Journal of Experimental Botany, Vol. 51, No. 345, pp. 739–745, April 2000
Water stress enhances b-amylase activity in cucumber
cotyledons
Daisuke Todaka, Hisashi Matsushima and Yukio Morohashi1
Department of Regulatory Biology, Faculty of Science, Saitama University, Urawa, Saitama 338–8570, Japan
Received 19 July 1999; Accepted 29 October 1999
Abstract
Cotyledons detached from 4-d-old cucumber (Cucumis
sativus L.) seedlings were subjected to water stress
(air-drying or PEG-treatment) to examine the effects
of the stress on carbohydrate metabolism. Amylolytic
activity in the cotyledon was increased about 6-fold
by water stress within 1 d. The substrate specificity
and the action pattern indicated that b-amylase is
responsible for the activity. Activities of azocaseinase,
malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating
that the effect of the stress on b-amylase is rather
specific. Cycloheximide-treatment strongly reduced
the enhancement of b-amylase activity. The hypocotyl
of cucumber seedlings also exhibited an increase in
the enzyme activity when subjected to water stress.
The major free sugars in cucumber cotyledons were
glucose, fructose, maltose, and sucrose; sucrose being
the most abundant. Sucrose content in excised,
unstressed cotyledons increased markedly during the
incubation. Changes in other free sugars were small
compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons
more sucrose and less starch accumulated than in
unstressed ones. Such results were discussed in
relation to the enhancement of b-amylase activity.
Key words: b-Amylase, cucumber cotyledon, starch,
sucrose, water stress.
Introduction
Plants are exposed to many types of environmental
stresses. Water stress is one of the most serious problems
for immotile plants and, in order to survive, plants show
numerous metabolic and molecular responses to water
stress (Hanson and Hitz, 1982; Ingram and Bartlets,
1996). Changes in carbohydrate metabolism have been
reported to occur in water-stressed bean leaves (Stewart,
1971). It has been shown that cytosolic glyceraldehyde3-phosphate dehydrogenase is strongly induced by water
stress in Craterostigma plantagineum and it has been
suggested that changes in the rate of glycolysis are important in coping with the stress ( Velasco et al., 1994).
Sucrose synthase and sucrose phosphate synthase genes
are reported to be up-regulated by drought (Ingram and
Bartlets, 1996). Sugars are the source of energy and
carbons needed for adaptive and/or defensive responses
to stresses. In addition, sugars such as raffinose and
sucrose are indicated to have important roles in protecting
cells from water stress; they are solutes available for
osmoregulation or function as protectants of macromolecules or membranes (Leopold, 1990; Bray, 1997).
It is, therefore, supposed that an increased supply of
sugars is required for water-stressed cells to survive. In
fact, water stress causes the increase in sucrose content
in bean leaves (Stewart, 1971) and a-amylase is induced
by water stress in barley leaves (Jacobsen et al., 1986).
The present paper reports that an enhancement of
b-amylase activity occurs in water-stressed cucumber
cotyledons and that sucrose accumulates under such
conditions.
Materials and methods
Plant material
Seeds of cucumber (Cucumis sativus L. cv. Tokiwajibae) were
soaked in water for 6 h at 28 °C, planted in moist vermiculite
and incubated at 28 °C in the dark. After 4 d cotyledons were
excised, 30 pairs were placed on two layers of 9 cm filter paper
(in Petri dishes), moistened with 10 ml of water or polyethylene
glycol 6000 (PEG) solution and then incubated at 28 °C in
the dark. Wet filter paper was replaced every day. In some
1 To whom correspondence should be addressed. Fax: +81 48 858 3584. E-mail: [email protected]
© Oxford University Press 2000
740 Todaka et al.
experiments, excised cotyledons were air-dried at 28 °C for 20 h
to approximately 65% of the initial fresh weight. Hypocotyl
parts (2 cm sections below the hook) of 3-d-old seedlings were
treated with 40% PEG in the same way as above, except for
the amount of PEG solution (7.5 ml instead of 10 ml ).
and layered on top of an activity gel containing 7.5% (w/v)
polyacrylamide and 0.25% (w/v) soluble potato starch in buffer
B. The gel-sandwich was incubated at 28 °C for 7 h or 18 h in
a closed chamber. Activity gel was stained with I -KI (0.056
2
and 0.56% [w/v], respectively, in 0.75 N HCl ).
Enzyme extraction
Cotyledons and hypocotyl sections were homogenized in
50 mM K-phosphate buffer (pH 6.8) containing 10 mM
2-mercaptoethanol (2-ME ) (6 ml per 30 pairs of cotyledons
and 3.5 ml per 45 sections) with a chilled mortar and pestle.
The homogenate was centrifuged at 10 000 g for 10 min, and
the supernatant was used as a crude enzyme preparation.
Action pattern of the enzyme
Products of starch hydrolysis by the enzyme, purified at the
step of hydroxylapatite column chromatography, were analysed
by TLC. The reaction mixture containing 0.25% (w/v) soluble
starch and the enzyme preparation (0.03 mmol maltose min−1)
in buffer A in a total volume of 0.2 ml was incubated at 30 °C
for 30 min. The hydrolysate was subjected to TLC after
removing unreacted polysaccharides by ethanol precipitation.
TLC was performed on a silica gel plate which was developed
twice with 75% (v/v) acetonitrile. Spots were visualized by
charring (5% [v/v] H SO in ethanol ).
2 4
Enzyme assays
Amylolytic activities in the crude extracts were assayed at 30 °C
at pH 6.8 (50 mM K-phosphate buffer) in the presence of
10 mM 2-ME by measuring the formation of reducing power
from potato soluble starch or b-limit dextrin prepared from
soluble starch (Garcia-Maya et al., 1980), by the method of
Bernfeld (Bernfield, 1955). The enzyme activity was expressed
as mmol maltose equivalent released min−1. As to b-amylase
activity, the degradation of p-nitrophenyl-maltopentaose
(PNPG5), a substrate specific for b-amylase of cereals
(Matheson and Seabourn, 1983), was determined using assay
kits purchased from Megazyme (Ireland). The activity was
expressed as the amount of PNPG5 hydrolysed min−1.
Proteolytic activity was determined using azocasein as substrate
according to a previous paper (Morohashi, 1982). Activities of
malate dehydrogenase and triose-phosphate isomerase were
assayed as described before (Morohashi and Shimokoriyama,
1975).
Determination of free sugars and starch
Free sugars were extracted from cotyledons with hot 80%
ethanol. After ether-treatment for removing lipids, extracts were
concentrated using a rotary evaporator. Sugars in the extract
were analysed with a HPLC system equipped with a refractive
index detector (Shimadzu). A Shim-pack CLC-NH column
2
(Shimadzu) was used for separating sugars. Sugars were eluted
with 75% (v/v) acetonitrile at a flow rate of 1 ml min−1 at 40 °C.
In order to determine starch contents, cotyledons were treated
with CHCl -methanol (351, v/v) to remove lipids, dried and
3
pulverized. The powder was subjected to starch analysis by the
method of Lustinec et al. (Lustinec et al., 1983).
Results and discussion
Partial purification of b-amylase
Air-dried cucumber cotyledons were homogenized in 50 mM
K-phosphate buffer (pH 5.5) containing 10 mM 2-ME (100 g
in 300 ml ) with a Waring blender. The homogenate was
centrifuged at 10 000 g for 10 min. The supernatant was filtered
through glass-wool to remove floating lipids. Solid (NH ) SO
4 2 4
was added to the filtrate to 50% saturation. After centrifugation,
precipitated proteins were dissolved in 10 mM K-phosphate
buffer (pH 6.3) containing 10 mM 2-ME (buffer A), dialyzed
against buffer A and applied to a DEAE-Toyopearl column
(2.6×25 cm) equilibrated with buffer A. After washing the
column with buffer A, the enzyme was eluted with a linear
NaCl gradient (0–0.2 M in buffer A). Fractions with amylolytic
activity were collected and subjected to glycogen precipitation
according to Silvanovich and Hill (Silvanovich and Hill, 1976).
The precipitate was dissolved in 10 mM K-phosphate (pH 6.8)
containing 10 mM 2-ME (buffer B) and applied to a hydroxylapatite column (1 cm×6 cm) equilibrated with buffer B. After
washing the column with buffer B, the amylolytic enzyme was
eluted with a linear gradient of K-phosphate (10–300 mM,
pH 6.8). Fractions with amylolytic activity were collected and
used for examining the action pattern of the enzyme. All steps
were performed at 4 °C.
Isoelectric focusing
Isoenzymes in the peak fraction eluted from the DEAEToyopearl column were separated by isoelectric focusing (IEF )
(pH range of 4–6.5) at 10 °C using an LKB Multiphor
apparatus according to the maker’s instruction. At the end of
focusing, the IEF gel was immersed in 50 mM K-phosphate
buffer (pH 6.8) containing 10 mM 2-ME for 20 min at 28 °C,
Changes in fresh weight
The fresh weight of excised cotyledons increased during
the experimental period (3 d) when incubated in water
( Fig. 1A). When the cotyledons were treated with PEG
at concentrations of 20% (w/v) or higher, the increase in
fresh weight was reduced or even decreased. As seen from
Fig. 1B, the changes in fresh weight reflect the changes in
water content in the tissues. Products of the hydrolysis
of reserves were shown to accumulate in excised mung
bean cotyledons, because translocation of the hydrolysis
products to growing tissues (axis) did not occur
(Morohashi, 1982). Sucrose was observed to accumulate
markedly in excised cucumber cotyledons as well (see
below). It is, therefore, supposed that water potential of
excised cucumber cotyledons would decrease. This probably causes water uptake by the cotyledon from the
incubation medium with higher water potential (water)
( Fig. 1). In cotyledons incubated in the media with lower
water potentials (30% or more concentrated PEG solutions), dehydration from the tissues occurs (Fig. 1), even
if the hydrolysis products accumulate.
Changes in amylolytic activities
Figure 2 shows the changes in total amylolytic activities
determined with soluble starch as the substrate. The
Water stress in cucumber 741
Fig. 1. Changes in fresh weight (A) and water content (B) of cucumber cotyledons. Cotyledons were excised from 4-d-old seedlings and treated
with PEG at concentrations of 0, 20, 30, 40, and 50% (w/v).
Fig. 2. Changes in amylolytic activity in cucumber cotyledons treated
with PEG at concentrations of 0, 20, 30, 40, and 50% (w/v). Substrate:
soluble starch. The means and standard errors of the means (vertical
bars) of three replicates are shown.
activity in control (water-treated) cotyledons changed
little during the experimental period. In contrast, the
activity increased markedly upon treatment with 30%
PEG and attained a level 6 times as high as the initial
level after 2 d. Thereafter, the activity declined. In
cotyledons treated with 40% or 50% PEG, the activities
increased much more rapidly than those in cotyledons
treated with 30% PEG and attained a maximum 12 h
after the beginning of the treatment at almost the same
level as that attained 2 d after the start of the treatment
with 30% PEG. The activity remained at this level during
the following 12 h and then declined. The increase in
amylolytic activity was also observed in cotyledons that
had been air-dried for 20 h to approximately 65% of the
initial weight (data not shown). It is, therefore, evident
that the increase in the activities in PEG-treated cotyledons is caused by water stress. The decline of the activities
after prolonged incubation in PEG solutions is presumably due to inappropriate physiological conditions caused
by severe water stress.
The amylolytic activities, as shown in Fig. 2, were
determined by assaying the formation of reducing power
from starch. a-Amylase, b-amylase, debranching enzyme,
and a-glucosidase are generally thought to be responsible
for such activities (Beck and Ziegler, 1989). Little activity
of debranching enzyme was detected in cucumber cotyledons when assayed with pullulan as the substrate (data
not shown). In order to examine the possibility of the
involvement of a-amylase, the hydrolysing activity against
b-limit dextrin was determined, but only a very low
activity ( less than 6% of total amylolytic activity) was
detected in PEG-treated cotyledons. Furthermore, no
amylolytic activity against starch azure, another aamylase-specific substrate, was present in the cotyledons
(data not shown). These results indicate that a-amylase
is not the major amylolytic activity induced by water
stress.
On the other hand, the degradation activities against a
b-amylase-specific substrate, PNPG5 (Matheson and
Seabourn, 1983), was found to be present in cucumber
cotyledons. It has been pointed out that, although PNPG5
is specific for cereal b-amylase, a-amylase from potato
tuber can accept this substrate (Nielsen et al., 1997). It
is, therefore, possible that PNPG5 degradation activities
detected in cucumber cotyledons are also due to a-amylase
activities. Since a-amylase is practically absent in cucumber cotyledons (see above), it is likely that PNPG5
degradation in cucumber is due to b-amylase action.
b-Amylase activity assayed using PNPG5 as the substrate
remained at a constantly low level in water-treated control
742 Todaka et al.
cotyledons (Fig. 3). In cotyledons treated with 30% PEG,
the activity increased during 2 d of the treatment to a
level about 6 times as high as the initial level. In cotyledons
treated with 50% PEG, the activity increased more rapidly
than that in cotyledons treated with 30% PEG and, after
12 h of the treatment, attained almost the same level as
that in cotyledons treated with 30% PEG for 2 d. The
activity in cotyledons treated with 50% PEG decreased
thereafter. This pattern of changes in b-amylase activities
was similar to that in total amylolytic activities (Fig. 2).
This indicates that b-amylase activity is mainly responsible
for the amylolytic activity detected in water-stressed
cotyledons. To confirm that b-amylase is the stress-induced
amylolytic activity, products of soluble starch digestion
were analysed by TLC (Fig. 4). As seen from Fig. 4, the
sole product was detected at the position of maltose. An
analysis by HPLC gave the same result (data not shown).
Although the presence of a-glucosidase activity was
not checked, the involvement of this enzyme is unlikely,
because free glucose could not be detected in the product
of starch hydrolysis by TLC (Fig. 4) and HPLC analyses
(data not shown). Taken together, the enhancement of
amylolytic activity in cucumber cotyledons by water stress
is mainly due to an increase in b-amylase activity.
It has been reported that water stress enhances the
activity of a-amylase in barley leaves and that the
enhancement is due to the synthesis of the enzyme protein
(Jacobsen et al., 1986). When cucumber cotyledons were
incubated in 40% PEG solution containing cycloheximide
(10−4 M ), the enhancement of amylolytic activities was
severely (about 80%) inhibited ( Table 1). This indicates
that the increase in activity caused by water stress is
dependent on de novo protein synthesis.
Fig. 4. TLC analysis of products formed from soluble starch by
b-amylase purified at the step of hydroxylapatite chromatography.
Lanes 1 and 2, glucose and maltose, respectively; lanes 3 and 4,
products obtained after reaction for 30 and 0 min, respectively.
Table 1. Effects of cycloheximide on the development of
b-amylase activity
Cotyledons excised from 4-d-old seedlings were treated with water,
PEG (40% [w/v]) or PEG+cycloheximide (10−4 M ) for 24 h. Substrate,
soluble starch. Shown is the mean of, and difference between (in
parenthesis), two replicates.
Treatment
Activity
(mmol maltose min−1 per pair of cotyledons)
Water
PEG
PEG+cycloheximide
0.05 (0.00)
0.29 (0.04)
0.09 (0.01)
Isoforms of b-amylase
Fig. 3. Changes in b-amylase activity in cucumber cotyledons treated
with PEG at concentrations of 0, 30 and 50% (w/v). Substrate: PNPG5.
The means and standard errors of the means (vertical bars) of three
replicates are shown.
Multiple forms of b-amylase have been reported in some
plants; for example, seven isoforms in soybean (Mikami
et al., 1982), four (Lundgard and Svensson, 1987) and
three isoforms in barley (Dreier et al., 1995), and two
isoforms in alfalfa (Doehlert et al., 1982), yam (Arai
et al., 1991), maize (Lauriere et al., 1992), and rye (Rorat
et al., 1995). In some plants, for example, pea (Lizotte
et al., 1990) and potato ( Vikso-Nielsen et al., 1997),
only a single form has been reported. To examine the
presence of isoforms of b-amylase in cucumber cotyledons, the peak fraction eluting from the DEAE-Toyopearl
column was subjected to IEF and subsequent iodinestaining for the detection of amylase activities (soluble
starch as the substrate). Three major bands with pIs of
approximately 5.7, 5.9 and 6.0, respectively, and three
minor ones with pIs of about 5.5, 6.2 and 6.3, respectively,
were detected in PEG-treated cotyledons (Fig. 5). These
bands did not appear when b-limit dextrin was used as
Water stress in cucumber 743
hydrolase, protease, in PEG-treated cotyledons was determined by using azocasein as the substrate. No stimulatory
effect of water stress was observed (data not shown).
Activities of malate dehydrogenase and triose-phosphate
isomerase were also not affected by water stress (data not
shown). Thus, b-amylase development resulting from
water stress seems to be a rather specific phenomenon.
Changes in contents of sugars and starch
Fig. 5. IEF gel of b-amylase in the peak fraction from DEAE-Toyopearl
chromatography. Lane 1, cotyledons treated with 40% (w/v) PEG for
1 d; lane 2, water control. The gel-sandwich was incubated at 28 °C for
7 h in the former case ( lane 1) and 18 h in the latter case ( lane 2).
the substrate (data not shown). When the enzyme
preparation from water-treated control cotyledons was
subjected to IEF analysis, three signals corresponding to
the three major bands present in PEG-treated cotyledons
were detected, but signals corresponding to the three
minor bands were barely detectable (Fig. 5). In this case,
the time of contact of an IEF gel with an activity gel was
prolonged to 18 h (7 h in the case of PEG-treated cotyledons) because of low enzyme activities. It is, therefore,
clear that the three forms in non-stressed cotyledons are
enhanced by water stress. On the other hand, the three
minor isoforms in stressed cotyledons seem to be newly
induced ones. The possibility, however, cannot be ruled
out that these isoforms were present in non-stressed
cotyledons as well, but undetectable because of their low
activities. An increase in b-amylase activity in barley
leaves subjected to osmotic stress was observed; in this
case, three isoforms in non-stressed leaves were enhanced
but no new isoform was induced (Dreier et al., 1995).
Major free (alcohol-soluble) sugars present in cucumber
cotyledons were glucose, fructose, maltose, and sucrose,
as revealed by HPLC analysis (data not shown). Among
them sucrose was the most abundant and changed most
markedly during incubation; changes in the contents of
other sugars were small relative to that of sucrose (Fig. 6).
Sucrose accumulated rapidly in water-treated cotyledons.
Cucumber cotyledons contain lipids as major reserves
(Bewley and Black, 1994). Reserve lipids are actively
metabolized to sucrose through gluconeogenesis and the
sucrose formed is transported to the growing axis (Bewley
and Black, 1994). In the present study, detached cotyledons were used for experiments (Materials and methods).
The removal of the sink (axis) brings about the accumulation of hydrolysis products in the source (cotyledon)
(Morohashi, 1982). This is why sucrose accumulated in
water-treated cucumber cotyledons.
Starch also accumulated in water-treated cotyledons
( Fig. 7). Sucrose is considered to be the primary substrate
Effects of water stress in the hypocotyl
In order to examine whether the stimulatory effects of
water stress on b-amylase activity were specific in the
cotyledons or seen in other tissues as well, the development of the enzyme activity in water-stressed hypocotyls
was studied using PNPG5 as the substrate. The activities
in hypocotyl sections treated with water and 40% PEG
for 1 d were 3.9 and 9.4 nmol PNPG5 min−1 hypocotyl
section−1, respectively. Thus, water stress caused an
increase in b-amylase activity in the hypocotyl, indicating
that the enhancement of the enzyme activity by water
stress is not limited to cotyledons.
Effects of water stress on other enzymes
It is of interest to know whether or not the effects of
water stress are b-amylase-specific. The activity of another
Fig. 6. Changes in sugar contents in cucumber cotyledons. Cotyledons
were excised from 4-d-old seedlings and treated with PEG at
concentrations of 0 (#), 30% ($) and 40% (w/v) (6). -----, Fructose;
––––, glucose; –-–-–, maltose; and ——, sucrose. The means of, and
differences between (vertical bars), two replicates are shown.
744 Todaka et al.
Fig. 7. Starch contents in cucumber cotyledons. Cotyledons excised
from 4-d-old seedlings were treated with water or PEG (30% or 40%
[w/v]) for 1 or 2 d. The means and standard errors of the means
(vertical bars) of three replicates are shown.
for starch synthesis (Duffus and Duffus, 1984). It has
been reported that the concentrations of sucrose and
starch increase in excised and illuminated leaves of
eggplants (Claussen et al., 1985). It is also known that
starch content increases under the conditions where
sucrose content is increased (e.g. exogenous application of
sucrose) (Nakamura et al., 1991). It is, therefore, probable
that a part of accumulated sucrose in excised cucumber
cotyledons is metabolized to starch.
More sucrose and less starch accumulated in PEGtreated cotyledons than in water-treated ones (Figs 6, 7).
It has been reported that water stress causes an active
conversion of starch to sucrose in bean leaves (Stewart,
1971). If this is also the case with cucumber cotyledons,
the phenomenon that the stress causes a decrease in starch
content and an increase in sucrose content is explainable.
In such a case, b-amylase induced by water stress is
possibly involved in starch degradation. However, it has
been pointed out that b-amylase of pea epicotyls is not
able to attack native starch without their prior digestion
by other enzymes (Lizotte et al., 1990). If cucumber
b-amylase is also unable to digest native starch, then
what is the physiological function of the b-amylase development in water-stressed cucumber cotyledons? Further
experiments are necessary to elucidate these points.
It was shown that exogenously supplied sucrose induces
the expression of b-amylase gene in leaf-petiole cuttings
of sweet potato (Nakamura et al., 1991). There is the
possibility that a similar mechanism is present in
cucumber cotyledons; the accumulated sucrose might be
responsible for the induction of b-amylase. This, however,
is unlikely in cucumber because no enhancement of
b-amylase activity occurred in control cotyledons (Figs
2, 3) in spite of the marked accumulation of sucrose
( Fig. 6).
An important question is what is the physiological
significance of sucrose accumulation in water-stressed
cotyledons. It is known that osmoregulation that involves
solute accumulation occurs in water-stressed cells,
resulting in a decrease in the osmotic potential of the
cells (osmotic adjustment) (Meyer and Boyer, 1981;
Matsuda and Rayan, 1990). A working hypothesis is that
sucrose might function as one of the osmotic components
in osmotic adjustment. Sucrose accumulation has been
reported to occur in bean leaves as a result of water stress
(Stewart, 1971). It has been reported that water stress
enhances the expression of a-amylase in barley (Jacobsen
et al., 1986). Although they did not determine the contents
of sugars, a-amylase induced by water stress may play a
role in increasing the contents of sugars. However, there
is no direct evidence for this working hypothesis at
present.
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