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Acidity has profound effects on the taste of apples (Malus × domestica). Malic acid is the predominant organic acid in apples. Differences in malic acid content are caused by differences in accumulation of malic acid in the vacuole. This accumulation may be caused by a gene that is responsible for transport of malic acid from the cytosol into the vacuole. Here, we provide evidence that a malic acid transporter gene at the top of chromosome 16 caused significant differences in malic acid concentration and pH of apples. The pH of apples in a segregating F1 population was mapped and at the pH locus (named henceforth Ma locus for malic acid), two putative malic acid transporter genes were detected. These genes show high homology to AtALMT genes that code for malate channel proteins located in vacuolar membrane in Arabidopsis. The expression of one of the candidate genes (Ma1) cosegregated clearly with malic acid content. The inheritance of at least one dominant allele of this gene sufficed for an increased expression level that likely caused the observed threefold increase of the malic acid concentration and the reduction of the pH from 4 to 3 in mature apples, compared to the presence of the recessive, lowly expressed allele only. Our results show that differences in fruit acidity were probably caused by differences in expression levels of alleles of a malic acid transporter gene.
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Differences in acidity of apples are probably mainly caused
by a malic acid transporter gene on LG16
Sabaz Ali Khan &Jules Beekwilder &Jan G. Schaart &
Roland Mumm &Jose Miguel Soriano &Evert Jacobsen &
Henk J. Schouten
Received: 5 September 2012 /Accepted: 10 October 2012 / Published online: 30 October 2012
#Springer-Verlag Berlin Heidelberg 2012
Abstract Acidity has profound effects on the taste of apples
(Malus ×domestica). Malic acid is the predominant organic
acid in apples. Differences in malic acid content are caused
by differences in accumulation of malic acid in the vacuole.
This accumulation may be caused by a gene that is respon-
sible for transport of malic acid from the cytosol into the
vacuole. Here, we provide evidence that a malic acid trans-
porter gene at the top of chromosome 16 caused significant
differences in malic acid concentration and pH of apples.
The pH of apples in a segregating F1 population was
mapped and at the pH locus (named henceforth Ma locus for
malic acid), two putative malic acid transporter genes were
detected. These genes show high homology to AtALMT genes
that code for malate channel proteins located in vacuolar
membrane in Arabidopsis. The expression of one of the can-
didate genes (Ma1) cosegregated clearly with malic acid con-
tent. The inheritance of at least one dominant allele of this
gene sufficed for an increased expression level that likely
caused the observed threefold increase of the malic acid
concentration and the reduction of the pH from 4 to 3 in
mature apples, compared to the presence of the recessive,
lowly expressed allele only. Our results show that differences
in fruit acidity were probably caused by differences in expres-
sion levels of alleles of a malic acid transporter gene.
Keywords Acidity .Apple .Malus ×domestica .Malic acid
transporter .Ma1 .pH
Apple (Malus ×domestica Borkh.) is an important fruit crop
of temperate regions. Its consumption is global and avail-
able throughout the year. The major part of the production is
consumed as fresh fruit, while a minor part is processed into
juices, concentrates, and purees. Apple represents a major
source of dietary antioxidants (Lee et al. 2003).
Acidity has profound effects on the perception of apple
fruit quality. A proper balance between sugar and acid
content is, therefore, important for a desired apple variety
(Visser et al. 1968). Malic acid is the predominant acid in
many fruits, especially in apple (Wu et al. 2007; Zhang et al.
2010). In apple approximately 80 to 90 % of organic acids is
malic acid (Nour et al. 2010). It was first isolated in 1785 by
Carl Wilhelm Scheele from apple juice (Jensen 2007). Malic
acid contributes to the sourness of fruits and is the source of
extreme tartness in confectionery. Malic acid added to food
products is denoted by E number E296 (
Malic acid is also known as carboxylic diacid. It has two
stereoisomeric forms (L- and D-enantiomers), though only
the L-isomer exists naturally (Yamamoto et al. 2001). The
salts and esters of malic acid are known as malates. Malate
is the predominant form of malic acid in many fresh fruits
(Yao et al. 2011).
Communicated by A. Dandekar
Electronic supplementary material The online version of this article
(doi:10.1007/s11295-012-0571-y) contains supplementary material,
which is available to authorized users.
S. A. Khan :J. G. Schaart :J. M. Soriano :E. Jacobsen :
H. J. Schouten (*)
Wageningen University and Research Centre, Plant Breeding,
P.O. Box 386, 6700 AJ Wageningen, The Netherlands
J. Beekwilder :R. Mumm
Wageningen University and Research Centre, Plant Research
International, Business Unit Bioscience,
P.O. Box 16, 6700 AA Wageningen, The Netherlands
J. M. Soriano
Inova Fruit BV,
Postbus 222, 4190 CE Geldermalsen, The Netherlands
Tree Genetics & Genomes (2013) 9:475487
DOI 10.1007/s11295-012-0571-y
Malate has a variety of important functions in plant life. It
has been defined as an essential storage carbon molecule
(Martinoia and Rentsch 1994), an intermediate in the tricar-
boxylic acid (TCA) cycle (Fernie et al. 2004), and a pH
regulator (Mathieu et al. 1986), and it also has control over
stomatal function (Lee et al. 2008).
Malic acid, in the form of its anion malate, occurs in all
metabolizing cells, as a key intermediate in the major
energy-producing biochemical pathway, known as the citric
acid cycle or Krebs cycle that occurs in the mitochondria. It
is also a part of the cytosolic pyruvate metabolism as an
intermediate between oxaloacetate and acetyl CoA. Both the
citric acid cycle and the pyruvate metabolism have exten-
sively been studied, and the enzymes involved in metabo-
lism of malate are well known. For example, PEPC is
considered to be an important enzyme involved in fruit
malic acid synthesis, while cyME is indicated as the key
enzyme in malic acid degradation (Berüter 2004; Yao et al.
2009). Yao et al. (2011) cloned the apple gene MdcyMDH,
encoding the cyMDH enzyme that catalyzes the reaction
from oxalacetic acid to malate. The expression level of this
gene was positively correlated with malate dehydrogenase
(MDH) activity throughout the fruit development, but not
with malate content (Yao et al. 2011). The activity of the
citric acid cycle is regulated based on requirements of ener-
gy, rather than on requirements of fruit acidity. In fact,
acidity of apples is considered to be determined by storage
of malate in the vacuole (Ulrich 1970). A detailed biochem-
ical analysis of two genetic variants of the apple variety
Usterapfelrevealed that transport of malic acid into the
vacuole plays an important role in malic acid accumulation
(Berüter 2004).
Acidity of apples is mainly caused by a single gene
(Brown and Harvey 1971; Nybom 1959;Visseretal.
1968; Visser and Verhaegh 1978). Maliepaard et al. (1998)
measured the pH of apples of a segregating F1 population
from the cross of apple cultivars Primaand Fiestaand
mapped pH as a monogenic trait at the top of linkage
group 16 (LG16). Maliepaard et al. named this map
position Ma locus. Although they had not measured or
mapped the malic acid content but just the pH, they
assumed that the segregation of pH was caused by
differences in malic acid content and, therefore, named
the genetic position as Ma locus. We used this population
as a starting point and added markers at this locus for
mapping the pH more precisely. We combined this
mapping information with the published genome se-
quence of apple (Velasco et al. 2010)fordetectionof
candidate genes at the Ma locus. We measured the
expression of these candidate genes in F1 progeny and
found a clear relationship between expression levels of
alleles of the putative apple malic acid transporter gene
(Ma1), malic acid content, and acidity of the apples. This
indicates that this gene caused the genetic segregation
of pH.
Materials and methods
Genetic mapping of the Ma locus
Maliepaard et al. (1998) were the first to construct genetic
linkage maps for apple, covering all 17 chromosomes. They
used a segregating F1 population of 164 genotypes from the
cross Prima×Fiesta.They measured the pH of mature
fruits of these progeny using bromocresol and pH indicator
paper (Maliepaard et al. 1998). The pH appeared to segre-
gate as a monogenic trait in a 3:1 fashion, and both parents
appeared to be heterozygous for the dominant trait for high
acidity (low pH). We improved the genetic linkage maps of
this population by adding DArT and simple sequence
repeats (SSR) markers (Schouten et al. 2011; Khan et al.
2012). Using the pH data from Maliepaard with the im-
proved genetic linkage maps, we mapped the pH again on
LG16 using JoinMap® 4.0 (Van Ooijn 2009).
pH was scored as a dominant marker. Both parents were
heterozygous for the Ma locus. Progeny that had a low pH
had received one dominant allele from Primaor one dom-
inant allele from Fiesta,or the dominant alleles from both
parents. From the pH data only, it could not be judged which
dominant allele(s) was inherited. Due to this uncertainty,
only progeny with high pH were used for mapping; as for
these progeny plants, it was clear that they inherited from
both parents the recessive alleles. This reduced the number
of progeny genotypes that could be used for mapping, and
therewith, this reduced the mapping accuracy. In spite of this,
a genetic window for the Ma locus could be drawn on LG16
using graphical genotyping. The cv. Golden Deliciousge-
nome sequence information was used (Velasco et al. 2010)to
find candidate genes for the pH in this genetic window.
Plant material for measurement of malic acid content
and expression of candidate genes
Progeny genotypes of the F1 segregating population from
the Prima×Fiestacross were selected based on their
genotype at the Ma locus. The genotypes selected for sam-
pling of fruits are shown in Table S1. Each genotype was
represented by two trees. These trees were located in the
experimental station, Randwijk, The Netherlands. In addi-
tion, both parents (Primaand Fiesta) of the population
were also included.
The progeny and parents were at full bloom from 26
April, 2010. Fruits were sampled at three developmen-
tal stages, i.e., 34 days after full bloom (DAFB), 60 DAFB,
and 95 DAFB. Per tree, eight fruits were sampled, giving 16
476 Tree Genetics & Genomes (2013) 9:475487
fruits per genotype. Fruits from different sides of each
individual tree were harvested to level out the effects of
the environmental factors such as sunlight during fruit de-
velopment. Fruits were peeled off, cut into small pieces, and
frozen in liquid nitrogen just after harvesting according to
method described by Khan et al. (2012). Care was taken to
exclude the seeds of the fruits. The knife was cleaned with
water and dried up with tissue paper before every next fruit.
Peel and flesh were separately put into polythene bags, then
shock-frozen in liquid nitrogen, and stored on dry ice. Once
all fruits were harvested, they were transferred to the labo-
ratory and stored at 80 °C.
The sizes (diameter) of the individual fruits were mea-
sured at each developmental stage using an electronic digital
caliper, model VWRI819-0012 (Control Company,
Friendswood, TX, USA). The average size at 34 DAFB
was 24 mm, at 60 DAFB, 40 mm, and at 95 DAFB,
62 mm (Table S1). The harvested fruit material was ground
as explained by Khan et al. (2012). This led to one sample
pertreepertissueperdevelopmental stage and to two
replicates (two trees) per genotype. The powder was collect-
ed in falcon tubes of 50 ml volume and stored immediately
at 80 °C in a freezer before any further processing.
In addition, mature fruits were included for malic acid
measurement. These fruits were harvested and stored in
autumn 2008, as described by Khan et al. (2012). For this
mature stage, fruits from the two individual trees per genotype
(four apples from each tree) were pooled before grinding to
make one representative sample of eight apples per genotype.
We used, for expression studies, fruits from 34, 60, and 95
DAFB. Malic acid was measured in fruits at 60 and 95 DAFB
and at mature stage, but not at 34 DAFB, as fruits at this stage
were too small for malic acid measurement.
Measurement of malic acid content
Extracts of flesh samples were prepared from the ground
powder according to a modified protocol by Lisec et al.
(2006). First, 250 mg frozen apple powder was transferred
to 2-ml cryocooled Eppendorf tubes, where 750 μl of meth-
anol including 25 μl internal standard (ribitol 0.5 mg/ml)
was added. Samples were briefly vortexed, sonified for
30 min, and centrifuged for 10 min at 21,000 rpm. Four
hundred microliters of the supernatant was transferred to a
new Eppendorf tube and 600 μldH
O and 300 μl chloro-
form were added. Thereafter, samples were again vortexed
for 20 s and centrifuged for 10 min at 21,000 rpm. Forty
microliters of the upper polar phase was then transferred to a
1.5-ml crimp neck glass vial containing a 0.1 ml micro-
insert. The samples were dried overnight in a SpeedVac®
concentrator (Thermo Scientific Savant SPD121P, Thermo
Scientific, Asheville, NC, USA). Samples were then capped
under an argon atmosphere.
The dried samples were derivatized online as described
by Lisec et al. (2006). First, 12.5 μl methoxyamine hydro-
chloride (20 mg/ml pyridine) was added to the samples and
incubated for 30 min at 40 °C with agitation. Then, the
samples were derivatized with 17.5 μlMSTFA(N-methyl-
N-trimethylsilyltrifluoroacetamide) for 60 min. An alkane
mixture was added to determine retention indices of
The derivatized samples were analyzed by a gas chroma-
tographytime-of-flight mass spectrometry (GCTOFMS)
system consisting of a Combi PAL autosampler (CTC
Analytics AG), an Optic 3 high performance injector
(ATAS GL International, Eindhoven, The Netherlands) and
an Agilent 6890 gas chromatograph (Agilent Technologies,
Amstelveen, The Netherlands) coupled to a Pegasus III
time-of-flight mass spectrometer (LECO, St. Joseph, MI,
USA). Two microliters of each probe was introduced into
the injector at a split flow of 20 ml min
, and a transfer
column flow of 1 mlmin
was used. The chromatographic
separation was performed using a ZB-5 capillary column
(30 m×0.32 mm × 0.25 μm), (Phenomenex, Utrecht, The
Netherlands) including a 10-m guardian column with heli-
um as carrier gas at a column flow rate of 1 ml min
. The
GC program started at 70 °C for 2 min, then rose by 10 °C
to 310 °C, and was maintained at this temperature for
5 min. The GC transfer line temperature was set to 270 °C.
The column effluent was ionized by electron impact at 70e
V. Mass spectra were acquired at 20 scans/s within a mass
range of m/z50 to 600, at a source temperature of 200 °C.
Raw data were processed by ChromaTOF software 2.0
(LECO, St. Joseph, MI, USA) and further by using
MetAlign software (Lommen 2009) to extract and align the
mass signals (s/n3). Mass signals that were below s/nof 3
were randomized between 2.4 and three times the calculated
noise value. Mass signals that were present in less than four
samples were discarded. Malic acid and the internal standard
ribitol were identified by comparing the retention time and the
mass spectra with those of authentic reference standards
(Sigma-Aldrich, St. Louis, MO, USA). The abundance of
malic acid was normalized to ribitol based on m/z133.
Expression analysis of the pH candidate genes
RNA isolation and cDNA synthesis
The expression profiles of the two candidate genes in the
genetic window were compared for low pH progeny, on the
one side, and high pH progeny, on the other side. In addi-
tion, expression profile of a third apple gene Cytosolic
malate dehydrogenase (MdcyMDH GenBank accession no.
DQ221207) was also studied. This gene is involved in the
synthesis of malate in cytosol of the plant cell (Yao et al.
2011) and is located on LG17 of apple. For the expression
Tree Genetics & Genomes (2013) 9:475487 477
analysis, total RNA was isolated from flesh of apple using
the method described by Asif et al. (2000) of the genotypes
mentioned in Table S1at developmental stages of 34, 60,
and 95 DAFB. At mature stage, the RNA was not of suffi-
cient quality to perform reliably quantitative real-time poly-
merase chain reaction (qRT-PCR). Each RNA sample
represented one developmental stage for one tree. The
RNA quantity was measured on a NanoDrop® ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington,
DE, USA). Further, the RNA quality and quantity were
evaluated by running 2 μl of the RNA sample on 1.5 %
agarose gel. Single-strand complementary DNA (cDNA) was
synthesized using iScriptcDNA Synthesis Kit (Bio-Rad,
Hercules, CA, USA) according to the manufacturer's manual.
Designing and testing of primers for qRT-PCR
For qRT-PCR, primers were designed for each candidate
gene using the online available primer-designing pro-
gram Primer3Plus (
primer3plus/primer3plus.cgi). The primer names, sequen-
ces, and amplicon sizes are given in Table 1.Primerswere
tested using qRT-PCR with iQ SYBR® green supermix
(Bio-Rad, Hercules, CA, USA). Two microliters of
cDNA, ten times diluted of the original cDNA obtained
with iScriptcDNA Synthesis Kit (Bio-Rad, Hercules,
CA, USA), was used in the reaction. PCR reactions were
performed in a C1000 Thermal Cycler, CFX96 Real-Time
System (Bio-Rad, Hercules, CA, USA). The PCR program
60 °C for 1 min, finishing with 95 °C for 10 s. The PCR
products were checked for quality by checking their clear
single peak in the melting curve and a clear band of the
expected amplicon size on 1.5 % agarose gel.
Checking the annealing sites of the primers
A normal PCR using genomic DNA from leaves was per-
formed to check the annealing of both qRT-PCR forward
and reverse primers in the two haplotypes of the progeny,
i.e., Ma and ma. For this purpose, two different individuals
belonging to each genotype class were used: Primaand
Fiesta(Mama), two genotypes from the F1 population, i.e.,
1988-001-014and 1988-001-015(MaMa)and1988-
001-02and 1988-001-03(mama). This confirmed the
amplification in both haplotypes of the gene (Ma and ma).
Possible differences in annealing of the qRT-PCR primers
on the two alleles of Ma1 and Ma2 could erroneously
suggest differences in expression levels of the alleles. In
order to check the annealing sites, PCR reactions were
performed in a GeneAmp® PCR System 9700 thermal cy-
cler (PerkinElmer, Freemont, CA, USA) in a final volume of
20 μl, containing 75 mM TrisHCl, pH 8.8; 20 mM
; 0.2 mM of each dNTP;
0.05 μM of each primer; 10 ng of genomic DNA; and
0.3 U of Super Taq DNA polymerase (HT Biotechnology,
Cambridge, UK). PCR products for both genes were se-
quenced at Greenomics (Wageningen, The Netherlands).
These obtained results confirmed the annealing of the
qRT-PCR primers in both haplotypes (data not shown).
The alignment of the sequenced product against the apple
genome sequence confirmed the presence of this fragment in
the Ma1 gene on LG16.
Additionally, different pairs of primers were developed
along Ma1 to check allele-specific information for Ma and
ma. PCR reactions and sequencing were performed as previ-
ously described. Information of primer sequences and single
nucleotide polymorphisms are given in Table S2and Fig. S1.
Performing qRT-PCR
The gene expression was measured for 216 cDNA samples.
As we studied simultaneously the expression profiles of 42
genes related to the phenylpropanoid pathway for the same
cDNA samples, we used the BioMarkSystem (Fluidigm,
San Francisco, CA, USA) (
Three 96.96 Dynamic Arrays of Integrated Fluidic Circuits
were used, comprising 48 primer pairs in two replicates. The
expression profiles of the genes related to the phenylpropa-
noid pathway will be reported elsewhere.
Table 1 Names, putative functions, and primer sequences of candidate genes used in qRT-PCR
Gene name Gene ID Gene function Forward primer
Reverse primer
size (bp)
Ma1 MDP0000252114
Malic acid
Ma2 MDP0000244249
Malic acid
MdcyMDH DQ221207
malate dehydrogenase
Derived from cv. Golden Deliciousgbrowse
GenBank accession number
478 Tree Genetics & Genomes (2013) 9:475487
Three reference genes were included for normalization
of the expression of target genes. These three genes were Actin
(GenBank accession DT002474), MdUBQ (GenBank accession
U74358), and mGAPDH (GenBank accession CN494000).
Eventually, only GAPDH (GenBank accession CN494000)
was used as reference gene, based on overall performance.
The Ct values for the candidate genes were normalized using
this reference gene, giving ΔCt values.
A pooled sample containing cDNA from all the samples
was used as reference sample. The expression of a gene in a
target sample was compared with the expression of that gene
in the pooled sample, giving ΔΔCt values. We named this as
relative expression of the target genes.
A dilution series of the reference sample was also included
which allowed calibration for each individual primer combi-
nation. The serial dilutions were as 1, 1/4th, 1/16th, 1/64th,
and 1/256th of the original sample concentration used.
PCR was performed using the following temperature pro-
file: 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 50 °C
for 30 s, and 72 °C for 1 min, finishing with 72 °C for 10 min.
PCR products were stained with GelRed(Biotium,
Hayward, CA, USA) and visualized under UV light. The
molecular sizes of the amplified fragments were estimated
by comparison with a 100-bp ladder.
Analysis of qRT-PCR data
The qRT-PCR data were analyzed using the Fluidigm®
Real-Time PCR Analysis Software version 3 (http:// Two main analyses were made, i.e.,
melting curve analysis and ΔΔCt values determination.
Subsequent analyses were performed using Microsoft Office
Excel 2010 and IBM SPSS Statistics.
Homology study between apple and Arabidopsis malate
transporter genes
To compare and study the evolutionary relationship of the
apple malate transporter (ALMT) genes and Arabidopsis
ALMT genes, a phylogenetic tree was constructed.
MetAlign of DNASTAR version 8.1 was used to construct
the phylogenetic tree using Kimura's method (Kimura 1980).
First, the protein sequences were aligned by ClustalW method
and then the bootstrapping analyses were performed using
1,000 boost trials.
Ma locus on LG16
The pH of the mature apples of the F1 progeny from the
Prima×Fiestacross showed a clear segregation as a
monogenic trait in a 3:1 ratio (Fig. 1). Maliepaard et al.
(1998) mapped this pH genetically on LG16 of apple. We
mapped the pH more precisely with JoinMap® 4.0 (Van
Ooijn 2009) by including additional markers (Schouten et
al. 2011; Khan et al. 2012). The results of this mapping are
shown in Fig. 2a, b.
Based on the genetic markers on LG16, the F1 progeny
could be divided into three genetic groups: one group of
progeny had inherited one copy of the dominant allele from
each parent for malic acid, giving homozygous dominant
genotypes (MaMa). The second group inherited one copy of
the dominant allele from one parent and one copy of the
recessive allele from the other parent, leading to heterozy-
gous genotypes (Mama). The third group represents the
homozygous recessive genotypes, lacking the dominant al-
lele (mama). Both parents are heterozygous. Figure 1shows
clearly that the genotypes that had one or two copies of the
dominant allele had a lower pH compared to the mama
The homozygous dominant progeny (MaMa) had, on the
average, a slightly lower pH compared to the heterozygous
progeny (Mama) (Fig. 1), but this difference was insuffi-
cient to distinguish the MaMa genotypes from the Mama
genotypes using the pH values only. Using graphical geno-
typing, the genetic window for the Ma locus was con-
structed for both parents (Fig. 2a, b).
Malic acid during apple fruit development
Although Wu et al. (2007) and Zhang et al. (2010) already
showed that malic acid is the main determinant of pH of
Fig. 1 Histogram of the F1 segregating population of Prima×
Fiestacross, sorted to pH of the ripe apple fruits. By means of genetic
markers at the top of linkage group 16 (LG16), the progeny has been
grouped into three classes, i.e., progeny that is homozygous for the
dominant allele for low pH (MaMa), heterozygous (Mama), and ho-
mozygous recessive (mama). Both parents are heterozygous. The three
genotype classes show a 3:1 Mendelian segregation. The mean pH
values of the three genotype classes differed significantly from one
another (ANOVA, LSD test, P<0.01), even when comparing the
MaMa group with the Mama group. However, on the basis of the pH
values only, it was not possible to distinguish MaMa genotypes from
Mama genotypes
Tree Genetics & Genomes (2013) 9:475487 479
apples, we wanted to confirm that the genetic segregation of
pH was caused by malic acid. Therefore, we measured malic
acid content in apple flesh at three different developmental
stages (Table S1)usingGCTOFMS. The results showed that
malic acid was clearly detectable in the apple flesh samples.
The mama group, lacking the dominant allele for acidity,
had lower levels of malic acid, as compared to the Mama
and MaMa groups (Fig. 3). The genetic segregation for
pH clearly resembled the genetic segregation for malic
Ch05e04z Ch05e04z
2498 2498
Marker name
Marker name
Physical position on ‘Golden
Delicious’ (kbp)
Physical position on ‘Golden
Delicious’ (kbp)
0 0
1 4
2 0 0
0 2 2
2 3
3 0
‘Golden Delicious’
Marker/gene name
Physical map (kbp)
‘Golden Delicious’
Marker/gene name
Physical map (kbp)
Fig. 2 Genetic window of the Ma locus on LG16 for both heterozy-
gous parents; aPrimaand bFiesta,the physical distances between
the markers on the sequenced genome of cv. Golden Deliciousare
given in c. The two putative malic acid transporter genes are given in
bold text. These genes reside at the middle of the genetic windows of
both parents. dThe fine mapping of the Ma locus on LG16 according
to Xu et al. (2011). The markers from Xu et al. (2011) are given in
italics. This fine mapping further confirms the position of our candi-
date malate transporter genes, i.e., Ma1 and Ma2 in the pH locus. (Xu
et al. 2011)
480 Tree Genetics & Genomes (2013) 9:475487
The level of malic acid at the unripe stages was rather
high as compared to the mature stage (Fig. 3; Table S1).
This means that during maturation, the acidity of the apples
decreased. Apple genotypes that were sour at the ripe stage
(Mama and MaMa genotypes) already contained more malic
acid at the earlier developmental stages compared to the
mama genotypes (Fig. 3). The sourness of these genotypes
was not the result of a slower breakdown of malic acid
during ripening but was a result of higher levels of malic
acid during the whole period of fruit development.
Two putative malic acid transporters were detected
in the genetic window of the Ma locus on LG16
The genetic windows of the Ma locus on LG16 were screened
for candidate genes using the sequence information of cv.
Golden Delicious(Velasco et al. 2010). Two candidate genes
were detected that may be responsible for the segregation of the
pH of the mature apple fruits (Table S3). These two candidate
genes are putatively annotated as malic acid transporters in the
cv. Golden Deliciousgenome browse annotation (http://
on this website as MDP0000244249 and MDP0000252114. In
view of similarity of these putative genes to the Arabidopsis
malic acid transporter genes, which are named AtALMT genes
(Kobayashi et al. 2007), we named these candidate genes in
M. ×domestica as Ma1 and Ma2 (Figs. 2and 6,Table1). No
structural genes for malic acid biosynthesis or degradation were
detected within these genetic windows (Table S3).
Only Ma1 showed positive effects on malic acid content
Figure 4shows the relationships between the expression of
the two putative malic acid transporter genes at the Ma
locus, i.e., Ma1 and Ma2, on one side, and malic acid
content, on the other side. No correlation could be
found between the gene expression and malic acid con-
tent for Ma2 (Fig. 4;r00.01; P00.94). However, the
relative expression of Ma1 showed a positive correlation
with the malic acid content (Fig. 4;r00.42; P00.004).
The mama genotypes that lack the dominant allele
showed a low expression of Ma1 and also low malic
acid content (Fig. 4).
The genotypes with one or two copies of the dominant
allele (Mama,MaMa) showed variable but higher expres-
sion levels and also higher levels of malic acid. This sug-
gests that Ma1 was responsible for the increased level of
malic acid in apple fruits.
Fig. 3 Malic acid content of apples at three different developmental
stages, i.e., 60 and 95 days after full bloom (DAFB) and at full maturity
of the fruits. The three genotype classes (MaMa,Mama, and mama) are
explained in Fig. 1. The malic acid content was normalized to ribitol.
The error bars refer to the standard deviations of the means and are
based on eight to 20 measurements per genotype class per develop-
mental stage, as shown in Table S1. The mama group was significantly
lower than the other two genotype groups. The Mama group did not
differ significantly from the MaMa group, but in the last stage, the LSD
test showed a statistically weak difference (P00.09)
Malic acid relative intensity
Malic acid relative intensity
0.0 0.
Malic acid relative intensity
.2 0.4 0.6 0
0.8 1.0 1.2
ative gene
lative gen
1.4 1.6 1.8
e expressio
e express
.4 2.6
ama Ma
ama Ma
ama Ma
ama Ma
Fig. 4 Relationships between gene expression and malic acid content
of three candidate genes at two different stages of apple fruit develop-
ment for three genotype classes (mama,Mama,andMaMa). The
different colors show different developmental stages as shown in the
legend. The malic acid content is normalized to ribitol
Tree Genetics & Genomes (2013) 9:475487 481
A third gene MdcyMDH (cytosolic malate dehydrogenase)
was included in the expression analysis. This gene was not in
the genetic window of Ma locus and rather located on LG17
but has been reported as a gene that is critical for malic acid
biosynthesis in apple (Yao et al. 2011). The expression of this
structural gene showed no significant correlation with malic
acid content (Fig. 4;r00.25; P00.14).
Relative expression of candidate genes Ma1,Ma2,
and MdcyMDH at three developmental stages
The relative expression of the three genes at three different
developmental stages is shown in Fig. 5. The mama class of
Ma1 showed lower expression at all three stages compared
to both the Mama and MaMa classes. The expression of the
MaMa group was, in general, the highest. For the other two
genes, i.e., Ma2 and MdcyMDH, no clear correlation was
found between the relative expression and pH genotype
groups (Fig. 5).
The apple genome has 27 putative malic acid transporters
Alignment of protein sequences of Arabidopsis (AtALMT
proteins: i.e., AtALMT1 (At1g08430) Hoekenga et al.
2006), AtALMT6 (Q9SHM1) (Hoekenga et al. 2006),
AtALMT9 (Q9LS46; Kovermann et al. 2007) to the protein
sequences of apple using online BLAST software (http:// indicated the
presence of 27 putative ALMT proteins in apple. We con-
structed a phylogenetic tree for these proteins, together with
AtALMT6, AtALMT9, petunia PH5 (a P-Type H
DQ334807; Verweij et al. 2008), and apple AttDT
(Q8LG88; Hurth et al. 2005)usingtheMegAlignof
DNASTAR version 8.1, according to the Kimura method
(Kimura 1980). We found that Arabidopsis AtALMT6 and
AtALMT9 have good homology with our candidate genes
Ma1 and Ma2 (Fig. 6).
Genetic position of the pH gene
Maliepaard et al. (1998) genetically mapped the pH of mature
apples using the segregating F1 population of Prima×
Fiestacross. They mapped it at the top of LG16. Liebhard
et al. (2003) found the same location for pH in a segregating
F1 population from a cross between other apple cultivars. We
used the same F1 population as Maliepaard et al. used. Since
the paper of Maliepaard et al., many other DNA markers were
added to the genetic map of this population (Schouten et al.
2011). We used this denser genetic map and further added
SSR markers that are located near the Ma locus on LG16
(Khan et al. 2012). This allowed us to map the Ma locus more
precisely. We focused on the pH of mature fruits, as the mature
stage is most relevant to eating quality. The pH appeared to
segregate very clearly (Fig. 1). The χ
tests for 1:1 and 3:1
segregation gave Pvalues of 0.000 and 0.21, which indicates
that the segregation deviated significantly from a 1:1 segrega-
tion and resembled a 3:1 segregation. The Ma locus could be
mapped as a monogenic trait at the top of LG16 (Maliepaard
et al. 1998).
Graphical genotyping was applied for setting the borders
of the genetic interval in which the gene that is responsible
for the segregation of the pH should reside (Fig. 2). As both
parents are heterozygous for the dominant allele for high
acidity, genetic windows could be made for both parents using
the pH values and genetic markers of the F1 population. The
Relative gene expression
Developmental stages
Ma1 mama
Relative gene expression
Developmental stages
Fig. 5 The expression of three candidate genes for the three different
pH genotype classes at three different stages of the fruits. The expres-
sion of a gene in a target sample was normalized for the expression of
the reference gene GAPDH in that sample and compared with the
expression in a pooled sample, giving the relative gene expression
482 Tree Genetics & Genomes (2013) 9:475487
genetic windows appeared to overlap to a large extent (Fig. 2).
As explained in Materials and methods,only a part of the F1
segregating population could be used for genetic mapping due
to the dominant inheritance in a 3:1 ratio.
Recently, Xu et al. (2011) mapped the pH and titratable
acidity of apple fruits of two populations that are not directly
related to the population we used. They found the same
major Ma locus on LG16. By developing and mapping eight
additional SSRs, they fine-mapped the Ma locus to a 150-kb
physical region on the published genome of apple cv.
Golden Delicious.This genetic window appeared to be
inside the window that we detected (Fig. 2).
The dominant allele for acidity increased the malic acid
content by a factor of three
The F1 population could be grouped into three classes, i.e.,
genotypes that were homozygous for the dominant allele for
lowpH(MaMa), the heterozygous group (Mama), and
genotypes that lacked the dominant allele (mama) and had,
therefore, a low acidity and high pH. From each of these
three groups, trees were selected for picking of apples at
different developmental stages. The apples were peeled off,
and the malic acid content in the apple flesh was measured.
The data confirmed the supposition from Maliepaard et al.
(1998) that malic acid was the important organic acid that
was responsible for the segregation of pH. Maliepaard et al.
assumed that malic acid was the organic acid that was
responsible for the clear differences in pH of the mature
fruits and, therefore, named this locus Ma (Maliepaard et al.
1998). However, they did not measure the malic acid levels
of the progeny but solely the pH.
The genotypes that had one or two copies of the domi-
nant allele (Mama,MaMa) and a pH of approximately 3.0
had three times higher malic acid content compared to the
mama genotypes that had a pH of approximately 4.0. This is
in agreement with several other investigations that indicated
that malic acid is the major organic acid in apples (Nour et
al. 2010; Wu et al. 2007; Zhang et al. 2010) and other crops
(review by Sweetman et al. 2009 and the references therein).
This suggests that the gene that was responsible for the
segregation of pH was indeed a gene that raised the malic
acid content strongly. The inheritance of the dominant allele
of this gene, Ma, coincided with an increase of the malic
acid content by a factor 3, compared to the inheritance of the
recessive allele ma. The addition of a second copy of the
Fig. 6 Phylogenetic tree of the
protein AtALMT1 (At1g08430)
in Arabidopsis, AtALMT6
(Q9LS46), petunia PH5 (a P-
Type H
-ATPase; DQ334807),
apple AttDT (Q8LG88), and 27
putative malic acid transporter
proteins in apple. The two
genes that reside at the genetic
locus for pH of ripe apples were
named as Ma1 and Ma2. The
numbers at each node represent
the bootstrap support values
(out of 1,000 replicates). The
units at the bottom represent the
number of substitution events
Tree Genetics & Genomes (2013) 9:475487 483
dominant allele slightly raised the malic acid content further
and, therewith, slightly lowered the pH, as can be seen in
Fig. 1, when comparing the Mama group with the MaMa
Two putative malic acid transporter genes reside
at the Ma locus
Thanks to the published genome sequence of the apple cv.
Golden Delicious(Velasco et al. 2010), we could align the
genetic markers at the locus on LG16 to the genome se-
quence and could scan the genetic windows for genes that
could be responsible for the differences in malic acid con-
tent and, therewith, pH (Table S3). No structural genes for
malic acid production or degradation were detected in these
genetic windows (Table S3). However, two putative malic
acid transporter genes appeared to be present at the centers
of the genetic windows for both parents (Fig. 2). These
genes show homology to the AtALMT malic acid transporter
gene family in Arabidopsis (Fig. 6).
Our findings are supported by the recent fine-mapping
results from Xu et al. (2011), as both candidate genes
appeared to reside in their smaller genetic region (Fig. 2).
Xu et al. (2011) found 44 putative genes in this region.
Although Ma1 and Ma2 are among these 44 genes, Xu et
al. (2011) did not mention these two genes as serious can-
didates. However, in a very recent paper, Bai et al. (2012)
describe a significant correlation between transcript abun-
dance for Ma1 and acidity of apples. This resembles our
findings, although Bai et al. used other parental plants.
Moreover, they sequenced alleles of Ma1 and detected a
premature stop codon in low acidity alleles. This stop codon
was not present in the high acidity alleles. This mutation
may be responsible for the abolished function of the low
acidity alleles (Bai et al. 2012). This would indicate that not
(only) the differences in transcript abundances of the alleles
of Ma1 may explain the segregation, but also that the differ-
ences in efficiency of the Ma1 allelic proteins may explain
the segregation.
The two candidate proteins show high homology to AtALMT
proteins that are located on vacuolar membrane
An ALuminium tolerance Malate Transporter gene (ALMT)
was first reported to have a function in the tolerance to toxic
aluminium in wheat (Sasaki et al. 2004). This followed the
idea that excretion of carboxylates results in chelation of Al
and consequently, rhizo deposition, preventing the toxic
cation from being taken up by the plant, as explained in
the review by Fernie and Martinoia et al. (2009). Later,
homologous genes were found in Arabidopsis (Hoekenga
et al. 2006), in Brassica napus (Ligaba et al. 2006), and rye
(Fontecha et al. 2007). Meyer et al. (2011) demonstrated
recently that AtALMT6 is a channel for transport of malate
into the vacuole.
Figure 6shows that the two candidate genes that we
detected at the Ma locus code for proteins that are highly
homologous to the Arabidopsis proteins AtALMT6 and
AtALMT9, but less to AtALMT1, and petunia PH5 (a P-
Type H
-ATPase). This makes sense as both the AtALMT6
and AtALMT9 are located on vacuolar membrane, but
AtALMT1 is located on cytoplasmic membrane (Kovermann
et al. 2007; Meyer et al. 2011; Hoekenga et al. 2006). In view
of this similarity, the candidate proteins probably are
located in vacuolar membrane, rather than on cytoplas-
mic membrane. This provides further evidence that these
are interesting candidates for malate accumulation in the
Figure 6shows that in the clade that harbors AtALMT6
and AtALMT9, there are other putative ALMT-like proteins
of apple. The underlying genes are located on LGs 6, 13,
and 14 and may be candidates for minor quantitative trait
loci (QTLs) for pH detected at other genomic regions (Xu et
al. 2011). The other ALMT-like proteins in apple are less
homologous to AtALMT6 and AtALMT9 and are less like-
ly candidates for malate accumulation in the vacuole.
The expression of one of these malic acid transporter genes
may be the limiting factor for the acidity of the fruits
The expression of both candidate genes was measured in the
three groups of genotypes. Ma1 appeared to have a low
transcript level in the mama group, a moderate expression
in the Mama group, and a high expression in the MaMa
group, while Ma2 did not show a clear differential expres-
sion among the three groups (Figs. 4and 5). This provides
evidence that Ma1 is the gene responsible for the segrega-
tion of pH in the F1 progeny.
The recessive allele of Ma1 is expressed at a low level
(Fig. 5) and is associated with a low level of malic acid
Fig. 7 The relationship between acidity of mature fruits of the F1
population and overall liking of these fruits in sensory evaluations,
derived from Maliepaard et al. (1998) and King et al. (2001)
484 Tree Genetics & Genomes (2013) 9:475487
(Fig. 5). The dominant allele is expressed more strongly
(Fig. 5), which may explain the increase in malic acid
content by a factor 3 (Fig. 3). Addition of a second copy
of the dominant allele further increased the transcript level
(Fig. 5) but raised the malic acid content only slightly and,
therewith, reduced the pH to a low extent (Figs. 1and 3).
This indicates that the expression level of Ma1 was the
limiting factor in the acidity of the fruits and that presence
of one copy of the higher expressed allele Ma was sufficient
to remove this limitation.
Genes for synthesis of malic acid are not associated with pH
Malic acid is an intermediate in the citric acid cycle, also
known as the tricarboxylic acid cycle or the Krebs cycle,
which is of central importance in all living cells. In eukaryotic
cells, the citric acid cycle occurs in the matrix of the mito-
chondrion. Yao et al. (2011) isolated and functionally ana-
lyzed the Malate dehydrogenase gene (MdcyMDH)from
apple. The enzyme malate dehydrogenase (MDH) is crucial
for malate synthesis in the cytosol. They localized the protein
in the cytoplasm and plasma membrane. The expression level
of MdcyMDH was positively correlated with MDH activity
throughout fruit development, but not with malate content. We
also found that MdcyMDH expression was not associated with
malic acid content (Figs. 4and 5). Further, this gene is not
located at the Ma locus on LG16, but on LG17, as detected by
aligning the DNA sequence to the published sequence of cv.
Golden Delicious.
Sweetman et al. (2009) have indicated that PEPC activity,
while linked to the synthesis of malate in normal acid
varieties, cannot explain the variation in malate levels seen
in the low acid fruits nor could NAD-MDH and NADP-ME
activities. Berüter (2004) studied the carbohydrate metabo-
lism in an apple variety and its mutant that differed in fruit
acidity. The high-acid genotype had five times more malic
acid compared to the low-acid genotype. However, Berüter
(2004) detected no difference in the catalytic activity of
enzymes involved in malate metabolism, such as PEP carbox-
ylase, MDH, and NADP malic enzyme. The rate of respiration
and the rate of malate synthesis were similar in both geno-
types. However, the uptake of
C malate was significantly
lower in excised tissue of lower acid fruit. These findings
suggest that low malate content in these fruits was not the
result of reduced ability to synthesize malate but of a restricted
ability to accumulate it in the vacuoles (Berüter 2004). This is
consistent with our findings, as we did not detect any gene for
malate biosynthesis at our genetic locus for pH but detected
there only putative malic acid transporter genes.
It is the storage of malic acid in plant cell vacuoles that
causes the change in pH in apple (Berüter 2004). Yamaki
(1984) isolated vacuoles from apple fruit flesh and detected
that more than 90 % of the malic acid, the main organic acid,
was located in the vacuole. Malate storage within the vacuole
allows the plant to accumulate this metabolite to very high
concentrations (up to >300 mM) and thus, maintain the con-
centration in the cytosol constant (Emmerlich et al. 2003).
Marker-assisted selection for the Ma locus may yield apple
cultivars with optimum acidity
Acidity of the fruits has a clear impact on overall liking by
consumers (Fig. 7). In conventional breeding of apples, due
to the long juvenile period of apple seedlings, it takes 6 to
8 years after making a cross to evaluate the progeny for fruit
quality. This leads to high costs for maintenance of progeny
over many years. Marker-assisted breeding, using sequence
information of the desired allele for acidity, allows selection
of young seedlings within one year after making the cross,
although these seedlings do not bear fruits yet. Early selec-
tion avoids the costs of growing of many seedlings over a
long period and discovering their poor acidity not until the
adult phase when they start to fruit. As Ma1 is the main
candidate gene for genetic segregation of the pH, we rec-
ommend using markers in or very close to this gene.
In this study, evidence has been provided that in apple,
the expression level of a malic acid transporter gene is the
limiting factor in malate accumulation in the vacuoles and
therewith, likely the major determining factor for acidity of
apple. This gene resides at the top of LG16. The genetic
inheritance of at least one dominant allele of this gene
sufficed for an increased expression level that led to a
threefold increase of the malic acid content and a reduction
of the pH from 4 to 3 in ripe apples, compared to inheritance
of the recessive, lowly expressed allele only.
Acknowledgments This project was financially supported by
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... This locus has been reported to control 17% to 42.3% of the variation in acid concentration in apple fruit . The gene underlying Ma, named Ma1, has been identified to encode an aluminum-activated malate transporter-like protein (Bai et al., 2012;Khan et al., 2013). A single nucleotide mutation from the guanine (G) to adenine (A) at position 1455 in the coding sequence of Ma1 results in a premature stop codon that truncates 84 amino acids at the C-terminus, causing low acidity (Bai et al., 2012;Li et al., 2020). ...
... Similar to other studies that investigated apple fruit acidity genetics, our analyses indicate that the Ma1 gene is a reliable predictor (P < 0.0001) of TA (Bai et al., 2012;Khan et al., 2013). Therefore, our study adds to the body of literature indicating that the Ma1 gene largely determines apple acidity (Bai et al., 2015;Brown and Harvey, 1971;Kouassi et al., 2009;Nybom, 1959;Visser and Verhaegh, 1978;Xu et al., 2012). ...
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The organic acid concentration in apple ( Malus × domestica ) juice is a major component of hard cider flavor. The goal of this study was to determine if the malic acid markers, Ma1 and Q8 , could classify the titratable acidity concentration in cider apple accessions from the United States Department of Agriculture Malus germplasm collection into descriptive classifications. Our results indicate that for diploid genotypes, the Ma1 marker alone and the Ma1 and Q8 markers analyzed together could be used to predict cider apple acidity ( P < 0.0001). Alone, the Ma1 marker categorized acidity into low (<2.4 g⋅L ⁻¹ ), medium (2.4–5.8 g⋅L ⁻¹ ), and high (>5.8 g⋅L ⁻¹ ) groups. The combination of Ma1 and Q8 markers provided more specificity, which would be useful for plant breeding applications. This work also identified a significant difference ( P = 0.0132) in acidity associated with ploidy. On average, the triploids accessions had 0.33 g⋅L ⁻¹ higher titratable acidity than the diploid accessions. Based on the results of this work, we propose a genetics-based classification system for cider apples with the acidity component defined by the Ma1 and Q8 markers.
... The TA values were calculated according to Sadler and Murphy [35], and the results are expressed as malic acid (%) [36]. In apple, malic acid is the predominant acid (80-90% of the organic acids) that contributes to sourness of fruits [35,37]. ...
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Apple fruits are high in phenolic compounds, sugar and dietary fiber content and are rich in malic acid and vitamins, with a significant impact on the organoleptic quality and its health-promoting properties. They can be turned out in value-added product such as apple chips due to the low cost of raw material. The aim of the study was to obtain apple chips, fat-free, healthy, traditionally dried and without added sugar, which can be easily obtained and capitalized economically, as well as the evaluation of their physico-chemical and sensory qualities. The apple chips were produced from three apple cultivars (‘Starkrimson’, ‘Golden Delicious’ and ‘Florina’) by drying the apple fruits in an oven and a dehydrator at 65 °C. To inactivate the browning enzymes, the apple slices were immersed in a solution of lemon salt (4%) for 7 min before drying. Apple chips were sensory-evaluated and relevant parameters were analyzed at defined intervals during storage at room temperature up to 21 days. The water activity (aw) values of apple chip samples dried in the oven ranged from 0.544 to 0.650, while for the samples dried in the dehydrator, aw values were between 0.374 and 0.426. During the storage, the pH of apple chips varied very little, while titratable acidity increased for all samples. Compared with fresh apple slices, it was observed that the total soluble solids (TSS) content of all dried apple chip samples decreased. Color parameters and browning and whitening indexes differed depending on the apple cultivars and dryer type used.
... Concerning malate, Bai et al. [13] indicated that Ma1, an aluminum-activated malate transporter like (ALMT-like) gene, could be the main determinant of malate content in apple fruit. Differences in fruit acidity were possibly caused not only through a single nucleotide mutation at base 1455 in the open reading frame (ORF), but also by differences in expression levels of Ma1 [13,14]. Further evidence held Ma1 responsible for the content of malic acid, and its conserved C-terminal domain for malate transport was identified [15]. ...
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Organic acid content in fruit is an important determinant of peach organoleptic quality, which undergoes considerable variations during development and maturation. However, its molecular mechanism remains largely unclear. In this study, an integrative approach of genome-wide association studies and comparative transcriptome analysis were applied to identify candidate genes involved in organic acid accumulation in peach. A key gene PpTST1, encoding tonoplast sugar transporter, was identified and the genotype of PpTST1 with a single-base transversion (G1584T) in the third exon which leads to a single amino acid substitution (Q528H) was associated with low level of organic acid content in peach. Overexpression of PpTST1His resulted in reduced organic acid content along with increased sugar content both in peach and tomato fruits, suggesting its dual function in sugar accumulation and organic acid content reduction. Two V-type proton ATPases interact with PpTST1 in yeast two-hybridization assay. In addition, the G1584T transversion appeared and gradually accumulated during domestication and improvement, which indicated that PpTST1 was under selection. The identification and characterization of PpTST1 would facilitate the improvement of peach fruit quality.
... Usually, red apples have higher pH values than that of green apples, and these parameters are very important for consumers since they appreciate the balance of sweetness and acidity [40]. pH of apples is mainly affected by the storage of malic acid in vacuoles, and a high amount of this organic acid can change an apple's pH [41,42]. Piagentini and Pirovani [43] found that red apples have a significantly (p < 0.05) lower amount of malic acid than green apples. ...
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The main aim of the study was to prepare the edible films based on carrageenan/chitosan and incorporate them into the following matrices: the natural extracts of Clitoria ternatea, Brassica oleracea, and Ipomea batatas. The films were characterized by TPC (total polyphenols content), antioxidant activity, and textural properties. Experimentally produced films were added in the packaging of freshly cut apple pieces, and the apple pieces were dipped into the films produced from carrageenan and chitosan. The appearance of the samples was monitored, as were antioxidant activity and total polyphenol content. The intelligent properties of films were evaluated too. The polymer type used for the preparation had the highest impact on the prepared films, and CHLCZ (red cabbage extract—Brassica oleracea) featured the best antioxidant activity. The intelligent properties were slightly confirmed in samples with the addition of red cabbage. The main finding was that the coating of fresh-cut apples emphasized the possibility to use a carrageenan matrix with the addition of extracts. The samples immersed in this coating type showed higher antioxidant activity as well as a superior color when compared to that of chitosan coated apple samples.
... In fruit trees, up to now just a few QTLs, loci and genes have been associated to acidity and sweetness. As an example, a locus called Ma on LG16 has been identified and characterised in apples, in which a transporter gene associated with low acidity was located (Khan et al. 2013). This association was attributed to a mutation-led truncation in aluminium activated malate transporter (ALTM) associated with low acidity in apple (Bai et al. 2015). ...
Background and Aims Identifying the genes that participate in the sweetness and acidity of the berry is key, because these traits are quintessential to the flavour and quality of fresh grapes and wine. In this study we focused on the identification of genomic regions that host genes associated with sweetness and acidity in tablegrapes. Methods and Results A highly saturated genetic map was prepared using a Ruby Seedless × Sultanina cross (RxS; n = 138) genotyped using an SNPlex-type platform, and quantitative trait loci (QTLs) were mapped. In the integrated map of this population, 1731 markers were distributed along the 19 linkage groups (LGs) of the species. Three significant QTLs, two in LG5 and one in LG8, were associated with fructose/glucose ratio, TA and tartaric acid concentration; these QTLs explained up to 20% of the phenotypic variance for these traits. Eight candidate genes located within the confidence intervals of the sugar and TA QTLs were chosen and their expression profiles analysed from flowering to berry ripening. A Genome-Wide Association Study (GWAS) analysis revealed the association of single-nucleotide polymorphisms in the same regions as QTLs. Conclusions Quantitative trait loci significant for sugar and acidity were identified on a tablegrape high-quality genetic map; furthermore, an association between acidity or sugar concentration and expression changes for candidate genes was also observed. Significance of the Study These findings could become the basis to develop selection tools for the breeding of these traits in tablegrape.
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Glucose is a preferred source of carbon and energy for plants. In addition to metabolic functions, glucose is a well-known signaling molecule that regulates plant growth and development through multiple pathways. In this review, the mechanisms by which glucose signaling regulates the accumulation of sugars and organic acids, as well as the ripening of fleshy fruit, are examined. An analysis of these complex molecular networks demonstrates the impact of glucose signal perception on fruit quality.
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Acidity is an important factor influencing the organoleptic quality of apple fruits. In this study, an apple pyrophosphate-energized proton pump (PEPP) gene was isolated and designated MdMa12. On the basis of a phylogenetic analysis in Rosaceae species, PEPP genes were divided into three groups, with apple PEPP genes most closely related to pear PEPP genes. Gene expression analysis revealed that high malic acid content was generally accompanied by high MdMa12 expression levels. Moreover, MdMa12 was mainly expressed in the fruit. A subcellular localization analysis suggested that MdMa12 is a mitochondrial protein. The ectopic expression and overexpression of MdMa12 in “Micro-Tom” tomato and apple calli, respectively, increased the malic acid content. One (MDH12) of four malate dehydrogenase genes highly expressed in transgenic apple calli was confirmed to encode a protein localized in mitochondria. The overexpression of MDH12 increased the malate content in apple calli. Furthermore, MdMa12 overexpression increased MdDTC1, MdMa1, and MdMa10 expression levels, which were identified to transport malate. These findings imply that MdMa12 has important functions related to apple fruit acidity. Our study explored the regulatory effects of mitochondria on the complex mechanism underlying apple fruit acidity.
Soluble sugars and organic acids play important roles in determining fruit taste. For apple, fructose is the most abundant soluble sugar, while the predominant organic acid is malic acid (Ma) that accounts for up to 90% of the total organic acids. The earliest genetic studies of inheritance have uncovered that apple fruit acidity is controlled by a single major locus on linkage group (LG) 16, designated Ma. Later, another important quantitative trait locus (QTL) for fruit acidity has been identified on LG. Besides these two QTLs, five other QTLs for apple fruit acidity are also found on LGs 2, 10, 13, 15, and 17. Sugar content is a heritable quantitative trait, and two QTLs for sugar content have been identified on LGs 1 and 3. Several strong candidate genes for fruit acidity, such as Ma1 and MdPP2CH encoding a protein phosphatase2C that inactivates H⁺-ATPases and Ma1 via dephosphorylation, have been reported. However, few candidates for sugar content have been identified. This review will cover these findings as well as assess their impact on pursuing genetic enhancement efforts to manipulate/modify these important components of fruit taste in apples.
Apples have distinctive quality attributes that may be defined by environmental conditions of the geographical regions where fruits are cultivated, such as temperature, solar radiation, photoperiod, and photothermic units. A three-year study was conducted to compare ‘Golden Delicious’ and ‘Red Delicious’ apples from two different regions, Washington, USA (WA) and Chihuahua, Mexico (CHIH). Apple samples were harvested weekly from early August to late October (~120-180 days after full bloom - DAFB), and analysed for quality parameters. Geographic environmental data were obtained, and photoperiod, solar radiation, degree-days and photothermal units were calculated. Results show quality differences between CHIH and WA apples. WA shows a ~5-week delay in apple bloom, possibly due to the lower temperatures presented in WA. Apples from both regions required the same photoperiod, ~ 2,222 h, to attain the beginning of the ripening stage, which took more days (three weeks) for CHIH apples, most likely attributed to the higher elevation of CHIH orchards (2,062 vs. 763 masl). The main distinctive quality differences found between WA and CHIH apples were firmness and aroma volatile compounds. CHIH apples presented substantially higher amounts of aroma compounds. WA apples showed greater firmness, probably due to lower photothermal units. Using all firmness data (both varieties, both growing zones) a remarkable correlation was found between firmness and photothermal units (R=0.89), which may suggest firmness could be improved by the manipulation of degree days and photoperiod, that is, temperature and light.
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In the version of this article initially published, in the Reagent Setup section, it was stated that 50% v/v Percoll should be made by diluting Percoll in 10× PBS at a 1:1 v/v ratio. Percoll should, in fact, be diluted in 2× PBS at a 1:1 ratio. The error has been corrected in the HTML and PDF versions of the article. In the version of this article initially published, a component (40 μl of 50 mg ml –1 BSA) was erroneously omitted from the 'Hybridization solution' recipe in the Reagent Setup section. The error has been corrected in the HTML and PDF versions of the article.
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The developmental changes of carbohydrates, organic acids, amino acids and phenolic compounds in ‘Honeycrisp’ apple flesh were investigated using GC–MS and HPLC. A total of 12 carbohydrates, 8 organic acids, 20 amino acids, and 18 phenolic compounds were identified and quantified. Each metabolite showed characteristic changes during fruit development, but in general, concentrations of most sugars and sugar alcohols either increased or remained unchanged whereas concentrations of most organic acids, amino acids and phenolic compounds decreased with fruit development, indicating that most sugars and sugar alcohols are synthesised and/or accumulate at a faster or similar rate relative to fruit growth whereas organic acids, amino acids and phenolics are synthesised and/or accumulate at a slower rate relative to fruit growth. On a whole fruit basis, the content of most metabolites increased with fruit development. In the flesh of mature ‘Honeycrisp’ apple, fructose and sucrose and sorbitol are the major sugars and sugar alcohol; malic acid is the major organic acid; aspartic acid, asparagine, glutamic acid, proline, threonine and γ-aminobutyric acid are the major amino acids; and procyanidin B1, procyanidin B2, chlorogenic acid, catechin and epicatechin are the major phenolic compounds, respectively.
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Apple fruit flavor is greatly affected by the level of malic acid, which is the major organic acid in mature apple fruit. To understand the genetic and molecular basis of apple fruit acidity, fruit juice pH and/or titratable acidity (TA) were measured in two half-sib populations GMAL 4595 [Royal Gala × PI (Plant Introduction) 613988] and GMAL 4590 (Royal Gala × PI 613971) of 438 trees in total. The maternal parent Royal Gala is a commercial variety and the paternal parents are two M. sieversii (the progenitor species of domestic apple) elite accessions. The low-acid trait segregates recessively and the overall acidity variations in the two populations were primarily controlled by the Ma (malic acid) locus, a major gene discovered in the 1950s (Nybom in Hereditas 45:332–350, 1959) and later mapped to linkage group 16 (Maliepaard et al. in Theor Appl Genet 97:60–73, 1998). The allele Ma has a strong additive effect in increasing fruit acidity and is incompletely dominant over ma. QTL (quantitative trait locus) analyses in GMAL 4595 mapped the major QTL Ma in both Royal Gala and PI 613988, the effects of which explained 17.0–42.3% of the variation in fruit pH and TA. In addition, two minor QTL, tentatively designated M2 and M3, were also detected for fruit acidity, with M2 on linkage group 6 of Royal Gala and M3 on linkage group 1 of PI 613988. By exploring the genome sequences of apple, eight new simple sequence repeat markers tightly linked to Ma were developed, leading to construction of a fine genetic map of the Ma locus that defines it to a physical region no larger than 150 kb in the Golden Delicious genome.
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Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65–82 kb containing 12–19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R 2 = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS1455 that targets the SNP1455 in Ma1 showed that the CAPS1455A allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for low pH or high acidity in apple.
Vacuoles of immature apple fruit (Malus pumila Mill. var. domestica Schneid.) were obtained by purification using Ficoll density gradient centrifugation after lysis of the protoplasts by both mild osmotic shock and the addition of EDTA and BSA. The recovery was about 35% of the protoplasts. The isolated vacuoles had a mean diameter of about 100 μm. The distribution of sugars, organic acids, phenolic compounds and amino acids in the vacuole, the cytoplasm and the free space was determined. Almost all of the fructose and glucose, the major sugars of the tissue, were found in the vacuole. Sorbitol was mainly located in the free space and the vacuole, and sucrose in the free space and the cytoplasm. More than 90% of the malic acid, the main organic acid, was located in the vacuole. Almost all of the phenolic compounds were also deposited in the vacuole. The volumes of the vacuole, the cytoplasm and the free space in the whole tissue were calculated from the cell numbers of the whole tissue, the volume of the isolated protoplasts, and the volume of the vacuoles present in the protoplast. The solute concentration in each compartment was estimated: vacuoles, 888 mm; cytoplasm, 37 mm; free space, 57 mm. How these compartmentations of solutes affected the translocation of sugars into the fruit and the cell expansion is discussed.
The names malic, maleic and malonic acid were all derived from the Latin word for apples. First, Swedish chemist Carl Wilhelm Scheele isolated malic acid from apple juice in 1785 which was named later by Lavoisier as "acide malique." In 1834, French chemist Théophile Jules Pelouze isolated "acide maléique" and "acide paramaléique". Both names were later Anglicized as maleic acid and para-maleic acid. In 1858, French chemist Victor Dessaignes prepared a third acid he named "acide malonique", a name that was later Anglicized as malonic acid.