Article

Chlorophyll degradation and resulting catabolite formation in stored Japanese bunching onion (Allium fistulosum L.)

Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
Journal of the Science of Food and Agriculture (Impact Factor: 1.71). 08/2008; 88(11):1981 - 1986. DOI: 10.1002/jsfa.3307

ABSTRACT

BACKGROUND: Loss of green colour is the main factor in quality deterioration of stored Japanese bunching onion (JBO, Allium fistulosum L.) leaves. Elucidation of chlorophyll (Chl) degradation has not been performed to date. In the present study, Chl catabolism and Chl-degrading enzyme activities in stored JBO leaves were investigated.RESULTS: Green leaves of JBO stored at 25 °C turned yellow within 3 days, whereas no changes occurred at 4 °C. Pheophytin (Phy) a, chlorophyllide (Chlide) a, pheophorbide (Pheide) a and C132-hydroxychlorophyll (OHChl) a were the main catabolites of Chl a and diminished at 25 °C concomitant with leaf yellowing, whereas no significant reductions were observed at 4 °C. The activities of Chl-degrading peroxidase and Mg-dechelation increased significantly at 25 °C but not at 4 °C.CONCLUSION: Chl degradation was accelerated and catabolite levels (mainly Phy a, Chlide a, Pheide a and OHChl a) were diminished in JBO leaves stored at 25 °C, suggesting that the catabolites are rapidly converted into their following forms. Activities of Mg-dechelation and Chl-degrading peroxidase were significantly enhanced during storage at 25 °C, indicating that these two enzymes have major roles in Chl degradation in stored JBO leaves. Copyright © 2008 Society of Chemical Industry

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Available from: Prasajith Kapila Dissanayake, Oct 25, 2015
Journal of the Science of Food and Agriculture J Sci Food Agric 88:19811986 (2008)
Chlorophyll degradation and resulting
catabolite formation in stored Japanese
bunching onion (Allium fistulosum L.)
Prasajith K Dissanayake,
1
Naoki Yamauchi
1,2
and Masayoshi Shigyo
1,2
1
The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
2
Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
Abstract
BACKGROUND: Loss of green colour is the main factor in quality deterioration of stored Japanese bunching onion
(JBO, Allium fistulosum L.) leaves. Elucidatio n of chlorophyll (Chl) degradation has not been performed to date.
In the present study, Chl catabolism and Chl-degrading enzyme activities in stored JBO leaves were investigated.
RESULTS: Green leaves of JBO stored at 25
C turned yellow within 3 days, whereas no changes occurred at 4
C.
Pheophytin (Phy) a, chlorophyllide (Chlide) a, pheophorbide (Pheide) a and C13
2
-hydroxychlorophyll (OHChl) a
were the main catabolites of Chl a and diminished at 25
C concomitant with leaf yellowing, whereas no significant
reductions were observed at 4
C. The activities of Chl-degrading peroxidase and Mg-dechelation increased
significantly at 25
C but not at 4
C.
CONCLUSION: Chl degradation was accelerated and catabolite levels (mainly Phy a,Chlidea,Pheidea and
OHChl a) were diminished in JBO leaves stored at 25
C, suggesting that the catabolites are rapidly converted
into their following forms. Activities of Mg-dechelation and Chl-degrading peroxidase were significantly enhanced
during storage at 25
C, indicating that these two enzymes have major roles in Chl degradation in stored J BO
leaves.
2008 Society of Chemical Industry
Keywords: chlorophyll degradation; Japanese bunching onion; chlorophyll catabolites; chlorophyll-degrading
enzymes
INTRODUCTION
The Japanese bunching onion (JBO, Allium fistulosum
L.), also referred to as Welsh onion or green onion,
belongs to the family Alliaceae and is of major
economic importance as a leafy vegetable in most
East Asian countries, particularly in Japan, China
and Korea.
1,2
Itcanberankedinninthplaceamong
vegetable crops produced in Japan.
3
The green leaves
and etiolated leaf sheaths are widely used in Japanese
dishes.
3
However, yellowing, i.e. loss of green colour,
which is the most significant attribute during the
buying stage, is one of the main factors in quality
deterioration of stored JBO and other leafy vegetables.
The highly perishable nature of leafy vegetables such
as spinach, parsley, leek and JBO means that they
have relatively short shelf-lives in terms of external
appearance as well as other quality parameters such as
microbial growth and nutritional content. The storage
life of JBO at 5
C is only about 1 week and high
temperatures cause more rapid yellowing and decay of
the leaves. Utilisation of very-low-temperature storage
(close to 0
C) and controlled atmospheric conditions
could extend the shelf-life by maintaining the green
colour of spinach, parsley and JBO.
4
The main causal
factor of quality deterioration or discolouration of
stored horticultural crops, especially green vegetables
such as broccoli, spinach, parsley and green beans, is
chlorophyll (Chl) breakdown.
5–8
However, for JBO
there have been no attempts at the elucidation of Chl
degradation as with other leafy vegetables.
Catabolism of Chl is a complex process taking place
in plants throughout their life-cycle and is particularly
prominent in the senescence and ripening stages of
fruits. The pathway for Chl breakdown consists of
several steps which have been elucidated in the last
few years.
9,10
In the first step of Chl degradation, Chl
a is degraded into chlorophyllide (Chlide) a by the
activity of chlorophyllase.
11,12
Elimination of Mg from
Chlide a to produce pheophorbide (Pheide) a,ina
reaction catalysed by Mg-dechelatase, is considered as
the second step.
13,14
In addition to that, instead of
removal of Mg from Chlide a,Tanget al.
15
showed a
direct removal of Mg from Chl a to form pheophytin
(Phy) a by Mg-dechelatase in Ginkgo biloba leaves.
Finally, Pheide a is decomposed to fluorescent Chl
catabolites, which are primarily colourless catabolites,
Correspondence to: Naoki Yamauchi, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
E-mail: yamauchi@yamaguchi-u.ac.jp
(Received 8 February 2008; revised version received 2 May 2008; accepted 4 May 2008)
Published online 14 July 2008
; DOI: 10.1002/jsfa.3307
2008 Society of Chemical Industry. J Sci Food Agric 00225142/2008/$30.00
Page 1
PK Dissanayake, N Yamauchi, M Shigyo
via red Chl catabolites, by both Pheide a oxygenase
and red Chl catabolite reductase.
10
These reactions,
which are thought to form the main pathway of Chl a
degradation, occur in the chloroplast.
10
However, the
degradation pathway of Chls remains to be clarified,
compared with the biosynthetic pathway of Chls.
On the other hand, peroxidase
16
and/or Chl
oxidase
17
are also reported to be involved in in vitro
Chl a oxidation to form C13
2
-hydroxychlorophyll
(OHChl) a. Further, findings of Yamauchi and
Watada
18
and Yamauchi et al.
19
also imply that Chl-
degrading peroxidase is involved in Chl degradation.
The aim of the present study was to investigate Chl
catabolism in JBO leaves during storage by analyses of
Chl a catabolite formation and Chl-degrading enzyme
activities.
MATERIALS A ND METHODS
Plant materials
The JBO (A. fistulosum L., cv. ‘Kujyo-hoso’) plants
used in this study were grown in an experimental
field with silver mulching at the Experimental Farm
of Yamaguchi University. A compound fertiliser was
applied before planting. The total amounts of the
three major nutrients in the basal dressing were 100 kg
N (as ammonium sulphate), 120 kg P (as calcium
superphosphate) and 100 kg K (as potassium chloride)
ha
1
. An additional fertiliser mixture, 65N:6P:19K
(80 kg ha
1
), was applied at weekly intervals with
irrigation to each plant to ensure uniform growth.
Healthy mature leaf blade tissues of the same growth
stage were harvested by cutting from the base of a
tiller in summer (July and August). All leaves were
thoroughly rinsed in flowing tap water to eliminate
any impurities on their surfaces and well dried using
moisture-absorbing paper. Green leaves of 20 cm
length were placed in perforated polyethylene bags
(0.03 mm thickness, 38 cm × 26.5 cm, with two 6 mm
holes) and stored at 25 or 4
C for 3 days in the dark.
Leaves were cut to 8.5 cm length for chemical analysis.
All experiments were replicated three times with two
leaves per replicate.
Determination of leaf surface colour and
chlorophyll content
The surface colour of leaves was evaluated by
measuring the hue angle (h
)
20
in an area 5 7 cm
from the leaf tip using a colorimeter (NF 777, Nippon
Denshoku, Tokyo, Japan), since the area closer to
the leaf tip was too slender (less than 1 cm) to
measure h
with the colorimeter. Chl content was
determined spectrophotometrically (U-2001, Hitachi,
Tokyo, Japan) using N,N-dimethylformamide.
21
High-performance liquid chromatography
analysis of chlorophyll catabolites
A 1 g portion of leaf tissue was homogenised in
an extraction mixture containing 8 mL of acetone
and 1 mL of 50 mmol L
1
4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES, pH 7.5) using
a mortar and pestle kept on ice. After homogenisation
the mixture was kept on ice for a further 5 min to allow
all pigments to absorb into the extraction solution.
Extracts were filtered first using Advantec #2 filter
paper (Advantec, Tokyo, Japan) and then through
a0.45
µm DISMIC filter (Advantec) prior to being
analysed for Chl a catabolites by high-performance
liquid chromatography (HPLC).
Pigments were separated in an HPLC system
(Hitachi, Tokyo, Japan) equipped with an L-
2130 pump with an automated gradient controller
(Hitachi) and an L-7420 UVvisible spectropho-
tometer (Hitachi) using a LiChrospher 100 RP-
18 column (250 mm × 4 mm; Cica Reagent, Tokyo,
Japan) and two solvents. Solvent A was 80:20 (v/v)
methanol/water and solvent B was ethyl acetate. Sol-
vent A was added to solvent B at a linear rate over
20 min until a 50:50 (v/v) mixture was attained. The
50:50 (v/v) mixture was kept isocratic for an additional
20 min. The flow rate was 1 mL min
1
and the sample
injection volume was 100
µL. The absorption spec-
trum of the pigments was recorded at 665 nm.
22
The
identification of Chl a and its catabolites was based on
the retention time and the visible absorption spectra.
Preparation of chlorophyll a and its catabolites
Chls were extracted from spinach (Spinacia oleracea
L.) with acetone, and Chls a and b were partially
purified by adding 1,4-dioxane and distilled water
to the acetone extract. The mixture was then
allowed to stand until a precipitate formed. The
mixture was subsequently centrifuged at 10 500 × g
for 15 min at 4
C and the precipitate was dissolved
in 40 mL of acetone, 6 mL of 1,4-dioxane and 14 mL
of distilled water. The mixture was again allowed
to stand until a precipitate formed and was then
centrifuged at 10 500 × g for 15 min. The precipitate
was dissolved in 10 mL of petroleum ether. Chl a
was separated by sucrose column chromatography
according to the method of Perkins and Roberts.
23
Chlide a was prepared by using a partially purified
chlorophyllase obtained by (NH
4
)
2
SO
4
precipitation
(20 40% saturation) from acetone powder of Citrus
unshiu peel, which has high chlorophyllase activity.
Phy a was prepared by adding two or three drops of
0.1 mol L
1
HCltoChla acetone solution. Pheide a
and pyropheophobide a were purchased from Wako
Pure Chemical Industries (Tokyo, Japan), and Tama
Biochemical (Tokyo, Japan) respectively.
Assay for chlorophyll catabolite formation
A 5 g portion of leaf tissue was homogenised in 10 mL
of extraction mixture containing 10 g L
1
Triton X-
100 and 100 mmol L
1
phosphate buffer (pH 7)
using mortar and pestle kept on ice. The extract was
filtered using two layers of Miracloth (Calbiochem,
San Diego, CA, USA). Tissue debris was precipitated
by centrifugation at 16 000 × g for 15 min at 4
C. A
1982 J Sci Food Agric 88:1981 1986 (2008)
DOI: 10.1002/jsfa
Page 2
Chlorophyll degradation in stored Japanese bunching onion
7 mL aliquot of supernatant was mixed with 0.7 mL
of Chl a (500
µgmL
1
) and incubated in a water bath
at 25
C in the dark for 0, 3 or 6 h. The reaction was
stopped by adding 2 mL of cold acetone to 0.5 mL
of extraction mixture after 0, 3 or 6 h. This mixture
was then filtered through a 0.45
µmDISMICfilter
(Advantec) and used for Chl a derivative analysis by
HPLC.
Chlorophyll-degrading enzyme assays
A 300 mg portion of leaf acetone powder was stirred for
1hat0
C in 7.5 mL of 10 mmol L
1
phosphate buffer
(pH 7) containing 50 mmol L
1
KCl and 1.2 g L
1
Triton X-100 for Chl-degrading peroxidase and Mg-
dechelatase assays. For chlorophyllase assay, 10 mmol
L
1
phosphate buffer (pH 7) containing 6 g L
1
3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesul-
fonate (CHAPS) was used. Subsequently, the extract
was filtered through two layers of Miracloth (Cal-
biochem) and the filtrate was centrifuged at 16 000 × g
for 15 min at 4
C. The supernatant was used as the
crude enzyme extract.
Chlorophyllase activity was determined by a
modification of the method of Amir-Shapira et al.
12
The reaction mixture contained 0.5 mL of crude
enzyme solution, 0.1 mL of 10 g L
1
CHAPS, 0.2 mL
of Chl a acetone solution (100
µgChla)and0.5mL
of 0.1 mol L
1
phosphate buffer (pH 8). The mixture
was incubated in a water bath at 25
C for 1 h and
the enzymatic reaction was stopped by adding 4 mL
of acetone. The remaining (non-degraded) Chl a was
extracted with 4 mL of hexane. Chlide a formed in
the acetone layer was assayed spectrophotometrically
(U-2001, Hitachian) by reading the absorbance at
667 nm. The activity was based on the increase in
absorbance by Chlide a at 667 nm.
24
Mg-dechelation activity was determined in two
ways. One was a modification of the method of
Suzuki and Shioi.
25
The reaction mixture contained
0.2 mL of crude enzyme solution, 0.3 mL of 98 nmol
L
1
chlorophyllin (Chlin) a and 0.75mL of 50mmol
L
1
Tris-HCl buffer (pH 7). Activity was measured
at 35
C by following the change in optical density
(OD) at 686 nm.
14
Chlin a was prepared according
to the procedure of Vicentini et al.
14
with slight
modifications. A 30 mL aliquot of Chl a acetone
solution was partitioned into 20 mL of petroleum
ether. After washing three times with 20 mL of distilled
water, the ether phase was separated and mixed with
2.5 mL of 300 g L
1
KOH in methanol to form Chlin
a. The solution in which Chlin a was allowed to
precipitate was centrifuged at 16 000 × g for 15 min at
4
C. The precipitate was brought to pH 9 by adding
2 mol L
1
tricine. In the other method the reaction
mixture contained 0.25 mL of 30
µmol L
1
Chlide a,
0.75 mL of 10 mmol L
1
phosphate buffer (pH 7)
and 0.2 mL of crude enzyme solution. Activity was
measured at 35
C by following the change in OD at
535 nm.
26
.
Chl-degrading peroxidase was determined as
described by Yamauchi et al.
27
The reaction mixture
contained 0.2 mL of Chl a acetone solution (100
µg
Chl a), 0.1 mL of 10 g L
1
Triton X-100, 0.1 mL of
5mmol L
1
p-coumaric acid, 1.5 mL of 0.1 mol L
1
citrate/0.2 mol L
1
phosphate buffer (pH 4.5), 0.2 mL
of enzyme solution and 0.1 mL of 3 g L
1
hydrogen
peroxide. Activity was determined spectrophotometri-
cally by measuring the decrease in Chl a at 668 nm at
25
C.
27
One unit of chlorophyllase was defined as a change
of 1
µg Chlide a formation min
1
, while one unit of
Chl-degrading peroxidase was defined as a change
of 1
µgChla degradation min
1
. Mg-dechelatase
activity was expressed as the increase in absorbance at
686 nm (OD
686 nm
)min
1
. Enzyme protein content
was assayed by the method of Bradford.
28
RESULTS AND DISCUSSION
Chlorophyll degradation
Green leaves of JBO stored at 25
C turned progres-
sively yellow starting from the leaf tip towards the base
during 3 days of storage, whereas leaves stored at 4
C
maintained their green colour apart from slight yel-
lowing at the tip. Leaf surface colour evaluated by h
was also significantly reduced in leaves stored at 25
C
in comparison with leaves stored at 4
C, in which no
significant changes in h
were observed (Fig. 1). Chl
a and b contents also diminished concomitantly with
surface colour reduction, but at a higher rate than the
h
change (Fig. 2). The rate of reduction of Chl a
in leaves stored at 25
C was significantly higher than
that in leaves stored at 4
C. The reduction of Chl b
followed a similar pattern.
The marketability of fresh JBO depends mainly on
the surface appearance of the leaves, especially their
green colour. Discolouration of the leaf surface during
storage naturally detracts from consumer preference.
The changes in h
of leaves stored at 25
Cwere
very large and similar to the pattern of Chl reduction
(Figs 1 and 2). Leaves stored at 4
C also showed
similar trends of change in h
and reduction of Chl
contents. These results suggest that the degradation
Figure 1. Changes in hue angle of Japanese bunching onion leaves
duringstorageat25and4
C. Vertical bars represent average values
with standard deviation (n = 3).
J Sci Food Agric 88:1981 1986 (2008) 1983
DOI: 10.1002/jsfa
Page 3
PK Dissanayake, N Yamauchi, M Shigyo
Figure 2. Changes in chlorophyll a and b contents of Japanese
bunching onion leaves during storage at 25 and 4
C. Vertical bars
represent average values with standard deviation (n = 3).
of Chls is the main causal factor of discolouration of
stored JBO. Similarly, in most leafy vegetables such as
parsley and spinach
6,7
and other green vegetables such
as broccoli
19
and green beans,
8
surface de-greening is
a result of Chl breakdown.
Formation of chlorophyll a catabolites
Phy a, Chlide a, Pheide a and OHChl a were detected
as the main catabolites of Chl a by HPLC analysis.
However, the levels of individual catabolites in the
leaves were very different. The level of Chlide a was
25% of that of Phy a at day 0, while the level of Pheide
a was 2.5% of that of Chlide a at day 0 (Fig. 3). The
level of Phy a in leaves stored at 4
C showed almost
no change over 3 days, whereas it decreased after the
first 2 days of storage at 25
C. The level of Chlide a at
4
C showed no differences between day 0 and day 3,
but at 25
C it diminished significantly during storage.
The level of Pheide a was unchanged during storage at
4
C, while it fell drastically on day 3 of storage at 25
C
following maintenance at the same level between days
0 and 2. In contrast, levels of OHChl a under both
storage conditions showed a diminishing trend during
storage, though the reduction was not significant at
4
C. The high content of Phy a indicated that it was
the main Chl catabolite in JBO leaves. However, there
has been no previous record of the formation of Phy
a as a main catabolite in leafy vegetables. Although
there was a mention of Phy a formation in stored
parsley leaves by Amir-Shapira et al.,
12
Yamauchi and
Watada
7
found no formation of Phy a in stored parsley
leaves. Tang et al.,
15
however, showed that Phy a was
the only catabolite in yellowing leaves of G. biloba.
Furthermore, Phy a accumulation in Langra mango
fruit has also been recorded.
29
The formation of Phy
a in stored leaves is still controversial, as it is not
described in the main pathway of Chl degradation.
30,31
Therefore further investigation of the formation of Phy
a in stored JBO leaves is necessary.
In almost all leafy vegetables, especially in spinach,
6
parsley,
7
broccoli
18
and radish cotyledons,
32
levels of
OHChl a show a trend of reduction during storage
similar to that found in JBO. Levels of Chlide a in
parsley, spinach and radish cotyledons are reduced
during storage after a slight increment, whereas a
continuous reduction of Chlide a in JBO during
storage at 25
C was observed. In general, all Chl a
catabolites in JBO diminished concomitantly with leaf
yellowing during 3 days of storage at 25
C, whereas
no significant reductions were recorded at 4
C.
Figure 3. Formation of chlorophyll a catabolites in Japanese bunching onion leaves during storage at 25 and 4
C. Vertical bars represent average
values with standard deviation (n = 3).
1984 J Sci Food Agric 88:1981 1986 (2008)
DOI: 10.1002/jsfa
Page 4
Chlorophyll degradation in stored Japanese bunching onion
Chlorophyll-degrading enzyme activities during
storage
On average, the activity of chlorophyllase showed
slight increases during storage at 25 and 4
C, but
no significant differences between the two storage
conditions were observed (data not shown). The
activity of Chl-degrading peroxidase at 25
Cincreased
significantly on day 2 of storage and remained at the
same level on day 3, while at 4
C it did not show any
significant changes during storage (Fig. 4).
Mg-dechelation activity was determined by two
different methods, one using Chlin a,anarticial
substrate, and the other using Chlide a,anative
substrate. Figure 5 shows the changes in Mg-
dechelation activity detected by the method using
Chlin a as substrate The activity of Mg-dechelation
in leaves stored at 25
C increased greatly from day
1 to day 3, while there were no differences in Mg-
dechelation activity in leaves stored at 4
C. Using the
method with Chlide a as substrate, the activity also
showed the same trends for the 25 and 4
Cstorage
conditions; the activity at 25
C was higher than that
at 4
C on day 3 (data not shown).
In different plant species, the activity of chlorophyl-
lase differs during senescence. In spinach
6
and barley
33
the activity of chlorophyllase is enhanced during senes-
cence, while decreases in chlorophyllase activity have
been found in green beans (Phaseolus vulgaris)
34
and
Figure 4. Chlorophyll-degrading peroxidase activity in J apanese
bunching onion leaves during storage at 25 and 4
C. Vertical bars
represent average values with standard deviation (n = 3).
Figure 5. Mg-dechelation activity in Japanese bunching onion leaves
duringstorageat25and4
C. Chlorophyllin a was used as substrate.
Vertical bars represent average values with standard deviation (n = 3).
broccoli florets (Brassica oleracea).
35
In JBO, despite
the slight increment in chlorophyllase activity with leaf
yellowing during storage, there were no differences in
activity between the two storage conditions. However,
the significant increase in Chl-degrading peroxidase
activity in JBO leaves at 25
C was concomitant with
the yellowing of leaf blades during storage. At 4
C,
there was neither a significant decrease in Chl a nor an
increase in Chl-degrading peroxidase activity during
storage. In most crops, including horticultural crops,
Chl declines during senescence concomitantly with an
elevation of peroxidase activity.
6,36,37
These findings
suggest that the activity of peroxidase has a significant
role in Chl degradation in JBO leaves during storage.
The increase in Mg-dechelation activity with leaf
yellowing at 25
C indicated that Mg-dechelation was
involved in the yellowing of JBO leaves by catalysing
the degradation of Chlide a to Pheide a during
storage. Furthermore, an increase in Mg-dechelation
activity was also reported in Chl degradation during
the ripening of strawberry fruits.
38
In contrast, a
decrease in Mg-dechelation activity was found in
oilseed rape cotyledons,
14
indicating that there are
differences in the behaviour of Mg-dechelation activity
among plants during senescence. Two types of Mg-
dechelation activity have been distinguished, one
associated with a heat-stable low-molecular-weight
compound known as a metal-chelating substance
(MCS), the other catalysed by an enzyme protein.
30,39
The Mg-dechelating protein acts only on the artificial
substrate Chlin a but not on the native substrate
Chlide a, whereas the MCS removes Mg from both
substrates.
25,26
Therefore these findings suggest that
an MCS might also be involved in removing Mg from
Chlide a in JBO.
Moreover, Tang et al.
15
showed the direct involve-
ment of Mg-dechelation in converting Chl a to Phy
a in yellowing G. biloba leaves, whereas Shioi et al.
39
reported that the release of Mg occurred only from
the de-phytylated compound, Chlide a. However, in
the present study the formation of Phy a was observed
during storage, suggesting that Mg-dechelation might
act directly on the removal of Mg from Chl a to form
Phy a in JBO leaves. Further study is necessary to
characterise the MCS in JBO leaves.
CONCLUSION
The present study showed that Chl degradation was
accelerated in JBO leaves stored at 25
C. Together
with diminishing Chl a, the levels of catabolites
that resulted during Chl degradation in JBO, mainly
Phy a, Chlide a, Pheide a and OHChl a, also
decreased during 3 days of storage at 25
C, suggesting
that the catabolites are rapidly converted into their
following forms. Activities of Mg-dechelation and
Chl-degrading peroxidase were significantly enhanced
during storage at 25
C, indicating that these two
enzymes have major roles in Chl degradation in stored
JBO leaves.
J Sci Food Agric 88:1981 1986 (2008) 1985
DOI: 10.1002/jsfa
Page 5
PK Dissanayake, N Yamauchi, M Shigyo
ACKNOWLEDGEMENTS
We thank Dr Cindy BS Tong (University of
Minnesota) for her gracious and critical reading of
the manuscript. This work was supported in part by
‘Knowledge Cluster Initiative’, MEXT.
REFERENCES
1 Kumazawa S and Katsumata H, Negi [Japanese bunching
onion], in Sosai-Engei Kakuron [Vegetable Crops],ed.by
Kumazawa S. Yokendo Press, Tokyo, pp. 280289 (1965).
(in Japanese).
2 Ford-Lloyd BV and Armstrong SJ, Welsh onion Allium fistu-
losum L., in Genetic Improvement of Vegetable Crops,ed.by
Kalloo G and Bergh BO. Pergamon, London, pp. 51 58
(1993).
3 Japanese Society for Horticultural Science, Horticulture in Japan
2006. Nakanishi Printing Co., Kyoto (2006).
4 Hardenburg RE, Watada AE and Wang CY, The commercial
storage of fruits, vegetables, and florist and nursery stocks.
USDA Agric Handbook 66:6470 (1986).
5 Costa ML, Civello PM, Chaves AR and Mart
´
ınez GA, Effect of
hot air treatments on senescence and quality parameters of
harvested broccoli (Brassica oleracea L var italica)heads.JSci
Food Agric 85:11541160 (2005).
6 Yamauchi N and Watada AE, Regulated chlorophyll degrada-
tion in spinach leaves during storage. JAmSocHortSci
116:5862 (1991).
7 Yamauchi N and Watada AE, Pigment changes in parsley
leaves during storage in controlled or ethylene containing
atmosphere. J Food Sci 58:616618,637 (1993).
8 Monreal M, Ancos BD and Cano MP, Influence of critical
storage temperatures on degradative pathways of pigments
in green beans (Phaseolus vulgaris cvs. Perona and Boby).
J Agric Food Chem 47:1924 (1999).
9H
¨
ortensteiner S, Chlorophyll breakdown in higher plants and
algae. Cell Mol Life Sci 56:330347 (1999).
10 Matile P, H
¨
ortensteiner S and Thomas H, Chlorophyll degrada-
tion. Annu Rev Plant Physiol Plant Mol Biol 50:6795 (1999).
11 Holden M, The breakdown of chlorophyll by chlorophyllase.
Biochem J 78:359 364 (1961).
12 Amir-Shapira D, Goldschmidt EE and Altman A, Chlorophyll
catabolism in senescing plant tissues: in vivo breakdown
intermediates suggest different degradative pathways for
Citrus fruit and parsley leaves. Proc Natl Acad Sci USA
84:19011905 (1987).
13 Langmeier M, Ginsburg S and Matile P, Chlorophyll break-
down in senescent leaves: demonstration of Mg-dechelatase
activity. Physiol Plant 89:347 353 (1993).
14 Vicentini F, Iten F and Matile P, Development of an assay
for Mg-dechelatase of oilseed rape cotyledons, using
chlorophyllin as the substrate. Physiol Plant 94:57 63 (1995).
15 Tang L, Okazawa A, Fukusaki E and Kobayashi A, Removal
of magnesium by Mg-dechelatase is a major step in the
chlorophyll-degrading pathway in Ginkgo biloba in the process
of autumnal tints. ZNaturforschC55:923926 (2000).
16 Yamauchi N and Minamide T, Chlorophyll degradation by
peroxidase in parsley leaves. JJpnSocHortSci54:265271
(1985).
17 Schoch S, R
¨
udiger W, L
¨
uthy B and Matile P, C13
2
-hydroxy-
chlorophyll a, the first product of the reaction of chlorophyll
oxidase. J Plant Physiol 115:8589 (1984).
18 Yamauchi N and Watada AE, Chlorophyll and xanthophyll
changes in broccoli florets stored under elevated CO
2
or ethylene-containing atmosphere. HortScience 33:114117
(1998).
19 Yamauchi N, Funamoto Y and Kanetsune Y, Involvement of
chlorophyll degrading enzymes with chlorophyll degradation
in stored broccoli (Brassica oleracea L.) florets. Food Sci Technol
Res 5:300303 (1999).
20 McGuire RG, Reporting of objective color measurements.
HortScience 27:12541255 (1992).
21 Moran R, Formulae for determination of chlorophyllous
pigments extracted with N,N-dimethylformamide. Plant
Physiol 69:13761381 (1982).
22 Yamauchi N, Harada K and Watada AE, In vitro chlorophyll
degradationinstoredbroccoli(Brassica oleracea L. var. italica
Plen.) florets. Postharv Biol Technol 12:239245 (1997).
23 Perkins HJ and Roberts DW, Purification of chlorophylls, pheo-
phytins and pheophorbides for specific activity determina-
tions. Biochim Biophys Acta 58:486498 (1962).
24 Tsuchiya T, Ohta H, Masuda T, Mikami B, Kita N, Shioi Y,
et al, Purification and characterization of two isozymes of
chlorophyllase from mature leaves of Chenopodium album.
Plant Cell Physiol 38:10261031 (1997).
25 Suzuki T and Shioi Y, Re-examination of Mg-dechelation
reaction in the degradation of chlorophylls using chlorophyllin
a as a substrate. Photosynth Res 74:217223 (2002).
26 Kunieda T, Amano T and Shioi Y, Search for chlorophyll
degradation enzyme, Mg-dechelatase, from extracts of
Chenopodium album with native and artificial substrates. Plant
Sci 169:177183 (2005).
27 Yamauchi N, Xia X and Hashinaga F, Involvement of flavonoid
oxidation with chlorophyll degradation by peroxidase in wase
satsuma mandarin fruits. JJpnSocHortSci66:283 288
(1997).
28 Bradford MM, A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
proteindye binding. Anal Biochem 72:248254 (1976).
29 Janave MT and Sharma A, Inhibition of chlorophyll degradation
in stay-green Langra mango (Mangifera indica L.) fruits.
BARC Newslett 273:80 86 (2006).
30 H
¨
ortensteiner S, Chlorophyll degradation during senescence.
Annu Rev Plant Biol 57:55 77 (2006).
31 Vicentini F, H
¨
ortensteiner S, Schellenberg M, Thomas H and
Matile P, Chlorophyll breakdown in senescent leaves:
identification of the biochemical lesion in a stay-green
genotype of Festuca pratensis Huds. New Phytol 129:247252
(1995).
32 Akiyama Y, Takahashi C and Yamauchi N, Pathway of
chlorophyll degradation in yellowing radish (Raphanus sativus
L.) cotyledons. J Jpn Soc Food Sci Technol 47:296301
(2000).
33 Sabater B and Rodr
´
ıguez MT, Control of chlorophyll degrada-
tion in detached leaves of barley and oat through effect of
kinetin on chlorophyllase. Physiol Plant 43:274276 (1978).
34 Fang Z, Bouwkamp JC and Solomos T, Chlorophyllase activ-
ities and chlorophyll degradation during leaf senescence in
non-yellowing mutant and wild type of Phaseolus vulgaris L.
JExpBot49:503 510 (1998).
35 Funamoto Y, Yamauchi N, Shigenaga T and Shigyo M, Effects
of heat treatment on chlorophyll degrading enzymes in
stored broccoli (Brassica oleracea L.). Postharv Biol Technol
24:163170 (2002).
36 Gong Y and Mattheis JP, Effect of ethylene and 1-
methylcyclopropene on chlorophyll catabolism of broccoli
florets. Plant Growth Regul 40:33 38 (2003).
37 Ketsa S, Phakawatmongkol W and Subhadrabhandhu S, Peel
enzymatic activity and colour changes in ripening mango
fruit. J Plant Physiol 154:363366 (1999).
38 Costa ML, Civello PM, Chaves AR and Mart
´
ınez GA,
Characterization of Mg-dechelatase activity obtained from
Fragaria × ananassa fruit. Plant Physiol Biochem 40:111118
(2002).
39 Shioi Y, Tomita N, Tsuchiya T and Takamiya K, Conversion of
chlorophyllide to pheophorbide by Mg-dechelating substance
in extracts of Chenopodium album. Plant Physiol Biochem
34:4147 (1996).
1986
J Sci Food Agric 88:1981 1986 (2008)
DOI: 10.1002/jsfa
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    • "P < 0.0001) and cultigen (F = 2.08; P = 0.0179), but there were no influences from their interaction. Values for chlorophyll b for cultigens are in close agreement with Dissanayake et al. [29] who reported values of ~17.00 mg chlorophyll b/100 g FM for the A. fistulosum cultigen “Kujyo-hoso.” Significant increases in chlorophyll b in response to UV-B radiation were found for cultigens “Feast,” “GA-C 76,” and “Shounan” (Table 5). "
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  • [Show abstract] [Hide abstract] ABSTRACT: The new olive cultivar 'Sikitita' was obtained from a cross between the 'Picual' and 'Arbequina' varieties. 'Sikitita' was selected for its features, making it particularly suited to high-density olive hedgerow orchards. From the standpoint of chloroplast pigment metabolism, the fruits of the 'Picual' and 'Arbequina' varieties have significant differences. It is therefore extremely interesting to analyze the descendants of both cultivars. With regard to chlorophyll catabolism, 'Sikitita' has proven to be a cultivar with low pigmentation and low levels of chlorophyllase activity. This is contrary to the findings obtained to date, where varieties with low pigmentation are a consequence of high chlorophyllase activity ('Arbequina') and highly pigmented fruits are due to low chlorophyllase activity ('Picual'). 'Arbequina' was, until recently, the only cultivar described that had developed a carotenogenic process, despite its anthocyanic ripening. However, from its father ('Arbequina'), the 'Sikitita' cultivar has inherited the pool of enzymes necessary to esterify xanthophylls at the chromoplast level. This makes 'Sikitita' a very interesting cultivar, with potential chemotaxonomic differences (such as esterified xanthophylls in the olive oils), and demonstrates the interest in genetic improvement programs for olive cultivars with different organoleptic characteristics.
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  • [Show abstract] [Hide abstract] ABSTRACT: To investigate the possible mechanism of color degradation of green prickleyashes (Zanthoxylum schinifolium Zucc.) in slow drying, changes in their peel colors, enzyme activities, content of chlorophylls (Chls) and derivatives were evaluated. The results showed the peel color changed from brilliant green to black-brown and Chls underwent a rapid degradation. Enzyme activities changed as follows: chlorophyllase activity decreased; chlorophyll-degrading peroxidase (Chl-POD) activity as well as pheophorbidase (Pheidase) exhibited a biphase trend displaying an inverted “V” phase, and the increase in Chl-POD resulted in the accumulation of C132-hydroxy-chlorophyll a. Based on the study of enzyme activities and Chl degradation, conclusions were drawn that Chl-POD and Pheidase were considered as the key enzymes to promote chlorophyll breakdown. Compared with slow drying, fast drying could inhibit the two key enzyme activities and blockade the chlorophyll-degrading pathway, which was proposed to process green prickleyashes.
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