Postharvest Biology and Technology 62 (2011) 161167

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Postharvest Biology and Technology

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Preharvest calcium applications inhibit some cell wall-modifying enzyme

activities and delay cell wall disassembly at commercial harvest of
Fuji Kiku-8 apples
Abel Ortiz a , Jordi Graell b , Isabel Lara a,
a
Departament de Qumica, Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain
b
Postcollita, Centre UdL-IRTA (XaRTA), Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain

a r t i c l e i n f o a b s t r a c t

Article history: To improve apple rmness during the postharvest chain, detailed knowledge of the biochemistry underly-
Received 23 February 2011 ing ripening-related cell wall disassembly, a very complex event, is required. Apple softening is reportedly
Accepted 30 April 2011 mediated by calcium loss from the middle lamella, and accordingly calcium applications are expected
to preserve fruit rmness. In this work, pre-harvest calcium sprays (7 weekly applications at 1.6%, w/v,
Keywords: 81123 days after full bloom) were applied to Fuji Kiku-8 apples, with the purpose of examining treat-
Apple
ment effects on cell wall metabolism during on-tree fruit maturation and ripening. Applied calcium
Cell wall
improved cell-to-cell adhesion as indicated by better preservation of the middle lamella and by higher
Fruit softening
Enzyme activity
contents of ionically bound pectins in treated fruit, leading to higher fruit rmness levels at commercial
Preharvest calcium sprays harvest. Matrix glycan breakdown was also delayed in response to calcium treatment. Calcium applica-
tions partially suppressed pectinmethylesterase, pectate lyase, -galactosidase, -l-arabinofuranosidase
and -xylosidase activities, without any apparent relationship with ethylene production rates.
2011 Elsevier B.V. All rights reserved.

1. Introduction apple rmness at the marketplace. Large differences have been

Texture, together with avour, is a major driver of preference for
apples (Malus domestica Borkh.) (Jaeger et al., 1998; Harker et al.,
2008). Firmness in apples is associated with juicy and crispy tex-
ture, whereas soft fruit can develop mealiness, a textural attribute
causing a starch-like sensation in the mouth, generally regarded
as unpleasant. Additionally, although ripening-related softening of
apple fruit displays a non-melting pattern, rmer fruit are more
resistant to physical damage during handling and extended storage,
and thus have higher commercial value.
Ripening-related softening of fruit is generally associated with
the dissolution of the middle lamella and with changes in the
composition, structure and linkages between cell wall polysaccha-
rides (Brummell and Harpster, 2001; Vicente et al., 2007; Goulao
and Oliveira, 2008). The action of a large number of cell wall-
localised proteins is responsible for such extensive modications in
these polymers, causing solubilisation, depolymerisation and rear-
rangements which ultimately affect cell wall strength and lead to
fruit softening (Brummell, 2006; Vicente et al., 2007; Goulao and
Oliveira, 2008). Detailed knowledge of the biochemistry of these
ripening-related events is required for eventual improvement of
Corresponding author. Tel.: +34 973 702526; fax: +34 973 702924.
E-mail address: lara@quimica.udl.cat (I. Lara).
reported among fruit species regarding the extent and enzyme
regulation of the modications in cell wall polysaccharides dur-
ing softening (reviewed in Goulao and Oliveira, 2008), which
means that this process should be studied specically for each
fruit type.
Although apple fruit softening has been the subject of many
studies (reviewed in Johnston et al., 2002), understanding of fac-
tors causing rmness loss is still poor. Apple softening is known
to be accompanied by increased pectin solubilisation, and by low-
ered contents of galactosyl and arabinosyl residues (Knee, 1973).
Apple softening has also been suggested to be mediated by cal-
cium loss from the pectin-rich middle lamella, and accordingly
calcium-treated apples are usually rmer that non-treated controls
(reviewed in Johnston et al., 2002), although preharvest calcium
applications do not always lead to higher rmness by the time of
harvest (Siddiqui and Bangerth, 1995). A recent work (Goulao et al.,
2007) has shown changes in a set of cell wall-modifying enzyme
activities during the development of Mondial Gala apple fruit, but
no data on simultaneous alterations in cell wall composition were
reported. Therefore, in this work we examined the temporal pattern
of changes in cell wall composition and in the activity of a range of
associated enzymes during on-tree maturation and ripening of Fuji
Kiku-8 apple fruit. Preharvest calcium sprays were also applied in
an attempt to evaluate the role of calcium in preservation of cell
wall structure and fruit rmness.

0925-5214/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2011.04.014
162 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167
2. Materials and methods
2.1. Plant material, calcium treatment and quality analysis
Fuji Kiku-8 apple (Malus domestica Borkh.) fruit, growing on
7-year-old trees grafted on M-9 EMLA rootstocks at the IRTA-
Experimental Station in Mollerussa (NE Spain), were sprayed
weekly with CaCl2 (1.6%, w/v). The treatment period was 23
June4 August 2008, corresponding to 81 and 123 days after full
bloom (dafb), respectively. Uniform and defect-free fruit samples
from treated and untreated trees were then picked weekly over
two months (11 August22 October), covering a dafb range of
130202. Commercial harvest took place at 195 dafb. Samples were
coded H1H10, corresponding to successive picking dates. At each
sampling date, rmness (expressed as N) was measured on two
opposite sides of 15 fruit, using an Effegi penetrometer equipped
with an 11-mm diameter convex tip. Soluble solids contents (SSC)
and titratable acidity (TA) were measured in juice pressed from
the whole fruit. SSC was determined with a hand-held refractome-
ter (Atago, Tokyo, Japan), and results expressed as % sucrose in an
equivalent solution. TA was determined by titrating 10 mL of juice
with 0.1 N NaOH to pH 8.1 using 1% (v/v) phenolphthalein; results
were given as g malic acid L1 . Starch hydrolysis was rated visu-
ally using a 110 EUROFRU scale (1, full starch; 10, no starch), after
dipping cross-section fruit halves in 0.6% (w/v) I2 1.5% (w/v) KI
solution for 30 s. Samples of esh tissue were also taken (3 repli-
cates 2 apples/replicate) at each picking date from treated and
untreated fruit, frozen in liquid nitrogen, freeze-dried, powdered,
and kept at 80 C until processing.
2.2. Determination of calcium content
Lyophilised tissue (1 g) was ashed in a mufe furnace at 500 C
for 2 h. Ashes were digested thereafter with 4 mL HCl:distilled
water (1:1, v/v) and heated at 70 C until complete sample dehy-
dration. Dried material was then resuspended in 2 mL HCl:distilled
water (1:1, v/v) for 15 min, ltered through Whatman 40 Ash-
less, and the ltrate diluted to 50 mL in distilled water. Samples
were then analysed by inductively coupled plasma emission spec-
troscopy (ICP-OES) in a Horiba Jobin Yvon ACTIVA spectrometer,
and results expressed as mg 100 gFW1 .
2.3. Extraction, fractionation and analysis of cell wall materials
Cell wall materials (CWM) were extracted in triplicate from
lyophilised tissue (3 g) according to Redgwell et al. (1992). Sam-
ples were homogenised in 20 mL phenol:acetic acid:water (2:1:1,
w/v/v) (PAW) for 20 min. After centrifugation of the homogenate at
4000 g and 4 C for 45 min, the pellet was resuspended in 10 mL
water and centrifuged again. The PAW and water wash super-
natants were combined and intensively dialysed (mol. wt. cut-off
7000) for 2 days against Milli-Q water at 4 C. The dialysate was
centrifuged at 4000 g and 4 C for 45 min to sediment out the
precipitate formed during the dialysis. The supernatant (hence-
forth, PAW-soluble fraction; PAWsf ) was recovered, lyophilised
and weighed. Yields of PAWsf were expressed as % (w/w) FW. The
pellet obtained after PAW extraction and water wash was subse-
quently washed twice in acetone, recovered by vacuum-ltration
through Whatman grade 4 paper lters, lyophilised and weighed
to determine yield of CWM, expressed as % (w/w) FW. For further
fractionation, CWM (100 mg) from each replicate were extracted
sequentially with water, 0.05 M cyclohexane-trans-1,2-diamine
tetra-acetate (CDTA), 0.05 M Na2 CO3 , and 4 M KOH as described
previously (Selvendran and ONeill, 1987), in order to fraction-
ate water-soluble pectin, non-covalently bound pectin, covalently
bound pectin and matrix glycans (hemicelluloses), respectively.
Each fraction was ltered through Miracloth, intensively dialysed
(mol. wt. cut-off 7000) for 2 days against Milli-Q water at 4 C,
lyophilised and weighed. Yields were expressed as % (w/w) CWM.
CDTA- and Na2 CO3 -soluble fractions were hydrolysed with sul-
phuric acid for further analysis. For hydrolysis, 1 mL of 12 M H2 SO4
was added to 3035 mg of fractions to be analysed. After pre-
hydrolysis for 1 h at 37 C, the suspension was diluted with 11 mL
distilled water and heated at 100 C for 2 h. Uronic acid content in
the hydrolysate was measured by the m-hydroxydiphenyl method
(Blumenkrantz and Asboe-Hansen, 1973) using galacturonic acid
as a standard. Total neutral sugars were estimated at 490 nm by
the phenolsulfuric acid assay (Dubois et al., 1956), with galactose
as the standard.
2.4. Extraction and assay of cell wall-modifying enzyme activities
For the extraction of polygalacturonase (exo-PG; EC 3.2.1.67 and
endo-PG; EC 3.2.1.15), pectinmethylesterase (PME; EC 3.1.1.11),
pectate lyase (PL; EC 4.2.2.2) and endo-1,4--d-glucanase (EGase;
EC 3.2.1.4) activities, a 10% (w/v) pulp homogenate was prepared
by homogenising 100 mg of freeze-dried pulp tissue in an extrac-
tion buffer prepared according to Lohani et al. (2004). PG activity
was determined on apple pectin (d.e. 7075%) as described previ-
ously (Pathak and Sanwall, 1998), with galacturonic acid (GalUA) as
a standard. One unit (U) of PG activity was dened as the liberation
of 1 mol of GalUA min1 . PME activity was measured according
to Hagerman and Austin (1986). For the assay, the reaction mixture
contained enzyme extract, apple pectin and bromothymol blue pre-
pared as described previously (Alonso et al., 1997). One unit (U) of
PME activity was dened as the decrease of one unit of A620 min1 .
PL activity was assayed with apple pectin as the substrate accord-
ing to Moran et al. (1968) as modied by Lohani et al. (2004). One
unit (U) of PL activity was dened as the increase of one unit of
A235 min1 . For the assessment of EGase activity, the DNS method
(Miller, 1959), with carboxymethylcellulose as the assay substrate,
was used to determine the amount of reducing sugars released,
with glucose as a standard. One unit (U) of EGase activity was
dened as the release of 1 mol of glucose min1 .
For the extraction of -galactosidase (-Gal; EC 3.2.1.23),
-xylosidase (-Xyl; EC 3.2.1.37) and -l-arabinofuranosidase
(AFase; EC 3.2.1.55) activities, a 10% (w/v) pulp homogenate was
prepared by homogenising 100 mg of freeze-dried pulp tissue in
an extraction buffer prepared according to previous work (Vicente
et al., 2005). -Gal, -Xyl and AFase activity assays were under-
taken in the crude extract as described in Vicente et al. (2005)
and Wei et al. (2010), respectively. One unit (U) of -Gal was
dened as the liberation of 1 mol of p-nitrophenol min1 from
p-nitrophenyl--d-galactopyranoside. One unit (U) of AFase or -
Xyl was dened as the release of 1 nmol of p-nitrophenol min1
from p-nitrophenyl--l-arabinofuranoside or p-nitrophenyl--d-
xylopyranoside, respectively.
Total protein content in the crude extracts was determined with
the Bradford (1976) method, using BSA as a standard. All analy-
ses were done in triplicate, and results were expressed as specic
activity (U mg protein1 ).
2.5. Microscopy observations
For microscopy observations, small pieces (1 mm3 ) of fruit cor-
tex were xed in freshly prepared 2% (w/v) p-formaldehyde and
2.5% (v/v) glutaraldehyde in 0.1 M Na-phosphate buffer, pH 7.2, at
4 C for 5 h. Samples were then washed three times in the same
buffer for 10 min at 4 C, post-xed with 2% osmium tetroxide for
2 h at 4 C, and rinsed again in Na-phosphate buffer, pH 7.2. Tissues
were dehydrated in a graded series of washes in ethanol (50100%,
v/v) and nally embedded in LR White resin and polymerised at
 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167 163

60 C for 48 h. For transmission-electron-microscopy (TEM) obser-

vations, ultra-thin (6090 nm thick) sections were cut and mounted
onto copper grids, counter-stained with uranyl acetate and lead
citrate for 30 min and 10 min, respectively (Reynolds, 1963), and
viewed in a Phillips (EM 301) electron microscope at 80 kV.

2.6. Statistical analysis

Results were treated for multiple comparisons by analysis of

variance (GLM-ANOVA), followed by the least signicant difference
(LSD) Fishers test at P 0.05 with the SAS software package (SAS
Institute, Cary, NC, USA, 1988).

3. Results and discussion

Calcium content decreased steadily throughout the experimen-

tal time, the strongest loss taking place during the second week of
September, between H4 and H5 stages (Table 1). Preharvest sprays
proved effective for enhancing the concentration of this element in
the tissues, as calcium content was consistently higher in treated
fruit, particularly at later maturity stages (H5H10), with increases
well above 25% in relation to untreated samples. Fruit weight and
SSC increased progressively with maturity stage, but no differences
were detected in response to calcium applications. In contrast, sig-
Fig. 1. Firmness (A) and yield of insoluble () and PAW-soluble () cell wall mate-
nicant differences were found for rmness (Fig. 1A) as well as for
rials (B) in esh of untreated and treated Fuji Kiku-8 apples at each sampling date.
TA and starch index (Table 1), although in this case only at advanced Points represent means of 15 (rmness) or three (cell wall materials) replicates.
maturity stages. With the aim of investigating the biochemical fac- Asterisks stand for signicant differences between treated and untreated fruit at
tors underlying higher rmness values around commercial harvest P 0.05 (LSD test).
in calcium-treated fruit, cell wall materials were extracted and
analysed. fruit, possibly in relation to higher rmness (Fig. 1A). For the PAW-
soluble fraction, treatment-related differences were detectable
3.1. Preharvest calcium sprays delayed cell wall disassembly in only at advanced (H7H10) maturity stages, while calcium appli-
mature fruit cations resulted in signicantly increased yields of insoluble CWM
already at the earlier (H4) phases. When the latter were fraction-
Decreased yields of the PAW-soluble fraction and higher con- ated, signicant differences in composition were found between
tents of insoluble cell wall materials (CWM) (Fig. 1B), indicative of treated and untreated fruit. Two pectin-enriched fractions were
delayed solubilisation of cell wall polymers, were found for treated recovered by extraction with 0.05 M CDTA and 0.05 M Na2 CO3 ,

Table 1
Fruit quality and calcium content of untreated and treated Fuji Kiku-8 apples at each sampling date.

Weight (g) SSC (%) TA (g L1 ) SIa (110) Ca2+ content (mg 100 g1 )

H1 August 11 Untreated 161.8 a 11.15 a 4.23 a 1.0 a 4.52 b

Calcium 166.0 a 11.51 a 4.38 a 1.0 a 5.25 a

H2 August 25 Untreated 186.9 a 11.17 a 4.34 a 1.3 a 4.29 b

Calcium 184.1 a 11.50 a 4.40 a 1.0 a 4.93 a

H3 September 2 Untreated 189.7 a 12.44 a 4.34 a 1.1 a 4.26 b

Calcium 188.6 a 12.14 a 4.45 a 1.0 a 5.06 a

H4 September 9 Untreated 207.7 a 12.02 a 4.16 a 2.7 a 4.38 a

Calcium 216.3 a 11.99 a 4.10 a 1.0 b 4.79 a

H5 September 16 Untreated 228.7 a 12.98 a 4.23 a 3.2 a 3.10 b

Calcium 215.7 a 13.37 a 4.04 a 3.4 a 4.27 a

H6 September 23 Untreated 224.3 a 14.05 a 3.93 a 3.3 a 3.00 b

Calcium 223.9 a 12.93 a 4.22 a 3.5 a 4.14 a

H7 September 30 Untreated 240.8 a 14.64 a 4.06 a 6.5 a 3.10 b

Calcium 223.7 a 14.56 a 4.04 a 6.1 a 4.18 a

H8 October 7 Untreated 240.5 a 14.72 a 2.94 b 6.7 a 3.27 b

Calcium 243.0 a 14.25 a 3.51 a 6.5 a 4.16 a

H9 October 15 Untreated 247.9 a 15.21 a 2.85 b 8.1 a 3.10 b

Calcium 247.4 a 15.07 a 3.56 a 7.1 b 3.92 a

H10 October 22 Untreated 252.9 a 15.47 a 2.61 b 9.0 a 2.92 b

Calcium 253.9 a 15.29 a 3.43 a 7.8 b 3.89 a

Values represent means of 15 (standard quality) or three (calcium content) replicates. Means followed by different letters within the same column for a given sampling date
are signicantly different at P 0.05 (LSD test).
a
EUROFRU 110 scale (1, full starch; 10, no starch).
164 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167

Table 2
Yield (% CWM) of fractions isolated from cell wall materials of untreated and treated Fuji Kiku-8 apples at each sampling date.

Watersf CDTAsf Na2 CO3sf KOHsf

Untreated Calcium Untreated Calcium Untreated Calcium Untreated Calcium

H1 August 11 0.335 aC 0.423 aD 10.575 aD 10.751 aF 25.789 aA 26.297 aA 10.812 aA 10.517 aAB
H2 August 25 0.614 aC 0.427 aD 11.619 aC 11.327 aE 25.145 aA 25.360 aB 10.897 aA 10.901 aA
H3 September 2 1.118 aB 0.835 aC 13.719 bA 14.352 aD 23.992 bB 25.113 aB 10.101 aB 10.590 aAB
H4 September 9 1.448 aA 0.985 bBC 12.617 bB 15.872 aAB 22.667 bC 23.619 aC 9.864 bB 10.612 aAB
H5 September 16 1.462 aA 1.331 aA 11.743 bC 15.399 aB 22.168 bC 23.416 aC 9.957 bB 10.576 aAB
H6 September 23 1.531 aA 1.383 aA 10.180 bDE 15.894 aA 22.386 aC 22.997 aC 9.257 bC 10.140 aBC
H7 September 30 1.582 aA 1.390 aA 9.537 bF 16.323 aA 21.765 aC 21.795 aD 8.928 aC 9.238 aDE
H8 October 7 1.553 aA 1.435 aA 9.271 bF 16.250 aA 20.917 aD 21.157 aD 8.270 bD 9.815 aCD
H9 September 15 1.574 aA 1.475 aA 9.583 bF 16.333 aA 20.278 aE 20.463 aE 7.639 bDE 8.715 aC
H10 September 22 1.557 aA 1.517 aA 9.716 bEF 14.931 aC 20.281 aE 20.475 aE 7.594 bE 8.199 aF

Values represent means of three replicates. Means followed by different lower-case letters within a row for a given fraction are signicantly different at P 0.05 (LSD test).
Means followed by different capital letters within a column are signicantly different at P 0.05 (LSD test).

respectively (Selvendran and ONeill, 1987). For untreated sam-
ples, the highest CDTAsf yields were found already at the H3
stage, and decreased thereafter (Table 2). Since this transient
increase in CDTAsf yields at the H3 stage coincided chronologi-
cally with a signicant decline in Na2 CO3sf contents, the question
arises whether a part of these covalently bound pectins were real-
located transiently to the CDTAsf after being depolymerised, in
agreement with observations for stone fruit species (Brummell
et al., 2004; Ortiz et al., 2010). These dynamics were altered in
treated fruit, where the highest CDTAsf yields were observed at
later maturity stages (H6H9). Moreover, calcium-treated samples
displayed higher values for this pectin-containing fraction dur-
ing almost the whole experimental time (Table 2), consistent with
the idea that it is mainly comprised of polymers inter-connecting
through calcium bridges. Accordingly, uronic acid content was
increased in calcium-treated apples in comparison with untreated
fruit (Table 3), indicating that applied calcium allowed better reten-
tion of pectic polymers in this fraction, and suggesting that higher
CDTAsf yields might have been a factor accounting for delayed
rmness loss in treated samples. Higher contents of ionically
bound pectins in calcium-treated fruit were also consistent with
TEM images showing better preservation of the middle lamella
in treated samples (Fig. 2) at both the H9 and H10 stages, when
cell-to-cell contact areas appeared largely lost in control fruit.
The Na2 CO3 -soluble fraction, enriched in pectins bound cova-
lently to the cell wall, decreased steadily during the period
considered, and was on the whole unaffected by calcium appli-
cations, with the exception of early (H3H5) maturity stages, for
which signicantly higher yields were observed for treated fruit
(Table 2). Calcium-treated samples retained higher contents of
uronic acids in this fraction than those untreated at some, though
not all, maturity stages considered (Table 3), coincident with higher
rmness levels (Fig. 1A), and indicative of better preservation of
cell wall structure. In agreement with this idea, treated samples
also retained signicantly more neutral sugars (Table 3), suggest-
ing reduced solubilisation of covalently bound pectins. If a part of
these pectins were actually reallocated transiently to the CDTAsf
after depolymerisation as observed for stone fruit (Brummell et al.,
2004; Ortiz et al., 2010), this would be also consistent with the
observation of lower content of neutral sugars in the CDTAsf in
calcium-treated as compared with untreated samples (Table 3),
possibly arising from lower solubilisation, and thus remobilisation,
of covalently bound pectins in treated fruit.
KOHsf yields, representative of hemicellulosic polymers, also
declined slowly throughout on-tree maturation. In contrast to the
Na2 CO3sf , the decline in KOHsf yields was signicantly delayed in
treated samples from as early as the H4 stage, approximately 45
days before commercial harvest. This suggests that solubilisation of
this fraction was a substantial contributor to rmness loss, which
disagrees with reports on fruit species displaying a melting pattern
of softening, such as peach (Prunus persica L. Batsch), nectarine (P.
persica L. Batsch var. nectarina), or melon (Cucumis melo L.). In these
fruits, hemicellulose breakdown has been suggested to contribute
to the onset of the softening process rather than to extensive rm-
ness loss (Wakabayasi, 2000; Brummell et al., 2004; Ortiz et al.,
2010), KOHsf yields generally showing no large variations during
the melting-like decrease in rmness, nor any apparent relation-
ship with rmness levels.

Table 3
Uronic acid and total neutral sugar contents (%, w/w) of the pectin-enriched fractions obtained from insoluble cell wall materials of untreated and treated Fuji Kiku-8 apples
at each sampling date.

Uronic acids Neutral sugars

CDTAsf Na2 CO3sf CDTAsf Na2 CO3sf

Untreated Calcium Untreated Calcium Untreated Calcium Untreated Calcium

H1 August 11 22.97 aEF 22.06 aE 37.75 aDE 36.96 aE 12.90 aD 12.58 aAB 44.14 aA 44.41 aA
H2 August 25 21.82 aF 22.37 aE 36.37 aE 39.09 aDE 14.37 aD 14.02 aAB 42.01 aAB 44.49 aA
H3 September 2 21.30 aF 21.76 aE 40.45 aD 41.41 aD 17.00 aCD 14.91 aA 45.00 aA 48.97 aA
H4 September 9 22.57 aF 23.98 aE 40.41 aD 41.08 aD 19.08 aBC 15.55 aA 38.56 bB 47.37 aA
H5 September 16 26.37 bDE 31.31 aD 54.53 bB 62.64 aB 22.96 aB 14.82 bAB 29.71 bC 35.79 aB
H6 September 23 26.69 bD 33.17 aD 64.98 bA 69.16 aA 21.02 aBC 13.62 bAB 23.74 bD 30.66 aC
H7 September 30 34.67 bBC 38.30 aC 61.91 aA 64.49 aB 29.80 aA 12.57 bAB 24.16 bD 31.06 aC
H8 October 7 32.76 bC 42.23 aB 62.76 aA 64.04 aB 28.25 aA 13.91 bAB 20.82 bDE 28.01 aCD
H9 October 15 37.51 bB 43.96 aB 53.51 bBC 57.31 aC 27.40 aA 12.01 bAB 19.63 bDE 27.31 aCD
H10 October 22 55.63 bA 62.62 aA 50.80 bC 54.28 aC 21.67 aB 10.54 bB 16.70 bE 23.91 aD

Values represent means of three replicates. Means followed by different lower-case letters within a row for a given fraction are signicantly different at P 0.05 (LSD test).
Means followed by different capital letters within a column are signicantly different at P 0.05 (LSD test).
 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167
Fig. 2. Transmission-electron-microscopy images of untreated (A and C) and calcium-treated (B and D) Fuji Kiku-8 apples picked at H9 (top) and H10 (bottom) maturity
stages (ml: middle lamella). Bars = 1 m.
3.2. Pectolytic and non-pectolytic enzyme activities were
modied in calcium-treated fruit
The temporal pattern of the different enzyme activities poten-
tially involved in cell wall disassembly is widely variable between
species and cultivars (Goulao and Oliveira, 2008). In this work,
diverse trends were observed for the enzyme activities consid-
ered throughout maturation and ripening of Fuji Kiku-8 apples.
In green fruit, cell wall pectin is generally highly methylated,
but the degree of methylesterication declines thereafter due to
PME-catalysed removal of methyl groups (reviewed in Brummell,
2006). This is consistent with the dynamics of PME activity in
untreated samples (Fig. 3A), where the highest levels were found
for H5 fruit, declining sharply thereafter. In contrast, PME activ-
ity levels in calcium-treated fruit were highest at early stages of
maturation (H1H4), and decreased steadily thereafter, with sig-
nicantly lower activities in comparison with the controls during
the rest of the experimental time. While demethylated pectins
are capable of calcium binding, pectin demethylation also gives
rise to regions of negatively charged side groups, which under
low calcium levels may result in electrostatic repulsion between
adjacent molecules, thus contributing to the weakening of the
cell wall (Grignon and Sentenac, 1991). In addition, demethylated
pectins are substrates for degradation by pectolytic enzymes such
as PG or PL (Brummell, 2006; Bennett and Labavitch, 2008). PG
165
activity peaked at the H3 stage (Fig. 3B) and rose again in ripen-
ing fruit (H7H10). Calcium applications had generally no effects
on PG activity, with the exception of very immature (H1H3)
fruit, for which signicantly lowered levels were found in treated
samples (Fig. 3B).
The observation of high PME and PG activity levels at early stages
of apple fruit maturation agrees with previous reports (Goulao
et al., 2007). Changes in the activity levels of some cell wall-related
enzymes during fruit ripening have been partially attributed to
variations in apoplast pH, which is known to decrease during the
process (Almeida and Huber, 1999), and is thus expected to affect
the activity of those enzymes having a pH optimum out of the actual
range. Furthermore, not only activity levels but the specic mecha-
nism of PME action on pectin has been reported to be pH-dependent
(Dens et al., 2000), leading to different inter-chain distributions of
the free carboxyl groups. At pH values close to neutrality, which
approximately correspond to those of the apoplast of unripe fruit,
PME acts by a single-chain mechanism, resulting in a block-wise
distribution of the new negative charges, which confers pectins
higher calcium afnity, and leads to cell wall stiftening. PME action
also contributes to localised lowering of apoplast pH, thus provid-
ing a regulation mechanism for other cell wall proteins (Goulao,
2010). Charged surfaces affect pectin hydration status, and may also
restrict the movement of basic proteins through the wall matrix as
well as their activity (Grignon and Sentenac, 1991).
166 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167
Fig. 3. Pectinmethylesterase (A), polygalacturonase and pectate lyase (B) activi-
ties in esh of untreated and treated Fuji Kiku-8 apples at each sampling date.
Points represent means of three replicates. Asterisks stand for signicant differences
between treated and untreated fruit at P 0.05 (LSD test).
Pectate lyases are thought to mediate pectin depolymerisa-
tion by -elimination, and their possible role in fruit softening
has received little attention until relatively recently. However, PL
expression or activity has been detected during ripening of a few
fruit species, including apple (Goulao et al., 2007) as well as straw-
berry (Fragaria ananassa Duch.) (Medina-Escobar et al., 1997),
banana (Musa acuminata Colla) (Domnguez-Puigjaner et al., 1997)
and nectarine (Ortiz et al., 2010). In this work, after a slight decline
between H1 and H2 stages, PL activity increased gradually until
the H6 stage, and remained steady thereafter (Fig. 3B). Activity lev-
els were signicantly reduced in response to calcium treatment
as early as the H3 stage, coincident with the rst hint of higher
CDTAsf yields in treated samples (Table 2). Although no or very lim-
ited pectin depolymerisation is believed to take place in apple fruit
(reviewed in Brummell, 2006; Goulao and Oliveira, 2008), results
suggest a possible key role for PL in cell wall disassembly.
Pectin solubilisation may also result from cleavage of the link-
ages to other cell wall components. It has also been suggested that
the highly branched structure of these polymers, arising from the
Fig. 4. -l-arabinofuranosidase and -galactosidase activities in esh of untreated
and treated Fuji Kiku-8 apples at each sampling date. Points represent means of
three replicates. Asterisks stand for signicant differences between treated and
untreated fruit at P 0.05 (LSD test).
abundant galactosyl- and arabinosyl-containing side-chains, may
determine cell wall porosity, and thus restrict the access of many
pectolytic enzymes to their substrates (Brummell and Harpster,
2001). In apple, loss of galactose has been found to occur mainly
before the onset of ripening, and a strong loss of highly branched
arabinans preceded fruit softening (Pena and Carpita, 2004). In this
work, a signicant increase in AFase activity was observed already
after the H4 stage, while a rise in -Gal activity levels was found
one week later (Fig. 4). In both cases, activity increased steadily
thereafter, and was signicantly inhibited at later stages of fruit
maturation and ripening (H6H10) in response to calcium applica-
tions, suggesting a potential role in the regulation of fruit softening.
AFase activity dynamics did not agree with previous reports for
Mondial Gala apples (Goulao et al., 2007), where decreasing lev-
els were observed during fruit development, followed by a sharp
increase in over-ripe fruit. Differences in arabinose content and
in cell wall integrity have been detected among apple cultivars
with different postharvest softening behaviour (Tong et al., 1999),
and indeed Mondial Gala and Fuji cultivars are rather dissimilar
regarding storage potential.
The KOH-soluble fraction is indicative of the hemicellulose frac-
tion, and is comprised mainly of xyloglucan polymers (Wakabayasi,
2000) which are susceptible to EGase-mediated breakdown. Con-
tradictory reports on the relevance of EGase activity for fruit
softening have been published (Goulao and Oliveira, 2008). Activity
levels were found to decrease progressively during fruit devel-
opment of Mondial Gala apples (Goulao et al., 2007), thus
de-emphasising its role in rmness loss. Contrarily, EGase activity
was higher in mature (H6H10) Fuji Kiku-8 apples in comparison
with earlier stages (Fig. 5), concomintantly with a decline in KOHsf
yields (Table 2). However, while calcium applications delayed the
decrease in the yields of this cell wall fraction, no signicant treat-
ment effects were observed for EGase activity, suggesting it was
not directly related to KOHsf yields. Alternatively, possible differ-
ences in EGase activity arising from pH variations in the apoplast
during the experimental time might have been masked due to the
use of a xed pH for enzyme assays. Matrix glycans are also suscep-
tible to cleavage by -Xyl (Brummell and Harpster, 2001). In spite
of a decreasing trend at early sampling dates, -Xyl activity lev-
els rose again as ripening progressed (Fig. 5), and were partially
suppressed in response to calcium applications, which suggests
a role in ripening-related cell wall disassembly of Fuji Kiku-8
apples. Other hemicellulose-acting enzymes such as xyloglucan
endotransglycosylase/hydrolases (XTH) were not explored in this
work, but XTH activity levels were found to remain unchanged
during ripening of Mondial Gala apples (Goulao et al., 2007).
The data thus indicate that applied calcium impacted cell wall
metabolism at different levels. A primary effect of the treatment
Fig. 5. -xylosidase and endo-1,4--d-glucanase activities in esh of untreated and
treated Fuji Kiku-8 apples at each sampling date. Points represent means of three
replicates. Asterisks stand for signicant differences between treated and untreated
fruit at P 0.05 (LSD test).
 A. Ortiz et al. / Postharvest Biology and Technology 62 (2011) 161167
was apparently related to the direct reinforcement of the cell wall
structure due to better preservation of the chelator-soluble frac-
tion, presumably mediated by the establishment of calcium bridges
between polymers. A secondary effect of calcium applications
was associated to partial inhibition of some cell wall-modifying
enzyme activities, which possibly led to delayed solubilisation
of cell wall polymers. A tertiary level of calcium effects might
have been exerted through limited access to substrates of partic-
ular enzymes arising from decreased AFase and -Gal activities,
which remove pectin side-chains and thus control to some extent
cell wall porosity, through the modulation of enzyme secretion
due to the protective role of calcium on cell membranes, or
through apoplast pH or charge changes owing to modications
in PME activity.
Calcium has also been shown to inhibit ethylene biosynthesis by
delaying ACO-catalysed conversion of ACC into ethylene (Lara and
Vendrell, 1998). Since many cell wall-modifying enzyme activities
are reportedly ethylene-dependent (Domnguez-Puigjaner et al.,
1997; Brummell and Harpster, 2001; Lohani et al., 2004; Wei et al.,
2010), partial suppression in calcium-treated samples might have
arisen from generally decreased ethylene production. However,
climacteric ethylene production by Fuji apples is characteristi-
cally moderate, and in this work it remained at undetectable levels
regardless of treatment (data not shown), which suggests that the
observed modications in enzyme activities potentially involved in
ripening-related cell wall disassembly were unrelated to changes
in ethylene production.
Acknowledgements
A. Ortiz was the recipient of a FPU grant from the Ministerio de
Ciencia e Innovacin (MICINN) of Spain. This work was supported
through the AGL2006-00345/ALI project, nanced by the Ministe-
rio de Educacin y Ciencia (MEC) of Spain. The authors are indebted
to P. Sopena and E. Comabella for technical assistance and to X.
Calomarde for microscopy observations.
References
Almeida, D.P.F., Huber, D.J., 1999. Apoplastic pH and inorganic ion levels in tomato
fruit: a potencial means for regulation of cell wall metabolism during ripening.
Physiol. Plant 105, 506512.
Alonso, J., Howell, N., Canet, W., 1997. Purication and characterisation of two
pectinmethylesterase from persimmon (Diospyros kaki). J. Sci. Food Agric. 75,
352358.
Bennett, A.B., Labavitch, J.M., 2008. Ethylene and ripening-regulated expression and
function of fruit cell wall modifying proteins. Plant Sci. 175, 130136.
Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determi-
nation of uronic acids. Anal. Biochem. 54, 484489.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein dye binding. Anal.
Biochem. 72, 248254.
Brummell, D.A., 2006. Cell wall disassembly in ripening fruit. Funct. Plant Biol. 33,
103119.
Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and
quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311340.
Brummell, D.A., Dal Cin, V., Crisosto, C.H., Labavitch, J.M., 2004. Cell wall metabolism
during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55,
20292039.
Dens, J.M., Baron, A., Renard, C.M.G.C., Pan, C., Drilleau, J.F., 2000. Different action
patterns for apple pectin methylesterase at pH 7.0 and 4.5. Carbohydr. Res. 327,
385393.
167
Domnguez-Puigjaner, E., Llop, I., Vendrell, M., Prat, S., 1997. A cDNA clone highly
expressed in ripe banana fruit shows homology to pectate lyases. Plant Physiol.
14, 10711076.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Anal. Chem. 28,
350356.
Goulao, L.F., 2010. Pectin de-esterication and fruit softening: revisiting a classical
hypothesis. Stewart Postharvest Rev. 1, 7.
Goulao, L.F., Oliveira, C.M., 2008. Cell wall modications during fruit ripening: when
a fruit is not the fruit. Trends Food Sci. Technol. 19, 425.
Goulao, L.F., Santos, J., de Sousa, I., Oliveira, C.M., 2007. Patterns of enzymatic activity
of cell wall-modifying enzymes during growth and ripening of apples. Posthar-
vest Biol. Technol. 43, 307318.
Grignon, C., Sentenac, H., 1991. pH and ionic conditions in the apoplast. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 42, 103128.
Hagerman, A.E., Austin, P.J., 1986. Continuous spectrophotometric assay for plant
pectin methyl-esterase. J. Agric. Food Chem. 34, 440444.
Harker, F.R., Kupferman, E.M., Marin, A.B., Gunson, F.A., Triggs, C.M., 2008. Eating
quality standards for apples based on consumer preferences. Postharvest Biol.
Technol. 50, 7078.
Jaeger, S.R., Andani, Z., Wakeling, I.N., MacFie, H.J.H., 1998. Consumer preferences
for fresh and aged apples: a cross-cultural comparison. Food Qual. Prefer. 9,
355366.
Johnston, J.W., Hewett, E.W., Hertog, M., 2002. Postharvest softening of apple (Malus
domestica) fruit: a review. N. Z. J. Crop Hortic. Sci. 30, 145160.
Knee, M., 1973. Polysaccharide changes in cell walls of ripening apples. Phytochem-
istry 12, 15431549.
Lara, I., Vendrell, M., 1998. ACC oxidase activation by cold storage on Passe-Crassane
pears: effect of calcium treatment. J. Sci. Food Agric. 76, 421426.
Lohani, S., Trivedi, P.K., Nath, P., 2004. Changes in activities of cell wall hydrolases
during ethylene-induced ripening in banana: effect of 1-MCP, ABA and IAA.
Postharvest Biol. Technol. 31, 119126.
Medina-Escobar, N., Crdenas, J., Moyano, E., Caballero, J.L., Munoz-Blanco, J., 1997.
Cloning, molecular characterization and expression pattern of a strawberry
ripening-specic cDNA with sequence homology to pectate lyase from higher
plants. Plant Mol. Biol. 34, 867877.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal Chem. 31, 426428.
Moran, F., Nasuno, S., Starr, M.P., 1968. Extracellular and intracellular polygalactur-
onic acid trans-eliminase of Erwinia carotovora. Arch. Biochem. Biophys. 123,
298306.
Ortiz, A., Seymour, G.B., Tucker, G.A., Lara, I., 2010. Cell wall disassembly during the
melting phase of softening in Snow Queen nectarines. Postharvest Biol. Technol.
58, 8892.
Pathak, N., Sanwall, G.G., 1998. Multiple forms of polygalacturonase from banana
fruits. Phytochemistry 48, 249255.
Pena, M.J., Carpita, N.C., 2004. Loss of highly branched arabinans and debranching of
rhamnogalacturonan I accompany loss of rm texture and cell separation during
prolonged storage of apple. Plant Physiol. 135, 13051313.
Redgwell, R.J., Melton, L.D., Brasch, D.J., 1992. Cell wall dissolution in ripening
kiwifruit (Actinidia deliciosa). Plant Physiol. 98, 7181.
Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron opaque stain
in electron microscopy. J. Cell. Biol. 17, 208212.
Selvendran, R.R., ONeill, M.A., 1987. Isolation and analysis of cell walls from plant
material. In: Glick, D. (Ed.), Methods of Biochemical Analysis, vol. 32. John Wiley
Interscience, New York, pp. 25153.
Siddiqui, S., Bangerth, F., 1995. Effect of pre-harvest application of calcium on esh
rmness and cell-wall composition of apples inuence of fruit size. J. Hortic.
Sci. 70, 263269.
Tong, C., Krueger, D., Vickers, Z., Bedford, D., Luby, J., El-Shiekh, A., Shackel, K.,
Ahmadi, H., 1999. Comparison of softening-related changes during storage of
Honeycrisp apple, its parents, and Delicious. J. Am. Soc. Hortic. Sci. 124,
407415.
Vicente, A.R., Costa, M.L., Martnez, G.A., Chaves, A.R., Civello, P.M., 2005. Effect
of heat treatments on cell wall degradation and softening in strawberry fruit.
Postharvest Biol. Technol. 38, 213222.
Vicente, A.R., Saladi, M., Rose, J.K.C., Labavitch, J.M., 2007. The linkage between cell
wall metabolism and fruit softening: looking to the future. J. Sci. Food Agric. 87,
14351448.
Wakabayasi, K., 2000. Changes in cell wall polysaccharides during fruit ripening. J.
Plant Res. 113, 231237.
Wei, J., Fengwang, M., Shi, S., Qi, X., Zhu, Z., Yuan, J., 2010. Changes and posthar-
vest regulation of activity and gene expression of enzymes related to cell wall
degradation in ripening apple fruit. Postharvest Biol. Technol. 56, 147154.