Journal of Plant Physiology 224–225 (2018) 75–85

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Antioxidant and enzymatic responses to oxidative stress induced by cold T

temperature storage and ripening in mango (Mangifera indica L. cv.
‘Cogshall’) in relation to carotenoid content
⁎
Rémy Rosaliea,b, Mathieu Léchaudelc, , Claudie Dhuique-Mayerd, Laurent Dufossée, Jacques Joasd
a
Centre de Coopération Internationale de Recherche Agronomique pour le Développement (CIRAD), UMR QualiSud, 7 Rue de l’Irat, 97410 Saint-Pierre, Ile de La Réunion,
France
b
Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments, Faculté des Sciences et Technologies, Université de La Réunion, Sainte-Clotilde, Ile de la
Réunion, France
c
CIRAD, UMR QualiSud, Station de Neufchâteau, Sainte-Marie, 97130 Capesterre-Belle-Eau, Guadeloupe, France
d
CIRAD, UMR Qualisud, TA B95/16, 73 rue Jean-François Breton, 34398 Montpellier Cedex 5, France
e
Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments, Université de La Réunion, ESIROI Agroalimentaire, Parc Technologique, 2 rue Joseph
Wetzell, 97490 Sainte-Clotilde, Ile de la Réunion, France

A R T I C LE I N FO A B S T R A C T

Keywords: The effects of 15 days of storage at 12 °C and 7 °C followed by fruit ripening at 20 °C on oxidative status, anti-
Ascorbate oxidant defense systems and carotenoid accumulation were studied for two successive years in mango fruits
Carotenoids (Mangifera indica L.) cv. Cogshall. Changes in the non-enzymatic (ascorbate) and enzymatic (SOD, CAT, APX,
Cold storage MDHAR, DHAR and GR) antioxidant systems, as well as oxidative parameters (H2O2 and MDA) and the contents
Mango
of the major carotenoids were measured for three maturity stages, at harvest and after ripening following cold
Reactive oxygen species
temperature storage. In control conditions (20 °C), ripening induced an increase in oxidation resulting in ROS
production and a decrease in ascorbate content. Fruit tissue protection was activated by means of antioxidant
and ascorbate regeneration enzyme systems. Carotenoid accumulated exponentially during ripening. Storage at
low temperatures increased respiration crisis intensity and therefore increased oxidation in the fruit pulp. Fruit
response to this increase varied according to the maturity stage, i.e., enzymatic responses in younger fruits were
very low in comparison to the control, whereas second harvest fruits had a significantly higher degree of en-
zymatic activity to cope with the oxidative stress. Carotenoid contents decreased with low temperatures and first
harvest fruits showed significantly lower values than the control, in opposition to second harvest fruits that
appeared not to be affected. We also suggest that, based on a review of the literature, a link can be made between
antioxidant system defense and carotenoid metabolism since ROS seems to play a central role as a stress signal in
plants.

1. Introduction common tropical fruit produced. Moreover, a part of this production is

Mastering tropical fruit quality and distribution is a major challenge
today, especially when fruit delivery is delayed by several weeks. Many
studies have investigated postharvest storage conditions and treatments
to enhance shelf life (Jin et al., 2014; Shivashankara et al., 2004;
Srinivasa et al., 2002). Cold storage is known to be the most common
way to prevent fruits from ripening and the most often used for trans-
port and/or long-term storage. This is the case for mango, one of the
major fruit productions worldwide and representing the second most
distributed via the export market and involves the management of both
ground and sea transport, requiring cold storage to prevent early ri-
pening. However, if cold temperatures make it possible to control ri-
pening, they can induce physiological disorders when the temperature
drops below the recommended threshold, which depends on the fruit
species and variety. Fruits will first attempt to adapt to the constraint of
the cold by setting up plant defense systems (Sivankalyani et al., 2016),
but if the constraint becomes too strong, chilling injuries will then
appear: scalds, softening, internal browning, electrolyte leakage and, in

Abbreviations: SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione
reductase; MDA, malondialdehyde; ROS, reactive oxygen species; CI, chilling injury; YPS, yellow point stage; RH, relative humidity; TBA, thiobarbituric acid; MTBE, methyl tertiobutyl
ether; HPLC, high pressure liquid chromatography; PDA, photodiode array; TTA, total titratable acidity; TSS, total soluble solid
⁎
Corresponding author.
E-mail address: mathieu.lechaudel@cirad.fr (M. Léchaudel).

https://doi.org/10.1016/j.jplph.2018.03.011
Received 7 November 2017; Received in revised form 13 March 2018; Accepted 15 March 2018
Available online 26 March 2018
0176-1617/ © 2018 Elsevier GmbH. All rights reserved.
R. Rosalie et al.
more severe cases, uneven ripening. It was observed that these symp-
toms generally appeared only after fruits returned to an ambient tem-
perature, i.e., 20 °C (Sivakumar et al., 2011). Consequently, it can be
deduced that they are influenced by both exposure time and cold in-
tensity, in addition to species or cultivar effects (Lurie and Crisosto,
2005) as demonstrated on mango by (Phakawatmongkol et al. (2004)).
Cold temperatures could therefore induce alterations in cell mem-
brane conformation and structure by increasing lipid saturation in
order to maintain membrane functionality (Sevillano et al., 2009). After
cold storage, membranes face disruptions that lead to ionic leakage and
accumulation of ROS linked to an increase in respiration rates related to
ripening (Kratsch and Wise, 2000). The membrane alteration is ob-
served through MDA content, which increases in fruits submitted to
cold temperatures along with the exposure time (Singh et al., 2013) and
this increase is correlated with superoxide (O2−) production (Zhou
et al., 2014). An increase of MDA content was positively correlated with
the apparition of chilling injury in mango peel and pulp for cultivar
Nam Dok Mai No. 4 (Junmatong et al., 2012). This ROS accumulation is
balanced in fruits by a complex antioxidant response involving both
non-enzymatic, i.e., ascorbate, and enzymatic systems such as super-
oxide dismutase, catalase, ascorbate peroxidase and the ascorbate-glu-
tathione cycle. Several studies have highlighted the involvement of
these systems during cold storage. In tomato, catalase (CAT) and glu-
tathione reductase (GR) responded positively to conservation at 4 °C
(Malacrida et al., 2006). In mandarin fruits, APX activity decreased
during storage at 2.5 °C, whereas SOD and CAT increased (Sala, 1998).
Since cold temperatures reduce fruit metabolism, they also have an
impact on fruit color. Malacrida et al. (2006) showed a decrease in
carotenoid synthesis, especially lycopene, which was four times lower
in ripe fruits that remained at 4 °C for four weeks. Carotenoid accu-
mulation might also be closely linked to fruit oxidation status, as pro-
posed by Fanciullino et al. (2013), who argued that the stress-related
production of ROS and its regulation closely control the synthesis and
the accumulation of carotenoid in fruits and leaves.
It is known that temperatures of around 12 °C are the optimum for
mango storage and that lower temperatures trigger alterations in fruit
quality (Medlicott et al., 1990). Studies dealing with the involvement of
oxidative status during cold storage are scarce in mango fruit compared
to other fruits. Most of the studies focus on cold exposure symptoms and
their alleviation by pre-treatments, e.g., cold shock (Zhao et al., 2006),
heat exposure, or dipping (Ding et al., 2007). Mango is known for its
high carotenoid content, especially for compounds involved in nutri-
tional value such as β-carotene, exhibiting pro-vitamin A activity. Its
accumulation decreases with storage temperature (Medlicott et al.,
1990) although no study regarding the impact of temperature on in-
dividual compounds was found.
Moreover, mangoes are often harvested before the suitable green
mature stage in order to ensure a longer shelf time, but this stage can
lead to reduced fruit quality (Medlicott et al., 1990) especially on young
fruits that were shown to be very sensitive to CI (Mohammed and
Brecht, 2002). The use of low temperatures for storage at excessively
early stages increased physiological disorders, loss of firmness and lack
of color development (Nunes et al., 2007). Harvest maturity stage at
harvest is also an important parameter that affects mango quality (Joas
et al., 2012) and it was shown that delayed harvest decreased fruit
ability to overcome oxidative stress induced by cold storage in plum
(Singh et al., 2013).
In this article, we aimed to evaluate the impact of both maturity
stage at harvest and storage temperature over two successive years on
the oxidative status of ‘Cogshall' mangoes, represented by oxidation-
related compounds (H2O2, MDA), antioxidant compounds (ascorbate,
carotenoids), ROS handling enzymes (SOD, CAT, APX) and ascorbate
regeneration enzymes (MDHAR, DHAR, GR). We also attempted to es-
tablish a possible link between carotenoid accumulation and ROS re-
sponse to a moderate stress linked to storage temperature.
76
Journal of Plant Physiology 224–225 (2018) 75–85
2. Material and methods
2.1. Plant and fruit material
This study was conducted on well-irrigated mango trees of cv.
Cogshall, a local variety subjected to exportation to France, grafted on
‘Maison Rouge’ rootstock, in Reunion Island (20°52’48”S, 55°31’48”E)
at the CIRAD Experimental Station, at an elevation of 95 m, during the
2012–2013 and 2013–2014 growing seasons. Chlorophyll fluorescence
parameters, i.e., minimal and maximal chlorophyll fluorescence (Fo and
Fm, respectively), and the variable chlorophyll fluorescence
(Fv = Fm − Fo), were measured on the fruit surface near the apex zone
where the first changes in peel color appear in order to assess the degree
of mango maturity, regardless of fruit growing conditions (Léchaudel
et al., 2010). Three maturity stages corresponding to three different
values of the variable chlorophyll fluorescence (Fv) parameter were
chosen: approximately 1400, 1250 and 950 for H1, H2 and H3, re-
spectively. H1 and H2 corresponded to two green mature and com-
mercial maturity stages. The third harvest stage, H3, corresponded to a
maturity stage when the fruit’s apex has started to lose its green color
and became yellow, referred to as the yellow point stage (YPS), a ty-
pical stage for this variety, indicating the onset of the climacteric pro-
cess. It was assumed that fruits harvested at the YPS presented the best
quality potential (Joas et al., 2005).
2.2. Low temperature storage
Forty fruits were selected from the first and second harvest stages
and separated into three groups: storage at 12 °C (12 fruits), storage at
7 °C (12 fruits) and 20 °C (16 fruits). Sixteen fruits were selected from
the third harvest stage and maintained at 20 °C. Four fruits were sam-
pled for analysis at harvest, corresponding to these H1, H2 and H3
stages. Four fruits were stored for ripening in controlled conditions
(20 °C and 90% relative humidity (RH)). Twelve fruits were stored at 12
and 7 °C and 90% RH for 15 days for the first and second harvest stages,
corresponding to the average storage time used for Cogshall mango
exportation. At the end of this period of storage, i.e. 15 days, fruits were
transferred for ripening in storage conditions corresponding to 20 °C
and 90% RH, and their external aspects were observed during ripening
to check external cold-induced defects, such as red spots, black spots,
and pitting or other general decays.
2.3. Postharvest and quality measurements
To ensure that ripe fruits had the same physiological age when
analyzed, respiratory metabolism and climacteric rise were used as
indicators (Joas et al., 2009). Each of the fruits was monitored daily to
follow the changes in respiration rate (RR). Respiration rate was ex-
pressed in terms of CO2 production at 20 °C (mmol kg−1 h−1) using a
closed system method. Fruits from each treatment were placed in in-
dividual 3 L airtight jars at 20 °C, and CO2 concentration was measured
every 20 min for 1 h by gas chromatography (GC) using an Agilent
M200 apparatus (SRA, Marcy l’Etoile, France) equipped with two
manifolds and two columns: Porapak Q (8 m), thermostated at 55 °C,
and MS-5A (4 m), thermostated at 60 °C, with helium and argon as
carrier gases, respectively.
Fruit sampling was done at the ripe stage, corresponding to R1, R2
and R3, for the three harvest stages H1, H2 and H3, respectively. Four
ripe fruits were analyzed three days after they had reached the highest
value of their respiration rate, corresponding to the “ready-to-eat”
stage.
Sampling conditions were established to limit matrix degradation
linked to light, temperature and enzymatic activity. Fruits were peeled,
cut, examined to check for the absence of internal cold-induced defects,
and part of the pulp was then ground in a Grindomix blender (Retsch,
Haan, Germany) to obtain a juice for the measurement of Total Soluble
R. Rosalie et al.
Solids content and Titratable Acidity; the other part was immediately
wrapped in an alumina sheet and dipped in liquid nitrogen. These
frozen pulps were ground in a Grindomix blender and the powders
obtained were stored in opaque plastic pots at −80 °C for future ana-
lysis.
A sample of each frozen powder was used for dry matter content
assessment, done by comparing its fresh mass and its dry mass recorded
after lyophilization, performed over 48 h at −52 °C. Lyophilization was
used to dry a part of each sample for carotenoid analysis. Lyophilization
was performed over 48 h at −52 °C on a sample of each frozen powder.
Dried mango powders were stored in plastic pots with a desiccation
pastille at −20 °C until analysis within three weeks.
Titratable acidity was measured using an automated titrimeter
(TitroLine easy, Schott, Mainz, Germany) with a 0.05N NaOH solution.
Total soluble solids were determined using a refractometer (Atago ATC-
1E; Tokyo, Japan).
2.4. Oxidative stress and antioxidant response measurements
Oxidant compounds (H2O2, MDA), antioxidant compounds (ascor-
bate, carotenoids), antioxidant enzymes (SOD, APX, CAT) and ascor-
bate-glutathione recycling enzymes (MDHAR, DHAR, GR) were de-
termined as follows.
Hydrogen peroxide content was assayed as described by Léchaudel
et al. (2013) using frozen mango pulp (0.25 g) homogenized in an ice
bath with 1 mL 0.1% (w:v) TCA. A reaction mixture was prepared with
the supernatant and KI. Absorbance readings of the triiodide ion (I3−),
resulting from the reaction between H2O2 and KI, were taken at 390 nm
by a microplate reader (BioTek Instruments, Inc.; Winooski, Vermont,
USA). H2O2 content was quantified according to a standard curve.
The thiobarbituric acid (TBA) test, which determines mal-
onyldialdehyde (MDA) as an end product of Ω-3 polyunsaturated lipid
peroxidation, was used to measure lipid peroxidation in fruits, as de-
scribed by Léchaudel et al. (2013). The amount of MDA–TBA complex
(red pigment) was calculated from the extinction coefficient:
155 mM−1 cm−1.
AsA content was measured according to the microplate-adapted
method developed by Léchaudel et al. (2013), omitting DHA content
determination. A reaction mixture was prepared with the supernatant
phosphate buffer (0.2 mM) and a reagent, including TCA (10%), or-
thophosphoric acid (42% v:v), 2.2-bipyridyl (4% w:v) dissolved in
ethanol (70%) and ferric chloride (3% w:v). The absorbance readings of
the Fe (2)-2,2-bipyridyl complex were taken at 525 nm with a micro-
plate reader. AsA content was established according to a standard curve
generated with commercial L-ascorbicacid.
All enzyme activities calculated from their kinetic reactions were
measured in a 200 μL volume at 25 °C, using a microplate reader. APX,
DHAR and MDHAR activities were measured using the method of
Murshed et al. (2002). SOD and CAT activities were measured using the
method described in Léchaudel et al. (2013). Proteins were quantified
using the Bradford assay (Bradford, 1976).
2.5. Carotenoid extraction and analysis
Carotenoid extraction was carried out according to the method of
Dhuique-Mayer et al. (2005) with slightly modifications. An aliquot
(0.5 g) of freshly lyophilized homogenized mango pulp was hydrated
with 2 mL of Ultrapure water and homogenized by magnetic stirrer
with 15 mL of an ethanol:hexane (4:3, v:v) mixture for 15 min. β-apo-8′-
carotenal (50 μL of 200 μg mL−1) was added as an internal standard.
The mixture was then centrifuged (8000 × g, at 4 °C, for 5 min). Or-
ganic phase (supernatant) were pooled after pellets were re-extracted
with solvent mixture for 15 min as previously to assess full discolora-
tion of the pellets. The organic phases were washed two times with
30 mL of 10% NaCl and three times with 25 mL of ultrapure water.
Then organic phase was dried using anhydrous Na2SO4 and evaporated
77
Journal of Plant Physiology 224–225 (2018) 75–85
to dryness under vaccuum at 37 °C. Carotenoid extract was dissolved in
20 mL of hexane.
10 mL of this solution were dried and dissolved in 50 μL di-
chloromethane and 450 μL of methanol:methyl‐tert-butyl ether (MTBE)
(1:1, v:v). This solution was diluted 4‐fold and filtered through a 0.45
membrane before HPLC analysis.
The remaining carotenoid extract (10 mL) was transferred in an
ambered flask and saponified by addition of 10 mL of 5% NaOH at room
temperature overnight under constant stirring. The reaction media was
then transferred in a separation funnel and washed three times by
20 mL of saturated NaCl solution. The hexanic phase was dried and the
aqueous phases were pooled together and washed three times with
dichloromethane. The dichloromethane phase was the added to the
dried hexanic remains and dried again under vaccuum at 37 °C. This
saponified extract was dissolved in 50 μL dichloromethane and 450 μL
of methanol:methyl‐tert-butyl ether (MTBE) (1:1, v:v). This solution was
diluted 4‐fold and filtered through a 0.45 membrane before HPLC
analysis.
Carotenoids were analyzed by HPLC (Dionex Ultimate 3000, Dionex
Co., Sunnyvale, CA, USA) coupled with a DAD detector. Carotenoids
were separated along a C 30 column (250 × 4.6 mm i.d., 5 μm YMC
Europ GmbH) thermostated at 30 °C. The mobile phases were A:
methanol:MTBE:water (96:2:2); and B: MTBE:methanol:water (80:18:2)
and isocratic system was used. Flow rate was 0.7 mL/min and injection
volume was 20 μL. Absorbance was then assessed at 209, 350, 450, and
470 nm using photodiode array detector.
2.6. Carotenoid identification
HPLC analyses were carried out with a Finnigan Surveyor plus
model, equipped of an autosampler, a PDA detector and quaternary
pump solvent delivery (Thermo Electron Corporation, San Jose, CA,
USA) on a saponified extract and a non-saponified/crude one.
Carotenoids were extracted as previously described above from freshly
lyophilized homogenized ripe mango pulp. Carotenoids were separated
along a C30 column (250 × 4.6 mm, 5 μm particle size), YMC (EUROP,
GmbH). The mobile phases were water/20 mM ammonium acetate as
eluent A/20 mM, ammonium acetate, methanol/20 mM, ammonium
acetate as eluent B and MTBE as eluent C. Flow rate was fixed at
0.7 mL/min and the column temperature was set at 25 °C. A gradient
program was performed: 0–2 min (40% A and 60% B, isocratic elution);
2–5 min (20% A and 80% B); 5–10 min (4% A, 81% B and 15% C);
10–60 min (4% A, 11% B and 85% C); 60–71 min (100% B); 71–72 min,
back to the initial conditions for equilibration. The injection volume
was 10 μl and the detection was monitored from 250 to 600 nm. After
passing through the flow cell of the diode array detector the column
eluate was split and 0.5 mL was directed to the ion trap of a LCQ mass
spectrometer fitted with an electrospray interface (Thermo Finnigan,
San Jose, California, USA). Experiments were performed in positive ion
mode. Scan range was 100–1000, scan rate: 1 scan s−1. The desolvation
temperature was set at 300 °C. The ionization source parameters were
set as followed: dry temperature 300 °C, source current 80 uA, nebuliser
pressure 20 psi, dry gas flow 75 L/min, capillary voltage 40 V, spray
voltage: 5 kV.
Identifications were performed using chromatrographic data and
uv-visible spectra treated by Chromeleon software (version 6.80).
Moreover, commercial standards of all-trans-violaxanthin was used to
confirm identification of this xanthophyll. Quantifications were estab-
lished from standard curves and expressed as violaxanthin or
ß‐carotene equivalents for xanthophylls and carotenes compounds, re-
spectively.
2.7. Statistical analysis
Analyses of variance were performed to assess the effect of the
storage temperature and the maturity stage at harvest on all the data
R. Rosalie et al.
Fig. 1. Effects of maturity stage at harvest and after ripening at 20, 12 and 7 °C on respiration rates (mmol CO2 kg−1 h−1) of mangoes from the 2012–2013 (A) and
2013–2014 (B) growing seasons. Plain lines represent the first harvest stage, dots lines represent the second harvest stage and dashed represent the third harvest
stage.
presented in the tables and figures. Multiple comparisons between
means of measurements at various maturity stages at harvest and after
ripening were performed using the Tukey test to evaluate whether or
not these means were significantly different. All statistical analyses
were computed using R software (Team, 2012).
3. Results
3.1. Changes in fruit characteristics and respiration rates in relation to
maturity stages and storage temperatures
Respiration rates, based on CO2 measurements (RR) during mango
ripening, were accumulated for two successive years. Some differences
concerning fruit respiration rates were observed, with a tendency of
fruits from the 2014 growing season to have lower respiration values;
nevertheless, fruits had the same behavior regardless of the growing
season (Fig. 1). The time until the maximum respiration rate measured
for both the first and second harvests was longer for fruits stored at 12
and 7 °C. Fruits from these two harvests took three days at room tem-
perature to reach maximum RR. RR values at harvest were relatively
low for H1 and H2 fruits (Fig. 1). At the end of the 15-days storage
period at 12 °C, fruits from H1 reached the highest RR, with a value of
4.2 mmoles CO2 kg−1 h−1, while those from H2 increased to
3.4 mmol kg−1 h−1. After storage at 7 °C, the RR of fruits from H1 had
increased to 3.3 mmol kg−1 h−1, while those from H2 reached
2.9 mmol kg−1 h−1. Maximum RR during ripening at 20 °C decreased
with the maturity stage at harvest by a factor of 0.77 at H3. Storage at
cold temperatures significantly increased the maximum RR values of
fruits from H1 and H2. It can be observed that the fruits of the 2014
78
Journal of Plant Physiology 224–225 (2018) 75–85
growing season harvested at H2 and then stored at 12 °C reached a
maximum RR that was similar to control fruits (Fig. 1B). Following fruit
peel and pulp observations, fruit showed no black or red spots and no
pitting regardless of the temperature conditions indicating that no
evidence of chilling injury symptoms was reported during this study.
Fruit weights depended on their harvest stage and the year of study
(Table 1). In 2013, fruits from the third harvest had the highest weight
(around 599 g), whereas first and second harvest weights were ap-
proximately 0.60 and 0.80 times this value (Table 1), respectively. In
2014, fruits were 0.77 times smaller at the third harvest (Table 1). Total
titratable acidity (TTA) of the flesh decreased along with the harvest
date from H1 to H3, with ripening reaching its lowest value at R3. Cold
storage did not significantly influence TTA values. Total Soluble Solid
content (TSS) in fruit pulp increased with harvest stage and sig-
nificantly with ripening, reaching the highest value at R3 with a score
of 17°Brix.
3.2. Changes in oxidative stress and antioxidant response in fruits in
relation to maturity stages at harvest
For fruits from early maturity stages at harvest (H1 and H2), H2O2
content remained relatively low, as did MDA content (Fig. 2). These two
compounds increased in fruits from the H3 stage, which corresponded
to the beginning of the respiration crisis, by 8-and 3-fold, compared to
the other stages, respectively.
Ascorbate content reached its highest values in fruits from the H1
stage. This content decreased with the maturity stage at harvest by
almost 3-fold in fruits from H3. Enzymes of the enzymatic antioxidant
system had low activity for H1 and H2 fruits (Fig. 3). At the H3 stage,
R. Rosalie et al. Journal of Plant Physiology 224–225 (2018) 75–85

Table 1
Effects of maturity stage at harvest and after ripening on fruit weight, Total Titratable Acidity (TTA), Total Soluble Solid (TSS) and dry matter (DMC) contents in the
pulp, according to temperature treatment for 2013 and 2014. Values are means ± sd n = 5 fruits.
Year Harvest At harvest Stored at 20 °C Stored at 12 °C Stored at 7 °C Storage Effect

2013
Weight (g) 1 384.09 ± 19.47 B a 395.07 ± 51.13 B ab 342.69 ± 31.26 B ab 323.66 ± 45.04 B b *
2 552.13 ± 52.59 A a 486.65 ± 31.26 B ab 434.23 ± 44.84 A b 463.89 ± 47.52 A b *
3 563.30 ± 52.34 A a 628.40 ± 155.43 A a – – –
TTA (meq 100 g−1) 1 77.53 ± 2.01 A a 13.95 ± 12.08 A b – – –
2 26.74 ± 2.37 B a 7.45 ± NA A b 4.42 ± 0.91 b 7.04 ± 1.56 b n.s
3 12.12 ± 4.38C a 7.45 ± 3.3 A a – – –
TSS (°Brix) 1 7.62 ± 0.75 B c 12.75 ± 1.71 C ab 14.50 ± 0.58 B a 12.00 ± 0.41 B b *
2 7.00 ± 0.82 B b 16.00 ± 0.58 B a 17.12 ± 0.85 A a 15.50 ± 0.58 A a n.s
3 15.80 ± 0.98 A b 19.75 ± 0.96 A a – – –
DMC (%) 1 14.30 ± 0.20 B a 13.5 ± 1.00 C a 13.20 ± 1.00 B a 12.70 ± 0.80 B a n.s
2 16.30 ± 0.80 B a 17.10 ± 0.80 B a 18.20 ± 0.70 A a 16.30 ± 1.30 A a n.s
3 21.30 ± 1.70 A a 22.2 ± 1.31 A a – –

2014
Weight (g) 1 346.57 ± 34.48 A a 370.11 ± 24.67 B a 371.52 ± 70.12 A a 358.77 ± 54.30 B a n.s
2 434.70 ± 100.51 A a 387.67 ± 49.26 B a 400.84 ± 53.19 A a 458.31 ± 86.91 A a n.s
3 – 460.15 ± 87.13 A – – –
TTA (meq 100 g−1) 1 32.47 ± 1.02 A a 5.02 ± 1.10 A b 4.25 ± 1.16 A b 3.60 ± 0.79 B b n.s
2 28.82 ± 3.53 A a 3.30 ± 0.31 A b 3.90 ± 0.74 A b 5.59 ± 0.62 A b ***
3 – 5.90 ± 3.67 A – – –
TSS (°Brix) 1 7.50 ± 2.35 A b 13.38 ± 1.11 B a 13.50 ± 0.50 A a 12.00 ± 1.00 B a n.s
2 8.88 ± 2.39 A b 14.62 ± 2.5 AB a 15.88 ± 1.65 A a 15.00 ± 0.82 A a n.s
3 – 17.25 ± 0.96 A – – –
DMC (%) 1 14.1 ± 0.4 A a 13.80 ± 0.30 A a 14.90 ± 1.30 A a 13.10 ± 1.30 B a n.s
2 16.9 ± 3.6 A a 17.30 ± 5.80 A a 16.20 ± 1.80 A a 16.00 ± 0.80 A a n.s
3 – 18.30 ± 1.20 A

Lowercase letters indicate that data for each harvest stage are significantly different at P < 0.05. Capital letters indicate that data for each temperature treatment are
significantly different at P < 0.05 according to Tukey's multiple comparison test. n.s., *, **, *** mean that the effect of temperature treatment is non-significant,
significant at P < 0.05, P < 0.01, and P < 0.001, respectively, for each maturity stage.

their activity dramatically increased by 2-, 10- and 10-fold for SOD, stages (Fig. 3F). Their values reached the one observed in fruits from
CAT and APX, respectively. It can be observed that CAT and APX ac- H3. In the case of the third harvest stage, ripening increased GR activity
tivities were barely detectable at H1 and H2. by a factor of 5.5.
For early maturity stages at harvest (H1 and H2), MDHAR and GR
activity was not detected, and DHAR activity was undetected at H1. At
3.4. Changes in oxidative stress and antioxidant response in ripe fruits in
H3, the activity of all the ascorbate regeneration enzymes was mea-
relation to previous storage temperatures
sured, and the highest activity was measured for MDHAR (Fig. 3).
Storage temperature did not have a significant impact on H2O2,
MDA and ascorbate content (Fig. 2). Enzymatic antioxidant systems
3.3. Changes in oxidative stress and antioxidant response in fruits in
were not influenced by cold storage with the exception of SOD and
relation to ripening
DHAR activity (Fig. 3). Protein content was also unaffected by tem-
perature treatments (Fig. 3G).
After ripening at 20 °C, H2O2 content increased in fruits at R1 and
Compared to fruit stored and ripened at 20 °C, a significant decrease
R2 compared to those at harvest, but remained non-significantly dif-
in CAT, APX and GR activity was observed for R1 fruits after two weeks
ferent from each other (Fig. 2A). MDA content doubled in ripe fruits at
of storage at 12 °C and ripening at 20 °C. MDHAR and DHAR activity
the R1 and R2 stages, and increased slightly between H3 and R3
remained equal to control values (Fig. 3). After storage at 7 °C, enzyme
(Fig. 2B). However, H2O2 content reached its highest values in fruits
activity for R1 fruits was equal to that of control fruits, except for the
from R3.
MDHAR activity, which decreased. These changes were significantly
Ascorbate content decreased by 0.25 and 0.30 times in fruits from
different for fruits from the second harvest. After storage at 12 °C and
R1 and R2, respectively, compared to their content at harvest (Fig. 2C).
ripening at 20 °C, fruits showed a significant increase in antioxidant
Values in fruits from H3 remained unchanged after ripening.
activity. CAT activity tripled, MDHAR and APX activity doubled,
During ripening, SOD activity increased by 0.25 and 1.13 times for
whereas GR activity increased by 16-fold (Fig. 3). After storage at 7 °C,
R1 and R2 fruits, respectively (Fig. 3A). At R3, this value remained the
CAT, APX and MDHAR activity nearly doubled and GR activity in-
highest. In the case of CAT and APX, the enzyme activities were mea-
creased 10-fold.
sured after ripening in fruits from the first and second harvest stages. It
can be observed that in both cases, the values were not significantly
different. Activity increased sharply in fruits from the third harvest 3.5. Changes in carotenoid composition in relation to maturity stages and
stage, by 4- and 7-fold for CAT and APX, respectively (Fig. 3B and C). storage temperatures
After ripening, a high increase in MDHAR activity was reported in
Fig. 3D. R1 and R2 fruits reached the same activity level as that mea- Cogshall mango pulp carotenoid extract was here characterized for
sured in H3 fruits, and the activity in R3 fruits was 50% higher than at the first time by HPLC‐MS analysis. The identification of both crude and
harvest. DHAR activity in R1 and R2 fruits was equal to that observed in saponified extracts through mass spectrometry combined with UV–vis
H2 fruits, whereas in R3 fruits, DHAR activity increased by 8-fold to spectra allowed the peak attribution presented in Table 2. The Peaks N°
reach the highest level of activity (Fig. 3E). GR activity was only 1 and 3 had similar mass (crude extract: 741, saponified extract: 601)
measureable after ripening for fruits from the first and second harvest and corresponded to dibutylated violaxanthin as described by Ornelas-

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R. Rosalie et al. Journal of Plant Physiology 224–225 (2018) 75–85

Fig. 2. Effects of maturity stage at harvest and after ripening for the three storage temperature treatments on the contents of hydrogen peroxide μmol gFW−1 (A),
malondialdehyde nmol gFW −1 (B), ascorbate mg 100 gFW −1 (C). At harvest, noted H, white bar corresponds to fruit from the first harvest, light one from the second
harvest and dark gray one from the third harvest. After ripening according to the different storage temperature treatments, noted R-1, R-2, R3, white bars correspond
to fruits from the first harvest, light gray bars correspond to second harvest fruits, and dark gray bars correspond to fruits from the third harvest. Values are
means ± sd n = 4 fruits.
At harvest, n.s., *, **, *** mean that the effect of the maturity stage is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively. After
ripening, n.s., *, **, *** mean that the effect of the storage temperature is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively, for each
maturity stage. Lowercase letters indicate that data are significantly different at P < 0.05.

Paz et al. (2007). Analysis of spectra allowed the attribution of the Peak
N° 1 to trans-violaxanthin dibutyrate and Peak N° 2 to 9-cis-violaxanthin
dibutyrate, on the basis of their % III/II and % AB/II ratios. The Peak N°
6 had a mass of 536 and a spectrum corresponding to α‐carotene while
the Peaks N° 7, 8 and 9 had a mass of 536 and spectra corresponding to
β‐carotene derivatives. Peaks N° 7 and 9 showed a slight shift at 340 nm
indicating a cis- conformation in the structure. Though the Peaks N° 7
and 9 were attributed respectively to 13- cis‐β‐carotene and 9‐cis‐β‐-
carotene. The Peak N° 8 corresponded to all-trans‐β‐carotene. Minor
carotenoids have also been identified basing on their molecular weights
and spectral analysis such as The Peak N° 2, neochrome, the Peak N° 4,
lutein and the Peak N° 5, β‐cryptoxanthin (Table 2). In this study we
focused on the four major carotenoid compounds: all‐trans‐violax-
anthine, 9-cis‐violaxanthine, α‐carotene and β-carotene.
Carotenoid content builds up along with the fruit harvest stage.
Carotenoid content remained low for the two first maturity stages and
dramatically increased at H3 (Fig. 4). Fruits from the first harvest had a
total carotenoid content of 17 and 9 μg gDM −1 for the 2013 and 2014

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R. Rosalie et al. Journal of Plant Physiology 224–225 (2018) 75–85

(caption on next page)

seasons, respectively. This content increased by 1.5-fold at the second started during ripening at 20 °C for the first harvest fruits, whereas ripe
harvest and again by 6.5-fold at the third harvest, which had already fruits reached 9-fold the harvest value at155 and 120 μg gDM −1 for
triggered carotenoid accumulation (Fig. 4). Carotenoid accumulation 2013 and 2014, respectively. For the second harvest, carotenoid

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R. Rosalie et al. Journal of Plant Physiology 224–225 (2018) 75–85

Fig. 3. Effects of maturity stage at harvest and after ripening at 20, 12 and 7 °C on the activities of (A) superoxide dismutase (U min−1 gFW−1), (B) catalase
(mmol min−1 gFW−1), (C) ascorbate peroxydase (μmol min−1 gFW−1), (D) monodehydroascorbate reductase (μmol min−1 gFW−1), (E) dehydroascorbate reductase
(μmol min−1 gFW−1), (F) glutathione reductase (μmol min−1 gFW−1), and the content in (G) protein (mg gFW−1). At harvest, noted H, white bar corresponds to fruit
from the first harvest, light one from the second harvest and dark gray one from the third harvest. After ripening according to the different storage temperature
treatments, noted R-1, R-2, R3, white bars correspond to fruits from the first harvest, light gray bars correspond to second harvest fruits, and dark gray bars
correspond to fruits from the third harvest. Values are means ± sd n = 4 fruits.
At harvest, n.s., *, **, *** mean that the effect of the maturity stage is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively. After
ripening, n.s., *, **, *** mean that the effect of the storage temperature is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively, for each
maturity stage. Lowercase letters indicate that data are significantly different at P < 0.05.

content increased with ripening by 7.8 and 9.5-fold for fruits of the
2013 and 2014 harvest, respectively. Fruits from the third harvest had
the highest content with 265 and 152 μg gDM −1 in 2013 and 2014,
respectively.
Carotenoids were the compounds that were the most affected by
storage temperature. Carotenoid content decreased according to the
storage temperature in R1 fruits, by 0.81 and 0.61-fold at 12 °C and
7 °C, respectively, and for both years. The storage temperature effect
was not significant for R2 fruits in the 2013 season (Fig. 4E). However,
R2 fruits stored at 7 and 12 °C had lower content than those at 20 °C.
The storage temperature effect on carotenoid content was confirmed in
2014, regardless of the harvests (Fig. 4F).
Results presented here for individual compounds are those obtained
in 2013. 2014 data had the same evolution except for R2 fruits after
storage at 7 °C.
At harvest, trans‐violaxanthin dibutyrate content was 1.65, 3.08 and
42.89 μg gDM −1 for the first, second and third harvests, respectively.
During ripening, the trans‐violaxanthin dibutyrate derivative increased
to reach 14.8-fold, 10.8-fold and 1.3-fold, respectively. cis-violaxanthin
dibutyrate content was 20–40% lower that its isomer, with a gradual
increase of content according to harvest stage after ripening. β-carotene
content also increased 10-fold with fruit age at harvest, from 3.08 to
31.45 μg gDM −1 at H1 and H3, respectively. This content continued to
increase after ripening at 20 °C to reach 11.6-fold, 6.65-fold and 1.3-
fold of the harvest content for R1, R2 and R3 respectively, making
β‐carotene the most abundant compound in mango (cv. “Cogshall”)
pulp for the R1 and R2 stages. α-carotene content remained low and
was not detectable at H1 and H2. It remained at approximately
2 μg gDM −1 after ripening and at H3.
R1 fruits were the most deeply affected by temperature storage.
Trans‐violaxanthin dibutyrate and cis‐violaxanthin dibutyrate content
was reached half of the value of the control at 12 °C and 7 °C.
α‐carotene content remained at low values, regardless of the tempera-
ture treatment. β‐carotene content was significantly affected by cold
storage at 7 °C when it reached 0.43 times the control value. It appears
that this compound was less affected than violaxanthin derivatives. R2
fruits from storage at 12 and 7 °C had lower contents than the control
fruits, and this difference was especially significant for β-carotene
(Fig. 4D).
4. Discussion
4.1. Effect of ripening on changes in respiration rates, oxidative stress and
antioxidant response in mango
Fruit respiration rates increased during ripening whether it started
on the tree (H3) or during post-harvest storage (R1 and R2). This in-
crease is known to increase ROS production due to mitochondrial ac-
tivity and is observable by the significant accumulation of H2O2 mea-
sured between fruit at harvest (green-mature stage) and in ripe fruit for
the first and second maturity stages at harvest, and by the increase in
MDA content (Pandey et al., 2013). In order to cope with this oxidative
stress, fruits increase their antioxidant response by consuming ascor-
bate and increasing their antioxidant enzymatic and ascorbate re-
generation systems. For green‐mature stages at harvest, most of the
antioxidant and regeneration enzyme activities were undetectable, with
the exception of SOD and DHAR. On the other hand, for fruits harvested
at a later maturity stage when a sharp increase in respiration rates was
measured, i.e., H3, fruits stabilized their oxidative status through the
action of their antioxidant enzymatic system as well as through their
ascorbate regeneration system that increased at this maturity stage. The
highest degree of enzyme activity of the enzymatic antioxidant defense
and ascorbate regeneration systems was observed in ripe fruits, e.g.,
plum, as of the beginning of ripening (Singh et al., 2012). Thus, ri-
pening induced a large increase in these enzymatic activities to cope
with the oxidative stress due to ripening and to stabilize the ascorbate
content.
4.2. Effect of temperature storage on changes in respiration rates, oxidative
stress and antioxidant response in mango
Despite the fact that no visual sign of CI symptoms was reported
during this study, storage temperature significantly influenced re-
spiration rates of fruits from both harvests, implying a higher ROS

Table 2
Chromatographic and spectral data obtained by HPLC-DAD-MS detection of the carotenoids in saponified mango cv. ‘Cogshall’.
Retention time min Peaks letters Carotenoida [M+H]+ m/z λmax nm*b % III/II % AB/II

15.80 1 violaxanthin/neoxanthin 601 328-416-440-468 86

30
16.64 2 neochrome 601 400-422-448 90
17.88 3 9-violaxanthin-cis-violaxanthin 601 328-412-436-464 84
10
21.25 4 lutein 569 420-444-472 47
28.54 5 β-cryptoxanthin 553 430-450-480 20
29.30 – Phytofluene – 331-348-368 68
28.09 – Phytoene – 276-286-298 –
31.26 6 α-carotene 536 422-448-472 34
32.47 7 13-cis- β-carotene – 340-416-444-466 –
35.74 8 β-carotene 536 452-478 12
36.95 9 9-cis-β-carotene 536 340-426-446-472 –

a
Tentative identification.
b
Main wavelength of maximum absorption are underlined.

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R. Rosalie et al. Journal of Plant Physiology 224–225 (2018) 75–85

Fig. 4. Effects of maturity stage at harvest and after ripening at 20, 12 and 7 °C on the contents in the various carotenoid compounds, (A) trans-violaxanthin
dibutyrate (μg gDW−1), (B) cis-violaxanthin dibutyrate (μg gDW−1), (C) α-carotene (μg gDW−1), (D) β-carotene (μg gDW−1), and in the total carotenoids content of
fruit from the 2012–2013 (E) and 2013–2014 (F) growing seasons. At harvest, noted H, white bar corresponds to fruit from the first harvest, light one from the second
harvest and dark gray one from the third harvest. After ripening according to the different storage temperature treatments, noted R-1, R-2, R3, white bars correspond
to fruits from the first harvest, light gray bars correspond to second harvest fruits, and dark gray bars correspond to fruits from the third harvest. Values are
means ± sd n = 4 fruits.
At harvest, n.s., *, **, *** mean that the effect of the maturity stage is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively. After
ripening, n.s., *, **, *** mean that the effect of the storage temperature is non-significant, significant at P < 0.05, P < 0.01, and P < 0.001, respectively, for each
maturity stage. Lowercase letters indicate that data are significantly different at P < 0.05.

production in cold-stored fruits. Fruits had a lower RR during storage
since the temperature slowed down their metabolism, but it dramati-
cally increased when they were transferred to ambient temperature
prior to ripening. This evolution is similar to the observations made on
tomato fruit (Malacrida et al., 2006) and pawpaw fruit (Archbold and
Pomper, 2003). Moreover, a previous study on mango reported that
fruits stored at 15 °C for 2 weeks showed no signs of CI and had higher
RR values during ripening, in comparison to fruits stored at 5 °C which
was partly inhibited (Nair and Singh, 2009). No significant difference in
H2O2 and MDA contents were observed between ripe fruits stored for
15 days at 7 °C and 12 °C and those stored at 20 °C. However, a slight
increase in MDA levels was observed for fruits from the first harvest
stored at 7 °C, which might suggest a possible initiation phase of CI. We
can also suppose that the time/temperature couple with 15 days of
storage at 12 or 7 °C was not sufficient to induce visible CI symptoms.
Junmatong et al. (2012) showed that mango cv. “Nam Dok Mai No. 4”
took at least 21 days at 5 °C to present deteriorations linked to CI, and
Ding et al. (2007) observed the same results for mango cv. “Zill” stored
at 5 °C. The tolerance to CI depends also on the variety
(Phakawatmongkol et al., 2004), with a probable impact on conditions
and time of initiation. Maturity stage at harvest can also influence fruit
ripening processes to alleviate CI in mangoes cv. Tommy Atkins
(Mohammed and Brecht, 2002), indicating that fruit ripening was in-
sufficient to reduce CI in immature fruit compared to mature one. In our
study, the ripening of the earlier maturity stage at harvest was not be
affected by low temperature, as cold temperature could suppressed
respiration and ethylene biosynthesis associated with the development
of this physiological disorder in mango (Nair et al., 2004; Nair and
Singh, 2009).
Ascorbate content was not significantly affected by cold storage
after 15 days and after ripening, as observed in mango cv. ‘Wacheng'
fruit (Zhao et al., 2006). These observations suggest that under these
conditions, the oxidant and antioxidant compounds of fruits are mainly
driven by their maturity stages at harvest and by the oxidation involved
during ripening. On the other hand, fruit antioxidant response varies
according to fruit maturity stage at harvest and the storage temperature
applied. Fruits stored at low temperatures, i.e., 7 °C, presented a higher
oxidative stress, represented through their higher maximum RR, than
those stored at 12 °C or 20 °C. In reaction to cold stress, fruits seemed to
activate enzymatic ROS scavenging responses by SOD, CAT and APX
and ascorbate regeneration enzymes. These responses seemed to be
sufficient to prevent fruits from deleterious oxidation incurred by low-

83
R. Rosalie et al.
temperature storage, as shown in mango (Zhao et al., 2006).
We observed that activities of CAT, APX and GR in fruits from the
first harvest were decreased after cold storage at 12 °C for CAT, APX
and GR, and at 7 °C for MDHAR. No evidence of an increase in enzy-
matic activity was measured at this temperature for which the ascor-
bate content seemed to be sufficient to act as a molecular scavenger. On
the contrary, fruits from the second harvest showed significantly higher
activity for CAT, APX, MDHAR and GR at 12 °C and 7 °C in comparison
to the control. This difference in fruit behavior reflects their ability to
adapt to cold temperatures and, consequently, their ability to balance
oxidation stress. These results are in agreement with Zhao et al. (2009)
who reported lower antioxidant activity for CAT and APX for mango cv.
‘Zihua’ green fruits during storage at 2 °C in comparison to pre‐yellow
fruits. However, few reports of fruit response during cold storage ac-
cording to their effective maturity stages at harvest were found in the
literature and these results highlight the importance of taking the
physiological age of fruit into account in order to develop a better
understanding of the impact of storage conditions on fruit metabolism.
4.3. Effect of temperature storage on carotenoid accumulation in mango
pulp
Carotenoid content of cv. “Cogshall” mangoes was close to those
observed for Kent mangoes by Ornelas-Paz et al. (2007), with trans-
violaxanthin diesters as major compounds, followed by β‐carotene and
cis-violaxanthin diesters. In this study, the ratio between these three
main compounds was 11/8/6 (R3, 20 °C) compared to 11/9/7 in cv.
‘Kent’. Concerning the total carotenoid content, that of fully ripe Cog-
shall mangoes is comparable to Kent (Ornelas-Paz et al., 2007) or ripe
Tommy Atkins (Mercadante and Rodriguez-amaya, 1998). Carotenoid
accumulation after cold storage was influenced by the fruit maturity
stage at harvest. Fruits from the first harvest were negatively affected at
7 and 12 °C for violaxanthin derivatives and at 7 °C for β-carotene for
both years. These results were coherent with those presented in the
literature, where fruits stored at low temperatures accumulated sig-
nificantly less carotenoids (Ding et al., 1998; Malacrida et al., 2006;
Talcott et al., 2005). Cold storage influenced carotenoid compounds
differently. It was observed that low temperatures slowed or blocked
carotenoid accumulation on tomato fruits (Gomez et al., 2009). Studies
of the changes in this content, once fruits are withdrawn from cold
storage, are scarce. Nevertheless, this phase is the most important be-
cause the ripening of climacteric fruits greatly influences the final
carotenoid content that will be available for the consumer. In this study,
we were able to observe for two consecutive years that β-carotene was
less down-regulated by cold temperature storage than xanthophyll
compounds in mango pulp of fruit from an early harvest, as observed in
papaya (Rivera-Pastrana et al., 2010) and in loquat (Ding et al., 1998).
Interestingly, second harvest fruit response was not impacted by pre-
vious exposure to cold at 12 °C. This highlights the fact that the meta-
bolism of these fruits was less affected at this storage temperature.
These results underline the negative impact of cold storage on the
accumulation of carotenoids in mango. After post-storage ripening, the
carotenoid content of fruits remained significantly lower for fruits
stored at 7 °C, particularly for early harvest fruit. Therefore, storage
conditions could affect the nutritional value of mango.
4.4. Interaction between oxidative status and carotenoid accumulation
Although we were able to determine how carotenoid accumulation
could be influenced by harvest stage and temperature, the interaction
between carotenoid accumulation and antioxidant enzyme activity
appears to be more complex. Some hypotheses for how this works in
plants were proposed by Bouvier et al. (1998) and Fanciullino et al.
(2013) in which ROS mediate the activation of the carotenogenesis
gene and where the global oxidative status influences carotenoid en-
zyme activity. Evidence of correlations between ROS and carotenoid
84
Journal of Plant Physiology 224–225 (2018) 75–85
metabolism was also observed by Lim et al. (2009) on chilling-sensitive
and tolerant pepper fruit, where increases in SOD, APX and CAT were
correlated with lycopene and β‐carotene accumulation during storage
at 1, 5 and 10 °C. A recent study in mango had pointed out the re-
lationship between ROS and the increase in nonenzymatic antioxidants
accumulation, but more for the phenolic metabolism than for the car-
otenoid one (Jiang et al., 2015). However, another study showed that in
mango pulp, the oxidative stress acted as signals triggering enzymatic,
i.e., SOD and CAT, and non-enzymatic, i.e., carotenoid, ascorbic acid
and total phenolics, antioxidants (Lopes et al., 2016).
In most studies, carotenoid accumulation is negatively influenced
by temperature, as observed through ripening-related gene activation in
cold-stored tomato fruit (Rugkong et al., 2011), and this influence is
somehow involved in ROS metabolism. Equilibrium is therefore in-
volved between the oxidative effect of ROS generated by abiotic stress
and fruit ripening, which dictates the ability of fruits to maintain a
healthy oxidative status. From this point of view, a link could be made,
taking the physiological age into account. We showed that early har-
vested fruits will respond to cold storage stress by using their high as-
corbate pool to act as molecular scavengers. On the other hand, an older
fruit will have already established its antioxidant metabolism and will
have a homogeneous response to stressful conditions. Moreover, in
early harvested fruits, the antioxidant enzymatic system appears to be
less efficient than that of later harvested fruits, resulting in a poor
carotenoid accumulation when fruits were kept at 12 or 7 °C, as shown
for pepper fruit stored at cold temperatures (Lim et al., 2009). On the
contrary, second harvest fruits exhibited higher enzymatic activity
during cold storage and, in parallel, higher carotenoid accumulation,
independently of storage temperature. As in this study, it has been
shown that harvest stage will define the final carotenoid content of the
ripe fruit (Ornelas-Paz et al., 2007; Talcott et al., 2005), but postharvest
conditions could disrupt carotenoid biosynthesis during ripening, de-
pending on the ability of fruit to manage cold stress (Gonzalez-Aguilar
et al., 2010).
5. Conclusions
Storage at low temperatures had an impact on mango cv. “Cogshall”
metabolism that was mitigated by the fruit harvest stage. The activity of
ROS scavenging and ascorbate recycling enzymes was more efficient for
second harvest fruits, allowing a better adaptation to low‐temperature
storage. On the other hand, antioxidant enzyme activity of fruits from
the first harvest did not clearly respond to low temperatures, but mainly
to the molecular scavenging provided by a higher initial ascorbate
content.
Total carotenoid contents were influenced by temperature treat-
ments, and when lower when storage temperature decreased.
Individual carotenoid accumulation also decreased with temperature.
Xanthophyll compounds were more susceptible than carotene to this
down-regulation. In this study, fruits were stored for two weeks at cold
temperatures, but it is probable that longer storage periods could in-
crease fruit response, with a higher negative impact on the final car-
otenoid content.
Since ROS metabolism and carotenogenesis are linked, further in-
vestigations into the interaction between the two metabolisms are ne-
cessary to determine how cold stress influences carotenoid synthesis.
Acknowledgments
The authors gratefully acknowledge C. Soria (CIRAD, UR HortSys,
Reunion Island) for technical assistance during the field experiments,
and J. Minier (CIRAD, UMR Qualisud, Reunion Island) and M.
Darnaudery (CIRAD, UR HortSys, Reunion Island) for assistance with
the biochemical analyses. We thank the Regional Council of Reunion
Island, France for funding the PhD of Rémy Rosalie, and G. Wagman for
revising the manuscript and improving the English.
R. Rosalie et al.
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