Blackwell Science, LtdOxford, UK

WBMWeed Biology and Management1444-61622003 Weed Science Society of Japan

31March 2003
076
Genes expressed in dormant
Echinochloa
seeds
T. Fukao
et al.
10.1046/j.1444-6162.2002.00076.x
Research PaperBEES SGML

Weed Biology and Management 3, 1520 (2003)

RESEARCH PAPER

Differential gene expression of the a-chain

of mitochondrial H+-transporting ATP synthase
between dormant and non-dormant seeds of paddy
Echinochloa weeds
TAKESHI FUKAO,1 SHOJI IDA,2 MARY E. RUMPHO,3 ROBERT A. KENNEDY4 and YUJI YAMASUE2*
1
Department of Horticultural Sciences, Program in Molecular and Environmental Plant Sciences, Texas A and M University,
College Station, Texas, USA, 2The Graduate School of Agriculture, Kyoto University, Kyoto, Japan, 3Department of
Biochemistry, Microbiology and Molecular Biology, and 4Department of Biology, University of Maine, Orono, Maine, USA
Gene expression was compared under favorable germination conditions between dormant and
non-dormant seeds of rice paddy Echinochloa weeds and a domesticated Echinochloa species
lacking dormancy. Two dormancy-specific cDNAs, Ecd1 and Ecd2, were identified by differential display. Northern blot analysis revealed that these genes were more strongly expressed in
dormant seeds than in non-dormant seeds. A database search for the Ecd1 sequence revealed
no significant homology with any known proteins, but the Ecd2 sequence was highly homologous with the a-chain of mitochondrial H+-transporting adenosine triphosphate (ATP) synthase in rice (Oryza sativa L.). These findings indicate that the gene encoding the enzyme
associated with conventional aerobic respiration is more abundantly transcribed in dormant
seeds.The results reported in the present study suggest that dormant seeds of paddy Echinochloa
weeds, which appear during the period when paddy soil becomes aerobic by drainage, may
maintain viability primarily by efficient conventional aerobic respiration, including ATP synthesis catalyzed by the mitochondrial H+-transporting ATP synthase.
Keywords: ATP synthase, differential gene expression, Echinochloa crus-galli var. formosensis,
E. oryzicola, E. utilis, seed dormancy.

INTRODUCTION
Seed dormancy is an adaptive characteristic that many
weed species display, resulting in enhanced survival
under unfavorable conditions (Li & Foley 1997). Dormant seeds do not germinate even under seemingly
favorable conditions for germination; dormancy,
thereby, allows seed germination to be dispersed in time.
In arable fields, numerous dormant weed seeds are
present in the soil-buried seed bank, constituting one of
the major factors contributing to the persistence of
weeds. Although seed dormancy has been studied from
physiological and biochemical approaches, mechanisms
*Correspondence to: Yuji Yamasue, Weed Science Laboratory, The
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,
Japan.
Email: yamasue@kais.kyoto-u.ac.jp
Received 15 May 2002; accepted 21 October 2002

regarding the imposition and maintenance are still

unknown in any species.
In Japan, the genus Echinochloa (Poaceae) includes one
domesticated species, E. utilis Ohwi et Yabuno (E. esculenta Scholz, 2n = 6X = 54, hereafter, utilis) and two
paddy weed species, E. oryzicola Vasing (E. phyllopogon
Stapf ex Kossenko, 2n = 4X = 36, hereafter, oryzicola)
and E. crus-galli Beauv. var. formosensis Ohwi (2n = 6X
= 54, hereafter, formosensis). Oryzicola is a progenitor
of the allohexaploidy utilis and formosensis, both of
which have identical genome constituents (Yabuno
1966). Both oryzicola and formosensis are obligate and
persistent weeds in flooded rice fields. These weed and
domesticated species of the Echinochloa complex are very
appropriate materials for studies of seed dormancy. First,
oryzicola and formosensis produce seeds (spikelets) with
very deep dormancy, while seeds of the domesticated
plant, utilis, genetically lack dormancy (Yamasue et al.

16

T. Fukao et al.

1992). Second, a relatively large volume of information

is available on the physiological and ecological behaviors
of the weed species, particularly oryzicola, because they
are highly troublesome in flooded rice fields (Arai &
Miyahara 1962; Kennedy et al. 1980; Rumpho &
Kennedy 1983; Yamasue & Ueki 1987; Yamasue et al.
1987; Kennedy et al. 1992).
In recent years, molecular approaches have been used to
isolate genes that are differentially expressed between
dormant and non-dormant seeds in several species, such
as wheat (Triticum aestivum L.), cheatgrass (Bromus secalinas
L.), wild oat (Avena fatua L.) and Arabidopsis thaliana
Heynh. (Ried & Walker-Simmons 1990; Morris et al.
1991; Girauadat et al. 1992; Goldmark et al. 1992; Dyer
1993; Johnson et al. 1995; Li & Foley 1995; Parcy et al.
1997). Several genes have been isolated using these
plants, but the identification of their gene products has
lagged. A few of the products identified have been found
to be related to desiccation tolerance or ABA signal
transduction, but any role that they may play in seed
dormancy has not been clarified.
In the present study, we used differential display to isolate seed dormancy-specific genes employing dormant
and non-dormant seeds of Echinochloa paddy weeds,
oryzicola and formosensis, and a domesticated species of
Echinochloa, utilis, which genetically lacks dormancy,
with an assumption that the genes are common between
the tetraploid and hexaploid weed species.
MATERIALS AND METHODS
Plant material
Seeds (spikelets) used for the present study were those of
our strains of oryzicola, formosensis and utilis. The
strains of oryzicola and formosensis originated from single seeds collected at a flooded rice field in Uji, Japan,
and that of utilis originated from a seed of the commercial Japanese barnyard millet (Kaneko Nursery Co. Ltd,
Tokyo, Japan). These individual strains were inbred
through several generations. The dormant seeds were
those stored for approximately six months under dry
conditions at -20C after harvesting. Non-dormant
seeds were those with dormancy broken by incubating
dormant seeds for a similar period under dry conditions
at 30C. Utilis seeds were also stored under the same
conditions as the non-dormant seeds.

tion test by placing 50 seeds on moistened filter paper for

14 days in the light at 30C. This was repeated three
times. A germination test was also conducted with dormant oryzicola and formosensis seeds treated with KCN
to artificially break dormancy. Five grams of seeds were
treated with 25 mL of 100 m KCN solution in the dark
for 48 h at 30C. After treatment, the seeds were thoroughly washed with distilled water and incubated under
the above germination conditions.
RNA extraction and differential display
Dormant and non-dormant seeds (5 g) of oryzicola and
formosensis, and non-dormant utilis seeds were imbibed
for 4 h at 30C prior to total RNA extraction. Separate
batches of dormant oryzicola and formosensis seeds were
imbibed for 48 h to serve as the control for the KCN
treatment. After imbibition or treatment with KCN,
seed samples were immediately frozen with liquid nitrogen and stored at -80C until total RNA extraction.
Total RNA was extracted from the seeds following the
SDS-phenol method of Ausubel et al. (1995a). Poly(A)+RNAs (mRNA) were prepared using Oligotex-dT30
(Takara Biochemicals Co. Ltd, Tokyo, Japan). Firststrand cDNA was synthesized following the method of
Yoshida et al. (1994). Second-strand cDNA synthesis was
conducted by PCR using 2010-mer RAPD primers
(OPA-1 to OPA 20, Operon Technologies Inc.,
Alameda, USA). The reaction mixture was composed of
45 mL Premix Taq (Takara Biochemicals Co. Ltd, Tokyo,
Japan), 1 mL cDNA solution and 1 m 10-mer primer.
Only one primer was used for each of the PCR reactions, which consisted of 45 cycles at 92C for 1 min,
35C for 1 min, and 72C for 2 min. The PCR products
were subjected to agarose gel electrophoresis and stained
with ethidium bromide.
Cloning of PCR fragments

Germination test

Specific PCR fragments from cDNA originating from

dormant seeds were excised from the agarose gels. The
gel slices were melted in 300 mL of TE buffer (10 m
Tris-HCl, pH 7.6, 1 m EDTA) at 70C for 5 min, and
frozen at -70C for 5 min. The frozen solution was
allowed to thaw at room temperature for 6090 min. Following centrifugation at 15 000 g for 1 min at 4C, the
PCR fragment was extracted from the supernatant once
with phenol : chloroform (1:1), once with chloroform,
and then precipitated with sodium acetate and ethanol.

To confirm if the sample seeds were in a dormant or

non-dormant state, seeds were subjected to a germina-

To clone the PCR fragments into vector DNA, a single

T was added to the 3 ends of blunt-cut vector DNA

Genes expressed in dormant Echinochloa seeds

(pBluescript II SK, Toyobo Co. Ltd, Tokyo, Japan) in
accordance with the T-A overhang method of Ausubel et
al. (1995b). The PCR fragment was ligated into the Ttailed vector using Ligation High (Toyobo Co. Ltd,
Tokyo, Japan). Both strands of the cDNA inserts were
sequenced, and then a homology search of cDNA
sequences was conducted using the amino acid database,
BLAST (NCBI, USA).

Table 1. Germination percentages of the seed samples

used for differential display
Species
Oryzicola

Formosensis

Northern blot analysis

Total RNA (20 mg per lane) or Poly(A)+-RNA (1 mg per
lane) was electrophoresed in 1% agarose gels containing
0.66% formaldehyde and stained with ethidium bromide. The RNA was transferred to a nylon membrane
(Hybond N+, Amersham Bioscience Corp., New Jersey,
USA) by capillary blotting in 20 SSC (0.3 sodium
acetate, 3 NaCl, pH 7.0). The 18S and 28S rRNA
bands were used to confirm equal loading in each lane.
Dig-labeled probes were synthesized from each insert
DNA using a Dig Labeling Kit (Boehringer Mannheim
Biochemica Ltd, GmbH, Germany). Hybridization,
washing, and signal detection were conducted with the
Dig-System (Boehringer Mannheim Co., Mannheim,
Germany).
RESULTS AND DISCUSSION
After 14 days exposure to favorable germination conditions, dormant oryzicola and formosensis seeds used in
the present study exhibited germination percentages of
zero and 0.7%, respectively (Table 1). This confirmed
that these seeds were dormant. On the contrary, dormancy breakage was confirmed in non-dormant oryzicola, formosensis and utilis seeds by measuring
germination percentages greater than 97% within five
days. Dormant oryzicola and formosensis seeds treated
with 100 m KCN for 48 h at 30C exhibited germination percentages of 85.7 and 80.7%, respectively, after
14 days. This indicates that KCN is effective in artificially breaking seed dormancy in these Echinochloa weeds
as reported by Yamasue et al. (1988).
Differential display was first carried out using cDNAs
from dormant, non-dormant and KCN-treated oryzicola seeds. Amplification of these cDNAs with a single
10-mer RAPD primer resulted in various banding patterns of PCR fragments. Of the 20 primers tested, eight
primers gave fragments specific to dormant oryzicola
seeds; they were not detected in the amplified products
of non-dormant or KCN-treated seeds. Banding patterns of PCR fragments derived from OPA-1 and OPA18 are shown in Fig. 1 as two examples of the many

17

Utilis

Dormant state

Germination
percentage (%)

Dormant
Non-dormant
Dormant/KCN-treated
Dormant
Non-dormant
Dormant/KCN-treated
Non-dormant

0.0c
99.3a
85.7b
0.7c
97.3a
80.7b
100.0a

Dormant seeds and non-dormant seeds were stored at -20C and 30C,
respectively. KCN-treated dormant seeds were exposed to 100 m KCN
for 48 h at 30C. For the germination test, all seeds were placed on wet
filter paper in Petri dishes for 14 days at 30C in the light.

Mean percentages of three replications.The means followed by the same

letter (i.e. a, b or c) are not significant according to Duncans multiple
range test at the 5% level.

(a)

(b)
1

kbps

1.4>
0.9>
0.6>
0.3>

kbps

1.4>
0.9>
0.6>
0.3>

Fig. 1. Differential display with (a) OPA-1 and (b) OPA18 primers in dormant, non-dormant and KCN-treated
oryzicola seeds. 1, dormant oryzicola (imbibed for 4 h); 2,
dormant oryzicola (imbibed for 48 h); 3, non-dormant
oryzicola (imbibed for 4 h); 4, KCN-treated oryzicola
(imbibed for 48 h). Arrows point to specific bands amplified only in dormant oryzicola seeds.

banding patterns obtained with 20 different primers. In

both gels, dormancy-specific bands apparent in lane 1
(4 h imbibition) disappeared in lane 2 (48 h imbibition),
indicating that mRNAs corresponding to these amplified cDNA fragments were not expressed 48 h after
imbibition. Differential display can detect a relatively
large number of false positive fragments (Johnson et al.
1995). To eliminate false positives in the present study,
differential display was also performed with hexaploidy
formosensis and utilis seeds using the same eight primers. The assumption is that dormancy-specific fragments

T. Fukao et al.

18

are common between the tetraploid and hexaploid Echinochloa weeds. Six of the eight primers that showed dormancy-specific fragments in oryzicola dormant seeds
produced the same fragments in non-dormant formosensis and utilis seeds.These fragments were excluded
from the candidates of dormancy-specific cDNAs. Two
fragments, Ecd1 and Ecd2, were amplified with the
remaining two primers (OPA-6 and OPA-18) in dormant seeds of both tetraploidy oryzicola and hexaploidy
formosensis, but neither was produced in their respective
non-dormant seeds or hexaploidy utilis seeds (Fig. 2).
To investigate transcriptional activity of the dormancyspecific genes in seeds, northern blot analysis was conducted using Ecd1 and Ecd2 as probes (Fig. 3). Probing
with Ecd2 revealed a greater signal with total RNA from
dormant formosensis or oryzicola compared to nondormant formosensis, oryzicola or utilis seeds (Fig. 3e
g). When total-RNAs were probed with Ecd1, three
bands were detected in both dormant and non-dormant

(a)
1

kbps

kbps
1.4>
0.9>
0.6>

1.4>
0.9>
0.6>

0.3>

0.3>

Ecd1

Ecd1

seeds (data not shown), suggesting there are three transcripts homologous to Ecd1 in both dormant and nondormant seeds of these Echinochloa weeds. In contrast,
different signal intensities were detected when poly
(A)+-RNAs were used as templates with Ecd1. Again,
transcriptional activity in dormant seeds was found to be
much higher than in their non-dormant counterparts
(Fig. 3a,b). No Ecd1 fragment was detected by differential display in utilis seeds, in contrast to dormant formosensis seeds (Fig. 2), but the intensities of Ecd1 signals
on northern blots were similar among the two seed samples (Fig. 3c). In KCN-treated formosensis seeds, the
transcript signal detected by Ecd1 was weaker than that
observed for dormant formosensis seeds (Fig. 3d), but no
difference was detected in Ecd2 signals between dormant
and KCN-treated dormant formosensis seeds (Fig. 3h).
The clones, Ecd1 and Ecd2, were sequenced and their
homology with known sequences was searched by
BLAST (NCBI, USA). Ecd1 did not show significant
homology with any known sequences. Ecd2, however,
contained high sequence identity with the a-chain of
H+-transporting ATP synthase in rice (score 250, Evalue 1e-65) and maize (score 249, E-value 2e-65)
(Table 2). In the present study, the activity of the H+transporting ATP synthase in dormant and non-dormant
seeds could not be measured due to difficulties in isolating mitochondrial membranes. There is, however, evidence to support the suggestion that dormant oryzicola
seeds respire via conventional aerobic respiration.

Ory
D ND

(b)
1

kbps

1.4>
0.9>
0.6>
0.3>

kbps

Ecd2

1.4>

Fms Utl
D ND

Fms
D ND

FMS
KCN

Ecd1

Ecd2

0.9>
0.6>

Ecd2

0.3>

Fig. 2. RT-PCR analysis with (a) OPA-6 and (b) OPA-18

primers in different Echinochloa weeds. 1, dormant oryzicola (imbibed for 4 h); 2, dormant oryzicola (imbibed
for 48 h); 3, non-dormant oryzicola (imbibed for 4 h);
4, KCN-treated oryzicola (imbibed for 48 h); 5, dormant
oryzicola (imbibed for 4 h); 6, dormant formosensis
(imbibed for 4 h); 7, non-dormant formosensis (imbibed
for 4 h); 8, utilis (imbibed for 4 h).

Fig. 3. Northern blot analysis of EcD1 and EcD2 expression in different Echinochloa weeds. Ory, oryzicola; Fms,
formosensis; Utl, utilis; D, dormant; ND, non-dormant
seeds; KCN, dormant seeds treated with KCN. The blots
contained 1 mg Poly(A)+-RNA (EcD1) or 20 mg of total
RNA (EcD2). Seeds of KCN-treated formosensis and the
control were imbibed for 48 h in KCN solution and distilled water, respectively, before extraction of RNA (d and
h). All others were imbibed for 4 h in distilled water (a, b,
c, e, f and g).

Genes expressed in dormant Echinochloa seeds

19

Table 2. Amino acid homology of Ecd2 in formosensis with the a-chain of mitochondrial H+-transporting ATP synthase
in rice (Oryza sativa L.) and maize (Zea mays L.)
Amino acid sequence
3

182
GDRQTGKTAVALDAMLNQQRWN-KGTDETKKLYCIYVAVGQKRSTVAQLVKTLEENDAMK
GDRQTGKTAIAIDTILNQKQMNSRGTNESETLYCVYVAIGQKRSTVAQLVQILSEANALE
GDRQTGKTAIAIDTILNQKQMNSRGTNESETLYCVYVAIGQKRSTVAQLVQILSEANALE
*********-*-*--***---*---*-*---***-***-***********--*-*--*-183
362
YTIIVAATASEAAPLQYIAPFSGCSMREWFRDNGKHALIIYDDLTKQAVAYRQMSLLLRR
YSILVAATASDPAPLQFLAPYSGCAMGEYFRDNGMHALIIYDDLSKQAVAYRQMSLLLRR
YSMLVAATASDPAPLQFLAPYSGCAMGEYFRDNGMHALIIYDDLSKQAVAYRQMSLLLRR
*---******--****--**-***-*-*-*****-*********-***************
363
482
PPGREAYPGDVFYLHSRLLERAAKMNDKLGGGSLTALPVIE
PPGREAFPGDVFYLHSRLLERAAKRSDQTGAGSLTALPVIE
PPGREAFPGDVFYLHSRLLERAAKRSDQTGAGSLTALPVIE
******-*****************--*--*-**-*******

Ecd2
Rice
Maize

Ecd2
Rice
Maize

Ecd2
Rice
Maize
The

* and _ indicates that amino acids are identical or one of them is different among the three species, respectively. Amino acid homology search was
carried out using BLAST (NCBI, USA). A part of the Ecd2 sequence (nucleotides 3482; translated with frame equals +3) was homologous with the achain of mitochondrial H+-transporting ATP synthase in rice (score 250; E-value 1e-65) and maize (score 249; E-value 2e-65).

(a)
Dormant seeds
shattered onto soil
Seeds
in soil

Seed germination

Dormant
Autumn

Flowering

Non-dormant
Winter

Spring

Summer

(b)
Rice harvesting

Soil

Intermittently
drained

Rice transplanting

Drained

Flooded

Fig. 4. Dormant period of (a) soil-buried Echinochloa

paddy weed seeds in contrast to (b) soil conditions in a rice
paddy field.

Dormant seeds absorb a greater amount of O2 and have

higher levels of activity of isocitrate dehydrogenase than
non-dormant seeds, and the respiratory quotient (RQ)
is very near to unity during imbibition under aerobic
conditions (Yamasue et al. 1987). Furthermore, the ratio
of ethanol : CO2 production by dormant seeds is
approximately 0.2, while non-dormant seeds show 0.8
under air, and 1.0 under nitrogen conditions (Yamasue
& Ueki 1987). In agreement with this data, greater gene
expression of the a-chain of the H+-transporting ATP
synthase observed in dormant seeds in the present study
suggests that dormant seeds of the Echinochloa paddy

weeds have greater mitochondrial enzyme activity than

non-dormant seeds and respire primarily through the
conventional aerobic respiration pathway.
No difference was found in the expression of Ecd2
between dormant and KCN-treated dormant formosensis seeds (Fig. 3h), even when dormancy of the KCNtreated seeds was broken by inhibition of aerobic
respiration. This indicates that dormancy breakage of
KCN-treated formosensis results from inhibition of
cytochrome oxidase activity, but not from low transcriptional activity of Ecd2 (homologous to mitochondrial
ATP synthase). Yamasue et al. (1988) previously demonstrated that KCN inhibits O2 absorption and cytochrome oxidase activity in dormant oryzicola seeds.
We postulate that aerobic respiration catalyzed by mitochondrial H+-transporting ATP synthase is metabolically
adaptive for dormant seeds of the Echinochloa paddy
weeds to generate ATP and maintain viability in the soilburied seed banks because the seeds are exposed to aerobic conditions during most of the period when they are
coincidentally dormant. In substrate consumption, aerobic respiration is far more efficient for seeds to maintain
viability for a long period under a dormant state compared to anaerobic fermentation.
A schematic illustrating the dormant period of Echinochloa weed seeds compared to soil conditions in a rice

20

T. Fukao et al.

paddy field of Japan is shown in Fig. 4.The innately dormant seeds of paddy Echinochloa weeds shatter and are
buried by cultivation in the intermittently drained soil of
rice fields in autumn (Arai & Miyahara 1962; Hirosue et
al. 2000). The seeds are gradually released from dormancy during the winter period. During the period
from seed burial to the next spring, the field is usually
drained and the soil is exposed to aerobic conditions.
The seeds become completely non-dormant when the
field is flooded with water in the late spring for transplanting rice seedlings. Non-dormant seeds of oryzicola
can germinate and grow under flooding conditions and
strict anoxia, supported by alcohol fermentation
(Kennedy et al. 1980; Yamasue et al. 1987). Differential
gene expression of ATP synthase between dormant
and non-dormant seeds may not be the determining factor leading to either a dormant or non-dormant state.
Rather, the differential expression observed in the
present study may be the result of the transition from
aerobic respiration in dormant seeds to anaerobic alcohol fermentation in non-dormant seeds.
ACKNOWLEDGMENT
The experiments reported herein were supported by a
Scientific Research Fund (C)(2) from The Ministry of
Science and Culture of Japan (No. 10660047).
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