Plant Cell Physiol. 31(2): 167-172 (1990)
JSPP © 1990
Lack of Functional Interrelationship between /5-amylase Photoregulation and
Chloroplast Development in Mustard (Sinapsis alba L.) Cotyledons
V. A. Manga and R. Sharma'
School of Life Sciences, University of Hyderabad, Hyderabad-500 134, India
Key words: /?-Amylase — Chloroplast development — Mustard — Phytochrome — SAN 9789 —
Sinapis alba L. — Subcellular localization.
/?-Amylase, a starch degrading enzyme is present in
many species of higher plants and in a few species of
microbes. In higher plants, bulk quantity of /?-amylase is
present in the organs which are involved in active metabolism of starch such as seeds, roots and tubers (Greenwood
and Milne 1968). The studies on in vivo regulation of /?amylase in cereal seeds have revealed that during seed germination, /?-amylase is activated from a preformed zymogen form. (Daussant 1977). The activated /?-amylase in
conjuction with a-amylase which is synthesized de novo initiate the degradation of starch stored in endosperm (Manners 1985). In comparison to seeds, in photosynthetic
tissues the in vivo regulation of /?-amylase activity and its
participation in starch metabolism is not well investigated.
In the cotyledons of mustard (Sinapis alba L.) seedlings, light initiates de novo synthesis of /?-amylase (Sharma and Schopfer 1982, 1987). The onset of above increase
in /J-amylase level correlates with the development of
various other photosynthetic processess in mustard seedlings (Frosch et al. 1976, 1977, Oelze-Karow and Mohr
1982). Moreover, in mustard /?-amylase is the sole amylolytic enzyme (Sharma and Schopfer 1982). In present
1
Abbreviations: DW, distilled water; RL, red light.
To whom correspondence should be addressed.
study, we investigated the possibility whether photoregulation of /ff-amylase has a functional link with chloroplast development and starch degradation in developing cotyledon.
Materials and Methods
Standard techniques for photomorphogenesis with
mustard seedlings were used (Mohr 1966), Mustard
(Sinapis alba L., 1977 harvest) seeds were germinated on
chromatographic papers soaked with DW or 0.4 nui SAN
9789 (Sandoz, Switzerland) solution at 25°C and were
either grown in darkness or were irradiated with continuous red (A max, 650 nm, 0.67 W • m~2) or white (2.4 W •
m" 2 ) light (Manga and Sharma 1985). The enzyme unit
Katal is defined as a mole of maltose released per second at
30°C. The extraction and assay of yS-amylase [EC 3.2.1.2]
activity was performed as described earlier (Manga and
Sharma 1985). The amount of starch present in the
cotyledons of light/dark grown seedling was estimated by
the method of Ching et al. (1983), and also by method of
Beutler (1978). These methods could detect up to a
minimum of 2//g/ml starch present in the crude extract.
Absorption spectroscopy—The in vivo absorption
spectrum of mustard cotyledons was recorded by monitoring absorbanbce of a single cotyledon in dual wavelength
167
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In the cotyledons of mustard seedlings light mediates an increase in /?-amylase [EC 3.2.1.2]
activity via agency of phytochrome. In order to understand the functional significance of above
photoresponse, the relationship between light induced yS-amylase increase, chloroplast development and starch content of cotyledon was investigated. The application of SAN 9789 a chlorosis
inducing inhibitor to mustard seedlings, though destroyed chloroplast, had no effect on light mediated increase in /?-amylase indicating the lack of functional interrelationship between chloroplast development and /?-amylase increase. The subcellular localization studies revealed that
/?-amylase is a cytosolic enzyme. Additionally, the increase in the level of /?-amylase had no relationship with in vivo starch level, which was present only in trace amounts. The noncorrelation
of the photoregulated /J-amylase increase with the starch content and its extra-chloroplastic localization indicates that /?-amylase does not participate in the mobilization of plastidic starch in
mustard cotyledon.
168
V. A. Manga and R. Sharma
homogenate were assayed for /?-amylase.
Sucrose density gradient centrifugation—The crude
homogenate obtained as described above was strained
through a single layer of muslin cloth and 1.0 ml of it was
layered on the top of a sucrose gradient (22 ml, 25-60%
w/w) in nitrocellulose centrifuge tubes. The tubes were
centrifuged at 83,000xg at 2°C in a SW 27 rotor in MSE
ultracentrifuge for 3 h. After centrifugation gradient was
fractionated by drawing 1 ml fractions from the top of gradient with a P-1000 Pipetman. The fractions were analyzed for various organelles markers and /?-amylase. The
density of the sucrose fractions was determined by the
following equation in an Abbe refractometer at 20°C:
(p = 2.7329 x n20°C 2.645.) The density of sucrose in each
fraction in conjunction with the marker enzyme data was
used to determine the organelle distribution in the fractions
(Hall and Moore 1983).
Results and Discussion
In mustard seedlings grown under light, cotyledons
expand, turn green and acquire the capacity to photosynthesize displaying characteristic features of photomorphogenesis (Mohr 1977, Kasemir 1983). In developing
mustard seedlings the onset of photoregulation of /?amylase activity is temporally very close to the development of various photosynthetic processes in cotyledons.
Since /?-amylase is the sole principal amylolytic enzyme of
mustard seedlings (Sharma and Schopfer 1982), it is likely
that the photoregulation of /?-amylase may be closely linked to the development of photosynthesis in the cotyledon
for the mobilization of starch in chloroplasts. The interrelationship between increase in /?-amylase and chloroplast
development was investigated by growing mustard seedlings with SAN 9789, a chlorosis inducing inhibitor. The
seedlings treated with SAN and grown in light are totally
photosynthetically inactive as the photodestruction of chlorophyll molecules by light initiates a cascade effect leading
to a complete degeneration of internal architecture of developing chloroplasts (ReiB et al. 1983, Oelmuller 1989). At
the same time, SAN does not have any significant effect on
other photomorphogenetic processes of seedlings (Jabben
and Dietzer 1978, Drumm-Herrel and Mohr 1982).
In mustard seedlings, SAN treatment leads to a marked reduction in the level of many photoregulated plastidic
enzymes such as NADP-glyceraldehyde 3-phosphate dehydrogenase (ReiB et al. 1983), ribulose bisphosphate carboxylase (Oelmuller and Mohr 1986), nitrite reductase (Rajasekhar and Mohr 1986) etc. However, at the same time
SAN has no effect on the level of cytosolic enzymes (ReiB et
al. 1983). Fig. 1 shows that SAN has no inhibitory effect
on the photoregulation of yS-amylase activity in mustard
cotyledons. In seedlings grown in SAN under continuous
RL the kinetics of light mediated rise in /S-amylase activity
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mode with At fixed at 750 nm and A2 scanning from 350 nm
to 750 nm using a specially designed cuvette in Hitachi 557
dual-wavelength spectrophotometer. The absorption spectrum was recoreded after positioning the cotyledon closest
to the photomultiplier tube to minimize the light scattering. Alternatively, cotyledons were homogenized with
80% acetone and chlorophyll and carotenoid content was
measured by the formula of Vernon (1960) and LiaaenJensen and Jensen (1971) respectively.
Differential centrifugation—20 pairs of cotyledons
harvested from 96 h old red light or dark grown seedlings
were homogenized on ice in a precooled mortar and pestle
with 2 ml Na-citrate buffer (20 mM, pH6.1) supplemented
with 0.4 M sucrose and 1 mM dithioerythritol. The homogenate after dilution to 7 ml with extraction buffer was strained through a single layer of muslin cloth to get rid of
cellular debris. A 500 /ul aliquot of homogenate was retained for determination of total /S-amylase activity present in
the crude extract. The crude homogenase was subjected to
sequential centrifugation for lOmin each at 500 xg and
2,000 x g to obtain pellets enriched in nuclei and plastids respectively. The pellets enriched in mitochondria and
ribosomes were isolated by centrifuging the supernatant
from last step at 20,000 x g for 30 min and 150,000 x g for
2 h respectively. The pellets so obtained were washed
twice with extraction buffer and were resuspended in 1 ml
of extraction buffer. A 100//I aliquot from each fraction
was used for the assay of /?-amylase activity.
Isolation of intact chloroplasts—40 pairs of cotyledons harvested from 96 h old red light grown seedlings
were homogenized in 6 ml of semi-frozen isolation medium
(0.33 M Sucrose, 0.05 M Tetra-sodium pyrophosphate,
2.0 mM EDTA, 5.0^M Ascorbate, pH 7.8). The homogenate was strained immediately through a single layer of
muslin cloth. The crude extract was centrifuged at
30,000 x g for 30 min at 4°C. The supernatent was decanted in one motion. The pellet was washed once by centrifugation. The final pellet was resuspended in a one ml of
resuspension buffer and loaded over a step gradient of sucrose solution (25%, 34% and 5 1 % Sucrose in 10 mM Tris,
pH 7.8). Chlorophyll was estimated from one aliquot
before loading. The gradient was centrifuged at 2,000 x g
for 15 min at 4°C. Intact chloroplasts were recovered as a
broad band at the interface of 34% and 5 1 % sucrose layers
and envelopes free chloroplasts at the interface of 24% and
34%. Both the bands were collected in two separate tubes
and diluted by dropwise addition of Tris (HC1 50 mM
pH 8.0) and 0.3 M sucrose simultaneously. The contents
were then centrifuged at 30,000 x g for 5 min at 4°C. The
pellet was washed once more and resuspended in 5 ml of
wash buffer (Hall and Moore 1983). Chlorophyll was
estimated in two pellets. Intactness of the chloropasts was
checked by ferricyanide reduction assay (Hall and Moore
1983). Intact and broken chloroplast fractions and crude
Photoregulation of /?-amylase
169
Fig. 1 Effect of SAN 9789 on the time course of fi-amylase activity in the mustard cotyledons.
Seedlings were grown in
SAN 9789 from the time of sowing in continuous red light (A) or
in darkness (A). The control seedlings were similarly grown in
distilled water in red light (•) or in darkness (O).
followed a similar profile to control seedlings grown without SAN. Similarly in seedlings grown in darkness with or
without SAN no difference in )S-amylase activity was apparent. The above results indicate that yS-amylase may be
a cytosolic enzyme in mustard cotyledons.
ReiB et al. (1983) reported that in mustard seedlings
SAN mediated inhibition of plastidic enzymes requires complete degeneration of chloroplasts. It is likely that under
red light SAN may not have completely destroyed chloroplast and thereby had no effect on /?-amylase activity. The
cotyledons of above seedlings were therefore spectroscopically scanned for possible presence of chloroplasts. The
in vivo absorption spectrum revealed a diminished peak of
400
500
600
WAVELENGTH I n m )
Fig. 2 In vivo absorption spectra of mustard cotyledon.
The
solid arrow indicates the position of chlorophyll peak. The seedlings were grown with DW or SAN under continuous red light
from the time of sowing. At 72 h from sowing, the in vivo absorption spectra of cotyledon was recorded for one set of seedlings (a),
while another set of seedlings was transferred to continuous white
light for 24 h. Thereafter, the cotyledons were harvested and absorption spectra recorded (b).
chlorophyll in comparison to DW grown control (Fig. 2a).
The quantitative estimation of photosynthetic pigments in
above seedlings revealed that SAN treatment totally
eliminated carotenoids both in red light and dark grown
seedlings (Table 1). However, this SAN/RL seedlings had
about 10% chlorophyll level of DW/RL seedlings, which
might signify incomplete destruction of chloroplasts.
Table 1 The relative amount of chlorophylls and carotenoids in the cotyledon of 72 h old mustard seedling
Pigment content
Chlorophyll
Treatment
ir cotyledon
Relativeamount
Carotenoid
, .
. , .
Relativeamount
fig/pair cotyledon
,0/
Red light
Control
5.09
100
SAN
0.46
9
0
Control
0
0
0.60
SAN
0
0
0
1.65
100
0
Dark
36
0
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TIME AFTER SOWING ( M
170
V. A. Manga and R. Sharma
S
0.2 -
650
WAVELENGTH
700
(nml
Since existence of above low amount of chlorophyll in
SAN/RL seedlings results due to inefficiency of red light
(0.67 W m ~ 2 ) in completing the photobleaching of cotyledon, the photodestruction of chloroplast was completed by
exposing above seedling to white light (Fig. 2b). Fig. 3
shows that white light exposure to above seedlings leads to
complete destruction of functional chloroplast as evident
by disappearance of chlorophyll peak. At the same time,
the chloroplast elimination did not inhibit the photoregulated rise in /?-amylase activity which followed identical profile to DW control seedlings (Fig. 4). Since above treatment completely eliminates chloroplasts (ReiB et al. 1983)
it can be concluded that in mustard cotyledon photoregulation of /?-amylase is not associated with chloroplast development.
Since in leaves the starch is localized only in the chloroplasts (Halmer and Bewley 1982) it is logical to assume a
plastidic location for /?-amylase. On subcellular localization of /?-amylase by differential centrifugation of cell ho-
TIME AFTER SOWING ( h >
Fig. 4 Time course of /?-amylase activity in the cotyledons of
SAN treated seedlings after transfer from red light to continuous
white light.
The seedlings were grown in SAN (A) or in distilled water (•) under continuous red light from the time of sowing.
At 72 h, the seedlings were transferred to continuous white light.
The solid arrow indicates the time of transfer of the seedlings
from continuous red light to white light.
mogenate about 95% of /?-amylase activity was recovered
exclusively in the final supernatant indicating a cytosolic localization of yS-amylase (data not shown). The sucrose
density gradient centrifugation of cell homogenate also confirmed the cytosolic localization of yS-amylase enzyme.
While organelles like mitochondria and chloroplasts migrated in the sucrose gradient the /?-amylase activity was
localized only in topmost fractions indicating a cytosolic localization of/9-amylase (Fig. 5). Since above methods may
yield partially broken chloroplasts, we isolated intact
plastids, as judged by ferricyanide reduction in a step gradient of sucrose (Table 2). However, after osmotic shock
lysis of above plastids no /?-amylase activity could be detected in above plastids confirming that /?-amylase in
mustard cotyledons is localized in extraplastidic compartment. The results with SAN are also more akin to a cytosolic localization of/ff-amylase, as ReiB et al. (1983) report-
Table 2 The relative amount of /?-amylase, chloropast recovery in different fractions of 96 h old mustard cotyledons and
ferricyanide reduction of intact chloroplast fraction
Fractions
Crude homogenate
Supernatent
Pellet
Intact chloroplast
/?-Amylase
activity
Chloroplast
recovery
Ferricyanide reduction
Before osmotic
shock
After osmotic
shock
100
—
—
—
98
—
—
—
100
—
—
11
No reduction
36
0.1
No activity
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Fig. 3 In vivo absorption spectra of a single cotyledon of 72 h
old mustard seedling grown with SAN under continuous red light
from the time of sowing (1).
The cotyledon was subjected to
three strong white light pulses each of 1 min duration, in situ in
the spectrophotometer. Immediately after each pulse the absorption spectra of the cotyledon was recorded (2, 3, 4). After 3rd
pulse, there was no further decrease in the peak height by subsequent white light pulses.
Photoregulation of fi-amylase
ed that SAN specifically destroys only plastidic enzymes
and has no effect on the photoregulation of cytosolic enzymes. Furthermore, when poly A + RNA from mustard
cotyledon is translated in vitro, and /?-amylase specific polypeptide is immunoprecipitated by /5-amylase specific antibody, it has a molecular mass identical to in vivo labelled
/?-amylase protein (58,000). Our experiments gave no indication of presence of N-terminal signal peptide which
is needed for translocation to chloroplasts (Sharma and
Schopfer 1987). All of these experiments clearly indicate
that in mustard cotyledons /?-amylase is a cytosolic enzyme
and its photoregulation by light is in no way linked to chloroplast biogenesis, another process which is also dependent
on light. The cytosolic localization of /?-amylase in mustard cotyledon is in anomaly with its logical function where
it should have been localized in chloroplast for the mobilization of starch.
The extent of /?-amylase participation in the starch degradation was investigated by studying the level of starch in
mustard cotyledons at 24 h intervals between 24-120 h
from sowing. However, no starch could be detected in
mustard cotyledons both in red light and dark grown seedlings even after using two different methods (Ching et al.
1983, Beutler 1978). All possible alterations like increasing the quantity of experimental material did not give any
positive results for the presence of starch in cotyledon up to
five days from sowing. Schafer (1983) reported that in developing mustard seed by the time of seed ripening the ab-
solute amount of starch falls to a very low level ( < 1 ng per
seed) due to its mobilization to synthesis of reserve fats.
It is likely that in mustard seedlings starch may be synthesized in plastids only during later phase of seedling
growth. Furthermore, the cytosolic localization of /?-amylase negates its possible role in plastidic starch degradation,
due to its spatial separation.
In summary, the photoregulation of /?-amylase has no
relation to chloroplast development and plastidic starch
mobilization in mustard cotyledons. In view of cytosolic
location of /J-amylase, the physiological significance of photoregulated increase in /S-amylase level in cytoplasm is not
clear, since it is not functionally related to initiation of photosynthesis and plastidic starch degradation in plants.
Perhaps, the photoregulation of /?-amyIase in mustard
cotyledon represents a situation of evolutionary relict.
However, recent reports showing the presence of soluble
maltodextrins in cytosols of few plants (Steup 1988) indicate that perhaps cytosolic /?-amylase may participate
in degradation of these dextrins in plants. Although it has
been reported that /?-amylase may also to some extent has
synthetic capability (Hehre et al. 1979) the existence of such
a catalytic action by /3-amylase in vivo seems to be remote.
The authors wish to thank the Dean, School of Life Sciences
for his support. This work was supported by a research grant
(F-3-115/86-SRII) to R. S. by University Grants Commission,
New Delhi. V. A. M. is recepient of FIP.
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