Postharvest Biology and Technology 128 (2017) 54–62

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Postharvest Biology and Technology

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

Internal disorders of ‘Rocha’ pear affected by oxygen partial pressure

and inhibition of ethylene action
Adriano Saqueta,b , Domingos Almeidab,*
a
Instituto Federal de Educação, Ciência e Tecnologia Farroupilha, Campus Panambi, Rua Erechim 860, 98280-000 Panambi, RS, Brazil
b
Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal

A R T I C L E I N F O A B S T R A C T

Article history:
Received 9 November 2016 Current technologies allowing the use of extremely low oxygen partial pressures (pO2), the introduction
Received in revised form 10 February 2017 of 1-methylcyclopronene (1-MCP), and the regulatory prohibition of diphenylamine are changing the
Accepted 12 February 2017 conventional storage protocols for pear cultivars. Internal disorders, in particular, severely damage pear
Available online 17 February 2017 quality during controlled atmosphere storage. ‘Rocha’ pear (Pyrus cummunis L.) was stored for 136 d at
0.5  C in air or under 3.0 and 0.5 kPa O2 with 0.6 kPa CO2. Fruits treated with 150 nL L 1 1-MCP were also
Keywords: stored at 3.0 and 0.5 kPa O2 after 32 d in air following the treatment. Internal disorders did not develop in
Adenylate energy charge fruit stored in air (20.8 kPa O2) or at 0.5 kPa O2 but affected 10.2% of the fruit stored in 3.0 kPa O2 after 136
Controlled atmosphere storage
d. 1-MCP increased disorder incidence at 0.5 and at 3.0 kPa O2. Four types of internal disorders occurred:
Fruit quality
core browning, white cavity, necrotic cavity, and flesh browning. Low O2 reduced ethylene production
Physiological disorders
Pyrus communis and respiration rates which were further reduced by the treatment with 1-MCP. ATP concentration and
adenylate energy charge were higher in fruit stored in air than in those at 3.0 and were generally lowest in
fruit at 0.5 kPa O2. The effect of pO2 on energy metabolism prevailed over that of 1-MCP treatment. The
linkage between ATP and adenylate energy charge (AEC) and the incidence of internal disorders was not
strong, since under the same pO2, 1-MCP enhanced the incidence of disorders with a negligible effect on
adenylate nucleotides or AEC. It was not possible to establish a threshold of ATP concentration or AEC
below which internal disorder develop. In conclusion, poststorage quality of ‘Rocha’ pear was better at
the extremely low pO2 of 0.5 kPa than at 3.0 kPa. 1-MCP was detrimental to internal disorders and blocked
poststorage softening of ‘Rocha’ pear stored at 0.5 kPa O2.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction fluorescence (Prange et al., 2003), respiratory quotient (Gasser

The main European pear cultivars in the world market are
suitable for long-term storage. At 1 to 0  C and relative humidity
higher than 90% pears can be stored in air for 3–6 months (Agar
et al., 2000; Wang and Sugar, 2013), after which time postharvest
life is limited by advanced ripening (Raese et al., 1999), decay
(Spotts et al., 2007), and superficial scald (Calvo et al., 2015).
Controlled atmosphere (CA) significantly extends the storage
period of pears. The recommended partial pressures of oxygen
(pO2) and carbon dioxide (pCO2) are specific for each cultivar and
growing region. CA-recommendations for pears are rapidly
evolving as new technologies allow more precise control of pO2
and pCO2, and the dynamic control of gas concentration based on
fruit physiological responses, namely changes in chlorophyll
* Corresponding author.
E-mail address: dalmeida@isa.ulisboa.pt (D. Almeida).
et al., 2008; Weber et al., 2015), and the ethanol accumulation in
the fruit or its release into the atmosphere (Veltman et al., 2003a).
CA extends the storage life of pear but alters the main causes of
postharvest life termination: internal disorders become a major
limiting factor under these conditions (Franck et al., 2007; Lum
et al., 2016).
‘Rocha’ pear grown in warm climates is sensitive to superficial
scald and internal disorders and both must be addressed to assure
poststorage quality. Diphenylamine (DPA), a standard postharvest
treatment until 2013, was effective in reducing the incidence and
severity of both storage disorders in ‘Rocha’ pear (Silva et al., 2010;
Almeida et al., 2016) and CA-storage recommendations were
developed for DPA-treated fruit. The recommended conditions
were pO2 of 2.5–3 kPa, and pCO2 lower than 0.7 kPa (Silva et al.,
2010; Almeida et al., 2016). However, under these CA-conditions
the incidence of internal disorders can be high in the absence of
DPA (Almeida et al., 2016).

http://dx.doi.org/10.1016/j.postharvbio.2017.02.005
0925-5214/© 2017 Elsevier B.V. All rights reserved.
 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
Pears are generally less tolerant than apples to very low pO2
(Streif et al., 2001; Thompson, 2010). This observation is consistent
with the lower internal air volume, higher density, and higher
resistance to O2 diffusion of pear in relation to apple (Ho et al.,
2006). Low pO2 in storage rooms changes the energy status of pear
fruit (Saquet et al., 2000; Veltman et al., 2003b), with detrimental
consequences in membrane phospholipids and enhancement of
internal disorders in ‘Conference’ pear (Saquet et al., 2003a) and
‘Braeburn’ apple (Saquet et al., 2003b). Therefore, storage
conditions that improve the levels of energy stored in the
adenylate nucleotides and the overall cell energy status in pear
have been related to lower incidence of internal disorders (Saquet
et al., 2000; Veltman et al., 2003b; Franck et al., 2007).
The ethylene action inhibitor 1-methylcyclopropene (1-MCP)
became an effective treatment to prevent superficial scald and
extend storage life in pear (Argenta et al., 2003; Isidoro and
Almeida, 2006; Villalobos-Acuña., et al., 2011; Almeida et al., 2016).
However, in contrast with apples, poststorage pear ripening can be
impaired by 1-MCP (Chiriboga et al., 2011).
The extension of storage period of ‘Rocha’ pear in the absence of
DPA requires the mitigation of superficial scald and internal
disorders. 1-MCP and ultra-low pO2 storage are two venues to
achieve these goals. This study aimed to evaluate the effect of pO2
and 1-MCP on internal disorders and on overall quality mainte-
nance of ‘Rocha’ pear during storage. ATP, ADP, and AMP were
assessed under several storage conditions in fruit with and without
ethylene action inhibition to address the relationship between cell
energy status and internal disorders.
Fig. 1. Internal disorders observed in ‘Rocha’ pear. Core browning (A), white cavity (B), necrotic cavity (C) and flesh browning (D).
55
2. Materials and methods
2.1. Fruit material
Pear (Pyrus communis L. ‘Rocha’) fruit were harvested at the
mature-green stage from an orchard located in Cadaval, Oeste
Region, Portugal. The maturity stage at harvest was measured in 3
replicates of 15 fruits each. Fruit had uniform size (60–65 mm), a
starch index of 8.2 (1–10 scale), flesh firmness of 52.4 N, total
soluble solids (TSS) of 11.2%, titratable acidity of 0.2% expressed in
malic acid equivalents, and a skin hue angle of 106.4 . After harvest
fruit were drenched with fludioxonil at 580 mg L 1 (Scholar1,
Syngenta, Basel, Switzerland) and cooled to 0.5  C.
2.2. 1-MCP treatment and storage conditions
Fruit were stored in 0.55 m3 cabinets at 0.5  C (0.3  C
fluctuation) and 90–93% relative humidity, in air or under two CA-
conditions: 0.5 kPa O2 or 3 kPa O2 with pCO2 below 0.6 kPa in each
instance (balance N2). The pO2 was lowered by flushing the
cabinets with N2 and the final pressure of 0.5 kPa O2 or 3 kPa O2 was
reached within 26 and 18 h, respectively.
1-MCP generated from SmartFreshTM (Agrofresh, Inc., Spring-
house, PA, USA) was applied at a dose of 150 nL L 1 for 24 h at
0.5  C. After the treatment with 1-MCP, the fruit were maintained
for 32 d in air before establishment of the CA-conditions indicated
above according to the commercial recommendations to prevent
ripening blockage. The storage temperature and gas partial
56 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
pressures inside the CA-storage rooms were daily monitored and
controlled by an automatic system (Isolcell, Laives, Italy).
2.3. Assessment of internal disorders
Fruit were assessed for internal disorders after 44, 96 and 136 d
in storage in 3 replicate batches of 60 fruit each. Fruit were
assessed immediately after removal from storage at 0.5  C and
after a subsequent 7-d shelf-life period at 20  C.
Fruit were cut transversely in 3 sections at the level of the seed
cavity and then cut longitudinally to visualize the extent of
disorder development. Internal disorders were classified in four
classes according to their symptomatology (Fig. 1): core browning,
white cavity, necrotic cavity, and flesh browning. The occurrence of
each disorder was expressed in percentage of affected fruit.
2.4. Evaluation of fruit quality characteristics
Skin color was measured in the CIE L*a*b* color space with a
tristimulus CR-400 chroma meter (Konica Minolta, Tokyo, Japan)
with the C illuminant. One measurement was made in the widest
part of the fruit in three replicates of 5 fruit each.
Flesh firmness was assessed with a penetrometer equipped
with an 8 mm probe (T.R. Turoni, Forli, Italy). The maximum force
to insert the probe 8 mm into the fruit flesh without skin was
recorded. Firmness was measured twice on opposite sides of each
fruit, in three replicated batches of 5 fruit each.
Juice was extracted from a 10 mm thick cylinder excised from
the equatorial region of the fruit and TSS and titratable acidity (TA)
measured in the juice. TSS was measured with a digital
refractometer (Hanna Instruments, Woonsocket, USA) and TA
determined by titration of an aliquot of 10 mL of juice diluted in
90 mL of distilled water, with 0.1 M NaOH until pH 8.1.
2.5. Ethylene and respiration measurements
Ethylene was measured in three replicate samples of 4 fruit
each. Fruit were sealed inside 2.15 L glass jars at 20  C for 2 h. A
headspace volume of 0.1 mL was removed with a glass syringe via a
rubber septum and injected into a gas chromatograph (Trace 1300,
Thermo Fisher Scientific Inc., Marietta, USA) fitted with a TG bond
alumina (Na2SO4) capillary column, 50 m long with 0.53 mm i.d.
(Thermo Fisher Scientific Inc., Marietta, USA). Helium was used as
carrier gas at a flow rate of 15 mL min 1. The injector temperature
was set at 160  C, the flame ionization detector at 180  C, and the
oven temperature held for 0.5 min at 100  C, followed by an
increase at 20  C min 1 to reach 180  C, and a holding of 0.5 min at
180  C.
Respiration, expressed by the release of CO2, was measured in
the same fruit samples used for ethylene determination. The
headspace CO2 concentration was measured with the infrared
sensor of an Oxycarb 6 gas analyzer (Isolcell, Laives, Italy) in a close
circulation circuit at a flow rate of 100 mL min 1.
2.6. Determination of adenylate nucleotides concentration
Tissue cylinders (18 mm thick and 40 mm length) were excised
from the core region of the fruit and seeds removed. The cylinders
were immediately frozen in liquid nitrogen and freeze-dried at
50  C and 100 kPa. Samples were fine powdered and used for
extraction. Three replicated extractions and measurements were
performed per each treatment.
Adenylate nucleotides (ATP, ADP and AMP) were extracted and
their concentrations determined by bioluminescence as described
by Saquet et al. (2003a) with adaptations (Saquet and Almeida,
2017). Briefly, 1 g of freeze-dried sample was extracted for 30 min
at 4  C with 10 mL of 5% trichloracetic acid, 2 mM of ethyl-
enediaminetetraacetic acid disodium salt dehydrate (EDTA). After
centrifugation at 21,000g for 30 min at 4  C, the supernatant was
diluted 30 fold with Tris-EDTA buffer (0.1 M Trizma Base and 2 mM
EDTA, pH 7.75). ATP was determined by the emission of
bioluminescence from 10 mL of extract mixed with 40 mL ATP
monitoring reagent (AMR; BioThema AB, Handen, Sweden) and
150 mL of tris-EDTA buffer (0.1 M Trizma Base and 2 mM EDTA, pH
7.75). Bioluminescence was measured at room temperature with a
Synergy 2 Multi-Mode Reader (BioTek, Winooski, VT, USA). ADP
was converted into ATP by incubation with 120 U mL 1 of pyruvate
kinase (PK; Sigma-Aldrich, St. Louis, MO, USA) prepared in a PEP
buffer for 30 min at room temperature. AMP was converted to ADP
and ADP converted to ATP with a mixture of adenylate kinase (AK;
Sigma-Aldrich, St. Louis, MO, USA) at 180 U mL 1 combined with
PK (120 U mL 1; Sigma-Aldrich, St. Louis, MO, USA) in PEP buffer
for 30 min at ca. 25  C. Total ATP concentration was determined as
described above and ADP and AMP calculated by difference. The
concentrations of adenylate nucleotides were expressed on a fresh
mass basis. AEC was calculated as [ATP] + 0.5 [ADP]/[ATP] + [ADP] +
[AMP] (Atkinson and Walton, 1967).
2.7. Data analysis
Data were subjected to one-way analysis of variance with the
storage treatment as a fixed factor. Means were separated by the
least significant difference (LSD) test at a=0.05. Analysis were
made with the software Action Stat (2014, São Carlos, SP, Brazil).
3. Results and discussion
3.1. Incidence of disorders
Superficial scald did not occur in any of the treatments. Even
though ‘Rocha’ is a susceptible cultivar, seasonal variability of
superficial scald incidence is high and orchard effects are strong.
Internal disorders were absent from fruit stored in air for 136 d
(Fig. 2). Fruit stored under 3 kPa O2 developed internal disorders:
4.4% were affected immediately after storage and the incidence
increased to 10.2% after 7 d in air at 20  C (Fig. 2). Fruit stored at
0.5 kPa O2 did not develop internal disorders during storage or
during the subsequent shelf-life period. The combined effect of 1-
MCP at 150 nL L 1 with low pO2 did not alter the incidence of
internal disorder in fruit stored under 3 kPa O2 but increased their
incidence under 0.5 kPa O2 (Fig. 2). It is not clear whether this effect
is due to the inhibition of ethylene action per se or to the delayed CA
Fig. 2. Occurrence of total internal disorders in ‘Rocha’ pear stored at 0.5  C for
136 d and following a 7 d shelf-life in air at 20  C. Untreated fruit were stored in air
or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa
O2. Vertical bars are standard deviation.
 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
regime in 1-MCP-treated fruit, although a delay in CA establish-
ment is reported to reduce the occurrence of internal disorders in
pear (Roelofs and de Jager, 1997).
A potential dilemma arises in optimizing conditions for long-
term CA-storage of ‘Rocha’ pear: If pO2 is reduced to levels that
provide an effective control of superficial scald, the risk of internal
disorder development increases. The inhibition of ethylene action
by 1-MCP can mitigate the occurrence of superficial scald, even at
150 nL L 1 (Isidoro and Almeida, 2006; Almeida et al., 2016), a
concentration that is 50% of the commercially recommended dose
for pear. However, an interaction is expected between 1-MCP
treatment and CA storage (see 3.3. and 3.4). Moreover, the effect of
1-MCP on pear internal disorders is not clear: It has been shown to
increase the occurrence of internal disorders during CA-storage in
‘Alexander Lukas’ (Hendges et al., 2015), but to alleviate it in ‘Abate
Fetel’ (Vanoli et al., 2016), and in ‘Rocha’ pear (Almeida et al., 2016).
When DPA was used as a postharvest treatment, storage of
‘Rocha’ pear was performed at pO2 of 2.5–3.0 kPa and pCO2 lower
than 0.7 kPa (Silva et al., 2010; Almeida et al., 2016). In the absence
of DPA, internal disorders developed in fruit stored under pO2 of
3 kPa, but not in air (20.8 kPa O2) or at the lower pO2 of 0.5 kPa
(Fig. 2). The absence of internal disorders under extremely low pO2
was reported for ‘d’Anjou’ pear maintained at 0.5 kPa O2 for 8
months (Mattheis and Rudell, 2011) and ‘Abbé Fétel’ pear stored for
7 months under dynamic controlled atmosphere with 0.8 kPa O2
and 0.45 kPa CO2 (Rizzolo et al., 2014). However, Mattheis et al.
(2013) have subsequently reported an aggravation of an internal
disorder described as pithy brown core in ‘d’Anjou’ pear stored at
0.5 kPa O2 in relation to 1.5 kPa O2.
3.2. Typology of internal disorders in ‘Rocha’ pear
Symptomatology differed among the internal disorders ob-
served in ‘Rocha’ pear. The confusing description, classification,
and terminology regarding pear internal disorders is recognized
(Franck et al., 2007) and there is still no consensus regarding their
nature and etiology. However, an individual analysis of the effects
of storage conditions on distinct symptomatologies is warranted
since the damages were differentially affected by pO2 or 1-MCP.
Four internal disorder symptomatologies were identified and
characterized during the storage period of ‘Rocha’ pear under CA
(Fig. 1): core browning, white cavity, necrotic cavity, and flesh
browning. Core browning refers to brown parenchyma tissue with
a maximum diameter of 15 mm restricted to the core region and
brown patches with soft and wet appearance (Fig. 1A). White
cavity describes holes in the fruit flesh without brown surface,
ranging in size from 2 to 35 mm, often with an elongate and narrow
shape (Figs. 1 B and 3); these cavities were often located radially
around the core region, but occasionally were also distributed
longitudinally reaching, sometimes, the proximal neck region.
Necrotic cavity consists of small cavities, not larger than 4 mm
diameter and 3 mm deep, with necrotic brown surface, always
located around the core region, with a typical ‘rosette’ appearance
(Fig. 1C). Flesh browning is brown tissue with firm and wet
appearance without cavity formation; the brown tissue was
frequently interspersed with bleached tissue and, in severe cases,
affected more than 80% of the fruit flesh (Fig. 1D).
Core browning, white cavity and flesh browning were the
internal disorders most frequently observed in ‘Rocha’ pear after
136 d of storage, affecting 2.3, 3.7, and 2.7% of the fruit, respectively
(Fig. 4). The higher incidence of core browning, white cavity,
necrotic cavity, and flesh browning was observed in fruit stored
under 3 kPa O2, with or without 1-MCP treatment (Fig. 4).
Core browning in pear has been described by the damage in the
core region and is considered a senescence-related disorder
(Larrigaudière et al., 2004). Other authors use a generic term to
57
Fig. 3. Initial stages of visible white cavities in ‘Rocha’ pear showing the absence of
necrotic tissues before the cavities enlarge to the symptom represented in Fig. 1B.
classify all internal disorders observed in fruit stored under CA-
storage, such as ‘brown heart’ (Streif et al., 2001; Saquet et al.,
2003a) or ‘core breakdown’ (Lammertyn et al., 2003), who used the
same term to brown tissue and the cavities combined. However,
core browning (Fig. 1A) observed in ‘Rocha’ pear was restricted to
the core region and seems to be unrelated to overall fruit
senescence. Considering the firmness (Fig. 5A) and skin color
(Fig. 5B) of fruit with this disorder, core browning cannot be
associated with senescent flesh breakdown. Core browning
incidence was higher in fruit stored under 3 kPa O2 than 0.5 kPa
O2 and its incidence was slightly aggravated by the treatment with
1-MCP at 150 nL L 1 under both CA-conditions (Fig. 4A).
Two distinct symptomatologies included cavities. White cavi-
ties were more common than necrotic cavities (Fig. 4B and C). The
occurrence of white cavities in CA-stored fruit was higher under
3.0 kPa O2 than under 0.5 O2 and in both instances the incidence
increased in 1-MCP-treated fruit. Cavity formation in apple is
associated with high pCO2 during CA-storage (Elgar et al., 1998;
Clark and Burmeister, 1999), and symptom expression explained
by dehydration of the tissue (Xuan et al., 2001; Lammertyn et al.,
2003). However, inconsistencies persist regarding the etiology of
the white cavity symptom. For example, large cavities were
reported in ‘Conference’ pear stored under 0.5 kPa O2 with pCO2
lower than 0.5 kPa (Saquet et al., 2000). White cavities occurred in
‘Rocha’ pear stored at 3 kPa O2 even with pCO2 of 0.6 kPa (Fig. 4B).
Even though these pCO2 are one order of magnitude higher than
those in air, 0.6 kPa CO2 is currently at the lowest range that can be
practically obtained in commercial CA-storage. Whether pCO2 of
about 0.6 kPa induces cavities remains unclear.
Pear cavities have been considered a late stage of development
of the brown tissue (Lammertyn et al., 2000; Lammertyn et al.,
2003; Franck et al., 2007). According to this hypothesis, disorder-
inducing conditions during CA-storage increase membrane per-
meability and the water leaked out of the cells diffuses and
evaporates from the fruit surface resulting in dry, brown, gas-
containing spaces that further dehydrate to form cavities (Xuan
et al., 2001; Lammertyn et al., 2003). However, the white cavities
observed in ‘Rocha’ pear (Fig. 3) were lined by white and
surrounded by apparently healthy tissue. The early stages of
white cavity formation occurred in white and not brown tissues
(Fig. 3) and enlarged while remaining white (Fig. 1 B). Instead, we
hypothesize that this type of cavity is caused by a programmed cell
death mechanism similar to that of aerenchyma formation under
hypoxic conditions in root tissues (Yamauchi et al., 2013).
Necrotic cavities were less frequent (Fig. 4C). The pattern of
occurrence of this disorder was clearly defined, i.e., restricted and
58 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62

Fig. 4. Occurrence of core browning (A), white cavity (B), necrotic cavity (C) and flesh browning (D) in ‘Rocha’ pear stored at 0.5  C for 136 d and following a 7 d shelf-life in
air at 20  C. Untreated fruit were stored in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

Fig. 5. Flesh firmness (A), skin color (B), acidity (C) and total soluble solids (D) in fruit stored at 0.5  C for 136 d and following a 7 d shelf-life in air at 20  C. Untreated fruit
were stored in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

uniformly distributed around the core region. We were not able to
associate the small and brown cavities around the core of the pear
fruit to a specific storage condition (Fig. 4C).
Flesh browning (Fig. 4D) was generally distributed in the flesh
of ‘Rocha’ pear, always without cavities. This symptom may result
from the development of core browning or coexist with it; the core
region of fruit affected by flesh browning also had an intense
brown coloration.
3.3. Poststorage fruit quality
After 136 d at 0.5  C followed by 7 d at 20  C, fruit stored in air
were soft with a firmness of 10 N (Fig. 5A). Firmness was better
retained during storage under low pO2 than in air, but subsequent
softening after removal from storage was fast; at the end of a 7-d
period in air at 20  C, the firmness of fruit stored under CA at 3.0 or
0.5 kPa O2 was similar to that of fruit previously stored in air
(Fig. 5A). 1-MCP slowed poststorage softening (Fig. 5A), an effect
that is very consistent in pear (Rizzolo et al., 2014; Almeida et al.,
2016). The combination of 1-MCP at 150 nL L 1 with storage under
0.5 kPa O2 inhibited poststorage softening at 20  C for 7 d (Fig. 5A).
Fruit yellowed slightly during storage in air but not under CA
(Fig. 5B). Poststorage yellowing was reduced in fruit treated with1-
MCP, and the combination of 1-MCP treatment and storage under
0.5 kPa O2 inhibited the subsequent decrease of hue angle during 7
d at 20  C (Fig. 5B). Effects of CA-storage and 1-MCP in maintaining
green skin in pears are well documented (Mattheis and Rudell,
2011; Gago et al., 2015; Rizzolo et al., 2015; Vanoli et al., 2016).
 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
Titratable acidity (Fig. 5C) was lower in fruit stored in air than in
fruit stored in CA, with or without 1-MCP treatment. TSS was
similar in all storage conditions (Fig. 5D).
Taken together, the physicochemical properties of ‘Rocha’ pear
after storage show that 3.0 and 0.5 kPa O2 prolong storage life while
allowing poststorage ripening within 7 d at 20  C. However, an
interaction between 1-MCP and pO2 effects is evident. Fruit treated
with 1-MCP at 150 nL L 1 and stored under 3.0 kPa O2 ripened
during shelf-life whereas poststorage ripening was inhibited
during 7 d in fruit previously stored at 0.5 kPa O2. An examination
of ethylene production and respiration rates may explain this
observation.
3.4. Ethylene production and respiration rate
Ethylene production was undetectable at harvest, increased
slightly during the first 45 d in storage, and at a higher rate
thereafter to reach a maximum at 96 d followed by a decline
(Fig. 6A). Ethylene production rate was highest in fruit stored in air
with a peak rate of 31.4 mg kg 1 h 1. Lower peaks were observed
under 0.5 and 3.0 kPa O2. 1-MCP at 150 nL L 1 combined with CA
further reduced the ethylene production rate; the peak rate of 1-
MCP-treated fruit stored under 3.0 and 0.5 kPa O2 was of 52.1 and
15.5% of that of untreated fruit under the same pO2, respectively.
The stronger suppression of ethylene production was observed in
1-MCP-treated fruit stored under 0.5 kPa O2 (Fig. 6A).
Fruit ripening during storage in air lead to lower ethylene
production rate during shelf-life in relation to that of fruit stored
under 0.5 kPa O2 (Fig. 6B). The combination of 1-MCP treatment
and CA storage strongly reduced ethylene production during
poststorage shelf-life, a suppression that was more intense in fruit
stored under 0.5 than under 3.0 kPa O2 (Fig. 6B). Fruit treated with
150 nL L 1 1-MCP and stored under 0.5 kPa O2 failed to recover the
ethylene emission remaining at 3.7 mg kg 1 h 1 even after 9 d
shelf-life at 20  C (Fig. 6B).
Fig. 6. Ethylene production rate of ‘Rocha’ pear during storage (A) and 9 d shelf-life
at 20  C after 136 d in storage (B). Untreated fruit were stored at 0.5  C in air or
under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2.
Vertical bars are standard deviation.
59
Respiration reached the highest rate after 96 d in storage in all
treatments, at the same moment of the maximum ethylene
production rate. Fruit stored in air for 96 d released CO2 at a higher
rate (35.1 mg kg 1 h 1) than fruit stored under 3.0 or 0.5 kPa O2
which respired at 23.1 and 13.3 mg kg 1 h 1, respectively (Fig. 7A).
Treatment with 1-MCP further reduced respiration at the end of
storage when 1-MCP-treated fruit respired at a rate of 5.1 mg kg 1
h 1 while air-stored fruit respired at 20.3 mg kg 1 h 1 (Fig. 7A).
In contrast with ethylene production, the respiration rate
during shelf-life at 20  C was similar in fruit previously stored
under low pO2 or in air (Fig. 7B). No residual effect of CA-storage on
the poststorage respiration rate of ‘Rocha’ pear was observed.
Respiration rate increased continuously during the shelf-life
period at 20  C (Fig. 7B). The rates were similar in fruit previously
stored in air or under 0.5 or 3 kPa O2, but lower in pear treated with
1-MCP. Respiratory metabolism is a source of chemical energy
stored in the cellular pool of adenylate nucleotides.
3.5. Changes in adenylate nucleotides and energy charge
Pear has a radial gradient in ATP concentration and AEC,
decreasing from the skin toward the core region (Almeida and
Saquet, in press). Since most internal disorders are located or
originate at or surrounding the core region (Fig. 1) the adenylate
nucleotides were quantified at the fruit core. ATP concentration at
harvest was 965.7 nmol g 1, accounting for 68.6% of the total
adenylates. ATP decreased in the first 44 d of storage and increased
thereafter, when the effect of storage conditions was observed
(Fig. 8A). After 136 d fruit stored in air had higher ATP
concentration than those stored in 0.5 or 3 kPa O2. Fruit treated
with 1-MCP and stored at 0.5 or 3 kPa O2 had the lowest
concentration of ATP at the end of storage but the effect of 1-
MCP was negligible in relation to that of pO2 (Fig. 8A). Storage
conditions affected ATP concentration in a way that paralleled their
Fig. 7. Respiration rate of ‘Rocha’ pear during storage (A) and 9 d shelf-life at 20  C
after 136 d in storage (B). Untreated fruit were stored at 0.5  C in air or under 0.5 or
3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars
are standard deviation.
60 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
Fig. 8. Changes in ATP (A), ADP (B) and AMP (C) in ‘Rocha’ pear during storage at
0.5  C. Untreated fruit were stored at 0.5  C in air or under 0.5 or 3.0 kPa O2 and 1-
MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard
deviation.
effect on respiration rate (Fig. 7A). A close relationship between
ATP concentration and respiration rate was observed during
storage of ‘Conference’ pear (Saquet et al., 2003a) and ‘Jonagold’
apple (Xuan and Streif, 2008), but not during ripening of ‘Rocha’
pear (Saquet and Almeida, 2017). 1-MCP has been shown to reduce
ATP concentration during storage of ‘Jonagold’ apple (Xuan and
Streif, 2008).
ADP concentration at harvest was 249.1 nmol g 1 (17.7% of total
pool) and changed irregularly during storage (Fig. 8B), as
previously observed in pome fruit (Saquet et al., 2000; Saquet
et al., 2003b). This erratic trend in ADP concentration is consistent
with the possibility of conversion of ADP to ATP and AMP by the
action of adenylate kinase or phosphorylated to ATP by ATP
synthase (Igamberdiev and Kleczkowski, 2015). The concentration
of AMP at harvest was 193.8 nmol g 1 (13.7% of the total pool of
adenylates) and decreased gradually during storage period with no
clear effect of storage conditions or 1-MCP treatment (Fig. 8C). A
decreasing trend in AMP concentration has been observed in
during storage of ‘Conference’ pear (Saquet et al., 2003a) and in
peach under chilling conditions (Jin et al., 2013), but AMP
accumulated in banana after exposition for 8–12 h to hypoxia
under 10 or 15 kPa O2 (Hill and ap Rees, 1995), and remained
relatively unaltered during ripening of ‘Rocha’ pear (Saquet and
Almeida, 2017).
The total pool of adenylates remained constant or declined
during the first 94 d in storage and increased thereafter (Fig. 9A). At
Fig. 9. Changes in total pool of adenylates (A) and AEC (B) in ‘Rocha’ pear during
storage at 0.5  C. Untreated fruit were stored at 0.5  C in air or under 0.5 or
3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars
are standard deviation.
the end of storage period, the total pool of adenylate nucleotides
was highest in fruit stored in air, followed by fruit stored at 0.5 and
3 kPa O2 with intermediary values. The lowest concentrations were
measured in 1-MCP-treated fruit (Fig. 9A).
In fruit stored in air the AEC decreased from 0.78 at harvest to
0.67 after 44 d in storage to increase thereafter to a maximum of
0.87 and reached 0.81 at the end of the storage period (Fig. 9B).
Fruit stored under low pO2 had lower AEC after the initial storage
period, with minor and inconsistent effect of 1-MCP (Fig. 9B).
Overall, the AEC in ‘Rocha’ pear under low pO2 ranged from 0.58 to
0.78 (Fig. 9B).
3.6. Adenylates nucleotides and the development of internal disorders
ATP concentration and AEC are frequently used to assess the
energy status of plant cells (Geigenberger et al., 2009) and have
been related to the development of internal disorders during CA
storage of apple and pear (Saquet et al., 2000, 2003a, 2003b;
Veltman and Peppelenbos, 2003; Veltman et al., 2003b). Evidence
that conditions improving fruit ATP concentration and AEC help
maintain cell integrity and reduce the occurrence of internal
disorders during CA storage of pear was first presented by Saquet
et al. (2000), and the argument was further supported by several
studies (Saquet et al., 2001; Veltman and Peppelembos, 2003;
Veltman et al., 2003b; Franck et al., 2003).
The argument that energy stored in the adenylate nucleotides is
a factor in reducing internal disorders is partially, but not fully,
supported by the results presented herein. Fruit stored in air
(20.8 kPa O2) had higher AEC than fruit stored under at 3.0 kPa O2
(Fig. 8B) and did not develop internal disorders (Fig. 2). However,
two experimental conditions disrupt the linkage between ATP or
AEC and disorder development. Fruit stored under the extremely
low pO2 of 0.5 kPa did not develop internal disorders in contrast
with fruit stored under 3.0 kPa O2 (Fig. 2) even if their ATP and AEC
remained at relatively similar levels throughout storage (Figs. 7 A
and 8 B). In addition, under the same pO2, 1-MCP enhanced the
 A. Saquet, D. Almeida / Postharvest Biology and Technology 128 (2017) 54–62
incidence of disorders (Fig. 2) with a negligible effect on adenylate
nucleotides or AEC (Figs. 7 and 8). It is hypothesized that a pO2 of
0.5 kPa significantly reduces the production of reactive oxygen
species so that the homeostatic balance between oxidative stress
and repair capacity is maintained during storage. It was not
possible to establish a threshold of ATP concentration or AEC below
which internal disorder develop.
Whether aspects of energy metabolism other than the equilibri-
um of the adenylate nucleotides play a role in unknown. The possible
use of reducing power stored in NADPH (Lum et al., 2016) to better
describe the cell energy status in relation to internal disorders is not
likely since NADPH was not affected by experimental conditions
during CA-storage of ‘Conference’ pear and ‘Jonagold’ apple,
irrespective of disorder incidence (Saquet et al., 2000).
4. Conclusions
Internal disorders in ‘Rocha’ pear did not develop after 136 d of
storage in air (20.8 kPa O2) or under 0.5 kPa but 10.2% of the fruit
were affected after storage at 3.0 kPa O2. 1-MCP at 150 nL L 1
combined with a 32 d delay in the pull down of pO2 enhanced the
incidence of internal disorders under 0.5 or 3.0 kPa O2. It was not
possible to relate ATP concentrations or AEC with internal disorder
development during CA-storage of ‘Rocha’ pear.
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