Journal of Experimental Botany, Vol. 49, No. 320, pp. 503–510, March 1998
Chlorophyllase activities and chlorophyll degradation
during leaf senescence in non-yellowing mutant and wild
type of Phaseolus vulgaris L.
Zhengyi Fang, John C. Bouwkamp1 and Theophanes Solomos
Department of Horticulture, University of Maryland at College Park, MD 20742, USA
Received 26 February, 1997; Accepted 15 October 1997
Abstract
Introduction
The activities of chlorophyllase, contents of pigments
including chlorophyll a and b, chlorophyllide a and b,
and phaeophorbide a during leaf senescence under
low oxygen (0.5% O ) and control (air) were investi2
gated in a non-yellowing mutant and wild-type leaves
of snap beans (Phaseolus vulgaris L.). Chlorophyllase
from leaf tissues had maximum activity when incubated at 40 °C in a mixture containing 50% acetone. In
both mutant and wild type, chlorophyllase activity was
the highest in freshly harvested non-senescent leaves
and decreased sharply in the course of senescence,
indicating that the loss of chlorophylls in senescing
leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered
in the mutant. The wild type had higher ratios of
chlorophyll a to chlorophyll b than the mutant and
chlorophyll a5b ratios increased during senescence in
both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were
detected, but no phaeophorbide a was found.
Chlorophyllide b had a greater accumulation than
chlorophyllide a in the early stage of senescence. Low
oxygen treatment not only delayed chlorophyll
degradation but also enhanced the accumulations of
chlorophyllide a and b and lowered the ratios of
chlorophyll a to chlorophyll b.
Leaf senescence involves degradation of proteins,
chlorophylls, nucleic acids, membranes, and subsequent
transport of some of the degradation products to other
parts of the plant (Buchanan-Wollaston, 1997; Noodén,
1988). The yellowing of the leaves due to chlorophyll
degradation is the most obvious visible symptom. In the
recent years, significant progress in the understanding of
chlorophyll degradation has been achieved. A four step
pathway of chlorophyll degradation was demonstrated
( Vicentini et al., 1995).
The initial step is the removal of phytol by chlorophyllase (Matile et al., 1989; Shimokawa et al., 1978).
Chlorophyllase is considered the first enzyme in the
pathway of chlorophyll degradation (Matile et al., 1997;
Hendry et al., 1987). However, the activity of chlorophyllase tends to decrease with senescence (MinguezMosquera et al., 1996; Yamauchi and Watada, 1991),
indicating that factors other than chlorophyllase activity
regulate chlorophyll degradation. Very recently, Matile
et al. (1997) reported in barley and oilseed rape leaves
that chlorophyllase is located in the inner envelope membrane of chloroplast. This finding supports the hypothesis
that chlorophyllase and chlorophyll are spatially separated, which prevents the hydrolysis of chlorophyll before
senescence (Fernandez-Lopez et al., 1991; Schoch and
Brown, 1987). Although it was found that chlorophyllase
activity was detectable in both thylakoid membrane and
chloroplast envelope in orange leaves (Brandis et al.,
1996), the authors explained that this result may not
Key words: Chlorophyllase, chlorophyll degradation,
chlorophyllides, non-yellowing mutant, Phaseolus vulgaris.
1 To whom correspondence should be addressed. Fax: +1 301 314 9308. E-mail: [email protected]
© Oxford University Press 1998
504
Fang et al.
represent a real association between chlorophyllase
and the chlorophyll–protein complexes. In addition, the
degradation of chlorophyll b is different from that of
chlorophyll a. Chlorophyll b is degraded by first being
converted to chlorophyll a (Scheumann et al., 1996; Ito
et al., 1993). This conversion is via 7-hydroxymethyl
chlorophyll a ( Ito et al., 1996) and is coupled with the
assembly of chlorophyll with apoproteins (Ohtsuka
et al., 1997).
The removal of magnesium from chlorophyllide by
Mg-dechelatase was found to be the second step in the
pathway of chlorophyll degradation (Owens and
Falkowski, 1982). Langmeier et al. (1993) suggested that
Mg-dechelatase was present before the onset of leaf
senescence and the activity of the dechelatase was evoked
as chloroplasts differentiated into gerontoplasts. The
appearance and maintenance of Mg-dechelatase activity
in senescent cotyledons required continuous cytoplasmic
protein synthesis (Langmeier et al., 1993).
The third step is oxygenolytic opening of porphyrin
macrocycle in phaeophorbide, which produces fluorescent
chlorophyll catabolites ( FCCs) (Ginsburg and Matile,
1993). The third step results in yellowing of senescent
leaves (Matile et al., 1996). The enzyme responsible for
the third step is phaeophorbide a oxygenase which is
located in gerontoplast envelope (Matile and
Schellenberg, 1996). This enzyme activity is detectable
only in senescent leaves (Hortensteiner et al., 1995). In
addition, the third step required oxygen, ATP, ferrodoxin,
NADPH, iron, and thylakoid and stromal proteins
( Hortensteiner et al., 1995; Vicentini et al., 1995; Ginsburg
et al., 1994; Ginsburg and Matile, 1993).
The final step includes the catabolism of FCCs to nonfluorescent chlorophyll catabolites (NCCs) ( Vicentini
et al., 1995) and the disposal of NCCs in vacuoles (Hinder
et al., 1996).
Non-yellowing or stay-green mutants provide a
particular useful tool to elucidate the mechanism of
chlorophyll degradation ( Thomas and Smart, 1993;
Matile, 1992). In a non-yellowing mutant of snap bean,
Ronning et al. (1991) found a lack of plastoglobuli in
the senescent leaf chloroplast and Bachmann et al. (1994)
reported that most of NCCs and FCCs were undetectable.
In the non-yellowing phenotype chlorophyll degradation
is not completely inhibited, since initial breakdown product chlorophyllides could be detected in the non-yellowing
mutant of Festuca pratensis ( Thomas et al., 1989).
Vicentini et al. (1995) reported, in the mutant of Festuca
pratensis, that both chlorophyllase and Mg-dechelatase
activities were as competent as the wild type, but the
mutant was deficient in phaeophorbide a oxygenase activity. Also, Thomas et al. (1996) recently demonstrated
that the phenotype of Gregor Mendel’s green pea is due
to a deficiency of the phaeophorbide a oxygenase.
The objective of this study was to investigate the
patterns of chlorophyllase activity and changes of pigment
contents during leaf senescence in a non-yellowing mutant
and a wild type of Phaseolus vulgaris L. under control
(air) and low oxygen. The low oxygen treatment was
included because of its retarding effects on senescence
(Solomos, 1988).
Materials and methods
Materials
Snap bean (Phaseolus vulgaris L.) cv. Sunray and its isoline
non-yellowing mutant were used as materials in this study. The
bean seeds were planted in 1 gallon pots filled with Sunshine
Mix No.1 media (contains 70–80% peat moss) and grown in a
greenhouse under regular culture conditions. Primary leaves,
which are defined as the first true unifoliate leaves, were
collected when the first trifoliate leaves were completely
expanded. The collected primary leaves were washed carefully
with distilled water and placed individually into sealed
desiccators. The petioles were inserted 1 cm deep into distilled
water.
Induction of senescence and low oxygen treatment
A stream of the appropriate gases was passed through the
sealed desiccators at a flow of 120 ml min−1. The leaves were
treated with 10 ppm ethylene in air for 2 d in a dark chamber
at 20 °C. Then the leaves were treated either with air or with
0.5% oxygen atmospheres for an additional 4 d with the same
condition as above. Leaf samples were collected at 0, 2, 4, and
6 d after treatment.
Measurement of pigments by HPLC
Leaf pigments were extracted by homogenizing 3 g leaves in
−20 °C cold acetone for 30 s at full speed with a Polytron
(Brikmann Instruments). The homogenate was filtered through
a sintered glass funnel and washed with cold acetone until the
residue was colourless. The residue, designated as acetone
powder, was dried in vacuo and stored at −20 °C. The acetone
extract from 3 g leaves was concentrated to 15 ml with a rotary
evaporator. This extract containing about 80% acetone was
then filtered with a Millipore filter (0.2 mm pores) for HPLC
analysis. HPLC pigment separation and determination was
based on the method of Yamauchi and Watada (1991) with
slight modifications. Before sample running, a C-18 Waters
Associate column was washed with solvent A (80% methanol
and 20% water) for 15 min. Then sample was run in a linear
gradient of solvent A and solvent B (ethyl acetate) at a flow
rate of 1 ml per min. The initial ratio of solvent A to B was
100%50%. The final ratio of solvent A to B is 50%550%. The
time for running from initial to final condition was set to
20 min and then an additional 25 min at the final condition
(50% solvent A and 50% solvent B). A single wavelength
detector at 658 nm was used for the measurements of chlorophyll
a and b, chlorophyllide a and b, and phaeophorbide a since the
absorbance peaks of these pigments are near 658 nm. Standard
chlorophyll a and b were separated by LK 6 silica gel 60A TLC
plates ( Whatman) in the solvent mixture (hexane:diethyl
ether:acetone, 60530520, by vol.). Chlorophyllide a and b were
obtained by the reactions of chlorophylls with chlorophyllase
under incubation conditions as described below. Phaeophorbide
a was prepared by adding one drop of 2 N HCl to the
chlorophyllide a solution. All standard pigments were prepared
in 80% acetone solutions for the analysis in HPLC and in
Chlorophyllase and chlorophyllides 505
Beckman DU-7 spectrophotometer. The retention time was
3 min for chlorophyllide b, 5 min for chlorophyllide a, 17 min
for phaeophorbide a, 24 min for chlorophyll b, and 26 min for
chlorophyll a. Each sample contained three replications.
Extraction of chlorophyllase and enzyme analysis
Chlorophyllase was extracted by using a modification of the
method of Fernandez-Lopez et al. (1992). 1 g acetone powder
was extracted with 50 ml of extraction buffer (41 mM TRIS,
320 mM glycine, 670 mM glucose, pH 7.8, 0.5% SDS (w/v), and
2% water-insoluble PVP) for 18 h at room temperature and
held for an additional 24 h at 4 °C. The extract was then filtered
through four layers of cheese-cloth and centrifuged at 10 000×g
for 20 min. Chlorophyllase was precipitated from the supernatant by adding (NH ) SO to achieve to 80% saturation and
4 2 4
centrifuging them at 20 000×g for 10 min. The pellet was
resuspended in 50 mM potassium phosphate buffer (pH 7.8)
and stored at −20 °C, and subsequently used for enzyme
analysis.
Before chlorophyllase activities were measured, investigations
on optimal incubation conditions for chlorophyllase activity
were carried out. The initial assay was based on the method of
Tanaka et al. (1982). A 2 ml reaction mixture containing 20 ml
0.1 M ascorbate, 100 ml chlorophyllase extract which contained
about 5 mg ml−1 of protein, 120 mM chlorophyll a and b and
1680 ml of an acetone/water mixture. To optimize the assay, the
following two variables were investigated using a single factor
design with three replications: (1) acetone concentrations in the
mixture were 10, 20, 30, 40, 50, 60, and 70% and (2) incubation
temperatures were 30, 35, 40, 45, and 50 °C. After 30 min
incubation, the reactions were stopped by adding a precooled
mixture of 2 ml acetone and 4 ml hexane followed by vigorous
shaking. The mixtures were centrifuged at 12 000×g for 10 min
at 4 °C. The lower phase (acetone) was used to measure the
production of chlorophyllide a by a DU-7 Beckman spectrophotometer. The quantity of chlorophyllide a was calculated from
the optical density (OD) at 665 nm using a 54.1 mM cm−1
extinction coefficient (Tanaka et al., 1982). Based on the above
optimization tests it was found that a reaction mixture with
50% acetone and incubated at 40 °C for 30 min was suitable for
assaying chlorophyllase activity, and these conditions were used
in all subsequent assays. Chlorophyllase activity was expressed
in production (nmol ) of chlorophyllide a g−1 fresh leaf h−1.
Each sample contained three replications.
Fig. 1. Effect of acetone concentration in the reaction mixture on
chlorophyllase activity. The relative activity of chlorophyllase was
calculated from the amount of chlorophyllide a produced in the reaction
mixture that was carried out at 40 °C for 30 min.
Results
Optimization of measurement of in vitro activity of
chlorophyllase from the leaves of snap beans
The enzymatic properties of chlorophyllase from snap
bean leaves have not been previously reported. In this
study, maximum in vitro activity of chlorophyllase was
found at 50% acetone containing mixture (Fig. 1) and
40 °C incubation temperature (Fig. 2). From Fig. 3, a K
m
value of this enzyme was estimated to be 9.2 mM
(chlorophyll a as substrate).
Chlorophyllase activity during leaf senescence in mutant
and wild type
As shown in Fig. 4, chlorophyllase activity was highest
in fresh leaves (day 0). After 2 d treatment with 10 ppm
Fig. 2. Effect of reaction temperature on chlorophyllase activity. The
relative activity of chlorophyllase was calculated from the amount of
chlorophyllide a produced. The assay mixture contained 50% (v/v)
acetone and was incubated for 30 min.
ethylene the activity declined to 39% and 48% of the
initial levels in mutant and wild type, respectively. At this
time the leaves had not begun to yellow. From day 2 to
day 6, the activity continued to decrease, indicating that
yellowing is not positively related to chlorophyllase activity. The data presented in Fig. 4 show that there is no
significant difference in chlorophyllase activities between
the mutant and wild type at any sampling time, suggesting
506
Fang et al.
based chlorophyllase activity (data not shown), due
probably to the rapid proteolysis during this period of
leaf senescence.
Pigment level changes during leaf senescence
Figures 5 and 6 show that during senescence in air
chlorophyll a and b contents declined quickly in the wildtype leaves while there were only small decreases of
chlorophyll a and chlorophyll b levels in the mutant
leaves. The losses of chlorophyll a and b, however, were
inhibited significantly by low oxygen treatment, especially
in the wild-type leaves (Figs 5, 6). At day 6 in the wild
Fig. 3. Effect of substrate (chlorophyll a) concentration on chlorophyllase activity. The relative activity of chlorophyllase was calculated
from the amount of chlorophyllide a produced. Assay mixture contained
50% (v/v) acetone and the reactions were carried out at 40 °C for 30 min.
Fig. 5. Chlorophyll a content in the non-yellowing mutant (M ) and
wild type ( WT ) snap bean leaves treated with control (air) or low
oxygen ( lo) atmosphere during senescence. The leaves were treated with
10 ppm ethylene for 2 d and then either with air or with 0.5% O for
2
4 d in the dark at 20 °C. The vertical bars are standard errors.
Fig. 4. Chlorophyllase activity during leaf senescence in a non-yellowing
mutant and wild type of snap bean. Chlorophyllase activity unit is nmol
of chlorophyllide a g−1 fresh weight leaf h−1. Reaction mixture
contained 50% (v/v) acetone and was incubated at 40 °C for 30 min.
Leaves were treated with 10 ppm ethylene for 2 d and then with air or
low oxygen for 4 d in the dark at 20 °C. The vertical bars are
standard errors.
that chlorophyllase activity is not blocked in the nonyellowing mutant. In addition, there is no significant
difference between air and the low oxygen on the final
day’s chlorophyllase activity. Chlorophyllase activity
based on the amount of protein was also expressed. These
patterns of chlorophyllase activity are very similar to the
patterns of those based on leaf fresh weight, except for
that from day 2 to day 6 the protein-based chlorophyllase
activity decreased more slowly than the fresh weight-
Fig. 6. Chlorophyll b content in the non-yellowing mutant (M ) and
wild type ( WT ) snap bean leaves treated with control (air) or low
oxygen ( lo) atmosphere during senescence. The leaves were treated with
10 ppm ethylene for 2 d and then either with air or with 0.5% O for
2
4 d in the dark at 20 °C. The vertical bars are standard errors.
Chlorophyllase and chlorophyllides 507
type, the low oxygen-treated leaves still had 0.85 mmol
chl a g−1 and 0.31 mmol chl b g−1 while the leaves held
in air had only 0.11 mmol chl a g−1 and 0.04 mmol chl
b g−1.
In the present study, no phaeophorbide a was detected
in either genotype at any time during senescence. The
results presented in Fig. 7 and Fig. 8 show that the mutant
accumulated appreciable amounts of chlorophyllide a and
chlorophyllide b, but the wild type did not. The kinetics
of chlorophyllide accumulation differs between a and b.
Chlorophyllide b increased earlier than chlorophyllide a
and reached a plateau level after 4 d. On the other hand,
the rise of chlorophyllide a level began later and continued
to increase up to 6 d. Figures 7 and 8 also show that the
pattern of chlorophyllide production during leaf senescence was influenced by low oxygen treatment. The low
oxygen treatment not only enhanced the accumulation of
chlorophyllide a and b in the mutant leaves, but also
made chlorophyllide a and b detectable in the wild-type
senescent leaves. A calculation of the ratio of chlorophyll
loss to chlorophyllide production on the final day is made
and listed in Table 1. It shows that the ratio is very low
in the wild type. In the mutant, the ratio of chlorophyll
a/chlorophyllide a is higher than the ratio of chlorophyll
b/chlorophyllide b and low oxygen treatment enhanced
the ratio especially in chlorophyll a/chlorophyllide a in
the mutant.
Discussion
Properties of chlorophyllase extracted from snap bean
leaves
Fig. 7. Chlorophyllide a content in the non-yellowing mutant (M ) and
wild type ( WT ) snap bean leaves treated with control (air) or low
oxygen ( lo) atmosphere during senescence. The leaves were treated with
10 ppm ethylene for 2 d and then either with air or with 0.5% O for
2
4 d in the dark at 20 °C. The vertical bars are standard errors.
Acetone concentration in the assay medium plays a
critical role in determining chlorophyllase activity
( Khamessan et al., 1993; Tanaka et al., 1982). In the
present study with snap bean leaves, it was found that
incubation with 50% acetone at 40 °C resulted in maximum chlorophyllase activity. In contrast, other studies
found optimal activity with 30% acetone at 30 °C in alga,
Citrus limon, and rye ( Khamessan et al., 1993; FernandezLopez et al., 1992; Tanaka et al., 1982). Thus the optimal
temperature and acetone concentration for snap bean leaf
chlorophyllase activity appear to be higher than those of
most other species. The purpose of adding acetone is to
solubilize water-insoluble chlorophyll, yet excessive acetone concentrations may cause protein precipitation which
would be expected to decrease enzyme activity (McFeeters
et al., 1971). Therefore, the range of optimal acetone
concentrations among species may be related to the
balance of solubilization and precipitation.
The role of chlorophyllase during leaf senescence
Fig. 8. Chlorophyllide b content in the non-yellowing mutant (M ) and
wild type ( WT ) snap bean leaves treated with control (air) or low
oxygen ( lo) atmosphere during senescence. The leaves were treated with
10 ppm ethylene for 2 d and then either with air or with 0.5% O for
2
4 d in the dark at 20 °C. The vertical bars are standard errors.
Chlorophyllase has been assumed to be located in
thylakoid membranes ( Tarasenko et al., 1986). The fact
that high activities of chlorophyllase exist in fresh bean
leaves, however, suggests that chlorophyllase may be
spatially separated from chlorophylls in non-senescent
tissues ( Fernandez-Lopez et al., 1991). This suggestion is
confirmed by a recent report by Matile et al. (1997), who
demonstrated that chlorophyllase is located in the chloroplast envelopes. In both the mutant and wild-type leaves
chlorophyllase activities decreased quickly during the
course of senescence. This result is same as that in soybean
primary leaves during senescence (Majumdar et al., 1991).
Low oxygen treatment delayed chlorophyll degradation
but did not affect chlorophyllase. These findings indicate
508
Fang et al.
Table 1. The ratio of chlorophyll loss to chlorophyllide production at day 6
The leaves were treated with 10 ppm ethylene for 2 d and then transferred to either control (air) or 0.5% O ( lo) for 4 d in the dark at 20 °C.
2
Chlorophyll a/chlorophyllide a
Chlorophyll b/chlorophyllide b
Chlorophylls a+b/chlorophyllides a+b
Mutant
(air)
Mutant
( lo)
Wild type
(air)
Wild type
( lo)
0.397
0.206
0.290
0.950
0.352
0.593
0
0
0
0.007
0.043
0.023
Table 2. The ratios of chlorophyll a to chlorophyll b during leaf senescence in a non-yellowing mutant and the wild type of snap bean
The leaves were treated with 10 ppm ethylene for 2 d and then were transferred to either control (air) or low oxygen (0.5% O ) ( lo) for 4 d in the
2
dark at 20 °C. Values represent means ±SE.
Days
Mutant
Wild type
0
2
4
6
1.898±0.045
1.940±0.043
2.144±0.041 (air)
2.474±0.082 (air)
1.934±0.042
2.338±0.038
2.902±0.022 (air)
2.908±0.040 (air)
1.952±0.036 ( lo)
2.327±0.020 ( lo)
that chlorophyllase activity does not regulate directly
chlorophyll degradation ( Fiedor et al., 1992; Rodriguez
et al., 1987). Since chlorophyllase activity is closely related
to yields of envelope markers (Matile et al., 1997) and
similar patterns of chlorophyllase activity were found in
the mutant and wild type, it suggests that the change in
the chloroplast envelope during senescence in the mutant
may be same as that in the wild type. This hypothesis is
consistent with the observation by Ronning et al. (1991),
who found that there was no appreciable differences in
chloroplast ultrastructure between the mutant and wild
type in snap bean. During leaf senescence, the earliest
change in cell structure is the breakdown of chloroplasts
(Gan and Amasino, 1997). The dramatic fall (more than
50%) of chlorophyllase activity in the first 2 d indicates
that such a change of chlorophyllase activity may be due
to the destruction of the chloroplasts ( Yamauchi and
Watada, 1993). Thus it is hypothesized that, with the
onset of senescence, chlorophyllase comes in contact with
chlorophyll due to its release from the envelope, caused
by the destruction of chloroplasts.
Protein degradation during the extraction of chlorophyllase in senescent leaves may contribute to the
decrease in chlorophyllase activity. However, the rapid
decrease of chlorophyllase activity in the first 2 d is
probably not caused by proteolysis since 88% of original
total protein amount was still present in the leaves on
day 2 (data not shown), indicating that at this stage
proteolysis was not strongly activated. But, by day 6 only
31% of the original protein remained. Such a rapid protein
hydrolysis may create artefacts in the determination of
chlorophyllase activity. It was not possible to separate
this influence since the recovery of chlorophyllase activity
in acetone powders or ammonium sulphate pellets was
not measured at any stage of senescence.
2.463±0.030 ( lo)
2.766±0.028 ( lo)
The effect of low oxygen on the leaf senescence
The current study found that low oxygen treatment not
only delayed chlorophyll degradation, but also resulted
in increases of chlorophyllide a and b levels. This result
agrees with the findings of Yamauchi and Watada (1993),
Peisker et al. (1989) and Thomas et al. (1989). In the
wild type although the loss of chlorophyll was delayed
significantly by low oxygen treatment, the amount of
chlorophyllide was very small. It may suggest that 0.5%
O concentration is not sufficiently low to inhibit com2
pletely the activity of the phaeophorbide a oxygenase or
that the retardation of chlorophyll degradation by hypoxia is not attributed to any of the three early enzymatic
steps in the pathway. In an earlier study, it was found
that in both mutant and wild type low oxygen (1.5% O )
2
treatment delayed the degradation of total proteins, but
did not affect the decrease in chlorophyllase activity
during senescence. Therefore, both the delay of chlorophyll degradation and the accumulation of chlorophyllides, which were caused by low oxygen treatment,
are not related directly to chlorophyllase activity. It was
observed that leaf yellowing processes were significantly
delayed by low oxygen treatment. Matile et al. (1989)
proposed and Ginsburg et al. (1994) confirmed that the
colour change step associated with tetrapyrrole ring
destruction is oxygen dependent. It is well established
that the storage of fresh fruits and vegetables under low
oxygen greatly extends their storage life by a suppression
in the induction of enzymes associated with their normal
ripening (Solomos, 1988). In the case of chlorophyll
degradation, however, it is not clear whether the retardation by low oxygen is due to the inhibition of the
synthesis of the phaeophoebide a oxygenase or due to the
decrease in its substrate, i.e. oxygen.
Chlorophyllase and chlorophyllides 509
Change of chlorophyll a/b ratio during senescence
Table 2 shows that (a) the ratio of chlorophyll a to b
increased in the course of senescence, (b) the wild type
had higher ratios than the mutant and (c) low oxygen
treatment lowered the ratios in the both genotypes.
According to the mechanism of conversion of chlorophyll
b to a (Scheumann et al., 1996; Ito et al., 1993), the
above results suggest that (a) the increased ratio of
chlorophyll a to b during senescence may be due to the
conversion of chlorophyll b to a; (b) the wild type may
have a more rapid conversion than the mutant; (c) low
oxygen treatment may slow the conversion. Falbel and
Staehelin (1996) reported that bottleneck in chlorophyll
a biosynthesis resulted in chlorophyll b deficiency.
Therefore, it is possible that the block of chlorophyll a
degradation in the non-yellowing mutant has a comparable effect on the chlorophyll a/b balance during
senescence.
Accumulation of chlorophyllides in the senescent mutant
leaves
The decrease amount of chlorophyll in senescent leaves
is not proportional to the level of chlorophyllide accumulation. The wild type had a rapid reduction of
chlorophylls, but there was no detectable amount of
chlorophyllides, indicating that breakdown of chlorophyllide is very rapid in the wild type. In contrast, the
non-yellowing mutant had a little decrease in chlorophyll
levels, but the accumulations of chlorophyllides were
high. This result may suggest that at least one step is rate
limited or even blocked in the mutant downstream in the
pathway. By comparing the patterns between chlorophyllide a and chlorophyllide b in the mutant, it was
found that more chlorophyllide b accumulated than
chlorophyllide a at day 2 and day 4 although chlorophyll
a was degraded more than chlorophyll b in this period.
This result is in agreement with hypothetical reaction
sequences proposed by Scheumann et al. (1996):
chlorophyll bchlorophyllide bchlorophyllide a
chlorophyll a.
Phaeophorbide a was not detectable in the bean leaves
In the present study, phaeophorbide a was detected
neither in the wild type nor in the mutant. This observation is reasonable for the wild type in that phaeophorbide
a may be catalysed to further breakdown compounds so
quickly that it does not accumulate to a detectable level.
The mutant may be deficient in Mg-dechalatase activity
since chlorophyllides are not quickly catalysed, which
results in a high level of accumulation. Low oxygen
treatment would also be expected to result in the accumulation of phaeophorbide since oxygen is required for the
breakdown of phaeophorbide (Ginsburg et al., 1994).
The current result, however, is not consistent with the
observation in a non-yellowing mutant of Festuca
pratensis, in which the existence of phaeophorbide a was
detected ( Vicentini et al., 1995). This result needs to be
confirmed in the non-yellowing mutants of other crops
such as pepper and tomato.
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