The Korean Society of Crop Science
J. Crop Sci. Biotech. 2009 (September) 12 (3) : 149 ~ 152
DOI No. 10.1007/s12892-009-0110-z
Effect of Light on Endogenous Levels of Gibberellin and
Abscisic Acid in Seed Germination of Photoblastic Weedy Rice
(Oryza sativa L.)
Sang-Yeol Kim1*, Sun-Joo Hwang2, In-Jung Lee2, Dong-Hyun Shin2, Sung-Tae Park1, Un-Sang Yeo1, Hang-Won Kang1
1Functional Crop Resource Development Division, Department of Functional Crop, National Institute of Crop Science, RDA,
Milyang 627-803, Korea
2Division of Plant Bioscience, College of Agriculture and Life Science, Kyungpook National University, Daegu 702-701, Korea
Received: July 21, 2009 / Accepted: September 18, 2009
? Korean Society of Crop Science and Springer 2009
The effect of red (R) and R/far-red (FR) lights on endogenous gibberellin (GA) and abscisic acid (ABA) content was first investi-
gated during the germination of photoblastic black-hulled weedy rice (PBWR) seeds. The R light-treated PBWR seeds germinated
after 36-48 h and germination was increased to 63% at 72 h. However, the FR light-treated seeds after R light treatment, suppressed
the R light effect showing only 11% germination even at 72 h after the light treatment. The PBWR seed treated with R light rapidly
increased the endogenous level of GA1 to about 200 times at 12 h before seed germination as compared with R/FR (control) which
suppressed the effect of R light. The contents of other GAs like GA12, GA53, GA19, GA20, and GA8 were not affected by the R light
irradiation. These results showed that the major biosynthetic pathway of GAs in PBWR seeds is the early 13-hydroxylation pathway
leading to GA1, which was suggested to be physiologically active in the PBWR seed germination. The decrease in the level of ABA
in the R light-treated seeds was greater than the R/FR light-treated seeds, indicating that the balance of endogenous GA1 and ABA is
responsible for the induction of germination in the PBWR seed.
Key words: abscisic acid, gibberellin, light, photoblastic weedy rice, seed germination
The two phytochrome-meditated straw-hulled and black-
hulled weedy rice lines were found by Chung and Paek (2003)
and Kim et al. (2009) with their germination characteristics
well-documented. The germination of the phytochrome meditat-
ed weedy rice seeds was photoreversible by R and FR light. The
R light induces seed germination and the FR light, given after R
light, suppresses the effect of R light. So far, most research is
focused on the effect of light on the photoreceptor phytochrome
but the exact physiological mechanism regulating the events
after irradiation is poorly investigated.
In various reports, hormone contents, especially those of GAs
and ABA have been associated with this control system. In the
seed germination of phytochrome meditated lettuce (Toyomasu
et al. 1993, 1994, 1998), rice (Choi et al. 1995) and Asteraceae
species (Plummer et al. 1997), the early 13-hydroxylation pathway
of GA metabolism leading to GA1 was reported. The GA1
content increased three to five times by R light treatment and
this effect was cancelled by subsequent FR light treatment
(Toyomasu et al. 1993). However, the contents of other GAs
such as GA19, and GA20 were not affected by the R light irradiation.
These results suggest that GA1 is the physiologically bioactive
GA in seed germination of photoblastic lettuce. The increase in
endogenous GA1 level was related with the increase in the ? -
amylase activity in rice (Choi et al. 1996).
An antagonistic effect between ABA and GA levels during
germination of dormant seeds was reported (Kobayashi et al.
1995; Toyomasu et al. 1994). The endogenous levels of ABA in
photoblastic lettuce seeds were decreased both by R light irradiation
and exogenously applied gibberellins. However, apart from
work with lettuce, information on endogenous GAs and ABA
Sang-Yeol Kim ( )
Endogenous GA and ABA in Photoblastic Weedy Rice
contents in the seed germination of phytochrome meditated
weedy rice has been limited. Therefore, this study was carried
out to quantify the endogenous GAs and ABA and know the
cause of enhancement of seed germination by R light in the
Materials and Methods
Mature weedy rice seeds were harvested in the fall from the
experimental field at the Department of Functional Crop,
Milyang, Korea. The seeds were stored at 4 ºC until utilization.
All experiments were conducted within 6 months after harvest.
Seeds were placed in a 9 cm-diameter Petri plates lined in a
Whatman #1 paper disc moistened with 7 ml of deionized water.
The plates were wrapped twice with aluminum foil and incubated
and allowed the seeds imbibed in the germination medium for
24 h at 25±0.5 ºC prior to imposing the light treatments. Two
light treatments such as R light (2.2 ? mol m-2s-1) and R light
followed by FR light (4.0 ? mol m-2s-1) as a control were used.
The treatment lasted for 10 min based on the previous experiment
by Kim et al. (2009). The germination was recorded at 12, 24,
36, 48, 60, and 72 h after light treatment. Treatments were repli-
cated four times with 100 seeds per replication.
For GA analysis, ten grams each of mature seeds were incubated
in a plastic box (19 mm width ? 29 mm length ? 2 mm depth)
containing 40 ml of deionized water at 25 ºC in the dark for 24
h. Light treatments were carried out as mentioned above.
Immediately after light treatment, the resultant light-treated
seeds were incubated in the dark at 25 ºC. Seeds were harvested
at chosen time (0, 12, 24, 36, 48, and 60 h). The tests were
performed in the temperature-controlled dark room within
25±0.5 ºC of limit set throughout experiment. All the above
procedures were carried out under dim green safe light. For
ABA analysis, one gram each of treated seeds was used.
The light source was from light emitting diode (LED, Good
Feeling Ltd, Korea) delivering approximately a 100 band width
at peaks of 665 and 735 nm for the R and FR components,
respectively. The spectral output for the light source was evaluated
with a LI 1800 spectroradiometer (Li-Cor, Inc. Lincoln, NE,
USA) at 2 nm intervals. were carried out under dim green safe
light. For ABA analysis, one gram each of treated seeds was used.
Analysis of endogenous gibberellins
The extraction of GAs followed the general procedure of Lee
et al. (1998). The powdered seeds were extracted with 80 and
100% (v/v) methanol (MeOH) and the two extracts were
combined and methanol was added to bring the combined
MeOH extract concentration to 60%.
High performance liquid chromatography
The GAs were chromatographed on a 3.9 ? 300 mm ? Bond
Pak C18 column (Waters, USA) and eluted at 1.5 ml min-1with
following gradient: 0 to 5 min, isocratic 28% MeOH in 1%
aqueous acetic acid; 5 to 35 min, linear gradient from 28 to 86%
MeOH; 35 to 36 min, 86 to 100% MeOH; 36 to 40 min, isocartic
100% MeOH. Up to 50 fractions of 1.5 ml each were collected.
Small aliquots (15 ?) from each fraction were taken and radioactivi-
ty was measured with liquid scintillation spectrometry
(Beckman, LS 1801). This was used to determine accurate reten-
tion times of each GA based on the elution of 2H-GA standard and
previously determined retention times of the labeled (deuterated)
Quantification of endogenous GAs
GAs were quantified using [17,17-2H2]-GA1, GA8, GA12,
GA19, GA20, GA53 (30 ng each) as internal standards
(purchased from Prof. Lewis N. Mander, Australian National
University, Canberra, Australia). The three prominent ions were
analyzed by GC-MS-SIM (Finnigan Mat GCQ) with dwell times
of 100 ms (Table 1). The endogenous GA contents were calcu-
lated from the peak area ratios, respectively.
Analysis of endogenous abscisic acid
The ABA content was analyzed using the method of
Browning and Wignall (1987), Qi et al. (1998), and Kamboj et
al. (1999) with modifications. The powdered seeds were extracted
with 30 ? of solution containing 95% isopropanol and 5%
glacial acetic acid, and 50 ng of [(±)-3,5,5,7,7,7-d6]-ABA
standard was added into extracts. ABA samples were methylated
with diazomethane for GC-MS analysis. For quantification using
selected ion monitoring, the Lab-Base (ThermoQuset,
Manchester, UK) data system software was used to monitor to
responses to ions of m/e 162 and 190 for Me-ABA and 166 and
194 for Me-[2H6]-ABA. The most prominent ion of ABA was
used for quantification. Quantification of ABA was based on the
peak area ratios of endogenous (non-deuterated, 190) to deuterated
(194) ABA. The GC-MS-SIM condition was similar to that of
GA except for oven condition.
Results and Discussion
The time course of germination of the PBWR seeds under
different light conditions is shown in Fig. 1. The R and R/FR
light treated seeds germinated 36-48 h after the light treatment
594, 238, 448
506, 448, 313
418, 375, 403
434, 374, 402
448, 251, 235
300, 240, 328
Table 1. HPLC fractions and GC-MS retention time of different gibberellins.
HPLC fractionGC-MS retention timePrincipal ions
JCSB 2009 (September) 12 (3) : 149 ~ 152
and the seeds germinated rapidly between 48-60 h in the R light
treatment. After 72 h light treatment, the germination of R treated
seed was 63% while it was only 12% in the R/FR light treatment.
These results indicate that a brief FR light irradiation, given
after the R light irradiation, cancelled the effect of R light.
Similar germination patterns reported in photoblastic lettuce
seed (Toyomasu et al. 1993) and straw-hulled weedy rice
(Chung and Paek 2003).
To investigate the cause of the increase in the seed germina-
tion by R light, and decrease in the germination by R/FR light,
the changes in endogenous GA and ABA contents were deter-
mined. In this study, the levels of GAs in the PBWR seeds were
determined from 0-60 h after the light treatment. This was done
because we focused on the changes of levels of GAs in the
PBWR seeds before and right after germination. Fig. 2 shows the
GA levels in R and R/FR treated PBWR seeds at 60 h after light
treatment. The endogenous levels of GA12, GA53, GA19, GA20, and
GA8 were not affected by the R light irradiation until 60 h after
light treatment. However, the endogenous GA1 content slightly
increased between 12-24 h, rapidly increased to 200-fold
between 24-36 h, and then further increased up to 48 h with R
light treatment as compared with R/FR treatments. These results
indicate that irradiation of R light induced physiologically active
Fig. 1. Time course of seed germination of photoblastic black-hulled weedy
rice treated with R and R/FR lights.
Fig. 2. Changes in endogenous gibberellin levels in the seed of photoblastic black-hulled weedy rice treated with R and R/FR lights.
Endogenous GA and ABA in Photoblastic Weedy Rice Download full-text
GA1 in the PBWR seeds at 12-24 h before seed germination since
the R light irradiation caused an increase of GA1 and seed germi-
nation. However, the increase in endogenous content of GA1 after
R light treatment was cancelled by subsequent FR light treat-
ment. Toyomasu et al. (1993) also found that bioactive GA1 was
increased in photoblastic lettuce seed germination after R light
treatment but it increased only three to five times as compared
with R/FR light treatment. In germinating seeds of lettuce and
Arabidopsis, the R light was shown to up-regulate the biosynthe-
sis of the bioactive GA1 and GA4 by inducing GA biosynthetic
genes (Toyomasu et al. 1998; Yamaguchi et al. 2001).
On the other hand, the endogenous ABA levels of PBWR
seeds irradiated with both R and R/FR (control) lights decreased
but the R treatment showed a greater decrease in ABA levels.
The ABA content was 10.2 ng g-1DW in 60 h maintained in
dark after 10 min R light irradiation (Fig. 3). This value was ca.
twice lower than the control with 22.6 ng g-1DW. The ABA
content decreased to 16.8 ng g-1DW at 60 h after R/FR treatment.
This result suggests that ABA content in the PBWR seed was
phytochrome controlled and the balance of endogenous GA1 and
ABA were responsible for the induction of germination in the
PBWR seed by the R light treatment. Similar antagonistic effect
between ABA and GA during seed germination was also reported
in photoblastic lettuce (Toyomasu et al. 1994) and pine seed
(Tillberg 1992). GA secreted by the embryo induces the
transcription of genes for ? -amylase and other hydrolytic
enzymes and the premature transcription of these genes is
suppressed by the presence of ABA in the germination of cereals
(Jacobsen et al. 1995). These results indicate that the balance of
endogenous GA1 and ABA levels is responsible for the induction
and decrease of germination in the PBWR seed treated with R
and R/FR lights.
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Fig. 3. Changes in endogenous abscisic acid levels in the seed of photo-
blastic black-hulled weedy rice treated with R and R/FR lights.