with dotted
lines) of folate levels. Data presented are mean of a minimum 3 biological replicates
(P3 ± SE).
614 P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619

6. the red ripe stage (RR/MG). In general, folate level was either
nearly similar to MG stage or declined at the RR stage, 63 acces-
sions showed declining folate content with ripening; while 11
accessions showed increased folate levels with ripening. Eight
accessions showed nearly no change (RR/MG = 0.95 to 1.05) in
folate content on ripening. The maximum reduction was observed
in accession EC7317 where the folate level in RR fruit was 28% of
MG stage. Contrary to this, three accessions showed more than
50% increase in the folate content during the transition from MG
to RR stage (Fig. 3).
3.4. Microbiological assay (MA) of total folate level
Total folate content of red ripe tissue of tomato natural
accessions was estimated by microbiological assay using L. casei
subspecies rhamnosus (strain ATCC 7469) which is an auxotrophic
strain and needs folate for growth. The examination of bacterial
growth in the presence of varying concentration of folic acid
revealed that the growth was linear in the range of 0.001–
0.009 ng/200 lL folate concentration. The correlation analysis
showed a good correlation value (r2
= 0.98) between bacterial
growth and folate levels. In view of this, the folate extracts
were diluted to fall within this range before microbiological assay.
During initial studies tomato fruit extracts were serially diluted to
determine 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 accessions
using above assay showed wide variation in levels ranging 4.5–
59.9 lg/100 g FW. The folate content in reference cultivar Arka
Vikas (AV) was 39.5 lg/100 g FW (Fig. 4A and B). Based on folate
levels, the accessions were classified into 3 groups. Fifty-four
accessions 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 accessions
showed more than 50 lg/100 g FW folate.
3.5. Comparison of microbiological assay and LC-MS estimation of
folate levels
In this study, we analyzed folate levels in tomato using both MA
and LC-MS. Though the accessions used for MA and LC-MS were
not grown concurrently, the plants were grown in same season
and 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. The
correlation in folate level in common accessions was analyzed at
the RR stage by LC-MS and MA (Fig. 5). However, only little corre-
lation was apparent between MA and LC-MS assay. Out of 49 acces-
sions, 26 accessions showed higher folate values and 23 accessions
showed nearly similar or lower values when estimated by MA than
by LC-MC. The analysis of the same sample from four different
accessions revealed that the folate value by MA was in the range
of 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 much
wider 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 microbial
assay to different folate forms. Taken together, it is apparent that
MA likely over/under estimates the folate levels compared to
LC-MS for red ripe fruits.
3.6. Single nucleotide polymorphism in folate biosynthesis pathway
genes
The folate biosynthesis in higher plants is distributed over three
compartments viz. mitochondria, plastids, and cytosol involving a
total of 11 enzymatic steps (Hanson & Gregory, 2011). In addition,
the vacuole presumably serves as a storage site of folates (Akhtar
et al., 2010). The folate precursor pterin is synthesized in the
cytosol by GTP cyclohydrolase I (Basset et al., 2002) and another
Fig. 3. The relative change in folate levels in tomato fruit during the transition from
mature green (MG) to red ripe (RR) stage (RR/MG). The majority of the accessions
showed decrease in folate level at the red ripe stage.
Fig. 4. Total folate content in fruits of tomato accessions at red ripe stage (160
accessions) using microbiological assay. Data are mean of a minimum 3 biological
replicates (P3 ± SE).
P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 615

7. precursor p-aminobenzoic acid (pABA) is synthesized in plastids by
aminodeoxychorismate synthase (Basset et al., 2004a) and amin-
odeoxychorismate lyase (Basset et al., 2004b). These precursors
are transported to mitochondria where end product tetrahydrofo-
late (THF) is synthesized and glutamylated. The polyglutamylation
of THF is assisted by folylpolyglutamate synthase (FPGS) that is
encoded 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 the
action of c-glutamyl hydrolases (GGH) which are encoded by three
genes in tomato (GGH1, GGH2, and GGH3), their activity is mainly
restricted 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 of
tomato. Presence of single nucleotide polymorphisms (SNP) was
investigated in these accessions in comparison with the reference
cultivar Arka Vikas. Since folate is a critical molecule essential for
plant survival, only limited polymorphism was observed in above
genes. Eleven accessions showed SNPs in GCHI gene while 12
accessions showed SNPs in ADCS gene. Seven accessions each
showed SNPs in ADCL1 and ADCL2 gene and were distributed
among three haplotypes. Maximum numbers of accessions showed
SNPs in FPGSm gene followed by GGH3 gene, but many of these
accessions shared a common haplogroup having similar SNP(s).
Interestingly, all the SNPs of FPGS and GGH were located in the
introns and SNPs in the ADCS gene were located in exons, while
SNPs 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 diverse
locations have been increasingly used as a resource to enrich the
commercial cultivars with desirable traits. Essentially this
approach uses a rigorous analysis of metabolite diversity among
accessions to identify natural genetic variants and introgression
of beneficial alleles into target cultivars. Examination of red ripe
fruits of 125 tomato accessions revealed folate content ranging
from 13.7 to 45.8 lg/100 g FW showing 3.4 fold variations within
the accessions. 5-CH3-THF was the major form present in both
red ripe and mature green fruits, though its level slightly declined
during ripening (Fig. 2). Though 5-CH3-THF is reported to be the
most prevalent folate form, our data point towards genotypic dif-
ferences among accessions for the differential accumulation of a
particular folate form. The relative contribution of each folate form
to 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 similar
variations were reported.
While 5-CH3-THF, 5-CHO-THF, and 5,10-CH+
THF were present
in both red ripe and mature green fruits, minor amounts of THF
was detected only at the mature green stage that too in few acces-
sions. Fourteen accessions showed very low (<20 lg/100 g FW) and
three accessions showed high folate level (>40 lg/100 g FW) by LC-
MS estimation. A commercially grown local tomato cultivar Arka
Vikas selected as a reference variety showed 17.2 lg/100 g FW
total folate level. The median folate levels in tomato fruits
(25.5 lg/100 g FW) was lower than other commonly consumed
fruits 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 and
reported similar variations in folate content. A similar study con-
ducted in 67 spinach accessions using HPLC-based estimation of
folate showed a range of 54–173 lg/100 g FW with 4 accessions
with folate content above 150 lg/100 g FW (Shohag et al., 2011).
For animals, 5-CH3-THF is reported to be the most bioavailable
form (Scott et al., 2000) making it the preferred form for the forti-
fication of food items (Scott et al., 2000). Therefore, accessions with
higher levels of 5-CH3-THF can be ideal parental lines for breeding
based 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 as
the second highest vitamer. The variance between our study and
Díaz 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 at
molecular and genetic level may identify the factors regulating
in 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 of
folate level in fruit at mature green and red ripe stage did not indi-
cate a fixed trend. Nonetheless, most accessions showed the
decline in folate levels during transition from mature green to
red ripe stage of ripening. While fruits of some accessions did
not 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 was
not directly or inversely related to total folate levels (Ramos-Parra
et al., 2013; Strålsjö et al., 2003).
Fig. 5. The correlation of folate levels determined by LC-MS and MA in fruits at the
red ripe stage in 49 tomato accessions.
Table 1
Folate content by LC-MS and microbiological assay (MA) in different accessions of
tomato at mature green (MG) and red ripe (RR) stage. Values are mean (±SE) of a
minimum of three biological replica (n P 3). The relative difference in folate levels
assayed by MA and LC-MS is given as percent value below the folate levels estimated
by MA, the level of folate estimated by LC-MS was taken as 100% for the respective
samples.
Fruit stage AV EC8372 BL1208 EC398405
Folate content by LC-MS
MG 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 assay
MG 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

8. Currently, little information is available about the influence of
seasonal variation on the folate levels in fruits. In strawberries har-
vested over a period of three years, the folate levels in a given
accession differed by ±20% (Strålsjö et al., 2003). Though the folate
levels in five tomato cultivars harvested over three years period
showed wide variations, the relative differences in folate levels
among the cultivars were nearly the same, irrespective of the sea-
son (Iniesta et al., 2009). In our study, tomato accessions were
grown in the open field during the season where climatic condition
showed only little variation. Barring Ailsa Craig which showed a
variation of 30%, the folate levels of Arka Vikas and Periakulam-1
cultivars in different season/growth conditions differed by only
20%. Considering above and earlier studies, it can be assumed that
observed wide variation in folate levels in tomato accessions are
mainly due to differences in their genotypes.
Compared to the animal system, where relative differences
between 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 the
folate content by measuring the proportional increase in turbidity
of culture in relation to exogenous folate levels. Though MA cannot
distinguish between different forms of folate, it does provide an
approximate estimation of total folate levels. A major limitation
of this assay is that L. casei growth response differs to different
forms 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 estimation
of total folate in food samples. Our results indicate that microbio-
logical assay was inaccurate, and it either overestimated the folate
level 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 both
assays (Fazili, Pfeiffer, & Zhang, 2007). On the other hand, in ready-
to-eat breakfast cereals, this assay overestimated folic acid levels
by 10–67% than the LC-MS based estimation (Phillips et al.,
2010). Given the wide range of differences observed between MA
and LC-MS assay, the latter assay more precisely estimates folate
levels. Moreover, LC-MS method also distinguishes different folate
species 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 for
folate estimations. It is desirable that MA estimation of folate levels
is 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 variation
among 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 show
polygenic inheritance, there are reports that variation in a single
allele may dramatically influence the level of a metabolite (Beló
et al., 2008; Harjes et al., 2008). In maize, a leucine-to-threonine
substitution in fatty acid desaturase 2 (fad2) gene at a conserved
position 71 negatively affected the activity of the FAD2 enzyme
leading to accumulation of oleic acid in the maize kernel (Beló
et al., 2008). In maize, considerable variation exists for carotenoid
accumulation 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 the
carotenoid 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 and
variation in metabolite levels may uncover key alleles that may
influence the level of a given metabolite. A genome-wide study
carried out using 96 Arabidopsis accessions indicated that the
genetic variation is a major component that controls the metabo-
lome variation (Chan, Rowe, Hansen, & Kliebenstein, 2010). In view
of this, polymorphism in selected folate pathway genes was exam-
ined using EcoTILLING.
Genomic DNA of 391 accessions was screened to score SNPs in
nine genes viz. GCHI, ADCS, ADCL1, ADCL2, FPGSm, FPGSp, GGH1,
GGH2, and GGH3 contributing to folate biosynthesis and removal
of glutamate tails in tomato. Our analysis showed one to several
SNPs in selected regions of above genes. Based on the size of frag-
ments, the accessions showing similar sized fragments were
grouped into respective haplotype. GCHI gene regulating pterin
biosynthesis showed polymorphism in 11 accessions grouped
under 8 haplotypes. Only 12 accessions harboured SNPs in ADCS
gene and belonged to three different haplotypes. Seven accessions
each showed SNPs in the exonic region of ADCL1 and ADCL2 gene.
The paucity of exonic SNPs in GCHI, ADCS, and ADCL genes may be
related to their critical role in the biosynthesis of folate. Since GCHI,
ADCS and ADCL genes act at the very initial steps of the folate
biosynthesis pathway, the SNP(s) affecting their function may be
lethal; therefore these genes are least likely to harbour genic
polymorphism.
Similar to above genes, little polymorphism was observed in
FPGS and GGH genes. Though 17 accessions showed SNPs in the
plastidial form of FPGSp gene, these were present only in 3 haplo-
types. Fifty-seven accessions showed SNPs in FPGSm gene and
formed 5 haplotypes. Similarly, GGH1 and GGH2 genes showed
two haplotypes while accessions harbouring SNPs in GGH3 gene
formed 3 haplotypes. Interestingly, observed polymorphism in
FPGS and GGH isoforms was restricted to intronic regions. Taken
together with the low frequency of the polymorphisms in GCHI,
ADCS and ADCL genes contributing to folate biosynthesis and
occurrence of polymorphism in the intronic region of FPGS and
GGH genes, it appears that the genes regulating folate biosynthesis
are recalcitrant to polymorphic changes. Since folate is one of the
critical vitamins that is needed for several metabolic reactions
including nucleic acid synthesis, it is likely that polymorphism in
these genes is not tolerated and may affect the optimal function
of plants.
Above observations are consistent with the literature reports
where no mutants have been reported in genes regulating folate
biosynthesis in plants, except for mutations in FPGS gene. In Ara-
bidopsis FPGS is encoded by three genes. However, their functions
are redundant, and phenotypes can be observed only with double
mutants (Mehrshahi et al., 2010) except fpgs1 (FPGSp) mutant that
has short root (Srivastava et al., 2011). The mutation in fpgs1 gene
that is located at splice junction releases chromatin silencing on a
genome-wide scale as it is indirectly essential for DNA and histone
methylation (Zhou et al., 2013). The mutation in another fpgs1
mutant (moots koom 2) located in C-terminal of protein leads to
exhaustion 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-Hernández
et al., 2014). Since in both mutants the mutations affecting FPGS1
function are located in the exonic region, it can be assumed that
intronic SNPs observed in our study may not affect the function.
Though most of the SNPs in folate biosynthesis genes were
located in introns, emerging evidences indicate that even intronic
SNPs can affect the gene function. Such an effect was reported
for 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
P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 617

9. 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 above
gene(s) remains to be established, the present study provides valu-
able information for the natural variation in the folate levels and
genes encoding above pathway in tomato accessions. Our study
highlights that wide range of variation in folate levels among
tomato accessions is not similarly reflected in SNPs present in
folate biosynthesis genes. In essence, our results indicate that the
folate level in fruits of tomato accessions are governed by a more
complex regulation at cellular homeostasis level, which remains
to be deciphered.
5. Conclusion
The present study examined the variation in folate levels in red
ripe 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. The
threefold variations in folate levels in accessions indicated geno-
type dependent regulation of folate levels. Accessions identified
with very high and very low folate levels were selected for future
breeding efforts to enhance folate levels in tomato. The limited
polymorphism in genes encoding folate biosynthesis pathway indi-
cated that due to essential requirement of folate for one-carbon
metabolism, the genes were recalcitrant to polymorphic variation.
In general our study provides valuable information for the natural
variation 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.), the
Council of Scientific and Industrial Research (research fellowship
to KT), University Grants Commission (research fellowship to PU
and SS).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
09.031.
References
Akhtar, T. A., Orsomando, G., Mehrshahi, P., Lara-Núñez, A., Bennett, M. J., Gregory, J.
F., III, & Hanson, A. D. (2010). A central role for gamma-glutamyl hydrolases in
plant folate homeostasis. The Plant Journal, 64, 256–266.
Basset, G. J. C., Quinlivan, E. P., Ravanel, S., Rébeillé, F., Nichols, B. P., Shinozaki, K., ...
Hanson, A. D. (2004a). Folate synthesis in plants: The p-aminobenzoate branch
is initiated by a bifunctional PabA-PabB protein that is targeted to plastids.
Proceedings of the National academy of Sciences of the United States of America,
101, 1496–1501.
Basset, G., Quinlivan, E. P., Ziemak, M. J., Díaz de la Garza, R., Fischer, M., Schiffmann,
S., ... Hanson, A. D. (2002). Folate synthesis in plants: The first step of the pterin
branch is mediated by a unique bimodular GTP cyclohydrolase I. Proceedings of
the National Academy of Sciences, 99, 12489–12494.
Basset, G. J. C., Ravanel, S., Quinlivan, E. P., White, R., Giovannoni, J. J., Rébeillé, F., ...
Hanson, A. D. (2004b). Folate synthesis in plants: The last step of the p-
aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate
lyase. The Plant Journal, 40, 453–461.
Beló, A., Zheng, P., Luck, S., Shen, B., Meyer, D. J., Li, B., ... Rafalski, A. (2008). Whole
genome scan detects an allelic variant of fad2 associated with increased oleic
acid levels in maize. Molecular Genetics and Genomics, 279, 1–10.
Chan, E. K. F., Rowe, H. C., Hansen, B. G., & Kliebenstein, D. J. (2010). The complex
genetic architecture of the metabolome. PLoS Genetics, 6, e1001198.
Delchier, N., Herbig, A. L., Rychlik, M., & Renard, C. M. (2016). Folates in fruits and
vegetables: Contents, processing, and stability. Comprehensive Reviews in Food
Science and Food Safety, 15(3), 506–528.
Díaz de La Garza, R., Gregory, J. F., & Hanson, A. D. (2007). Folate biofortification of
tomato fruit. Proceedings of the National academy of Sciences of the United States
of America, 104, 4218–4222.
Fazili, Z., Pfeiffer, C. M., & Zhang, M. (2007). Comparison of serum folate species
analyzed by LC-MS/MS with total folate measured by microbiologic assay and
Bio-Rad radioassay. Clinical Chemistry, 53, 781–784.
Freisleben, A., Schieberle, P., & Rychlik, M. (2002). Syntheses of labeled vitamers of
folic acid to be used as internal standards in stable isotope dilution assays.
Journal of Agricultural and Food Chemistry, 50, 4760–4768.
Goyer, A., & Navarre, D. A. (2007). Determination of folate concentrations in diverse
potato germplasm using a trienzyme extraction and a microbiological assay.
Journal of Agricultural and Food Chemistry, 55, 3523–3528.
Hanson, A. D., & Gregory, J. F. III, (2011). Folate biosynthesis, turnover, and transport
in plants. Annual Review of Plant Biology, 62, 105–125.
Harjes, C. E., Rocheford, T. R., Bai, L., Brutnell, T. P., Kandianis, C. B., Sowinski, S. G., ...
Stapleton, A. E. (2008). Natural genetic variation in lycopene epsilon cyclase
tapped for maize biofortification. Science, 319, 330–333.
Iniesta, M. D., Perez-Conesa, D., Garcia-Alonso, J., Ros, G., & Periago, M. J. (2009).
Folate content in tomato (Lycopersicon esculentum). Influence of cultivar,
ripeness, year of harvest, and pasteurization and storage temperatures.
Journal of Agricultural and Food Chemistry, 57(11), 4739–4745.
Kader, A. A., Perkins-Veazie, P. & Lester, G. E. (2004). Nutritional quality and its
importance in human health. US Dept. Agric, Agric Handbook 66. <http://www.
ba.ars.usda.gov/hb66/nutritionalQuality.pdf>.
Kang, B., Gu, Q., Tian, P., Xiao, L., Cao, H., & Yang, W. (2014). A chimeric transcript
containing Psy1 and a potential mRNA is associated with yellow flesh color in
tomato accession PI 114490. Planta, 240, 1011–1021.
Keurentjes, J. J. B., Fu, J., Ric de Vos, C. H., Lommen, A., Hall, R. D., Bino, R. J., ... van der
Plas, L. H. W. (2006). The genetics of plant metabolism. Nature Genetics, 38,
842–849.
Lucock, M. (2000). Folic acid: Nutritional biochemistry, molecular biology, and role
in disease processes. Molecular Genetics and Metabolism, 71, 121–138.
Mehrshahi, P., Gonzalez-Jorge, S., Akhtar, T. A., Ward, J. L., Santoyo-Castelazo, A.,
Marcus, S. E., ... Lara-Núñez, A. (2010). Functional analysis of folate
polyglutamylation and its essential role in plant metabolism and
development. The Plant Journal, 64, 267–279.
Mohan, V., Gupta, S., Thomas, S., Mickey, H., Charakana, C., Chauhan, V. S., ... Sharma,
R. (2016). Tomato fruits show wide phenomic diversity but fruit developmental
genes show low genomic diversity. PLoS One, 11(4), e0152907. http://dx.doi.org/
10.1371/journal.pone.0152907.
Moldt, J., Pokorny, R., Orth, C., Linne, U., Geisselbrecht, Y., Marahiel, M. A., &
Batschauer, A. (2009). Photoreduction of the folate cofactor in members of the
photolyase family. Journal of Biological Chemistry, 284(32), 21670–21683.
Orsomando, G., de la Garza, R. D., Green, B. J., Peng, M., Rea, P. A., Ryan, T. J., ...
Gregory, J. F. (2005). Plant c-glutamyl hydrolases and folate polyglutamates
characterization, compartmentation, and co-occurrence in vacuoles. Journal of
Biological Chemistry, 280, 28877–28884.
Phillips, K. M., Ruggio, D. M., Ashraf-Khorassani, M., Eitenmiller, R. R., Cho, S., Lemar,
L. E., ... Perry, C. R. (2010). Folic acid content of ready-to-eat cereals determined
by liquid chromatography-mass spectrometry: Comparison to product label
and to values determined by microbiological assay. Cereal Chemistry, 87, 42–49.
Phillips, K. M., Ruggio, D. M., Ashraf-Khorassani, M., & Haytowitz, D. B. (2006).
Difference in folate content of green and red sweet peppers (Capsicum annuum)
determined by liquid chromatographyÀmass spectrometry. Journal of
Agricultural and Food Chemistry, 54, 9998–10002.
Piironen, V., Edelmann, M., Kariluoto, S., & Bed}o, Z. (2008). Folate in wheat
genotypes in the HEALTHGRAIN diversity screen. Journal of Agricultural and Food
Chemistry, 56, 9726–9731.
Quinlivan, E. P., Hanson, A. D., & Gregory, J. F. (2006). The analysis of folate and its
metabolic precursors in biological samples. Analytical Biochemistry, 348,
163–184.
Ramos-Parra, P. A., García-Salinas, C., Hernández-Brenes, C., & Díaz de la Garza, R. O.
I. (2013). Folate levels and polyglutamylation profiles of papaya (Carica papaya
cv. Maradol) during fruit development and ripening. Journal of Agricultural and
Food Chemistry, 61, 3949–3956.
Rébeillé, F., Ravanel, S., Jabrin, S., Douce, R., Storozhenko, S., & Van Der Straeten, D.
(2006). Folates in plants: Biosynthesis, distribution, and enhancement.
Physiologia Plantarum, 126, 330–342.
Reyes-Hernández, B. J., Srivastava, A. C., Ugartechea-Chirino, Y., Shishkova, S.,
Ramos-Parra, P. A., Lira-Ruan, V., ... Díaz de la Garza, R. I. (2014). The root
indeterminacy-to-determinacy developmental switch is operated through a
folate-dependent pathway in Arabidopsis thaliana. New Phytologist, 202,
1223–1236.
Scott, J., Rébeillé, F., & Fletcher, J. (2000). Folic acid and folates: The feasibility for
nutritional enhancement in plant foods. Journal of the Science of Food and
Agriculture, 80, 795–824.
Scott, J. M., Weir, D. G., & Kirke, P. N. (1995). Folate and neural tube defects. Folate in
Health and Disease, 1, 329–360.
Shabalina, S. A., Spiridonov, N. A., & Kashina, A. (2013). Sounds of silence:
Synonymous nucleotides as a key to biological regulation and complexity.
Nucleic Acids Research, 41, 2073–2094.
618 P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619

10. Sharma, S., Tyagi, K., Narasu, M., Sreelakshmi, Y., & Sharma, Rameshwar (2011).
Mismatch cleavage by CEL I endonuclease: A tool for rapid detection of
homozygous and heterozygous mutants. The IUP Journal of Genetics & Evolution,
4, 2.
Shohag, M., Wei, Y.-Y., Yu, N., Zhang, J., Wang, K., Patring, J., ... He, Z.-L. (2011).
Natural variation of folate content and composition in spinach (Spinacia
oleracea) germplasm. Journal of Agricultural and Food Chemistry, 59,
12520–12526.
Sreelakshmi, Y., Gupta, S., Bodanapu, R., Chauhan, V. S., Hanjabam, M., Thomas, S., ...
Mohan, V. (2010). NEATTILL: A simplified procedure for nucleic acid extraction
from arrayed tissue for TILLING and other high-throughput reverse genetic
applications. Plant Methods, 6, 3.
Srivastava, A. C., Ramos-Parra, P. A., Bedair, M., Robledo-Hernández, A. L., Tang, Y.,
Sumner, L. W., ... de la Garza, R. I. D. (2011). The folylpolyglutamate synthetase
plastidial isoform is required for postembryonic root development in
Arabidopsis. Plant Physiology, 155, 1237–1251.
Storozhenko, S., De Brouwer, V., Volckaert, M., Navarrete, O., Blancquaert, D., Zhang,
G. F., ... Lambert, W. (2007). Folate fortification of rice by metabolic engineering.
Nature Biotechnology, 25, 1277–1279.
Strålsjö, L. M., Witthöft, C. M., Sjöholm, I. M., & Jägerstad, M. I. (2003). Folate content
in strawberries (Fragaria  ananassa): Effects of cultivar, ripeness, year of
harvest, storage, and commercial processing. Journal of Agricultural and Food
Chemistry, 51, 128–133.
Tyagi, K., Upadhyaya, P., Sarma, S., Tamboli, V., Sreelakshmi, Y., & Sharma, R. (2015).
High performance liquid chromatography coupled to mass spectrometry for
profiling and quantitative analysis of folate monoglutamates in tomato. Food
Chemistry, 179, 76–84.
Waller, J. C., Akhtar, T. A., Lara-Núñez, A., Gregory, J. F., McQuinn, R. P., Giovannoni, J. J.,
& Hanson, A. D. (2010). Developmental and feedforward control of the expression
of folate biosynthesis genes in tomato fruit. Molecular Plant, 3, 66–77.
Wilson, S., & Horne, D. (1982). Use of glycerol-cryoprotected Lactobacillus casei for
microbiological assay of folic acid. Clinical Chemistry, 28, 1198–1200.
Zhang, G. F., Maudens, K. E., Storozhenko, S., Mortier, K. A., Van Der Straeten, D., &
Lambert, W. E. (2003). Determination of total folate in plant material by
chemical conversion into para-aminobenzoic acid followed by high
performance liquid chromatography combined with on-line postcolumn
derivatization and fluorescence detection. Journal of Agricultural and Food
Chemistry, 51(27), 7872–7878.
Zhou, H. R., Zhang, F. F., Ma, Z. Y., Huang, H. W., Jiang, L., Cai, T., ... Zhu, J.-K. (2013).
Folate polyglutamylation is involved in chromatin silencing by maintaining
global DNA methylation and histone H3K9 dimethylation in Arabidopsis. Plant
Cell, 25, 2545–2559.
P. Upadhyaya et al. / Food Chemistry 217 (2017) 610–619 619