Download Structural and chemical differences in the cell wall regions in

Journal of the Science of Food and Agriculture
J Sci Food Agric 88:1277–1286 (2008)
Structural and chemical differences
in the cell wall regions in relation to scale
firmness of three onion (Allium cepa L.)
selections at harvest and during storage
Timothy W Coolong,1∗ William M Randle2 and Louise Wicker3
1 Department
of Horticulture, University of Kentucky, Lexington, KY 40546, USA
of Horticulture and Crop Science, Ohio State University, Columbus, OH 43210, USA
3 Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA
2 Department
Abstract
BACKGROUND: Firmness in vegetables is an important textural attribute affecting consumer attitudes toward
freshness and quality. Firmness, structural carbohydrates, polygalacturonase (PG), and pectin methylesterase
(PME) activity were measured in three onion (Allium cepa L.) lines at harvest and after 4, 8, and 12 weeks of
storage.
RESULTS: The high-dry-matter onion, MBL87-WOPL, had the firmest bulbs at harvest and delayed softening
during storage. MBL87-WOPL had the thickest cell wall/middle lamella region, and highest levels of dry matter
and total uronic acid. Furthermore, MBL87-WOPL had the lowest levels of PG and PME activity during storage.
Pegasus, a poor-storing cultivar, had the softest bulbs at harvest, lowest levels of uronic acid, and thinnest cell
wall/middle lamella. A good storing, moderately firm onion cultivar (MSU4535B) presented intermediate levels of
firmness and total uronic acid content. Differences in uronic acid in water-soluble pectin accounted for much of
the difference in total uronic acid among lines. Cellulose concentrations were similar among all lines at harvest.
In addition, cellulose concentrations decreased in all lines during storage. Transmission electron microscopy
performed on bulbs at harvest and after 12 weeks of storage indicated that degradation of the middle lamella had
occurred during storage, leading to cell separation.
CONCLUSION: Our results suggest that differences in onion scale firmness at harvest may be due to differences in
water-soluble pectin uronic acid concentrations. Furthermore, the rate of bulb softening during storage at 6.6 ◦ C
was greater in onion lines with higher levels of PME and PG activity in storage.
 2008 Society of Chemical Industry
Keywords: cellulose; uronic acid; pectin methylesterase; polygalacturonase; middle lamella
INTRODUCTION
Onions (Allium cepa L.) have been cultivated by
humans for thousands of years. Though primarily
consumed for their flavor, the textural characteristics
of onions also contribute to the consumer’s perception
of bulb quality.1,2 Texture is an aggregate quality
composed of several measurable parameters, including
firmness, crispness, elasticity, and gumminess.3 Owing
to ease of use and cost-effectiveness, firmness values in
fruits and vegetables are often measured using a handheld or press-mounted penetrometer.4 Measurements
of firmness in fruit and vegetables can be affected by a
number of factors including cell size and arrangement,
cell wall and middle lamella strength and cell turgor.4,5
The physiochemical properties of the cell wall
and middle lamella in fruits and vegetables have
been extensively investigated.6 However, unlike fruits
such as apple, for which the relationship between
cell wall/middle lamella and firmness has been
thoroughly investigated, much less is known about
the physiochemical properties of onion.2,4,5,7 There
have been several studies reporting on the cell
wall chemistry of onion.8 – 11 One study correlated
mechanical properties of bulbs to cell wall chemistry,
demonstrating that the tensile strength of tightly
packed epidermal cells was many times greater than
that of loosely spaced parenchyma cells in bulb scales
in the onion cultivar ‘Sturon’.10 In addition, the hard,
papery, outer scales of the bulbs had significantly
higher concentrations of galactose-rich pectin than
softer interior scales. This suggests that the physical
arrangement of cells and chemical composition of the
cell wall/middle lamella could affect the mechanical
properties of bulbs.10 Recent studies have indicated
∗
Correspondence to: Timothy W Coolong, Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA
E-mail: [email protected]
(Received 5 October 2007; revised version received 27 December 2007; accepted 3 January 2008)
Published online 31 March 2008; DOI: 10.1002/jsfa.3219
 2008 Society of Chemical Industry. J Sci Food Agric 0022–5142/2008/$30.00
TW Coolong, WM Randle, L Wicker
that significant softening occurs in onion tissue during
refrigerated storage.12
The composition of pectic polysaccharides may
affect the texture of onion.10 Therefore it is possible
that changes in firmness during storage could
be affected by the activity of pectinases. Pectin
methylesterase (PME) (EC 3.1.1.11) is responsible
for demethylesterification of polygalacturonic acid
chains. Once demethylesterified these polymers can
bind to free calcium ions, resulting in cross-linking of
adjacent pectin chains, increasing firmness. However,
if native polygalacturonases (PG) (EC 3.2.1.15)
are present, the galacturonic acid chains may be
hydrolyzed, resulting in depolymerization of the pectin
and potential softening.13,14 PME activity, which has
been detected in fresh and dehydrated onion tissue,
has been proposed to affect the firmness of thermally
processed onion.15,16 This suggests that PME may
play an active role in modifying the texture of onion.
Although PG activity has not been investigated in
onion, it has been correlated to a loss of cellular
adhesion in the roots of Leek (Allium porrum L.), a
species related to onion.17 Thus, although there are
many factors that may influence texture in fruits and
vegetables, it is probable that PME and PG activity
may affect texture in onion tissue during storage.18 – 20
The roles of pectin, cellulose, PME, PG, and
cell wall/middle lamella architecture in relation to
onion scale firmness at harvest and during storage
are unknown. Therefore, the objectives of this study
were to determine the relationship between firmness
at harvest and during storage and uronic acid
concentrations in pectin, cellulose content, and PME
and PG activity. Cell wall/middle lamella architecture
was also investigated to determine whether changes in
firmness could be correlated to changes in cell wall
morphology.
Plants were watered daily and fertilized twice weekly
with a half-strength Hoagland’s #2 nutrient solution.21
Ten weeks after transplantation, supplemental overhead lighting with an average canopy light intensity of
277 µmol m−2 s−1 (Basic Quantum Meter, Spectrum
Technologies, Plainfield, IL, USA) was provided to
induce bulbing in the long-day onion line. All onions
were subjected to 17 h day lengths.
Onions were harvested when 50–70% of the plants
in a given line displayed soft pseudostems. On 3 May
2006 bulbs from ‘Pegasus’ were harvested and placed
in nylon mesh bags and cured at 36 ◦ C for 72 h. Each
bag contained 15 bulbs and four bags were harvested
from each of the four replications. Bags were placed
into refrigerated storage, 6.6 ± 1.4 ◦ C and 82 ± 4.2%
relative air humidity. Bulbs from MBL 87-WOPL
and MSU4535B were harvested on 25 May 2006
and 16 June 2006, respectively, and were cured and
placed into storage as described previously. Due to
the presence of disease, only three replications of
MBL 87-WOPL were harvested and stored. Bulbs
were analyzed at harvest and after 4, 8, and 12 weeks
in storage. All subsequent analyses were conducted
on 15 bulb composite samples with four replications
of Pegasus and MSU4535B and three replications of
MBL 87-WOPL.
MATERIALS AND METHODS
Plant material
Three onion selections with contrasting firmness
values were selected for the current study: (a) the
short-day cultivar ‘Pegasus’ (Seminis seeds, Oxnard,
CA, USA) represented a soft, poor-storing bulb
grown for the sweet onion market; (b) the longday breeding line, MSU4535B, has good storage
potential and is moderately firm; and (c) the open
pollinated selection MBL 87-WOPL could be used
primarily for dehydration and is very firm. On
18 November 2005 seeds from each variety were
seeded into 200-cell plastic plug trays. Seedlings were
greenhouse grown, watered, and fertilized as needed.
After 6 weeks, seedlings were transplanted into boxes
(2.45 m × 1.22 m × 0.15 m) containing Fafard 52 Mix
(Fafard Inc., Agawam, MA, USA). Plants were evenly
spaced 8.75 cm on center and each box held 96 plants
of each of the three lines. Four boxes were spaced
throughout the greenhouse in completely randomized
design with four replications.
Soluble solids and firmness
Total soluble solids (TSS) were measured by taking
1–2 mm longitudinal slices from bulbs at each
sampling time and crushing them in a pneumatic press.
Several drops of the juice were placed on a hand-held
refractometer (Kernco, Tokyo, Japan) to determine
TSS. Bulb scale firmness was measured by cutting a
2 × 4 cm slice from the first fully fleshy scale (usually
the second or third scale from the outside of the
bulb) at the equatorial region of each bulb. Firmness
was measured as the force required to penetrate the
scale using a 1 mm diameter probe coupled to a fruit
penetrometer mounted on a motorized press operated
at a speed of 1.5 mm s−1 (Model 327, McCormick
Fruit Tech, Yakima, WA, USA). Firmness for each
2 × 4 cm slice was measured three times and averaged.
1278
Sprouting and rooting
As an indicator of the state of dormancy, the
percentage of bulbs that could resume root or shoot
growth was measured. To do this, 2–3 cm thick crosssections of 15 bulbs containing the root plate and
apical meristem were placed in boxes filled with Fafard
Super Fine Germinating Mix (Fafard Inc.). Bulb slices
were watered daily and maintained in a greenhouse
with day/night temperature set points of 28/20 ◦ C
under natural light intensities and photoperiods. After
10 days the percentage of bulbs exhibiting rooting and
sprouting was recorded.
Alcohol-insoluble solids, pectin fractioning, and
total pectin determination
The alcohol-insoluble solids (AIS) residue was
prepared from onion tissue according to a modification
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Differences in the cell wall regions in relation to scale firmness in Allium cepa L.
of the method of Huber and Lee.22 Longitudinal slices,
5–10 mm thick, were cut from bulbs. Slices were
homogenized in a blender for 60 s with four volumes
(w/v) of 95% ethanol. Two more volumes of 95%
ethanol were added and the homogenate was boiled at
100 ◦ C for 20 min with slow stirring. The homogenate
was cooled in an ice-water bath for 30 min. The cooled
residue was filtered under vacuum through glass fiber
filters (APFF, 0.7 µm, Millipore, Billerica, MA, USA).
Based on the initial sample weight the residue was
sequentially washed with six volumes of 95% ethanol,
four volumes of 100% ethanol, and four volumes of
acetone. The residue was dried overnight in a fume
hood. The dried AIS residue was weighed and ground
to a fine powder using a coffee grinder and stored at
−20 ◦ C until analysis.
The AIS was sequentially fractioned into water-,
chelator-, acid-, and alkali-soluble pectin.23 Approximately 30 mg of onion AIS was extracted at 60 ◦ C
for 90 min in 40 mL of 0.05 mol L−1 sodium acetate
buffer, pH 5.2, to obtain water-soluble pectin (WSP).
The WSP was obtained by centrifuging the extract
at 30 000 × g for 15 min and filtering the supernatant
through one layer of Miracloth (CalBiochem, EMD
Biosciences, San Diego, CA, USA). The remaining
pellet was resuspended in 40 mL of 0.05 mol L−1
sodium oxalate, 0.05 mol L−1 ammonium oxalate,
and 0.05 mol L−1 sodium acetate pH 5.2 and incubated for 90 min at 60 ◦ C to obtain chelator-soluble
pectin (CSP). The remaining pellet was again suspended in 40 mL of HCL pH 2.5 and incubated for
90 min at 60 ◦ C. The extract was centrifuged and filtered as previously to obtain the acid-soluble pectin
(ACSP). The remaining pellet was resuspended in
40 mL of 0.05 mol L−1 NaOH and incubated for
90 min at 60 ◦ C, centrifuged, and filtered as previously
in order to obtain the alkaline-soluble pectin (AKSP).
The uronic acid content of each pectin fraction was
determined using the m-hydroxydiphenol method.24
Fructans with a degree of polymerization longer than
three are co-extracted in the AIS. Although fructans
do react with the m-hydroxyphenol used to determine uronic acid concentrations, their contribution
to the total absorption of each fraction was minimal and did not significantly affect the results of the
assay.
Total uronic acid content of the AIS was also
determined.25 Approximately 5 mg of AIS was
weighed into a 50 mL beaker to which 5 mL of
concentrated cold sulfuric acid was added. The
beaker was stirred in an ice bath for approximately
10 min until the AIS dissolved. Then 5 mL of cold
deionized water was added in 1 mL increments. After
10 min cold deionized water was added to bring
the solution to a volume of 25 mL in a volumetric
flask. An aliquot of the solution was analyzed for
galacturonic acid.24 Galacturonic acid content of
samples was estimated from a linear regression
of galacturonic acid standards at concentrations of
0–20 µg.
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Cellulose
Cellulose concentrations in the AIS residue were
determined.26 Approximately 30 mg of AIS residue
was weighed into glass test tubes and dissolved with
3.0 mL of 10:1 (80% acetic acid:concentrated nitric
acid) and heated in a boiling water bath at 100 ◦ C
for 30 min, centrifuged for 10 min at 5000 × g and
the supernatant was discarded. The pellet was washed
with 10 mL of deionized water and centrifuged at
5000 × g for 10 min and repeated twice. The washed
pellet was dissolved in 10 mL of 67% sulfuric acid,
mixed and diluted to 100 mL with deionized water.
An aliquot of the solution was pipetted into a glass
test tube to which 3.5 mL of deionized water and
10 mL of cold anthrone reagent (0.2 g anthrone in
100 mL concentrated sulfuric acid) (Sigma, St Louis,
MO, USA) was added. Tubes were mixed and placed
in a boiling water bath for 18 min. After cooling
in an ice bath for 5 min, samples were read on
a spectrophotometer at 620 nm and concentrations
calculated based on a standard curve developed using
purified cellulose (Sigma). Spiked samples containing
purified cellulose averaged 90% recovery.
Enzyme activity and protein determination
All enzyme extractions and purification steps were
performed in a cold room at 4 ◦ C. Enzyme extracts
were boiled for 5 min prior to assaying to serve as
blanks for each sample. PG activity was determined
according to a modification of the 2-cyanoacetamide
method.27 PG was extracted from approximately 15 g
fresh tissue obtained from the equatorial region of
bulbs using a 6 mm cork borer and homogenized
in 45 mL of cold 1 mol L−1 NaCl solution pH 6.0
for 1 min using a Waring blender at high speed
(Waring Laboratory and Science, Torrington, CT,
USA). The homogenate was extracted overnight at
4 ◦ C. The homogenate was filtered through two layers
of Miracloth and centrifuged at 4 ◦ C and 10 000 × g
for 10 min. A 0.5 mL aliquot of the supernatant
was concentrated and reducing sugars removed using
centrifugation and filtration at 12 000 × g and 4 ◦ C
for 1 h (Microcon YM-10, Millipore). Then 0.1 mL
of 50 mmol L−1 sodium acetate pH 4.4 was added
to the retentate and the solution was reconcentrated
by centrifugation at 12 000 × g and 4 ◦ C for 40 min.
The retentate was redissolved in 0.1 mL 50 mmol L−1
sodium acetate pH 4.4 and galacturonic acid content
determined. One unit of PG activity was determined
to be the amount of enzyme that released 1 µmol of
reducing sugar (galacturonic acid) 60 s−1 .
PME activity was determined by titration at pH
7.5.28 PME was extracted from 15 g of fresh tissue
homogenized with 60 mL of ice-cold extraction buffer
(0.1 mol L−1 NaCl, 0.25 mol L−1 Tris-base, pH 8.0)
for 1 min using a Waring blender at high speed.
The homogenate was extracted for 3 h at 4 ◦ C, after
which the mixture was filtered through two layers
of Miracloth and centrifuged for 10 min at 4 ◦ C and
10 000 × g. The supernatant was collected and 30%
1279
TW Coolong, WM Randle, L Wicker
ammonium sulfate added. The solution was allowed to
precipitate overnight. The dispersion was centrifuged
for 15 min at 4 ◦ C and 10 000 × g and the supernatant
collected. The enzyme assay was carried out using a
pH stat titrator (Brinkmann, Westbury, NY, USA).
0.4 mL of extract was added to 20 mL of a solution
of 1.0% high-methoxy citrus pectin with 0.1 mol L−1
NaCl at pH 7.5 and 37 ◦ C. The assay was conducted
for 20 min. One unit of PME activity was determined
to be the amount of enzyme that released 1 µmol of
carboxylic acid 60 s−1 .
Protein analysis
Total protein concentrations used in each enzyme
assay were determined using the bicinchoninic acid
method.29 A commercially available kit was used
and manufacturer’s instructions followed using bovine
serum albumin protein as a standard (Pierce BCA
Protein Assay Kit, Pierce, Rockford, IL, USA).
Microscopy
Samples were analyzed using light microscopy and
transmission electron microscopy (TEM) to determine
whether any structural changes could be observed in
the cell wall/middle lamella region between cultivars
at harvest and during storage. All samples were
prepared in a cold room at 4 ◦ C. At harvest and
after 12 weeks of storage, bulbs were sampled. Five,
1–2 mm3 samples were cut from each of 15 bulbs
and immediately placed into Sorensen’s phosphate
buffer, pH 7.2, with 4% glutaraldehyde fixative for 8 h
(EMS, Hatfield, PA, USA).30 The fixative solution
was decanted and samples washed twice for 30 min
and once overnight in Sorensen’s phosphate buffer.
Samples were decanted and post-fixed in Sorensen’s
phosphate buffer containing 1% osmium tetraoxide
for 2 h. Samples were washed for 20 min in Sorensen’s
buffer and repeated twice. Samples were dehydrated
in a graded ethanol series (20%, 30%, 50%, 70%,
95%,100% ethanol) for 20 min at each step. Propylene
oxide was used as a transitional solvent prior to
embedding. Samples were embedded in Spurr’s lowviscosity embedding resin (EMS).31
Sections for light (1–2 µm) and TEM (90 nm)
microscopy were cut on a Reichert Jung ultracut
E ultramicrotome (Reichert Microscope Services,
Depew, NY, USA) using a diamond knife (Diatome
Histo 5100 and Ultra 45◦ ; Diatome, Hatfield, PA,
USA). Sections for light microscopy were dried on
glass slides and stained with 0.5 g L−1 toluidine
blue (Sigma) and visualized on an Olympus Bx51
microscope (Olympus America Inc., Center Valley,
PA, USA). Cell wall/middle lamellar thickness was
determined by measuring cell walls of four cells per
bulb at points where adjacent cells made contact
(20–25 measurements per bulb) and averaging them.
Ultrathin sections were cut for TEM and placed on
200 mesh copper grids and stained with uranyl acetate
and lead citrate. Sections were visualized on a Zeiss
902A TEM (Carl Zeiss Microimaging, Thornwood,
1280
NY, USA). Five samples for each onion line per
sampling time were tested.
Statistical analysis
All data was subjected to the GLM procedure testing for significance of main effects and interactions
between cultivar and storage time using SAS statistical software (SAS v. 9.1.3, SAS institute, Cary,
NC, USA). Percentage data were transformed using
arc–sin transformations. Data were considered significant when P < 0.05.
RESULTS
Firmness
Onion scale firmness was affected by cultivar and
storage time (Fig. 1). There was also a significant
interaction among cultivars and storage time for onion
scale firmness. The interaction was the result of
firmness decreasing at different rates in the cultivars
during storage. The high-dry-matter line MBL87WOPL had the highest average firmness at harvest
(4.17 N), which decreased by 0.34 N over 12 weeks in
storage. The cultivar Pegasus had the lowest average
firmness readings at harvest (2.96 N). Scale firmness
decreased by 0.44 N after 12 weeks in storage. The
long-day line, MSU4535B, had an average firmness of
3.75 N at harvest and an average decrease of 0.52 N
in scale firmness during storage.
Dry matter, TSS, and weight loss
The percentage dry matter in bulbs differed significantly by cultivar and storage time (Fig. 2(A)). Bulb
dry matter concentrations in MBL87-WOPL ranged
from 15% to 18%. The fresh-market sweet onion
Pegasus had levels of bulb dry matter between 9% and
10.5%. A significant interaction between cultivar and
storage time regarding dry matter concentration was
Figure 1. Firmness of onion (Allium cepa L.) scales in measured in
newtons (N) at harvest, 4, 8, and 12 weeks of storage. Each data point
represents the mean (± s.e.) of four replications for Pegasus and
MSU4535B and three replications of MBL87-WOPL onions.
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Differences in the cell wall regions in relation to scale firmness in Allium cepa L.
monitored. This resulted from the increase in dry matter content in MBL87-WOPL during storage, while
the other cultivars presented slight differentiation.
Total TSS was affected by cultivar, and was highest
in MBL87-WOPL and lowest in Pegasus (Fig. 2(B)).
Total TSS was also affected by storage time. Onion
line and storage time significantly interacted to affect
bulb TSS. While TSS declined slightly during storage
in Pegasus and MSU4535, TSS increased slightly in
MBL87-WOPL, resulting in a small, but significant
interaction. Weight loss in bulbs, measured as a percentage of fresh weight at harvest, differed among lines
and increased during storage (Fig. 2(C)). There was
also a significant interaction between onion line and
storage time for weight loss. Much of this interaction
is the result of the large weight loss that occurred
in MBL87-WOPL between 4 and 8 weeks of storage,
compared to the smaller weight losses incurred by
the other cultivars during that time. MSU4535B lost
the least amount of its harvest weight during storage
(6%), while MBL87-WOPL had the largest weight
loss, eventually losing 16% of its initial weight after
12 weeks in refrigerated storage.
Rooting and sprouting
Breaking of dormancy as measured by the ability of
the harvested bulbs to resume growth by sprouting
new shoots or roots was affected by cultivar and
storage duration (Fig. 3). There was also a significant
interaction between cultivar and storage duration
regarding sprouting and rooting. At harvest the onion
cultivar Pegasus exhibited rooting or sprouting in
roughly 60% of bulbs, while the other varieties
required 8 weeks of storage to reach similar levels.
Pectins and cellulose
Total uronic acid content differed among onion
lines, but did not change during storage (Fig. 4(A)).
No interactions were present. Average total uronic
acid content ranged from 51 to 54 mg g−1 dry
weight in MBL87-WOPL, followed by MSU4535B
with 42–47 mg g−1 dry weight, and Pegasus, with
36–38 mg g−1 dry weight. There was a significant
interaction between cultivar and storage time affecting
WSP uronic acid concentrations. The interaction
was the result of a significant increase in WSP
uronic acid concentrations between harvest and
4 weeks storage in MSU4535B, while the WSP
uronic acid concentrations in other onion lines
remained unchanged. Second, there was an increase
in WSP uronic acid concentrations in MBL87-WOPL
between 8 and 12 weeks of storage, while the WSP
uronic acid concentration in other cultivars remained
stable. Although there was a significant interaction
between onion line and storage duration for average
WSP uronic acid concentrations, it is important to
note the significant differences in WSP uronic acid
concentrations among the different onion lines at
harvest and during storage. The difference in WSP
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Figure 2. (A) Percentage dry matter content; (B) percentage total
soluble solids (TSS); (C) percentage fresh weight loss from harvest.
Each data point represents the mean (± s.e.) of four replications for
Pegasus and MSU4535B and three replications of MBL87-WOPL
onion (Allium cepa L.) lines measured at harvest, 4, 8 and 12 weeks of
storage.
uronic acid concentrations accounted for much of the
differences observed for total pectin concentrations.
Storage time and cultivar interacted to affect CSP
uronic acid concentrations (Fig. 4(C)). This interaction was the result of some cultivars experiencing
larger changes in storage than others. There was also a
significant decrease in CSP uronic acid concentration
during storage, most noticeably in MBL87-WOPL.
The ACSP uronic acid concentration differed
among onion lines and during storage, though no
interactions were present (Fig. 4(D)). Pegasus and
MBL87-WOPL had the highest concentrations of
1281
TW Coolong, WM Randle, L Wicker
changes in the gross thickness of the cell wall and
middle lamella region were not observed during
storage. Light micrographs of onion cells illustrate
visible differences in cell wall/middle lamella thickness
at harvest for the onions tested (Fig. 6(A–C)).
Changes to the middle lamella region were observed
frequently during storage using TEM (Fig. 7(A–F)).
In many cases the middle lamella appeared to be
disrupted in adjacent cells. Structures resembling
carbohydrate chains were frequently observed in the
middle lamella after cell separation (Fig. 7(B,C)).
Figure 3. Percentage of bulb slices exhibiting sprouting or rooting
after 10 days of incubation measured at harvest, 4, 8, and 12 weeks of
storage. Each data point represents the mean (± SE) of four
replications for Pegasus and MSU4535B and three replications of
MBL87-WOPL onions (Allium cepa L.).
ACSP uronic acid during storage, while MSU4535B
had the least. The ACSP uronic acid concentration
decreased in all onions during storage. The AKSP
uronic acid fraction, composed primarily of hemicellulose, was not affected by onion line or storage time
and averaged 17.3 mg g−1 dry weight for all cultivars.
Cellulose concentrations decreased in all lines during storage, falling by nearly 40% in MBL87-WOPL.
There was also significant interaction between storage time and cultivar for bulb cellulose concentrations
(Fig. 4(E)).
Enzyme analysis
Storage time and onion line interacted to affect
PME activity (Fig. 5(A)). Bulb PME activity varied
widely in Pegasus during storage, resulting in a
cultivar × storage interaction. Bulb PME activity
decreased significantly during storage for MSU4535B
and MBL87-WOPL. Although similar to Pegasus at
harvest, MBL87-WOPL consistently had the lowest
level of PME activity during storage, averaging
between 0.1 to 0.2 units mg−1 protein. Onion type
and storage time also interacted to affect PG activity
(Fig. 5(B)). The interaction was the result of an
increase in PG activity in Pegasus accompanied by
a decrease in PG activity in MSU4535B after 8 weeks
in storage. In addition to the interaction, there were
significant differences in PG activity among onion
lines, with Pegasus and MSU4535B having the highest
activities and MBL87-WOPL having the lowest level
of activity.
Microscopy
Differences in cell wall/middle lamella thickness were
observed in onions at harvest (Table 1). Significant
1282
DISCUSSION
Bulb firmness was highest in MBL87-WOPL, followed
by MSU4535B and Pegasus at harvest. In addition,
MBL87-WOPL remained firm for the first 8 weeks
of storage, whereas both MSU4535B and Pegasus
experienced declines in firmness during the first
8 weeks of storage. Such results prompted the
following questions. What properties at harvest could
be responsible for the differences in bulb firmness, and
what could account for the varying rates of softening
between MBL87-WOPL and Pegasus or MSU4535B
during storage?
Firmness at harvest correlated well with bulb dry
matter content and TSS. The differences in dry
matter content, which may be due differences in
fructan concentration, could result in an increase
in cell water potential and cell turgor, which may
lead to firmer plant tissue and increased postharvest
lift.13,32,33,34 In addition, nearly 90% of weight loss
in onion bulbs during storage has been attributed to
water loss.35 Thus it may be possible that changes in
firmness could be due to water loss and changes in
turgor. However, MBL87-WOPL was the firmest at
harvest and throughout storage, and experienced the
largest percentage of weight loss of cultivars tested
(Fig. 2(C)). The weight loss that occurred between 4
and 8 weeks of storage did not lead to a concomitant
decrease in firmness.
Uronic acid concentrations in pectin correlated well
with bulb firmness levels at harvest. Pectin accounts
for about 30% of the polysaccharides in the primary
cell wall and middle lamella.36 At harvest, MBL87WOPL had the highest total uronic acid concentration
and had the firmest bulbs. Pegasus had the lowest total
uronic acid concentration at harvest and produced the
Table 1. Mean cell wall and middle lamella thickness for three onion
(Allium cepa L.) lines measured at harvest and after 12 weeks in
storage using light microscopy. Each mean represents five individual
bulb replications
Onion
MBL87-WOPL
MSU4535B
Pegasus
Harvest (µm)
12 weeks (µm)
4.19aa
3.07ab
2.29b
3.81a
3.91a
1.97b
a
Different letters in the same column indicate significant differences at
P < 0.05.
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Differences in the cell wall regions in relation to scale firmness in Allium cepa L.
Figure 4. (A) Total uronic acids (UA); (B) uronic acids in water-soluble pectin (WSP); (C) uronic acid in chelator-soluble pectin (CSP); (D) uronic acid
in acid-soluble pectin (ACSP); (E) uronic acids in alkaline-soluble pectin (ASP); (F) cellulose, measured at harvest, 4, 8, and 12 weeks of storage.
Data points represent the mean (± SE) in mg g−1 dry weight (DW) of four replications for Pegasus and MSU4535B and three replications of
MBL87-WOPL onions (Allium cepa L.).
Figure 5. Enzyme activities measured for three cultivars of onion (Allium cepa L.) at harvest, 4, 8, and 12 weeks of storage. Activities of (A) pectin
methylesterase (PME) and (B) polygalacturonase (PG). Each data point represents mean (± SE) in activity units mg−1 protein for four replications of
Pegasus and MSU4535B and three replications of MBL87-WOPL. One activity unit is equivalent to 1 µmol of product produced 60 s−1 from a given
substrate.
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
1283
TW Coolong, WM Randle, L Wicker
Figure 6. Light micrographs illustrating cell walls and middle lamella (CW/ML) regions of three onion (Allium cepa L.) lines at harvest: (A) Pegasus;
(B) MSU4535B; (C) MBL87-WOPL. All micrographs were taken at 200× magnification.
Figure 7. Transmission electron micrographs of onion (Allium cepa L.) parenchyma cell wall (CW) and middle lamella (ML) regions at harvest and
after 12 weeks of storage. (A) MBL87-WOPL at harvest; (B) MBL87-WOPL after 12 weeks of storage; (C) close-up of B, showing carbohydrate
chains between adjacent cells; (D) MSU4535B at harvest; (E) MSU4535B after 12 weeks of storage; (F) MSU4535B at 12 weeks of storage.
Micrographs for A, B, D–F were taken at 7000× magnification (bar = 2 µm). Micrograph for C was taken at 22 000× magnification (bar = 1 µm).
softest bulbs. Much of the difference in total uronic
acid concentrations can be attributed to differences
in WSP uronic acids among the three cultivars.
Although there were significant differences among
onions in concentrations of CSP and ACSP uronic
acids, they were generally minor. The uronic acids
isolated in the WSP fraction are generally shorter
and less branched than the other pectin fractions.11,37
Nonetheless, WSP uronic acids contribute to cellular
adhesion and the strength of the middle lamella. 37
It is possible that differences in WSP uronic acids
at harvest may be responsible for the measured
differences in onion scale firmness. Light microscopy
(Fig. 6(A–C)) and subsequent measurements of the
middle lamella suggest that the structure of the middle
lamella contributed to the observed differences in scale
firmness among the onion lines measured (Table 1).
MBL87-WOPL had the thickest cell wall/middle
lamella, highest concentration of WSP and was
firmest at harvest. Pegasus had the softest scales at
1284
harvest, thinnest cell wall/middle lamella and lowest
concentration of uronic acids in the WSP fraction.
No differences were monitored in cellulose concentrations among the cultivars at harvest. Because
cellulose is the principal structural unit of the primary
cell wall, it would be expected to be most concentrated in the firmest bulbs.38 It has been reported that
higher concentrations of cellulose were detected in a
firm onion cultivar ‘Pukekohe Longkeeper’ than in a
softer ‘Houston Grano’ bulb.39 However, there were
no differences in cellulose concentrations among the
cultivars tested in this experiment. Furthermore, all
cultivars experienced a significant decrease in cellulose
concentrations between harvest and 4 weeks of storage,
but only bulbs from Pegasus and MSU4535B underwent significant softening during the same period. This
suggests that changes in cellulose concentration were
not responsible for loss of firmness during storage.
Firmness in the three onion lines tested in this
experiment declined significantly during storage.
However, Pegasus and MSU4535B had a greater rate
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
Differences in the cell wall regions in relation to scale firmness in Allium cepa L.
of softening in storage than MBL87-WOPL. Results
obtained with TEM suggested that degradation of
the pectin-rich middle lamella during storage was
common. As such, the pectin-modifying enzymes
PME and PG were measured.
There was significant variation in the level of
PME and PG activity in Pegasus and MSU4535B
bulbs during storage, resulting in a significant storage
duration × variety interaction. However, the level of
PME and PG activity in these two lines was generally
higher than in MBL87-WOPL during storage. The
level of PME and PG activity in MBL87-WOPL
was relatively stable and low for the duration of the
experiment. The combined actions of PME and PG
in plants results in the degradation of galacturonic
acid polymers, resulting in cell separation along the
middle lamella.40 Thus, the higher levels of PME
and PG activity in Pegasus and MSU4535B could
have led to a decrease in cellular adhesion, resulting
in the rapid softening that occurred in these bulbs.
Previously, PG activity was linked to disruption in
cellular adhesion in roots of A. porrum.17 Although it
is clear that many enzymes are involved in softening
of fruits and vegetables, our data suggest that PME
and PG activity during storage may be related to rate
of softening in onion bulbs.18,19 Comparisons with
rooting and sprouting indices suggest that changes in
dormancy were not directly related to PME and PG
activity.
Decreases in onion scale firmness during storage
also appeared to be related to the integrity of the middle lamella. Previous results obtained using an environmental scanning electron microscope demonstrated
that onion cells, mechanically separated using a specially designed tensometer, fractured within the middle
lamella material and not at the middle lamella cell wall
interface.41 Using TEM, changes were observed in the
structure of the middle lamella during storage. After
12 weeks of storage, disruption of the middle lamellar
region was frequently observed (Fig. 7). In some samples structures resembling carbohydrate chains were
observed between adjacent cells. Since TEM was only
performed on samples at harvest and after 12 weeks in
storage we cannot speculate on the rate of degradation
observed during storage. However, the results demonstrate that degradation of the middle lamella during
storage was common in the onions tested.
Our results indicate that uronic acid concentration
in the WSP may be a factor affecting bulb firmness
in the onions analyzed. Pegasus had the lowest
concentration of uronic acids in the WSP and the
softest bulbs at harvest, while MBL87-WOPL had
the highest concentration of WSP uronic acids and
were the firmest bulbs at harvest. Furthermore, light
microscopy of onion cells indicated large differences in
cell wall/middle lamella region, with MBL87-WOPL
having the thickest cell wall/middle lamella region
and Pegasus the thinnest. In addition, degradation
of the middle lamella during storage appeared to be
relatively common. To our knowledge this is the first
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa
attempt to correlate bulb softening to changes in pectin
and cellulose concentrations as well as PME and PG
activity in onion during storage. The results obtained
here should provide researchers with more insight
regarding what factors affect bulb texture at harvest
and during storage.
ACKNOWLEDGEMENTS
We would like to thank Dr Michael Havey for
graciously providing seeds for MSU4535B and Ms
Beth Richardson for her expertise in microscopy.
REFERENCES
1 Randle WM and Lancaster JE, Sulphur compounds in alliums
in relation to flavour quality, in Allium Crop Science: Recent
Advances, ed. by Rabinowitch HD and Currah L. CABI,
Wallingford, UK, pp. 329–356 (2002).
2 Waldron KW, Parker ML and Smith AC, Plant cell walls and
food quality. Comp Rev Food Sci Food Safety 2:101–119
(2003).
3 Rosenthal AJ, Relation between instrumental and sensory
measures of food texture, in Food Texture Measurement and
Perception, ed. by Rosenthal AJ. Aspen, Gaithersburg, MD,
pp. 1–17 (1999).
4 Edwards M, Vegetables and fruit, in Food Texture Measurement
and Perception, ed. by Rosenthal AJ. Aspen, Gaithersburg,
MD, pp. 259–281 (1999).
5 DeEll JR, Khanizadeh S, Saad F and Ferree DC, Factors
affecting apple fruit firmness: a review. J Am Pomol Soc
55:8–27 (2001).
6 Waldron KW, Smith AC, Parr AJ, Ng A and Parker ML, New
approaches to understanding and controlling cell separation
in relation to fruit and vegetable texture. Trends Food Sci
Technol 8:213–221 (1997).
7 Van Buren JP, The chemistry of texture in fruits and vegetables.
J Texture Stud 10:1–23 (1979).
8 Matsuura Y, Matsubara K and Fuchigami M, Molecular composition of onion pectic acid. J Food Sci 65:1160–1163
(2000).
9 Ng A, Smith AC and Waldron KW, Effect of tissue type and
variety on cell wall chemistry of onion (Allium cepa L.). Food
Chem 63:17–24 (1998).
10 Ng A, Parker ML, Parr AJ, Saunders PK, Smith AC and
Waldron KW, Physiochemical characteristics of onion (Allium
cepa L.) tissues. J Agric Food Chem 48:5612–5617 (2000).
11 Redgwell RJ and Selvedran RR, Structural features of cellwall polysaccharides of onion Allium cepa. Carbohydr Res
157:183–199 (1986).
12 Coolong TW and Randle WM, The effects of calcium chloride
and ammonium sulfate on onion (Allium cepa L.) bulb quality
at harvest and during storage. HortScience 43:465–471 (2008).
13 Gomez-Galindo F, Herppich W, Gekas V and Sjoholm I, Factors affecting quality and postharvest properties of vegetables:
integration of water relations and metabolism. Crit Rev Food
Sci Nutr 44:139–154 (2004).
14 Micheli F, Pectin methylesterases: cell wall enzymes with
important roles in plant physiology. Trends Plant Sci
6:414–419 (2001).
15 Kim JC, Firmness of thermal processed onion as affected by
blanching. J Food Process Preserv 30:659–669 (2006).
16 Garcia E, Alviar-Agnew M and Barrett DM, Residual pectinesterase activity in dehydrated onion and garlic products. J
Food Process Preserv 26:11–26 (2002).
17 Peretto R, Favaron F, Bettini V, Delorenzo G, Marini S,
Alghisi P, et al, Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum
L. Planta 188:164–172 (1992).
1285
TW Coolong, WM Randle, L Wicker
18 Brummell DA, Cell wall disassembly in ripening fruit. Funct
Plant Biol 33:103–119 (2006).
19 Brummell DA and Harpster MH, Cell wall metabolism in fruit
softening and quality and its manipulation in transgenic
plants. Plant Mol Biol 47:311–340 (2004).
20 Smith CJS, Watson CF, Morris PC, Bird CR, Seymour GB,
Gray JE, et al, Inheritance and effect on ripening of antisense
polygalacturonase genes in transgenic tomatoes. Plant Mol
Biol 14:369–379 (1990).
21 Hoagland DR and Arnon DI, The water culture method for
growing plants without soil. Calif Agric Expt Stn Circ 347
(1950).
22 Huber DJ and Lee JH, Comparative analysis of pectins from
pericarp and locular gel in developing tomato fruit, in
Chemistry and Function of Pectins, ed. by Fishman ML
and Jens JJ. American Chemical Society, Washington, DC,
pp. 141–156 (1986).
23 De Vries JA, Voragen AGJ, Rombouts FM and Pilnik W,
Extraction and purification of pectins from alcohol insoluble
solids from rip and unripe apples. Carbohydr Polym 1:117–127
(1981).
24 Blumenkrantz N and Asboe-Hansen G, New method for
quantitative determination of uronic acids. Anal Biochem
54:484–489 (1973).
25 Ahmed AER and Labavitch JM, A simplified method for
accurate determination of cell wall uronide content. J Food
Biochem 1:361–365 (1977).
26 Updegraff DM, Semimicro determination of cellulose in
biological materials. Anal Biochem 32:420–424 (1969).
27 Gross KC, A rapid and sensitive spectrophotometric method
for assaying polygalacturonase using 2-cyanoaceamide.
HortScience 17:933–934 (1982).
28 Banjongsinsiri P, Kenney S and Wicker L, Texture and distribution of pectic substances of mango as affected by infusion of pectinmethylesterase and calcium. J Sci Food Agric
84:1493–1499 (2004).
29 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,
Provenzano MD, et al, Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 (1985).
1286
30 Dawson RMC, Elliot DC, Elliot WH and Jones KM, Data for
Biochemical Research (3rd edn). Oxford University Press,
Oxford (1989).
31 Spurr AR, A low viscosity epoxy resin embedding medium for
electron microscopy. J Ultrastruct Res 26:31–43 (1969).
32 Kopsell DE and Randle WM, Onion cultivars differ in pungency
and bulb quality changes during storage. HortScience
32:1260–1263 (1997).
33 Van Laere A and Van Den Ende W, Inulin metabolism in dicots:
chicory as a model system. Plant Cell Environ 25:803–813
(2002).
34 Rutherford PP and Whittle R, Methods of predicting the longterm storage of onions. J Hortic Sci 59:349–356 (1984).
35 Komochi S, Bulb dormancy and storage physiology, in Onions
and Allied Crops, Vol. 1, ed. by Rabinowitch HD and
Brewster JL. CRC Press, Boca Raton FL, pp. 89–111 (1990).
36 Willats WGT, McCartney L, Mackie W and Knox JP, Pectin:
cell biology and prospects for functional analysis. Plant Mol
Biol 47:9–27 (2001).
37 Ridley BL, O’Neill MA and Mohnen D, Pectins: structure,
biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57:929–967 (2001).
38 Carpita NC and Gilbeaut DM, Structural models of primary cell
walls in flowering plants: consistency of molecular structure
with the physical properties of the walls during growth. Plant
J 3:1–30 (1993).
39 O’Donoghue EM, Somerfield SD, Shaw M, Bendall M, Hedderly D, Eason J, et al, Evaluation of carbohydrates in
Pukekohe Longkeeper and Grano cultivars of Allium cepa.
J Agric Food Chem 52:5383–5390 (2004).
40 Jarvis MC, Briggs SPH and Knox JP, Intercellular adhesion
and cell separation in plants. Plant Cell Environ 26:977–989
(2003).
41 Donald AM, Baker FS, Smith AC and Waldron KW, Fracture
of plant tissues and walls as visualized by environmental
scanning electron microscopy. Ann Bot 92:73–77 (2003).
J Sci Food Agric 88:1277–1286 (2008)
DOI: 10.1002/jsfa