Journal of Environmental Biology
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Effects of light on the feedback control of GA-20 oxidase gene
homolog in DongJinByeo seedlings
April 2006, 27(2) 367-371 (2006)
For personal use only
Commercial distribution of this copy is illegal
JaeHong Kim1, InSun Yoon2 and MiYoung Lee1
1Division of Life Science, Soonchunhyang University, Asan PO Box 97, Chungnam, 336-600, Korea
2National Institute of Agricultural Biotechnology, Suwon, 441- 707, Korea
(Received: 27 July, 2004 ; Accepted: 21 March, 2005)
Abstract: The effects of gibberellin (GA) on the expression of GA-20 oxidase gene homolog were examined in light-grown seedlings
and dark-grown seedlings of DongJinByeo. The growth rates of the stems of etiolated seedlings were faster than those of green
seedlings. However, upon addition of GA to these seedlings, the stem growth rates of green seedlings were faster than those of
etiolated seedlings. To understand the molecular mechanism of GA gene regulation in DongJinByeo, total RNA from DongJinByeo
was hybridized with cDNA of GA-20 oxidase gene homolog. Greater accumulation of transcript of GA-20 oxidase gene homolog was
observed in green seedlings than in etiolated seedlings. However, upon addition of GA, higher accumulation of the gene transcript
was found in etiolated seedlings than in green seedlings, indicating that expression of the transcript of GA-20 oxidase gene homolog
might be inhibited by light. These results suggest that light might regulate feedback control of the transcript of GA-20 oxidase gene
homolog in DongJinByeo.
Key words: GA-20 oxidase gene, Light, Feedback control, DongJinByeo .
Gibberellins (GA) are tetracyclic diterpenoid
phytohormones with an essential role in plant growth and
development. They are involved in the regulations of growth
responses in higher plants, including stem elongation, seed
germination, mobilization of seed reserves, fruit set and flower
induction (Hooley, 1994; Swain and Olszewski, 1996; Carrera
et al., 1999). GAs are synthesized from isopentenyl
pyrophosphate via geranylgeranyl pyrophosphate (Hedden and
Kamiya, 1997; Lange, 1998). The formation of ent-kaurene
from geranylgeranyl pyrophosphate, with copalyl
pyrophosphate as an intermediary is the first committed step of
GA biosynthesis. This reaction is catalyzed by the enzymes
ent-copalyl diphosphate synthase and ent-kaurene synthase,
which have been cloned in many plant species (Carrera et al.,
1999). ent-Kaurene is metabolized to GAs by monooxygenases
and 2-oxoglutarate-dependent dioxygenase enzymes
responsible for successive oxidation of C-20, with loss as CO2
and the formation of C-19 GAs. GA-20 oxidase is suggested to
be one of the important points of regulation in the GA-
biosynthesis. The final step of bioactive GA synthesis, from
GA53/GA12 to GA1/GA4, is catalyzed through two parallel
pathways (i.e. early-13-hydroxylation and non-13-hydroxylation
pathways) by two soluble 2-oxoglutarate-dependent
dioxygenases in the cytosol, GA-20 oxidase and GA-3 oxidase
from rice (Sakamoto et al., 2004). GA-20 oxidase is a
multifunctional enzyme and has been cloned and expressed
from various plant species. Various cDNA clones were isolated
from a number of plants, such as pumpkin, Arabidopsis,
spinach, pea, rice, and French bean (Lange et al., 1994;
Phillips et al., 1995; Xu et al., 1995; Martin et al., 1996; Wu et
al., 1996; Garcia-Martínez et al., 1997; Toyomasu et al., 1997;
Sakamoto et al., 2004). Comparison of these cDNAs has
indicated highly conserved domains in all GA-20 oxidase
proteins. Moreover, bioactive GAs have been reported to control
their own synthesis through a negative-feedback mechanism of
the GA-20 oxidase and 3β-hydroxylase genes (Chiang et al.,
1995; Xu et al., 1995; Martin et al., 1996; Vidal et al., 2003).
However, the information about GA-20 oxidase transcript
regulation by GA as well as light in DongJinByeo is not
available. The effects of light and GA on the transcript levels of
genes encoding GA-20 oxidase homolog in DongJinByeo
(Oryza sativa cv. Dong-Jin) seedlings were examined in this
Materials and Methods
Experimental plant: The seeds of DongJinByeo were soaked
in a 1% sodium hypochlorite for 15 min, washed with sterilized
water three times. The sterilized seeds were immersed in
sterilized water and grown in a growth chamber at 28 °C for 3
days until germination. The sprouts were planted in flower pots
containing vermiculite and then cultivated with sufficient
watering for upto 10 days under total darkness for etiolated
seedlings and under a 12hr light/12hr dark cycle for green
seedlings. Seedlings were harvested at indicated days and
frozen in liquid N2, and then stored at –80 °C until used.
Application of gibberellin: To investigate the feed-back
regulation of GA-20 oxidase gene in DongJinByeo, seedlings
grown in pots were watered with 500 ml aqueous solution
containing10-6 M gibberellic acid (GA3) for 6 days.
Polymerase chain reaction and plasmid: The complete open
reading frame of a rice gibberellin-20 oxidase cDNA (U50333)
was generated by PCR with the genomic DNA isolated from
T65/Tall rice as a template. Sense primers used for PCR of
Kim et al.
Fig. 1: Effects of gibberellin on the heights of green seedlings and etiolated seedlings of DongJinByeo. The heights of these
seedlings were measured at the indicated cultivation time without gibberellin (A) and with gibberellin (B).
GA-20 oxidase was 5'-ATGAGCATGGTGGTGCAGCAG-3',
and antisense primers for GA-20 oxidase was 5'-
CTAGGAGTATATTGTTGGTTG-3'. E. coli XL-1 blue was used
for transformation and the transformation was carried out
basically as described by Sambrook and Russel (2001). GA-20
oxidase cDNA fragment was ligated with pGEM-T vector which
had been cut with EcoR1 to the formation of pGOX20. The XL-
1 blue transformed with pGOX20 was grown overnight in Luria-
Bertani media containing 50 µg/ml ampicillin, and grown until
OD=0.4 at 560 nm. Plasmid pGOX20 was isolated according to
the alkali lysis method as described by Sambrook and Russel
(2001). The isolated plasmid was treated with EcoRI, and the
GA-20 oxidase gene homolog was purified by using ultrafree-
DA column (Millipore).
Total RNA extraction: Total RNA was isolated from the
seedlings by using Trizol reagent (Gibco BRL). 100 mg of the
seedlings were homogenized in 1 ml of Trizol reagent, and the
homogenized samples were incubated for 5 min at 30°C to
permit complete dissociation of nucleoprotein complexes. After
adding chloroform, the sample was centrifuged at 12,000 × g for
15 min at 4°C, and then the aqueous phase was transferred to a
fresh tube. Total RNA was precipitated from the aqueous phase
by mixing with isopropyl alcohol. RNA pellet was washed once
with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml
of Trizol reagent used for the initial homogenization. RNA pellet
was briefly air-dried, and then dissolved in RNase-free water.
After incubating for 10 min at 60°C, the RNA was quantified
Northern hybridization: 20 µg of total RNA was loaded per
lane and run out on agarose/formaldehyde gels. The RNA was
blotted onto a nitrocellulose filter and hybridized with a
radioactively labeled (Rediprime kit, Amersham Life Science)
GA-20 oxidase gene homolog. 1.2 Kb GA 20 oxidase gene
homolog was used as a probe. Unless stated otherwise,
Control of GA-20 oxidase gene homolog
Fig. 2: Time course of the gibberellin effect on the growth of 8 day-old DongJinByeo seedlings grown under light condition (A) and
dark condition (B). The plant heights were measured after 12, 24, 36 and 48 hr treatment of 10-6 M gibberellin.
membranes were hybridized overnight at 65 ºC in a solution
containing 0.5 M Na2HPO4 (pH 7.2), 7% SDS, 1 mM EDTA at
42 ºC with 50% formamide, 5×SSC, 50 mM Na2HPO4 (pH 6.3),
1×Denhardt’s, 0.1% SDS, 0.1 mg ml-1 denatured salmon sperm
DNA, and with random primed 32P-labelled cDNA fragment as a
probe. Membranes were washed three times with 2×SSC, 0.1%
SDS, then additionally with 0.1×SSC, 0,1% SDS at 42 ºC and
0.1×SSC, 0,1% SDS at 65 ºC. The membranes were then
autoradiographed with an intensifier screen for either one or
Results and Discussion
It has been reported that expression of many plant
genes is regulated via interaction of hormones and light.
However, it is not known exactly how light signal mediated by
plant phytochromes or hormones is transduced to bring about
changes in gene expression. Moreover, detailed information
concerning the direct relationship of light and gibberellin is
limited. Light was also reported to modulate promoter activity as
well as transcript levels of various genes through interacting with
several hormonal signals (Yi et al., 2003). In the rice genome,
four GA-20 oxidase-like genes were reported, three of which
(OsGA2ox1, OxGA2ox2, and OsGAox3) were identical to the
genes reported earlier (Sakamoto et al., 2001; Sakai et al.,
2003), whereas seven copies have been previously reported in
Arabidopsis (Schomburg et al., 2003). GA-deficient mutant and
GA biosynthesis inhibitors were very useful when GA-20
oxidase gene regulation had to be examined (Hedden, 2003).
Ancymidol was reported to block the conversion of ent-kaurene
into ent-kaurenoic (Coolbaugh et al., 1978) and prohexadione
was known to inhibit dioxygenases blocking GA biosynthesis at
the 3β-hydroxylation step that converts GA20 into GA1
Kim et al.
2 4 6 days
2 4 6
2 4 6 days 2 4 6 days
Fig. 3: The effect of gibberellin on the accumulation of transcript level of GA-20 oxidase gene homolog under light and dark
(Nakayama et al., 1990). In potato, inhibition of GA
biosynthesis by application of ancymidol or prohexadione
resulted in a much stronger accumulation of StGA20ox1 and
StGA20ox3 transcripts than of transcript StGA20ox2 (Carrera
et al., 1999). The feedback control of GA-20 oxidase gene by
active GA was also reported in several plants through GA
biosynthesis inhibitors similarly shown in potato (Carrera et al.,
1999; Vidal et al., 2003).
Fig. 1 shows the effect of gibberellin on the height of
light-grown DongJinByeo and dark-grown DongJinByeo. The
heights of the etiolated seedlings were higher than those of
green seedlings without GA. However, upon addition of GA to
these seedlings, the growth rates of green seedlings were
faster than those of dark seedlings. Moreover, the heights of
GA-treated green seedlings were much higher than those of
untreated green seedlings. Time course of gibberellin effect on
the growth of the seedlings under light condition and dark
condition was shown in Fig. 2. Stem elongation effect of GA
began to occur at the green seedlings following GA treatment
for 12 hours, while at etiolated seedlings following GA
treatment for 24 hours. About 11% and 13% stimulations of the
growths were shown in the green seedlings and etiolated
seedlings, respectively, after 48 hour treatment of 10-6 M
gibberellin. Longer treatments or higher concentrations of GAs
were required to visualize notable effect of gibberellin as
reported in patato (Carrera et al., 1999).
In order to investigate how light and GA affect the
transcript level of GA-20 oxidase gene in DongJinByeo, we
analyzed the level of the GA-20 oxidase transcript expression
with and without light through northern hybridization. Fig. 3
indicates the differential accumulation of GA-20 oxidase
transcripts with and without light. The expression levels of GA-
20 oxidase transcripts in green seedlings were higher than
those in etiolated seedlings in the absence of GA. However, the
transcript levels in green seedlings were decreased greatly
upon addition of GA. Thus the transcript levels in green
seedlings were lower than in etiolated seedlings following
gibberellin treatment. These results indicate that expression of
GA-20 oxidase is regulated by negative-feedback control by the
biosynthetic end-product GA in the light, while expression of
GA-20 oxidase is regulated by positive-feedback control by GA
in the dark. In many species, the expression of GA-20 oxidase
is subject to feed-back regulation by the content of active GAs
(Hedden and Kamiya 1997; Toyomasu et al., 1997). The
evidence comes mainly from application experiments and work
with GA biosynthetic mutants, where the contents of active GAs
have been altered substantially. The levels of expression of all
GA-20 oxidase mRNAs increased greatly in the ga1 dwarf
mutant compared with wild-type plants. Treatment of wild-type
plants with inhibitors of GA biosynthesis (ancymidol or
prohexadione) increased the levels of expression of the
StGA20ox1 and StGA20ox3 transcripts, but had little effect on
the StGAox2 mRNA in potato (Carrera et al., 1999). In lettuce,
two GA20x genes have been isolated and described as subject
to feed-back regulation by applied GA1, but only one of them
(LsGA20ox2) seems subject to feed-back regulation by the
increase in endogenous GA1 following red-light irradiation
(Toyomasu et al., 1998). The transcript levels of genes
encoding GA-20 oxidase, as well as those encoding GA-3
oxidases are generally subject to feed-back regulation (Hedden
and Kamiya 1997; Cowling et al., 1998), whereas the
expression of the genes encoding GA-2 oxidase, a GA-
inactivating enzyme, is under feed-forward regulation (Thomas
et al., 1999). In agreement with this hypothesis, we also have
Control of GA-20 oxidase gene homolog
found that the application of GA reduced the transcript levels of
GA-20 oxidase gene homolog in light-grown DongJinByeo
seedlings, while GA increased the transcript levels in dark-
grown seedlings. More detailed experiments will be needed to
make sure how light and GA interact in the molecular levels
and regulate GA-20 oxidase gene multilaterally in DongJinByeo
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Asan PO Box 97, Chungnam, 336-600, Korea
Tel. & Fax: +82-041-530-1355
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