Вы находитесь на странице: 1из 11

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

Contents lists available at ScienceDirect

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
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,
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
CIRAD, UMR QualiSud, Station de Neufchâteau, Sainte-Marie, 97130 Capesterre-Belle-Eau, Guadeloupe, France
CIRAD, UMR Qualisud, TA B95/16, 73 rue Jean-François Breton, 34398 Montpellier Cedex 5, France
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


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
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

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

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

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

more severe cases, uneven ripening. It was observed that these symp- 2. Material and methods
toms generally appeared only after fruits returned to an ambient tem-
perature, i.e., 20 °C (Sivakumar et al., 2011). Consequently, it can be 2.1. Plant and fruit material
deduced that they are influenced by both exposure time and cold in-
tensity, in addition to species or cultivar effects (Lurie and Crisosto, This study was conducted on well-irrigated mango trees of cv.
2005) as demonstrated on mango by (Phakawatmongkol et al. (2004)). Cogshall, a local variety subjected to exportation to France, grafted on
Cold temperatures could therefore induce alterations in cell mem- ‘Maison Rouge’ rootstock, in Reunion Island (20°52’48”S, 55°31’48”E)
brane conformation and structure by increasing lipid saturation in at the CIRAD Experimental Station, at an elevation of 95 m, during the
order to maintain membrane functionality (Sevillano et al., 2009). After 2012–2013 and 2013–2014 growing seasons. Chlorophyll fluorescence
cold storage, membranes face disruptions that lead to ionic leakage and parameters, i.e., minimal and maximal chlorophyll fluorescence (Fo and
accumulation of ROS linked to an increase in respiration rates related to Fm, respectively), and the variable chlorophyll fluorescence
ripening (Kratsch and Wise, 2000). The membrane alteration is ob- (Fv = Fm − Fo), were measured on the fruit surface near the apex zone
served through MDA content, which increases in fruits submitted to where the first changes in peel color appear in order to assess the degree
cold temperatures along with the exposure time (Singh et al., 2013) and of mango maturity, regardless of fruit growing conditions (Léchaudel
this increase is correlated with superoxide (O2−) production (Zhou et al., 2010). Three maturity stages corresponding to three different
et al., 2014). An increase of MDA content was positively correlated with values of the variable chlorophyll fluorescence (Fv) parameter were
the apparition of chilling injury in mango peel and pulp for cultivar chosen: approximately 1400, 1250 and 950 for H1, H2 and H3, re-
Nam Dok Mai No. 4 (Junmatong et al., 2012). This ROS accumulation is spectively. H1 and H2 corresponded to two green mature and com-
balanced in fruits by a complex antioxidant response involving both mercial maturity stages. The third harvest stage, H3, corresponded to a
non-enzymatic, i.e., ascorbate, and enzymatic systems such as super- maturity stage when the fruit’s apex has started to lose its green color
oxide dismutase, catalase, ascorbate peroxidase and the ascorbate-glu- and became yellow, referred to as the yellow point stage (YPS), a ty-
tathione cycle. Several studies have highlighted the involvement of pical stage for this variety, indicating the onset of the climacteric pro-
these systems during cold storage. In tomato, catalase (CAT) and glu- cess. It was assumed that fruits harvested at the YPS presented the best
tathione reductase (GR) responded positively to conservation at 4 °C quality potential (Joas et al., 2005).
(Malacrida et al., 2006). In mandarin fruits, APX activity decreased
during storage at 2.5 °C, whereas SOD and CAT increased (Sala, 1998). 2.2. Low temperature storage
Since cold temperatures reduce fruit metabolism, they also have an
impact on fruit color. Malacrida et al. (2006) showed a decrease in Forty fruits were selected from the first and second harvest stages
carotenoid synthesis, especially lycopene, which was four times lower and separated into three groups: storage at 12 °C (12 fruits), storage at
in ripe fruits that remained at 4 °C for four weeks. Carotenoid accu- 7 °C (12 fruits) and 20 °C (16 fruits). Sixteen fruits were selected from
mulation might also be closely linked to fruit oxidation status, as pro- the third harvest stage and maintained at 20 °C. Four fruits were sam-
posed by Fanciullino et al. (2013), who argued that the stress-related pled for analysis at harvest, corresponding to these H1, H2 and H3
production of ROS and its regulation closely control the synthesis and stages. Four fruits were stored for ripening in controlled conditions
the accumulation of carotenoid in fruits and leaves. (20 °C and 90% relative humidity (RH)). Twelve fruits were stored at 12
It is known that temperatures of around 12 °C are the optimum for and 7 °C and 90% RH for 15 days for the first and second harvest stages,
mango storage and that lower temperatures trigger alterations in fruit corresponding to the average storage time used for Cogshall mango
quality (Medlicott et al., 1990). Studies dealing with the involvement of exportation. At the end of this period of storage, i.e. 15 days, fruits were
oxidative status during cold storage are scarce in mango fruit compared transferred for ripening in storage conditions corresponding to 20 °C
to other fruits. Most of the studies focus on cold exposure symptoms and and 90% RH, and their external aspects were observed during ripening
their alleviation by pre-treatments, e.g., cold shock (Zhao et al., 2006), to check external cold-induced defects, such as red spots, black spots,
heat exposure, or dipping (Ding et al., 2007). Mango is known for its and pitting or other general decays.
high carotenoid content, especially for compounds involved in nutri-
tional value such as β-carotene, exhibiting pro-vitamin A activity. Its 2.3. Postharvest and quality measurements
accumulation decreases with storage temperature (Medlicott et al.,
1990) although no study regarding the impact of temperature on in- To ensure that ripe fruits had the same physiological age when
dividual compounds was found. analyzed, respiratory metabolism and climacteric rise were used as
Moreover, mangoes are often harvested before the suitable green indicators (Joas et al., 2009). Each of the fruits was monitored daily to
mature stage in order to ensure a longer shelf time, but this stage can follow the changes in respiration rate (RR). Respiration rate was ex-
lead to reduced fruit quality (Medlicott et al., 1990) especially on young pressed in terms of CO2 production at 20 °C (mmol kg−1 h−1) using a
fruits that were shown to be very sensitive to CI (Mohammed and closed system method. Fruits from each treatment were placed in in-
Brecht, 2002). The use of low temperatures for storage at excessively dividual 3 L airtight jars at 20 °C, and CO2 concentration was measured
early stages increased physiological disorders, loss of firmness and lack every 20 min for 1 h by gas chromatography (GC) using an Agilent
of color development (Nunes et al., 2007). Harvest maturity stage at M200 apparatus (SRA, Marcy l’Etoile, France) equipped with two
harvest is also an important parameter that affects mango quality (Joas manifolds and two columns: Porapak Q (8 m), thermostated at 55 °C,
et al., 2012) and it was shown that delayed harvest decreased fruit and MS-5A (4 m), thermostated at 60 °C, with helium and argon as
ability to overcome oxidative stress induced by cold storage in plum carrier gases, respectively.
(Singh et al., 2013). Fruit sampling was done at the ripe stage, corresponding to R1, R2
In this article, we aimed to evaluate the impact of both maturity and R3, for the three harvest stages H1, H2 and H3, respectively. Four
stage at harvest and storage temperature over two successive years on ripe fruits were analyzed three days after they had reached the highest
the oxidative status of ‘Cogshall' mangoes, represented by oxidation- value of their respiration rate, corresponding to the “ready-to-eat”
related compounds (H2O2, MDA), antioxidant compounds (ascorbate, stage.
carotenoids), ROS handling enzymes (SOD, CAT, APX) and ascorbate Sampling conditions were established to limit matrix degradation
regeneration enzymes (MDHAR, DHAR, GR). We also attempted to es- linked to light, temperature and enzymatic activity. Fruits were peeled,
tablish a possible link between carotenoid accumulation and ROS re- cut, examined to check for the absence of internal cold-induced defects,
sponse to a moderate stress linked to storage temperature. 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. Journal of Plant Physiology 224–225 (2018) 75–85

Solids content and Titratable Acidity; the other part was immediately to dryness under vaccuum at 37 °C. Carotenoid extract was dissolved in
wrapped in an alumina sheet and dipped in liquid nitrogen. These 20 mL of hexane.
frozen pulps were ground in a Grindomix blender and the powders 10 mL of this solution were dried and dissolved in 50 μL di-
obtained were stored in opaque plastic pots at −80 °C for future ana- chloromethane and 450 μL of methanol:methyl‐tert-butyl ether (MTBE)
lysis. (1:1, v:v). This solution was diluted 4‐fold and filtered through a 0.45
A sample of each frozen powder was used for dry matter content membrane before HPLC analysis.
assessment, done by comparing its fresh mass and its dry mass recorded The remaining carotenoid extract (10 mL) was transferred in an
after lyophilization, performed over 48 h at −52 °C. Lyophilization was ambered flask and saponified by addition of 10 mL of 5% NaOH at room
used to dry a part of each sample for carotenoid analysis. Lyophilization temperature overnight under constant stirring. The reaction media was
was performed over 48 h at −52 °C on a sample of each frozen powder. then transferred in a separation funnel and washed three times by
Dried mango powders were stored in plastic pots with a desiccation 20 mL of saturated NaCl solution. The hexanic phase was dried and the
pastille at −20 °C until analysis within three weeks. aqueous phases were pooled together and washed three times with
Titratable acidity was measured using an automated titrimeter dichloromethane. The dichloromethane phase was the added to the
(TitroLine easy, Schott, Mainz, Germany) with a 0.05N NaOH solution. dried hexanic remains and dried again under vaccuum at 37 °C. This
Total soluble solids were determined using a refractometer (Atago ATC- saponified extract was dissolved in 50 μL dichloromethane and 450 μL
1E; Tokyo, Japan). 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
2.4. Oxidative stress and antioxidant response measurements analysis.
Carotenoids were analyzed by HPLC (Dionex Ultimate 3000, Dionex
Oxidant compounds (H2O2, MDA), antioxidant compounds (ascor- Co., Sunnyvale, CA, USA) coupled with a DAD detector. Carotenoids
bate, carotenoids), antioxidant enzymes (SOD, APX, CAT) and ascor- were separated along a C 30 column (250 × 4.6 mm i.d., 5 μm YMC
bate-glutathione recycling enzymes (MDHAR, DHAR, GR) were de- Europ GmbH) thermostated at 30 °C. The mobile phases were A:
termined as follows. methanol:MTBE:water (96:2:2); and B: MTBE:methanol:water (80:18:2)
Hydrogen peroxide content was assayed as described by Léchaudel and isocratic system was used. Flow rate was 0.7 mL/min and injection
et al. (2013) using frozen mango pulp (0.25 g) homogenized in an ice volume was 20 μL. Absorbance was then assessed at 209, 350, 450, and
bath with 1 mL 0.1% (w:v) TCA. A reaction mixture was prepared with 470 nm using photodiode array detector.
the supernatant and KI. Absorbance readings of the triiodide ion (I3−),
resulting from the reaction between H2O2 and KI, were taken at 390 nm 2.6. Carotenoid identification
by a microplate reader (BioTek Instruments, Inc.; Winooski, Vermont,
USA). H2O2 content was quantified according to a standard curve. HPLC analyses were carried out with a Finnigan Surveyor plus
The thiobarbituric acid (TBA) test, which determines mal- model, equipped of an autosampler, a PDA detector and quaternary
onyldialdehyde (MDA) as an end product of Ω-3 polyunsaturated lipid pump solvent delivery (Thermo Electron Corporation, San Jose, CA,
peroxidation, was used to measure lipid peroxidation in fruits, as de- USA) on a saponified extract and a non-saponified/crude one.
scribed by Léchaudel et al. (2013). The amount of MDA–TBA complex Carotenoids were extracted as previously described above from freshly
(red pigment) was calculated from the extinction coefficient: lyophilized homogenized ripe mango pulp. Carotenoids were separated
155 mM−1 cm−1. along a C30 column (250 × 4.6 mm, 5 μm particle size), YMC (EUROP,
AsA content was measured according to the microplate-adapted GmbH). The mobile phases were water/20 mM ammonium acetate as
method developed by Léchaudel et al. (2013), omitting DHA content eluent A/20 mM, ammonium acetate, methanol/20 mM, ammonium
determination. A reaction mixture was prepared with the supernatant acetate as eluent B and MTBE as eluent C. Flow rate was fixed at
phosphate buffer (0.2 mM) and a reagent, including TCA (10%), or- 0.7 mL/min and the column temperature was set at 25 °C. A gradient
thophosphoric acid (42% v:v), 2.2-bipyridyl (4% w:v) dissolved in program was performed: 0–2 min (40% A and 60% B, isocratic elution);
ethanol (70%) and ferric chloride (3% w:v). The absorbance readings of 2–5 min (20% A and 80% B); 5–10 min (4% A, 81% B and 15% C);
the Fe (2)-2,2-bipyridyl complex were taken at 525 nm with a micro- 10–60 min (4% A, 11% B and 85% C); 60–71 min (100% B); 71–72 min,
plate reader. AsA content was established according to a standard curve back to the initial conditions for equilibration. The injection volume
generated with commercial L-ascorbicacid. was 10 μl and the detection was monitored from 250 to 600 nm. After
All enzyme activities calculated from their kinetic reactions were passing through the flow cell of the diode array detector the column
measured in a 200 μL volume at 25 °C, using a microplate reader. APX, eluate was split and 0.5 mL was directed to the ion trap of a LCQ mass
DHAR and MDHAR activities were measured using the method of spectrometer fitted with an electrospray interface (Thermo Finnigan,
Murshed et al. (2002). SOD and CAT activities were measured using the San Jose, California, USA). Experiments were performed in positive ion
method described in Léchaudel et al. (2013). Proteins were quantified mode. Scan range was 100–1000, scan rate: 1 scan s−1. The desolvation
using the Bradford assay (Bradford, 1976). temperature was set at 300 °C. The ionization source parameters were
set as followed: dry temperature 300 °C, source current 80 uA, nebuliser
2.5. Carotenoid extraction and analysis pressure 20 psi, dry gas flow 75 L/min, capillary voltage 40 V, spray
voltage: 5 kV.
Carotenoid extraction was carried out according to the method of Identifications were performed using chromatrographic data and
Dhuique-Mayer et al. (2005) with slightly modifications. An aliquot uv-visible spectra treated by Chromeleon software (version 6.80).
(0.5 g) of freshly lyophilized homogenized mango pulp was hydrated Moreover, commercial standards of all-trans-violaxanthin was used to
with 2 mL of Ultrapure water and homogenized by magnetic stirrer confirm identification of this xanthophyll. Quantifications were estab-
with 15 mL of an ethanol:hexane (4:3, v:v) mixture for 15 min. β-apo-8′- lished from standard curves and expressed as violaxanthin or
carotenal (50 μL of 200 μg mL−1) was added as an internal standard. ß‐carotene equivalents for xanthophylls and carotenes compounds, re-
The mixture was then centrifuged (8000 × g, at 4 °C, for 5 min). Or- spectively.
ganic phase (supernatant) were pooled after pellets were re-extracted
with solvent mixture for 15 min as previously to assess full discolora- 2.7. Statistical analysis
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. Analyses of variance were performed to assess the effect of the
Then organic phase was dried using anhydrous Na2SO4 and evaporated storage temperature and the maturity stage at harvest on all the data

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

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

presented in the tables and figures. Multiple comparisons between growing season harvested at H2 and then stored at 12 °C reached a
means of measurements at various maturity stages at harvest and after maximum RR that was similar to control fruits (Fig. 1B). Following fruit
ripening were performed using the Tukey test to evaluate whether or peel and pulp observations, fruit showed no black or red spots and no
not these means were significantly different. All statistical analyses pitting regardless of the temperature conditions indicating that no
were computed using R software (Team, 2012). evidence of chilling injury symptoms was reported during this study.
Fruit weights depended on their harvest stage and the year of study
3. Results (Table 1). In 2013, fruits from the third harvest had the highest weight
(around 599 g), whereas first and second harvest weights were ap-
3.1. Changes in fruit characteristics and respiration rates in relation to proximately 0.60 and 0.80 times this value (Table 1), respectively. In
maturity stages and storage temperatures 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
Respiration rates, based on CO2 measurements (RR) during mango date from H1 to H3, with ripening reaching its lowest value at R3. Cold
ripening, were accumulated for two successive years. Some differences storage did not significantly influence TTA values. Total Soluble Solid
concerning fruit respiration rates were observed, with a tendency of content (TSS) in fruit pulp increased with harvest stage and sig-
fruits from the 2014 growing season to have lower respiration values; nificantly with ripening, reaching the highest value at R3 with a score
nevertheless, fruits had the same behavior regardless of the growing of 17°Brix.
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 3.2. Changes in oxidative stress and antioxidant response in fruits in
and 7 °C. Fruits from these two harvests took three days at room tem- relation to maturity stages at harvest
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 For fruits from early maturity stages at harvest (H1 and H2), H2O2
period at 12 °C, fruits from H1 reached the highest RR, with a value of content remained relatively low, as did MDA content (Fig. 2). These two
4.2 mmoles CO2 kg−1 h−1, while those from H2 increased to compounds increased in fruits from the H3 stage, which corresponded
3.4 mmol kg−1 h−1. After storage at 7 °C, the RR of fruits from H1 had to the beginning of the respiration crisis, by 8-and 3-fold, compared to
increased to 3.3 mmol kg−1 h−1, while those from H2 reached the other stages, respectively.
2.9 mmol kg−1 h−1. Maximum RR during ripening at 20 °C decreased Ascorbate content reached its highest values in fruits from the H1
with the maturity stage at harvest by a factor of 0.77 at H3. Storage at stage. This content decreased with the maturity stage at harvest by
cold temperatures significantly increased the maximum RR values of almost 3-fold in fruits from H3. Enzymes of the enzymatic antioxidant
fruits from H1 and H2. It can be observed that the fruits of the 2014 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

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 – –

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-

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

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

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

16.64 2 neochrome 601 400-422-448 90
17.88 3 9-violaxanthin-cis-violaxanthin 601 328-412-436-464 84
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 –

Tentative identification.
Main wavelength of maximum absorption are underlined.

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

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

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

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

References stress response in the dwarf tomato cultivar Micro-Tom. Physiol. Plant. 127, 10–18.
Medlicott, A., Sigrist, J., Sy, O., 1990. Ripening of mangos following low-temperature
storage. J. Am. Soc. Hortic. Sci. 115, 430–434.
Archbold, D.D., Pomper, K.W., 2003. Ripening pawpaw fruit exhibit respiratory and Mercadante, A.Z., Rodriguez-amaya, D.B., 1998. Effects of ripening, cultivar differences,
ethylene climacterics. Postharvest Biol. Technol. 30, 99–103. and processing on the carotenoid composition of mango. J. Food Chem. 8561,
Bouvier, F., Backhaus, R.A., Camara, B., 1998. Induction and control of chromoplast- 128–130.
specific carotenoid genes by oxidative stress. J. Biol. Chem. 273, 30651–30659. Mohammed, M., Brecht, J.K., 2002. Reduction of chilling injury in ‘Tommy Atkins’
Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram mangoes during ripening. Sci. Hortic. 95, 297–308.
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, Murshed, R., Lopez-Lauri, F., Sallanon, H., 2002. Microplate quantification of enzymes of
248–254. the plant ascorbate-glutathione cycle. Anal. Biochem. 383, 320–322.
Dhuique-Mayer, C., Caris-Veyrat, C., Ollitrault, P., Curk, F., Amiot, M.J., 2005. Varietal Nair, S., Singh, Z., 2009. Chilling injury during storage affects respiration rate and fruit
and interspecific influence on micronutrient contents in citrus from the quality in kensington pride mango fruit. Acta Hortic. 820, 737–743.
Mediterranean area. J. Agric. Food Chem. 53, 2140–2145. Nair, S., Singh, M., Tan, S.C., 2004. Chilling injury in relation to ethylene biosynthesis in
Ding, C.K., Chachin, K., Hamauzu, Y., Ueda, Y., Imahori, Y., 1998. Effects of storage ‘Kensington Pride’ mango fruit. J. Hortic. Sci. Biotechnol. 79, 82–90.
temperatures on physiology and quality of loquat fruit. Postharvest Biol. Technol. 14, Nunes, M.C.N., Emond, J.P., Brecht, J.K., 2007. Quality curves for mango fruit (cv.
309–315. Tommy Atkins and Palmer) stored at chilling and nonchilling temperatures. J. Mol.
Ding, Z.S., Tian, S.P., Zheng, X.L., Zhou, Z.W., Xu, Y., 2007. Responses of reactive oxygen Catal. B Enzym. 30, 104–120.
metabolism and quality in mango fruit to exogenous oxalic acid or salicylic acid Ornelas-Paz, J.D.J., Yahia, E.M., Gardea-Bejar, A., 2007. Identification and quantification
under chilling temperature stress. Physiol. Plant. 130, 112–121. of xanthophyll esters, carotenes, and tocopherols in the fruit of seven Mexican mango
Fanciullino, A.L., Bidel, L.P.R., Urban, L., 2013. Carotenoid responses to environmental cultivars by liquid chromatography-atmospheric. J. Agric. Food Chem. 55,
stimuli: integrating redox and carbon controls into a fruit model. Plant Cell Environ. 6628–6635.
37, 1–17. Pandey, V.P., Singh, S., Jaiswal, N., Awasthi, M., Pandey, B., Dwivedi, U.N., 2013. Papaya
Gomez, P., Ferrer, M.A., Fernandez-Trujillo, J.P., Calderon, A., Artes, F., Egea-Cortines, fruit ripening: ROS metabolism, gene cloning, characterization and molecular
M., et al., 2009. Structural changes, chemical composition and antioxidant activity of docking of peroxidase. J. Mol. Catal. B Enzym. 98, 98–105.
cherry tomato fruits (cv. Micro-Tom) stored under optimal and chilling conditions. J. Phakawatmongkol, W., Ketsa, S., Van Doorn, W.G., 2004. Variation in fruit chilling injury
Sci. Food Agric. 89, 1543–1551. among mango cultivars. Postharvest Biol. Technol. 32, 115–118.
Gonzalez-Aguilar, G.A., Villa-Rodriguez, J.A., Ayala-Zavala, J.F., Yahia, E.M., 2010. Rivera-Pastrana, D.M., Yahia, E.M., Gonzalez-Aguilar, G.A., 2010. Phenolic and car-
Improvement of the antioxidant status of tropical fruits as a secondary response to otenoid profiles of papaya fruit (Carica papaya L.) and their contents under low
some postharvest treatments. Trends Food Sci. Technol. 21, 475–482. temperature storage. J. Sci. Food Agric. 90, 2358–2365.
Jiang, Z., Zheng, Y., Qiu, R., Yang, Y., Xu, M., Ye, Y., et al., 2015. Short UV-B exposure Rugkong, A., McQuinn, R., Giovannoni, J.J., Rose, J.K.C., Watkins, C.B., 2011. Expression
stimulated enzymatic and nonenzymatic antioxidants and reduced oxidative stress of of ripening-related genes in cold-stored tomato fruit. Postharvest Biol. Technol. 61,
cold-stored mangoes. J. Agric. Food Chem. 63, 10965–10972. 1–14.
Jin, P., Zhu, H., Wang, L., Shan, T., Zheng, Y., 2014. Oxalic acid alleviates chilling injury Sala, M., 1998. Involvement of oxidative stress in chilling injury in cold-stored mandarin
in peach fruit by regulating energy metabolism and fatty acid contents. Food Chem. fruits. Postharvest Biol. Technol. 13, 255–261.
161, 87–93. Sevillano, L., Sanchez-ballesta, T., Flores, F.B., 2009. Physiological, hormonal and mo-
Joas, J., Caro, Y., Léchaudel, M., 2005. Ripening behaviour of mango (Cv Lirfa) in relation lecular mechanisms regulating chilling injury in horticultural species. Postharvest
to carbon nutrition stress and harvest period. Acta Hortic. 687, 401–404. technologies applied to reduce its impact. J. Sci. Food Agric. 89, 555–573.
Joas, J., Caro, Y., Lechaudel, M., 2009. Comparison of postharvest changes in mango (cv Shivashankara, K.S., Isobe, S., Al-Haq, M.I., Takenaka, M., Shiina, T., 2004. Fruit anti-
Cogshall) using a Ripening class index (Rci) for different carbon supplies and harvest oxidant activity, ascorbic acid, total phenol, quercetin, and carotene of Irwin mango
dates. Postharvest Biol. Technol. 54, 25–31. fruits stored at low temperature after high electric field pretreatment. J. Agric. Food
Joas, J., Vulcain, E., Desvignes, C., Morales, E., Léchaudel, M., 2012. Physiological age at Chem. 52, 1281–1286.
harvest regulates the variability in postharvest ripening: sensory and nutritional Singh, S.P., Singh, Z., Swinny, E.E., 2012. Climacteric level during fruit ripening influ-
characteristics of mango (Mangifera indica L.) cv. Coghshall due to growing condi- ences lipid peroxidation and enzymatic and non-enzymatic antioxidative systems in
tions. J. Sci. Food Agric. 92, 1282–1290. Japanese plums (Prunus salicina Lindell). Postharvest Biol. Technol. 65, 22–32.
Junmatong, C., Uthaibutra, J., Boonyakiat, D., Faiyue, B., Saengnil, K., 2012. Reduction Singh, Z., Singh, R.K., Sane, V.A., Nath, P., 2013. Mango – postharvest biology and bio-
of chilling injury of ‘Nam Dok Mai No. 4’ mango fruit by treatments with salicylic technology. Crit. Rev. Plant Sci. 32, 217–236.
acid and methyl jasmonate. J. Agric. Sci. 4, 126–136. Sivakumar, D., Jiang, Y., Yahia, E.M., 2011. Maintaining mango (Mangifera indica L.) fruit
Kratsch, H.A., Wise, R.R., 2000. The ultrastructure of chilling stress. Plant Cell Environ. quality during the export chain. Food Res. Int. 44, 1254–1263.
23, 337–350. Sivankalyani, V., Sela, N., Feygenberg, O., Zemach, H., Maurer, D., Alkan, N., 2016.
Léchaudel, M., Urban, L., Joas, J., 2010. Chlorophyll fluorescence: a nondestructive Transcriptome dynamics in mango fruit peel reveals mechanisms of chilling stress.
method to assess maturity of mango fruits (Cv. ‘Cogshall’) without growth conditions Front. Plant Sci. 7, 1579.
bias. J. Agric. Food Chem. 58, 7532–7538. Srinivasa, P.C., Baskaran, R., Ramesh, M.N., Prashanth, H.K.V., Tharanathan, R.N., 2002.
Léchaudel, M., Lopez-Lauri, F., Vidal, V., Sallanon, H., Joas, J., 2013. Response of the Storage studies of mango packed using biodegradable chitosan film. Eur. Food Res.
physiological parameters of mango fruit (transpiration, water relations and anti- Technol. 215, 504–508.
oxidant system) to its light and temperature environment. J. Plant Physiol. 170, Talcott, S.T., Moore, J.P., Lounds-Singleton, A.J., Percival, S.S., 2005. Ripening associated
567–576. phytochemical changes in mangos (Mangifera indica) following thermal quarantine
Lim, C.S., Kang, S.M., Cho, J.L., Gross, K.C., 2009. Antioxidizing enzyme activities in and low-temperature storage. Food Chem. Toxicol. 70, 337–341.
chilling-sensitive and chilling-tolerant pepper fruit as affected by stage of ripeness Team, R.D.C, 2012. R: A Language and Environment for Statistical Computing.
and storage temperature. J. Am. Soc. Hortic. Sci. 134, 156–163. Zhao, Z., Jiang, W., Cao, J., Gu, Y., 2006. Effect of cold-shock treatment on chilling injury
Lopes, M.M.A., Silva, E.O., Canuto, K.M., Silva, L.M.A., Gallão, M.I., Urban, L., et al., in mango (Mangifera indica L. cv. ‘Wacheng’) fruit. J. Sci. Food Agric. 86, 2458–2462.
2016. Low fluence pulsed light enhanced phytochemical content and antioxidant Zhao, Z., Cao, J., Jiang, W., 2009. Maturity-related chilling tolerance in mango fruit and
potential of ‘Tommy Atkins’ mango peel and pulp. Innov. Food Sci. Emerg. Technol. the antioxidant capacity involved. J. Sci. Food Agric. 89, 304–309.
33, 216–224. Zhou, Q., Ma, C., Cheng, S., Wei, B., Liu, X., Ji, S., 2014. Changes in antioxidative me-
Lurie, S., Crisosto, C.H., 2005. Chilling injury in peach and nectarine. Postharvest Biol. tabolism accompanying pitting development in stored blueberry fruit. Postharvest
Technol. 37, 195–208. Biol. Technol. 88, 88–95.
Malacrida, C., Valle, E.M., Boggio, S.B., 2006. Postharvest chilling induces oxidative