ORIGINAL ARTICLE

The metabolic fate of citric acid as affected by cold storage

in blood oranges
Angela Roberta Lo Piero & Luca Lo Cicero & Ivana Puglisi
Received: 21 September 2012 / Accepted: 11 February 2013
#Society for Plant Biochemistry and Biotechnology 2013
Abstract Citrus fruit are natural source of several phyto-
chemicals useful in the maintenance of a satisfactory status
of the human health. As citric acid is one of the main
determinant of citrus fruit quality, researches focusing the
elucidation of citrate metabolism have been constantly car-
ried out, often leading to a deeper understanding of the
biochemical and physiological changes occurring during
fruit development and ripening. Lower interest has been
directed to the knowledge of the metabolic fate of citrate
during fruit storage, although the temperature controlled
conservation is a spread and, in many cases, mandatory
practise. In this work, we evaluated the expression of the
main enzymes involved in the citrate metabolism, such
citrate synthase, citrate lyase and both the cytosolic and
mitochondrial isoforms of aconitase, in blood oranges
subjected to cold storage (4 C15 days). Total acidity
(TA) and total soluble solid (TSS) were also determined to
correlate such fruit quality parameters with gene expression.
The results suggest that cold storage influences both the
analytical parameters and gene expression, in particular, a
strong induction of the transcription of all the selected gene
was observed simultaneously with the sudden reduction in
acidity. These findings suggest that aconitase gene products
localized in the cytosolic compartment are strongly impli-
cated in the consumption of the citrate released from vacu-
oles and meanwhile mitochondrial aconitase is involved in
the catabolism of organelle-localized citrate. The expression
of citrate lyase turned out to be also induced during cold
storage of blood oranges. The alternative breakdown of
citrate through the citrate lyase, which is ruled out during
fruit development and ripening, might be a pathway exclu-
sively activated in response to cold storage and therefore
correlated with the sharp cold induction of the flavonoid
biosynthesis which is supplied by the citrate lyase reaction
products.
Keywords Citrus sinensis
.
Citric acid
.
Cold storage
.
Blood orange
.
Gene expression
.
Real time RT-PCR
Abbreviations
CitSyn citrate synthase
CitLya citrate lyase
Aco aconitate hydratase
TA total acidity
TSS total solubile solid
Introduction
The interest on the biological processes regulating citrus
fruit development and ripening are related to its importance
for human diet as supply of ascorbic acid, fibre and several
phytochemicals with healthy promoting features (Tadeo et
al. 2008). Citrus fruits undergo to non climacteric ripening
which implicates both a sharp decrease in the active growth
and a certain number of biochemical and physiological
changes leading to edible fruits (Iglesias et al. 2007). During
ripening there is a decline in titratable acidity mostly due to
catabolism of citric acid, the main organic acid of citrus
juice, and an increase in sugars. The total soluble solids to
titratable acidity ratio is commonly known as maturity index
(Iglesias et al. 2007). Previous studies comparing acidless
and acidic varieties have reported considerable evidences
that citrate synthase is not responsible for different acid
accumulation thus suggesting that the acidity is a process
subjected to the control of many coordinated enzymes
(Sadka et al. 2000). According to recent hypothesis citrate
Angela Roberta Lo Piero suggested the research direction, designed the
experiments, carried out data analysis and wrote the manuscript; Luca
Lo Cicero and Ivana Puglisi performed both experimental work and
data analysis.
A. R. Lo Piero (*)
:
L. Lo Cicero
:
I. Puglisi
Dipartimento di Scienze delle Produzioni Agrarie e Alimentari
(DISPA), Via S. Sofia 98,
95123 Catania, Italy
e-mail: rlopiero@unict.it
J. Plant Biochem. Biotechnol.
DOI 10.1007/s13562-013-0197-7
is released from the vacuole into the cytosol (Shimada et al.
2006) and then metabolized into isocitrate by citosolic
aconitase (Cercs et al. 2006; Terol et al. 2010). A prevalent
role in acidity loss of an alternative citrate breakdown into
oxaloacetate and acetylCoA catalyzed by citrate lyase is
ruled out since a decrease in the ATP:citrate lyase mRNA
level during fruit ripening has been reported (Cercs et al.
2006). Although the role of citrate levels in determining
citrus fruit taste during fruit developmental stages has been
extensively investigated (Cercs et al. 2006; Terol et al.
2010; Iglesias et al. 2007), the processes related to citrate
metabolism during fruit postharvest storage are still poorly
understood. In this respect, cooling to non freezing temper-
ature is by far the most important postharvest practise in
achieving shelf-life extension of fresh fruits and for the
preparation of ready-to-eat products whose consumption is
widely increasing as a consequence of their convenience
and freshness (Amarowicz et al. 2009; Plaza et al. 2011).
In addition, cold quarantine treatments, which involve the
exposure of fruit to low temperature for a period of 10
16 days, represent the accepted procedure for Medfly disin-
festations of citrus fruits required by the regulatory agencies
of most importing countries (Schirra et al. 2004). In previ-
ous work, the transcriptome analysis based on subtractive
hybridization was performed in order to emphasize the
overall induction in gene expression after the exposure of
blood oranges [(Citrus sinensis) L. Osbeck Tarocco Sciara]
to low temperature (Crif et al. 2011). Among the clones
specifically expressed in the cold treated samples the EST
enconding the ATP:citrate lyase turned out to be selectively
induced by cold suggesting that citrate might be catabolized
under cold storing conditions through the citrate lyase reac-
tion (Crif et al. 2011). In the cytosol, citrate lyase gives rise
to oxaloacetate and acetyl CoA. This latter molecule under
the shape of malonyl CoA, through the reaction catalysed by
chalcone synthase becomes part of the flavonoid skeleton.
On the other side, oxaloacetate is converted into phospho-
enolpyruvate which may be channeled to plastids and there
consumed by the phenylalanine biosynthesis pathway.
Therefore, it has been proposed that cold stress induces
transcriptome modifications oriented towards the enhance-
ment of the flavonoid biosynthesis pathway in blood or-
anges at the expenses of cytosolic citrate levels (Crif et
al. 2011). The results are supported by the findings that the
analyses of gene expression show that the amount of tran-
scripts of selected genes belonging to the anthocyanin bio-
synthesis pathway (chorismate mutase, CM1; phenylalanine
ammoni a l yase, PAL; chal cone synt hase, CHS;
dihydroflavonol-4-reductase, DFR; anthocyanidin synthase,
ANS, UDP-Glucose Flavonoid glucosyl transferase, UFGT;
and glutathione S-transferase, GST) (Lo Piero et al. 2005a;
Cotroneo et al. 2006; Lo Piero et al. 2009) sharply increases
during cold storage. Similarly, the anthocyanin levels of
fruit exposed to cold reach values eight times higher than
that observed in the time zero sample (Crif et al. 2012) thus
confirming that cold storage enhances fruit nutraceutical
properties and might favor the marketing of bioactive
fruits useful in the prevention of some degenerative diseases
(De Pascual-Teresa and Sanchez-Ballesta 2008; de Pascual-
Teresa et al. 2010). In this work, considering that citrate is
extremely important for the taste of citrus fruit affecting the
sourness as well as the sweetness by masking the sugar
taste, we evaluated the expression of main enzymes in-
volved in the citrate metabolism, such citrate synthase,
citrate lyase and both the cytosolic and mitochondrial
isoforms of aconitase, in blood oranges subjected to cold
storage. Total acidity (TA) and total soluble solid (TSS)
were also determined in juice extract to correlate such fruit
quality parameters with gene expression.
Materials and methods
Plant material and storage conditions
Blood [(Citrus sinensis) L. Osbeck Tarocco Sciara] oranges
were harvested in January 2010 from an approximately
15 year old tree grown at the orchard of the Centro di
Ricerca per l'Agrumicoltura e le Colture Mediterranee in
the territory of Palazzelli (Italy). Fruits harvested in January
were still at immaturity stage since this variety reaches the
fully ripe status in late February-March. Once transferred in
the laboratory, the oranges were washed with distilled water,
gently dried with paper towels and then left to dry at room
temperature for 3 h. Storage, samplings and further fruit
handling was performed according to (Crif et al. 2012).
Briefly, the oranges were randomly placed in two boxes one
of them stored in a ventilated cold room at 4 C in darkness.
The remaining box was placed in a temperature-controlled
device kept at 25 C, in darkness (control samples). Sam-
plings were carried out before storage (time 0) and every
3 days for a total storage period of 15 days.
Measurement of gene expression by real-time quantitative
RT-PCR
Real-time PCR, was performed using the SuperScript III
Platinum two-step qRT-PCR kit. To minimize mRNA loss
and avoid DNA contamination, isolated polyA
+
RNA was
used as a template for first-strand synthesis before RT-PCR.
Pure mRNA was prepared from orange flesh using the
Quickprep mRNA purification kit (GE Healthcare,
Piscataway, NJ). Reverse transcription of mRNA (1 g)
was achieved by following the manufacturers protocol.
The relative quantitation of gene expression between cold
treated and control orange samples was calculated using the
J. Plant Biochem. Biotechnol.
comparative threshold (C
T
) method (Heid et al. 1996) as
detailed in Crif et al. (2011).
The nucleotide sequences used in this paper are in Genbank
under the following accession numbers: FK826648.1 (ATP:
citrate lyase, CitLya), GQ372880.1 (citrate synthase, CitSyn),
FN552254 (aconitate hydratase, Aco1), FN552256 (aconitate
hydratase, Aco2), FN552255 (aconitate hydratase, Aco3),
AY498567 (EF-1 partial cds).
Analytical methods
Juices extracted with a domestic blender were assayed for
total acidity (TA) and total soluble solid (TSS) according to
standard methods (http://www.dpi.nsw.gov.au/__data/
assets/pdf_file/0020/320294/Citrus-maturity-testing.pdf).
The data is expressed as a percentage (%).
Statistical analysis
For analytical parameters, data set are reported as average
values SD (standard deviation) of three independent ex-
periments performed upon sample triplicates each consisting
of four fruits. With regards to the analysis of gene expres-
sion, for each gene, three independent triplicates of quanti-
tative PCR experiments were performed to generate an
average C
T
and to calculate standard deviation (SD), each
triplicate prepared by mRNA samples independently isolat-
ed from four fruit (Lo Piero et al. 2011).
Results and discussion
Figure 1 shows the results obtained after determining the
TA, TSS and TSS/TA of Tarocco Sciara orange fruits during
storage at 4 and 25 C for 15 days. The total acidity of fruits
stored at 25 C remained unchanged for the most of the
storage time starting to decline at the twelfth days of stor-
age; in samples stored at 4 C higher acidity values were
registered up to the ninth day of storage then a distinct
acidity loss is observed (Fig. 1a). A continuous increase in
TSS occurred in Tarocco Sciara fruits stored at 25 C,
whereas in cold stored fruits the TSS parameter decreases
after the ninth day reaching values around 1.5 % less than
that observed in the 25 C stored samples (Fig. 1b). Because
of the increase in TSS and the simultaneous decrease in TA,
the TSS/TA ratio (maturity index) of the 25 C samples
begins to rise from 4.7 (12 days) to 6.1 (15 days) (Fig. 1c).
On the contrary, the concomitant decrease of both TSS and
TA leads to slighter increase of the maturity index during the
storage at 4 C. Increase of the TSS/TA ratio during
prolonged storage at both 8 and 22 C has been observed
in the blood orange, and, high TSS/TA ratio has generally
been considered as a quality index for orange fruits although
its increase during storage can also be accompanied by the
development of off-flavors due to formation of ethanol in
the fruit (Rapisarda et al. 2001). Therefore, the slowdown of
the biochemical modifications leading to the increase of the
maturity index might represent a favorable consequence of
cold storage preceding the marketing phase. To understand
Fig. 1 Change in TA a, TSS b, TSS/TA c Tarocco Sciara fruits during
storage at 4 C and 25 C
J. Plant Biochem. Biotechnol.
the role of different genes involved in citrate metabolism
during cold storage (4 C for 15 days) in blood orange and
their importance in the control of fruit quality parameters,
their expression was determined by real time RT-PCR in
flesh tissues. We focused the interest upon citrate synthase
(CitSyn), catalyzing the condensation of oxaloacetate and
acetyl CoA, and citrate lyase (CitLya). Afterwards, the
analysis was carried out on aconitase which catalyzes the
reversible isomerization of citrate to isocitrate. Two
isoforms of aconitase have been detected in all eukaryotic
cells: mitochondrial aconitase involved in tricarboxylic acid
cycle and cytosolic aconitase that participates in cytosolic
citrate metabolism (Cercs et al. 2006; Sadka et al. 2000)
and in the glyoxylate cycle (Hayashi et al. 1995). Recently,
sequence analyses identified three active aconitase genes in
citrus (Terol et al. 2010) named CcAco1, CcAco2 and
CcAco3, all of them were considered in this work. In details,
Fig. 2 shows the expression pattern of CitSyn (Fig. 2a),
CitLya (Fig. 2b), Aco1 (Fig. 2c), Aco2 (Fig. 2d) and Aco3
(Fig. 2e) which are expressed at low levels rather constantly
along the whole experimental period at 25 C (Fig. 2).
Conversely, after 36 days of low temperature exposure,
the levels of all considered gene start to sharply increase
reaching the maximum value after 15 days of storage, the
aconitase genes, especially Aco3 (Fig. 2e) showing the
highest values of relative expression. It has been suggested
that the Aco3 gene might have organelle localization and
therefore be involved in the tricarboxylic acid cycle taking
place in the mitochondrial matrix (Terol et al. 2010). Both
the decrease in TSS registered in cold stored fruit (Fig. 1b)
and the induction of Aco3 expression suggest that the met-
abolic fate of the citrate is not the sugar synthesis which
involves reactions that partly take place in the glyoxysomes.
On the contrary, it might be channeled in the tricarboxylic
acid cycle functioning as energy source thus confirming
previous data reading that localizes Aco3 in the mitochon-
dria rather than in the glyoxysomes (Terol et al. 2010). In
this respect, the Aco3 gene product might be also involved
in the breakdown of the newly synthesized citrate achieved
by the CitSyn catalyzed reaction as the expression of this
gene is also induced by cold (Fig. 2a). However, we cannot
exclude that the decrease of TSS might be also linked to
sugar utilization in the glycosylation of newly synthesized
anthocyanidins catalyzed by UDP-glucose-flavonoid-
glucosyl transferse (UFGT) whose levels are strongly in-
creased by cold storage (Lo Piero et al. 2005b). The
Fig. 2 Analysis of the expression of a citrate synthase (CitSin), b
citrate lyase (CitLya) c aconitase isoform 1 (Aco1), d aconitase isoform
2 (Aco2) e aconitase isoform 3 (Aco3) in response to cold storage in
blood orange flesh (Tarocco Sciara). The relative quantitation of gene
expression between orange samples was calculated by real time RT-
PCR using the comparative threshold (CT) method; each value repre-
sents the mean value of three replications each composed of three
independently isolated mRNA sample SE (or SD)
b
J. Plant Biochem. Biotechnol.
induction of the aconitase isoforms (Fig. 2c, d and e) was
coincident or slightly preceding the acidity peak observed at
9 days storage in the cold stored samples and the rise in gene
expression was concomitant with the reduction of acid
levels (Fig. 1a). These latter observations resemble the bio-
chemical changes that occur in citrus fruit during develop-
ment and ripening (Cercs et al. 2006) and therefore seem to
be modifications joining both situations. Moreover, the ob-
served enhancement of Aco1 and Aco2 expression is prob-
ably related to the aconitase activity localized in the
cytosolic compartment and implicated in the consumption
of the citrate released from vacuoles (Cercs et al. 2006;
Terol et al. 2010). In this respect, microarray data suggested
that cytosolic citrate is then sequentially metabolized to
isocitrate, 2-oxoglutarate and glutamate. Thereafter, gluta-
mate is both utilized for glutamine production and catabo-
lized through the gamma-aminobutirate shunt (GABA)
(Cercs et al. 2006). The occurrence and the sub cellular
localization of CitLya in plant is not fully understood, there-
fore there are some evidence that the enzyme is distributed
between the cytosol and the plastids in several plant species
(Rangasamy and Ratledge 2000). Different studies have
provided evidence that CitLya in plastids functions to sup-
ply acetylCoA for fatty acids synthesis, whereas it is thus
reasonable to assume that cytosolic CitLya might be in-
volved in the supply of acetyl CoA for several metabolic
pathways (Rangasamy and Ratledge 2000). Wherever it is
localized, the citrate consumption through the CytLya reac-
tion to yield both acetylCoA and oxaloacetate seems not to
be involved in the acidity loss during citrus fruit ripening
(Cercs et al. 2006). However, a major role of this gene
turned out in blood oranges subjected to cold storage. The
induction of CytLya expression, observable in Fig. 2b, con-
firms previous data showing that the transcription of this
gene is specifically enhanced under low temperature and
demonstrating that the increased expression is positively
correlated with the enhanced rate of the anthocyanin bio-
synthesis pathway (Crif et al. 2011).
In conclusion, the results of this study indicate that cold
storage influences the maturity index of orange fruits which
is lower than that observed in control samples (25 C). This
is achieved by the simultaneous decrease of TSS and TA
registered from the ninth day of storage on, and it might
represent a favorable characteristic obtainable by the post
harvest cold storage which might precede fruit marketing.
The overall analysis of the analytical parameters and gene
expression indicates that, after the ninth day of cold storage,
both sugars and mitochondrial citrate might be used as
energy source. This hypothesis is supported by both the
decrease of TSS and the induction of Aco3 gene expression.
The strong induction of the Aco1 and Aco2 expression
accounts for the cytosolic reduction of citrate to produce
isocitrate, this being the first step involved in a reaction
sequence responsible of the acidity loss occurred at 4 C.
A pivotal role of CytLya, which is not involved in citrus
development and ripening (Cercs et al. 2006), at least in
citrus non pigmented varieties, has to be assumed during
cold storage. The breakdown of citrate through the CitLya
reaction might be correlated with the strong induction of
flavonoid biosynthetic pathway which is feeded by the
reaction products of CitLya, and for this reason it might be
a pathway exclusively activated in response to cold storage
(Crif et al. 2011). As concern the orange fruit stored at 25 C,
the increase in the maturity index achieved by the concomitant
increase of TSS and acidity (TA) reduction underlies that
gluconeogenesis could be an active pathway in that condition.
Moreover, the basal levels of Aco gene expression appear to
be sufficient to get the acidity loss registered after 15 days.
Finally, these results integrate the picture of the metabolic
modifications occurring during cold exposure of blood or-
anges which associate the increase in anthocyanin content
(Crif et al. 2012) with a reduction of citrate level which
otherwise might mask the sugar taste. An intriguing point will
be explored in the near future to assess whether or not the
utilization of citrate by CytLya in citrus fruit stored at low
temperature is prerogative of the pigmented varieties.
Acknowledgments Financial support was provided by the University
of Catania, Fondi del Bilancio Universitario, Progetti di Ricerca di
Ateneo (PRA), 2009, assigned to Dr. Angela Roberta Lo Piero. Thanks
are due to Prof. Carmela Maria Lanza which kindly provided the
refractometer used in TSS determination and to Dr. Giuseppe
Reforgiato Recupero who supplied the materials.
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