Journal of Experimental Botany, Vol. 47, No. 297, pp. 577-582, April 1996
Journal of
Experimental
Botany
Metabolic activities in germinated ancient lotus seeds
Yuri Maeda1, Takao Minamikawa1'3 and Ben-Mei Xu 2
1
2
Department of Biology, Tokyo Metropolitan University, Minami-ohsawa, Hachioji, Tokyo 192-03, Japan
Beijing Botanical Garden, Institute of Botany, Academia Sinica, Xiangshan, Beijing, China
Received 9 September 1995; Accepted 14 December 1995
Radiocarbon dating using seed samples from this collection revealed their age to be 580 + 70-years-old (Zhang
Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi)
et ai, 1985). This age was supported by a document
were unearthed in a western suburb of Beijing in 1984,
describing the location where the seeds were found as a
and determined to be 580 + 70-years-old by radiocarflower garden of an administrative noble family in the
bon dating. Pretreatment with concentrated sulphuric
early Ming era. The seed of Taizi lotus is surrounded by
acid for 6 h promoted 75% of the seeds tested to
an extremely hard pericarp and thus, in a strict sense, the
germinate. Over 3 weeks of incubation after the addidispersal unit should be called a dry fruit. It is necessary
tion of water, storage starch, total soluble sugar and
to puncture the hard external structures to allow imbibiglobulin in the cotyledons decreased whereas albution to occur; this can be done by piercing the hilum with
min and soluble a-amino nitrogen increased. These
a sharp object or incubating for 6 h in concentrated
changes in cotyledonary components were similar to
sulphuric acid. Taizi lotus showed a germinability higher
the changes seen in four present-day Indian lotus varithan 90% after pretreatment with sulphuric acid in 1984.
eties, although the content of reducing sugar in Taizi
Biochemical studies in 1988 showed that the ATP content
lotus was significantly lower. The polypeptide comof Taizi lotus was similar to that of modern cultivars (Gu
positions in extracts from the cotyledons and embryet al, 1988).
onic axis of Taizi seeds, as analysed by SDS-PAGE,
In this paper an attempt has been made to examine
were similar to those of present-day Jiangxi seeds.
fundamental metabolic activities maintained in ancient
Taizi lotus also showed the ability to incorporate radioTaizi seeds and to report on analyses of cotyledonary
active leucine into the trichloroacetic acid (TCA)-insolcomponents before and after germination. Preliminary
uble fraction of a cotyledonary extract during the 24 h
analyses were carried out with seeds of the present-day
post-imbibition period. The incorporation was inhibIndian lotus cultivar collected at Jiangxi to minimize the
ited when seeds of Taizi lotus were allowed to rehynumber of Taizi lotus seeds to be used. The Jiangxi lotus
drate for 24 h in the presence of a-amanitin or
has morphological features similar to the Taizi lotus and
cycloheximide.
was readily available. The cotyledonary components of
three other present-day Indian lotus varieties were also
Key words: Ancient lotus, Nelumbo nucifera, protein synexamined and the data compared with those obtained
thesis, seed germination, seed viability.
from the Taizi lotus. Analytical data suggest that the
ancient lotus maintains the ability to metabolize storage
components in the cotyledons after germination. The
polypeptide compositions in extracts from the cotyledons
Introduction
and embryonic axis of Taizi seeds, as analysed by
Seeds of the Taizi lotus (Nelumbo nucifera Gaertn cv.
SDS-PAGE, were similar to those of present-day Jiangxi
Taizi) were unearthed from a pond deposit (1.2-1.7 m in
seeds. The ancient lotus also showed activity of protein
depth) at Taizhou village in the western suburbs of Beijing
synthesis comparable to that of the Jiangxi lotus, as
in 1984. There was no record or remembrance that lotus
determined by the incorporation of a labelled amino acid
plants had been grown in this area in modern times.
into the TCA-insoluble fraction of cotyledons.
Abstract
3
To whom correspondence should be addressed. Fax: +81 426 77 2559.
Abbreviations: CBB, Coomassie brilliant blue R; LSU and SSU, large and small subunits of Rubisco; PAGE, polyacrylamide gel electrophoresis;
Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; TCA, trichloroacetic acid.
© Oxford University Press 1996
578
Maeda et al.
Materials and methods
Plant materials
Seeds of Taizi lotus were collected in a suburb of Beijing, China
in 1984 (Zhang et al., 1985). Present-day Indian lotus seeds
that are morphologically similar to seeds of Taizi lotus were
collected at Jiangxi, Hebei, Shanghai, and Harbin in 1990 to
1991, and are given the name of their location. Seeds were
stored in a cold room conditioned at 0-4 °C until use. Before
the seeds were set to germinate, they were immersed in
concentrated sulphuric acid at room temperature for 6 h (Gu
et al., 1988), washed thoroughly with running tap water
overnight, then placed semi-submerged in a plastic plate at
25 °C under a 10 h light/14 h dark photopenod. Plants grown
for 1-3 weeks were collected, the cotyledons and shoots were
separated, and stored at — 20 °C until use. Fully expanded
leaves were collected from lotus plants grown in the campus of
Tokyo Metropolitan University.
Analytical methods
Chopped cotyledons (3 g) were ground with 15 ml of 0.1 M
sodium borate buffer (pH 8.0) in an ice-cold mortar and pestle.
The homogenate was centrifuged at 20000 xg for 15 min. The
precipitated residue was re-extracted with 15 ml of the same
buffer, and the supernatant was combined with the first
supernatant. An aliquot (4 ml) of the extract was mixed with
1 ml of 40% TCA solution and centrifuged. Contents of a-amino
nitrogen, reducing and total sugars in the TCA-soluble solution
were determined by the methods of Yemm and Cocking (1955),
Somogyi (1952) and Dubois et al. (1956), respectively. Another
aliquot (15 ml) of the extract was dialysed for 48 h against
20 mM sodium citrate-phosphate buffer (pH 5.0) containing
2 mM 2-mercaptoethanol. The precipitate formed during dialysis
was collected by centrifugation and re-dissolved in 5 ml of
0.1 M sodium borate buffer (pH 8.0) containing 2 mM mercaptoethanol. This protein fraction was designated 'globulin'
and consisted of the major storage proteins, while the protein
fraction remaining in the supernatant was designated 'albumin'.
Protein content was measured by the method of Bradford
(1976) using bovine serum albumin as a standard. Denaturing
gel electrophoresis was conducted according to the procedure
of Laemmli (1970). The insoluble residue of the initial borate
extraction was washed with 50% and absolute ethanol, and
dried. Starch in the powder was hydrolysed by refluxing it in
2% HC1 for 3 h, and the amount of reducing sugar released
was measured (Somogyi, 1952). The results presented are the
means of duplicate measurements in an experiment using four
or more seeds.
Embryonic axes of the dry seed or shoots of growing seedlings
were extracted with 10 vols of the SDS-PAGE sample buffer
(Laemmli, 1970). The homogenate was boiled for 3 min and
centrifuged at 7000xg for 15 min. Proteins in the supernatant
were analysed by SDS-PAGE on a 15% gel and immunoblotting
using rabbit polyclonal antisera raised against LSU and SSU
of Rubisco from French bean. The immunoblotting was
conducted as described elsewhere (Tanaka et al, 1993). Protein
bands were visualized by a Vectastain ABC kit (Vector
Laboratories). The antisera were kindly supplied by T. Yoshida
of our laboratory.
the cotyledons (a) and that incorporated into TCA-insoluble
material (b) were determined as described previously
(Minamikawa et al., 1983). The rate of protein synthesis was
presented as % incorporation (bx 100 a~!).
Results and discussion
Morphological changes after germination
The lotus seeds tested exhibited the emergence of the first
shoot from the hard pericarp 3-7 d post-imbibition under
the growth conditions used in the present study. Thus,
when the emergence of a shoot was observed by 7 d after
the addition of water, the seed was defined as a germinated
seed. Germination rates were 75% (12/16) for Taizi, 48%
(54/112) for Jiangxi, 100% (8/8) for Shanghai, 100%
(8/8) for Harbin, and 62.5% (5/8) for Hebei. The embryonic axes in dry (quiescent) Jiangxi seeds were often
found to be covered with fungal mycelia and their cotyledons were brownish. These seeds are thought to have lost
germinability.
The second shoot of all the varieties tested sprouted
7-10 d post-imbibition. The third shoot of Taizi sprouted
2-3 weeks post-imbibition, but no third shoot was
observed during this period for Jiangxi (Fig. 1) and the
other three lotuses. Taizi lotus may have adapted to
relatively shallow water as compared to Jiangxi and the
other three present-day lotuses. The growth rate of Taizi
shoots was much lower than those of the present-day
lotuses. The first shoot of Taizi lotus grew to about 7 cm
and 20 cm 1 week and 3 weeks post-imbibition, respectively, whereas those of Jiangxi grew to about 10 cm and
30 cm. The other three present-day lotuses exhibited
growth rates similar to that of Jiangxi (data not shown).
However, the fresh weight of Taizi shoots 3 weeks post-
Labelling experiments
Chopped cotyledons (0.2 g) were allowed to absorb L-[U14
C]leucine solution ( l x l O 6 cpmrnl" 1 . 11.5 GBqmmol"1;
DuPont/NEN). At the time intervals indicated, cotyledons were
washed with water, and both the radioactivity taken up by
Fig. 1. Development of shoots in germinated lotus seeds (Jiangxi and
Taizi). The seeds were grown under the conditions described in
Materials and methods. Two shoots of Jiangxi and three shoots of
Taizi sprouted during the 3-week post-imbibition period.
Metabolic activities in ancient lotus
imbibition was comparable to those of Jiangxi and
Shanghai, and was higher than the other two lotuses
(Figs 2, 3). The cotyledon fresh weight of Jiangxi lotus
increased due to rehydration for the first week and
decreased slightly thereafter.
Changes in cotyledonary components after germination
The amounts of major components in cotyledons of dry
seeds and 1-, 2- and 3-week-old germinated seeds of
Jiangxi lotus were measured, for which a larger number
of seeds were available for investigation (Fig. 2). The
same analysis was conducted using cotyledons of dry and
3-week-old germinated seeds of Taizi lotus and the other
Weeks
Fig. 2. Changes in growth parameters and contents of cotyledonary
components before and after germination of present-day lotus seeds
(Jiangxi). (A) Fresh weight of cotyledons. (B) Fresh weight of shoot.
(C) Content of starch in cotyledons. (D) Contents of soluble sugars in
cotyledons. Contents of total and reducing sugars are presented as
equivalents to g sucrose and glucose, respectively. (E) Content of
protein in cotyledons. The globulin and albumin fractions were
separated as described in Materials and methods. (F) Content of
soluble a-amino nitrogen in cotyledons. The content is expressed as
equivalent to mg L-leucine.
579
three present-day varieties (Fig. 3). The content of reserve
starch in dry cotyledons of Jiangxi lotus decreased gradually after germination and was reduced to less than half
at 3 weeks post-imbibition. Similar patterns of reserve
starch consumption were observed for Taizi and the other
three present-day lotuses. The content of total and reducing sugars in Jiangxi lotus showed some increase after 1
week probably due to the hydrolysis of reserve starch,
and then decreased in the second week. For the other
four lotuses, the content of total sugar decreased by 3
weeks post-imbibition, but that of reducing sugar changed
little. It should be noted that the content of reducing
sugar in cotyledons of Taizi lotus was much lower than
the other present-day lotuses, with a negligible amount
in 3-week-old germinated seeds (Fig. 3).
Globulin content in cotyledons of Jiangxi lotus
decreased gradually and the soluble a-amino nitrogen
content increased as the growth of shoots proceeded
(Fig. 2). Albumin content was very low as compared to
globulin levels. Albumin also decreased for the initial 2
weeks, but it increased thereafter probably due to partial
solubilization of the globulin fraction. Figure 4A presents
SDS-PAGE patterns of the globulin and albumin fractions from cotyledons of dry and germinated Jiangxi
seeds. The intensities of several major bands of globulin
gradually decreased until 3 weeks post-imbibition, but no
new globulin bands were detected in this fraction after 3
weeks post-imbibition. Most of the polypeptides in a
range of 15-30 kDa detected in the albumin fraction from
dry cotyledons disappeared by 1-2 weeks post-imbibition,
and the intensities of the bands of about 35 kDa were
increased. In the third week of post-imbibition, most of
the polypeptides in a range of 35-70 kDa disappeared,
and 15-30 kDa polypeptides increased again to a limited extent.
The other three present-day lotuses also showed a
Fig. 3. Changes in growth parameters and contents of cotyledonary components before and after germination of lotus seeds (Taizi, Shanghai,
Hebei, and Harbin). Growth parameters and contents of cotyledonary components were determined as described in the legend to Fig. 2.
580
Maeda et al.
decrease in the globulin content and an increase in the
albumin and soluble a-amino nitrogen contents during 3
weeks post-imbibition. Taizi lotus showed similar trends,
but the albumin and soluble a-amino nitrogen contents
in cotyledons of 3-week-old germinated Taizi seeds were
much lower than the others (Fig. 3). The SDS-pattern
of the globulin fraction from Taizi lotus showed decreased
intensities of several bands of 23-50 kDa during the 3
weeks of study (Fig. 4B). A decrease in several bands of
14-50 kDa was observed in the albumin fraction from
Taizi lotus during the same period.
Thus, in addition to a lower level of reducing sugar
compared to the present-day lotus varieties, low contents
of albumin and soluble a-amino nitrogen in cotyledons
of 3-week-old germinated seeds are another feature of
Taizi lotus (Fig. 3). It remains unclear whether these
differences in cotyledonary components between Taizi
lotus and the four present-day varieties are due to the
inherent characteristic or are ascribable to a change
caused by a long-term quiescence in the pond deposit.
The question shouid be solved once the first filial generation of Taizi lotus are available for analysis.
Gel-electrophoresis patterns of polypeptides from shoots
before and after germination
It was noted that the embryonic axes of the dry (quiescent)
seed of Taizi and the four present-day lotuses were
A. Jiangxi
Globulin
greenish, and therefore it was expected that Rubisco
subunits would be detected in extracts from the embryonic
axes, since this protein comprises as much as half of the
total soluble proteins in mesophyll cells of leaves (Peoples
and Dalling, 1988; Feller and Fischer, 1994). After
SDS-PAGE, at least 30 polypeptides were detected on
the gel for extracts from embryonic axes of both Taizi
and Jiangxi seeds, and some of them disappeared in
extracts from 3-week-old shoots (Fig. 5A). Among them
were two conspicuous polypeptides of 54 kDa and
15 kDa, respectively, that apparently corresponded to
LSU and SSU of Rubisco with respect to their molecular
masses. However, the 54 kDa polypeptide was slightly
smaller than the LSU (55 kDa) from 3-week-old shoots
and fully expanded leaves of Taizi and Jiangxi lotuses,
and both the 54 kDa and 15 kDa polypeptides hardly
reacted with the antisera against the subunits of Rubisco
from French bean (Fig. 5B). Thus, it was concluded that
these polypeptides differ from the Rubisco subunits of
lotus leaves in terms of molecular mass and immunoreactivity. They were probably synthesized in the embryonic
axes during seed maturation and degraded in the course
of germination because only slight amounts were present
in the extracts from 3-week-old shoots. In the 3-week-old
shoots in which primary leaves were not yet expanded, a
small amount of the Rubisco LSU was detected by
immunoblotting (Fig. 5B), although the SSU was hardly
Albumin
B. Taizi
Globulin
Albumin
kDa
3
Weeks
0
Weeks
Fig. 4. SDS—polyacrylamide gel electorophoresis patterns of globulin and albumin fractions from cotyledons of dry and germinated lotus seeds
(Jiangxi and Taizi). The cotyledonary extracts were separated into globulin and albumin fractions as described in Materials and methods. The
fractions that were lyophilized and treated with 20 /xl each of the SDS-sample buffer were separated on a 1 mm thick 12.5% gel. The globulin and
albumin fractions corresponding to 10 /A and 0.5 ml (Jiangxi) and 15 /il and 0.3 ml (Taizi) of the extracts, respectively, were loaded on to each
lane. The gels were stained with CBB.
Metabolic activities in ancient lotus
A. CBBstain
Jiangxi
Dry 3W Full
Taizi
Dry 3W Full
581
kDa
16
Incubation time (h)
-15(SSU)
B. Immunoblot
Jiangxi
Taizi
Dry 3W FuU Dry 3W Full
-LSU
-SSU
Fig. 5. SDS-polyacrylamide gel electrophoresis patterns (A) and immunoblotting of Rubisco (B) for extracts from shoots of dry and
germinated lotus seeds (Jiangxi and Taizi). Proteins were extracted
from embryonic axes of dry seeds (Dry), shoots of 3-week-old seedlings
(3W) and fully expanded leaves (Full). (A) Extracts (20 mg protein
per lane) were analysed by SDS-PAGE on 15% gel, and the gel was
stained with CBB. (B) After extracts (4mg protein per lane) were
separated by SDS-PAGE, immunoblotting was performed using
antisera against the Rubisco LSU and SSU.
detectable, presumably because of the lower titre in the
antiserum against the SSU. The total protein patterns on
the gels were similar between Taizi and Jiangxi lotuses.
24
Fig. 6. Time-course change in incorporation rate of L-[U-14C]leucine
into the TCA-insoluble fraction from cotyledons of germinating lotus
seeds (Jiangxi). Dry cotyledons were allowed to absorb L-[U- 14 C]
leucine for 24 h. At the time intervals indicated, radioactivities taken
up by cotyledons ( • ) and those incorporated into the TCA-insoluble
fraction were determined. Per cent incorporation (O) is represented as
incorporation cpm x 100/uptake cpm. Experimental details are described
in Materials and methods.
9.8 x 104 cpm of the precursor was taken up by the
cotyledons and 0.9% of the radioactivity taken up was
found in the TCA-insoluble fraction. This incorporation
rate was comparable to that of Jiangxi lotus.
Preliminary experiments with inhibitors of eukaryotic
transcription and translation indicated that a-amanitin at
concentrations of 0.1, 1 and 5 ju.gml""1 inhibited the
incorporation of L-[U-14C]leucine into the TCA-insoluble
fraction by 35, 52 and 72%, respectively, during the initial
24 h of imbibition, while cycloheximide showed a weak
inhibition at 10 figml"1 and an almost total inhibition
at 50 (ngml"1. Both 0.1-5 jugml"1 a-amanitin and 1-50
txg ml" l cycloheximide had practically no effect on the
uptake of the labelled leucine by the cotyledons. Based
on these data, inhibitor experiments with Taizi lotus were
performed; a-amanitin at a concentration of 5 figml"1
and cycloheximide at 50 /^g ml" 1 exhibited 69% and 88%
inhibition of incorporation of the radioactivity into the
TCA-insoluble fraction, respectively, during the 24 h
post-imbibition (Fig. 7). Under the same conditions,
a-amanitin and cycloheximide showed 43% and 91%
inhibition, respectively, with Jiangxi lotus.
In vivo synthesis of protein
Possible factors contributing to the maintenance of viability
in aged lotus seeds
L-[U-14C]leucine taken up by the cotyledons of Jiangxi
lotus rose at a rapid rate for the initial 2 h probably due
to 'physical rehydration' (Fig. 6). After the initial 2 h,
the uptake of the radioactivity increased at a slower rate
until 24 h. The incorporation rate (% incorporation) of
radioactivity into the TCA-insoluble fraction had an 8 h
lag period, after which it increased at a constant rate
until the end of the experimental period. When dry
cotyledons of Taizi lotus were allowed to absorb the
labelled leucine solution for 24 h as described above,
The results presented here indicate that the ancient Taizi
lotus seed maintains the activity of protein synthesis in
an early stage of germination and also the ability to
mobilize storage materials such as starch and globulin in
the cotyledon to support shoot growth. In addition,
appreciable activities of starch phosphorylase in extracts
from cotyledons of dry and 3-week-old germinated seeds
of Taizi lotus were recently detected as well as in the
present-day lotus varieties (Minamikawa et cil., 1995).
Earlier, Priestley and Posthumus (1982) described the
582 Maeda et al.
Jiangxi
Taizi
15
0.9
1.0
0.6
10
D
0.5'
0.5
thawing for nearly 600 years. Thus, the factors that
maintained the ability of the embryo to differentiate upon
rehydration as well as the metabolic activities that are a
prerequisite for the differentiation may have been largely
attributable to both the hard pericarp and the ideal
environment in which the fruits were embedded.
0.3
Acknowledgements
Con
Ama
CH
0
0
Con
Ama
CH
Fig. 7. Effects of a-amanitin and cycloheximide on incorporation rate
of L-[U-14C]leucine into the TCA-insoluble fraction from cotyledons
of germinating lotus seeds (Jiangxi and Taizi). (A) Chopped cotyledons
(0.2 g) were allowed to absorb L-[U-14C]leucine solution ( l x l O 6
cpm m l " ' , 11.5 GBq mmol"') in the presence and absence of a-amanitin
or cycloheximide. After a 24 h incubation, the cotyledons were washed
with water, and both the radioactivity taken up by the cotyledons (a)
and that incorporated into the TCA-insoluble material (b) were
determined. The rate of protein synthesis was presented as %
incorporation (b x 100a~'). Con, control; Ama. a-amanitin (5 ^g ml" 1 ),
and CH, cycloheximide (50 ^g ml" 1 ).
analysis of the fatty acid contents of Pulantien lotus
seeds. These ancient seeds were collected in 1952 at
Pulantien, China, and radiocarbon dating for one of the
viable seeds indicated an age of about 446 years. Four
seeds were available for the analysis, and three of them
were viable but lacked vigour. The results showed that
lipids of the unimbibed seeds were still highly polyunsaturated, suggesting that they had undergone little atmospheric autoxidation.
The dry lotus fruit has a hard pericarp which surrounds
the seed within. Microscopic observations showed that
the pericarp is composed of an outermost epidermis, two
thick layers of paliside cells with an acid-resistant fibrous
light zone sandwiched between them, and an inner parenchymatous cell layer (Zhang et al., 1985). The Taizi lotus
seeds have lost the outermost epidermis probably due to
microbial decomposition. The palisade cell layers and the
light zone between them are likely to play an essential
role in preventing the passage of water and gas between
the inside and its environment.
The Taizi lotus seeds had been buried under a pond
deposit at the moderately cold north-eastern area of
China. The muddy deposit under water might suppress
microbial growth through low temperatures and low
oxygen pressures. This environment may have contributed
to protecting the pericarp of the lotus fruit from microbial
decomposition and physical damage due to freezing and
Our thanks to Dr Andrew D Powell for his assistance with the
preparation of the manuscript. We are indebted to Tomoe
Yoshida of our laboratory for providing antisera against
subunits of French bean Rubisco.
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