Content uploaded by Yue-ie Hsing
Author content
All content in this area was uploaded by Yue-ie Hsing on Mar 16, 2014
Content may be subject to copyright.
A Novel Class of Gibberellin 2-Oxidases Control
Semidwarfism, Tillering, and Root Development in Rice W
Shuen-Fang Lo,
a,b
Show-Ya Yang,
a
Ku-Ting Chen,
b
Yue-Ie Hsing,
c
Jan A.D. Zeevaart,
d
Liang-Jwu Chen,
a,1,2
and Su-May Yu
b,1,2
a
Institute of Molecular Biology, National Chung-Hsing University, Taichung 402, Taiwan, Republic of China
b
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, Republic of China
c
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan, Republic of China
d
Department of Energy Plant Research Laboratory and Department of Plant Biology, Michigan State University,
East Lansing, Michigan 48824-1312
Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes
of GA2oxs inactivate GAs through 2b-hydroxylation: a larger class of C
19
GA2oxs and a smaller class of C
20
GA2oxs. In this
study, we show that members of the rice (Oryza sativa)GA2ox family are differentially regulated and act in concert or
individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C
20
GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing
these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of
two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary
bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C
20
GA2oxs
(motif III) was found to be important for activity of these GA2oxs. Moreover, C
20
GA2oxs were found to cause less severe
GA-defective phenotypes than C
19
GA2oxs. Our studies demonstrate that improvements in plant architecture, such as
semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or
modified C
20
GA2oxs.
INTRODUCTION
Gibberellins (GAs) are a class of essential hormones controlling a
variety of growth and developmental processes during the entire
life cycle of plants. Plants defective in GA biosynthesis show
typical GA-deficient phenotypes, such as dwarfism, small dark
green leaves, prolonged germination dormancy, inhibited root
growth, defective flowering, reduced seed production, and male
sterility (King and Evans, 2003; Sakamoto et al., 2004; Fleet and
Sun, 2005; Tanimoto, 2005; Wang and Li, 2005). Therefore, it is
important for plants to produce and maintain optimal levels of
bioactive GAs to ensure normal growth and development. The
bioactive GAs synthesized by higher plants are GA
1
,GA
3
,GA
4
,
and GA
7
(Hedden and Phillips, 2000). Most genes encoding
enzymes catalyzing GA biosynthesis and catabolism have been
identified (Graebe, 1987; Hedden and Phillips, 2000; Olszewski
et al., 2002; Sakamoto et al., 2004; Yamaguchi, 2008).
A major catabolic pathway for GAs is initiated by a 2b-
hydroxylation reaction catalyzed by GA2ox (Figure 1). C
19
GA2oxs identified in various plant species can hydroxylate the
C-2 of active C
19
-GAs (GA
1
and GA
4
)orC
19
-GA precursors (GA
20
and GA
9
) to produce biologically inactive GAs (GA
8
,GA
34
,GA
29
,
and GA
51
, respectively) (Sakamoto et al., 2004). Recently, three
novel C
20
GA2oxs, including Arabidopsis thaliana GA2ox7 and
GA2ox8 and spinach (Spinacia oleracea) GA2ox3, were found to
hydroxylate C
20
-GA precursors (converting GA
12
and GA
53
to
GA
110
and GA
97
, respectively) but not C
19
-GAs (Schomburg
et al., 2003; Lee and Zeevaart, 2005). The 2b-hydroxylation of
C
20
-GA precursors to GA
110
and GA
97
renders them unable to be
converted to active GAs and thus decreases active GA levels.
The class C
20
GA2oxs contain three unique and conserved
amino acid motifs that are absent in the class C
19
GA2oxs (Lee
and Zeevaart, 2005).
The physiological functions of GA2oxs have been studied in a
variety of plant species. Arabidopsis GA2ox1 and GA2ox2 are
expressed in inflorescences and developing siliques, consistent
with a role of GA2ox in reducing GA levels and promoting seed
dormancy (Thomas et al., 1999). Study of the pea (Pisum sativum)
slender mutant, where the SLENDER gene encoding a GA2ox
had been knocked out, showed that GA levels increased dur-
ing germination, and resultant seedlings were hyperelongated
(Martin et al., 1999). More recently, a dwarf phenotype was also
found to correlate with reduced GA levels in two Arabidopsis
mutants in which GA2ox7 and GA2ox8 were activation tag-
ged, and ectopic overexpression of these two genes in trans-
genic tobacco (Nicotiana tabacum) led to a dwarf phenotype
(Schomburg et al., 2003). These studies demonstrated that
1
These authors contributed equally to this work.
2
Address correspondence to ljchen@nchu.edu.tw or sumay@imb.sinica.
edu.tw.
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Liang-Jwu Chen
(ljchen@nchu.edu.tw) and Su-May Yu (sumay@imb.sinica.edu.tw).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.108.060913
The Plant Cell, Vol. 20: 2603–2618, October 2008, www.plantcell.org ª2008 American Society of Plant Biologists
GA2oxs are responsible for reducing the level of active GAs in
plants. C
20
GA2oxs have also been shown to be under photo-
periodic control in dicots. In long-day rosette plants, such as
spinach, plants grow vegetatively and do not produce a stem
under short photoperiod due to deactivation of GA
53
to GA
97
.
However, upon transfer to long days, stem elongation and
flowering are initiated due to upregulation of GA 20-oxidase
(GA20ox), which converts GA
53
to GA
20
and further to bioactive
GA
1
by GA 3-oxidase (GA3ox) (Lee and Zeevaart, 2005).
Among the several rice (Oryza sativa) mutant phenotypes
caused by GA deficiency, increased tiller growth has been
extensively studied. We found that rice mutants overexpressing
GA2oxs exhibit early and increased tiller and adventitious root
growth. Rice tillering is an important agronomic trait for grain
yield, but the mechanism underlying this process remains mostly
unclear. The rice tiller is a specialized grain-bearing branch that
normally arises from the axil of each leaf and grows indepen-
dently of the mother stem (culm) with its own adventitious roots.
The MONOCULM1 (MOC1) gene, which encodes a GRAS family
nuclear protein and is expressed mainly in the axillary buds, is an
essential regulator of rice tiller bud formation and development
(Li et al., 2003). Two transcription factors, O. sativa homeobox
1(OSH1) and TEOSINTE BRANCHED1 (TB1), have been pro-
posed to act downstream of MOC1 in promoting rice tillering
(Li et al., 2003). OSH1 is a rice homeobox gene that is expressed
during early embryogenesis and is considered a key regulator of
meristem initiation (Sato et al., 1996). Rice TB1 is an ortholog of
the maize (Zea mays)tb1 gene that is expressed in axillary
meristems and regulates outgrowth of this tissue (Hubbard et al.,
2002). In wild-type rice, OSH1 and TB1 mRNAs are detected in
both the axillary and apical meristems of tiller bud; expression of
both OSH1 and TB1 fell significantly and neither could be
detected in meristems in the moc1 mutant, in which only a
main culm without any tiller developed due to defects in the
formation of tiller buds (Li et al., 2003). However, the rice TB1 has
also been shown to function as a negative regulator for lateral
branching in rice (Takeda et al., 2003), a function similar to the
maize TB1 (Hubbard et al., 2002).
In this study, functions of the rice GA2ox family were char-
acterized through genetic, transgenic, and biochemical ap-
proaches, with emphasis on three genes encoding C
20
GA2oxs
that have three unique and conserved motifs. We showed that
C
20
GA2oxs play pleotropic roles regulating rice growth and
architecture, particularly tillering and root systems that may favor
grain yield.
RESULTS
TheRiceGA2oxFamily
A total of 10 putative GA2oxs (see Supplemental Table 1 online)
were identified by BLAST search of the National Center for
Biotechnology Information (NCBI), The Institute for Genomic
Research (TIGR), and Rice Genome Automated Annotation
System (RiceGAAS) databases with conserved domains in
2-oxoglutarate–dependent oxygenases, a family of GA-modifying
enzymes, and nucleotide sequences of four partially character-
ized rice GA2oxs(GA2ox1 to GA2ox4) (Sakamoto et al., 2001,
2004; Sakai et al., 2003), and two uncharacterized GA2oxs
(GA2ox5 and GA2ox6) (Lee and Zeevaart, 2005). Four other
GA2oxs, designated here as GA2ox7 to GA2ox10, were identi-
fied in this study.
Mapping of GA2oxs in the rice genome sequence revealed that
seven GA2oxs clustered on chromosomes 1 and 5 and others
located on chromosomes 2, 4, and 7 (see Supplemental Figure
1A online). Amino acid sequence comparison (see Supplemental
Table 2 online) generated a phylogenetic tree among the rice
GA2ox family (see Supplemental Figure 1B online) and 19
GA2oxs from eight dicot plant species (see Supplemental Table
3 online), which revealed that rice GA2ox5, GA2ox6, and GA2ox9
are more closely related to the Arabidopsis GA2ox7 and GA2ox8
and spinach GA2ox3 (Figure 2). Only these six GA2oxs contain
the three unique and conserved motifs (Lee and Zeevaart, 2005)
(see Supplemental Figure 2 online).
Figure 1. Schematic Diagram of GA Metabolism and Response
Pathways.
Conversion of GA
12
and GA
53
to GA
110
and GA
97
, respectively, by
2b-hydroxylation was demonstrated experimentally only for GA2ox6 in
this study. GA2ox5, GA2ox6, and GA2ox9 are proposed to have similar
functions due to the presence of three conserved motifs unique to C
20
GA2oxs. Inactivation of C
19
-GA precursors, GA
1
and GA
4
by the C
19
GA2oxs, GA2ox1 and GA2ox3, in rice was demonstrated experimentally
(Sakamoto et al., 2001; Sakai et al., 2003). Bioactive GA positively
regulates germination, stem and root elongation, and flower develop-
ment but negatively regulates OSH1 and TB1 that control tillering. GA
also negatively regulates adventitious root development.
2604 The Plant Cell
Differential Expression of GA2ox Is Associated with Flower
and Tiller Development and Seed Germination
Growth of the rice cultivar Tainung 67 used in this study can
be divided into vegetative, reproductive, and ripening phases
(Figure 3A). To understand the role that individual GA2oxs may
play in rice growth, their temporal expression profiles during the
rice life cycle were examined. As leaves have been shown to be a
major site of GA biosynthesis (Choi et al., 1995), mRNAs were
purified from leaves at different growth stages ranging from 5 to
100 d after imbibition (DAI) and analyzed by RT-PCR. Genes
GA2ox1 to GA2ox9 were differentially expressed in leaves, and
their expression was also temporally regulated (Figure 3B).
However, mRNAs of GA2ox10 were not detected in any tissue
at any growth stage, indicating that GA2ox10 could be a pseudo-
gene or its mRNA level was too low to be detected.
Based on temporal mRNA accumulation patterns, GA2oxs
could be classified into two groups. As can be seen in Figure 3B,
for one group excluding GA2ox2 and GA2ox6, accumulation
of their mRNAs in leaves was detected prior to the transition
from vegetative to reproductive growth phases. By contrast, for
Figure 2. Phylogenetic Tree Based on the Comparison of Plant GA2oxs.
Amino acid sequences of 29 GA2oxs from nine plant species (see
Supplemental Table 3 online). Plant species: At, Arabidopsis thaliana;
Cm, Cucurbita maxima;Ls,Lactuca sativa;Nt,Nicotiana sylvestris;Pc,
Phaseolus coccineus; PaPt, Populus alba 3P. tremuloides;Ps,Pisum
sativum; So, Spinacia oleracea. The scale value of 0.1 indicates 0.1
amino acid substitutions per site.
Figure 3. Differential Expression of Two Groups of GA2oxs Regulates
Flower and Tiller Development.
(A) Developmental phases during the life cycle of rice. The timeline is
measured in days after imbibition (DAI).
(B) Temporal expression patterns of GA2oxs in rice. The last fully
expanded leaves were collected from rice plants at different develop-
mental stages. Total RNA was isolated and analyzed by RT-PCR using
GA2ox and GA3ox2 gene-specific primers (see Supplemental Table 4
online). The 18S rRNA gene (rRNA) was used as a control.
(C) Tiller development during the life cycle of rice. A total of eight plants
were used for counting tiller number, and error bars indicate the SE of the
mean at each time point.
Rice GA 2-Oxidase Family 2605
another group including GA2ox2 and GA2ox6, their mRNAs
accumulated in leaves after the phase transition from vegetative
to reproductive growth. GA2ox6 mRNA could also be detected in
leaves at early seedling stage and transiently at high level during
the active tillering stage. Since expression of most GA2oxs
terminated after the active tillering stage, the pattern of tiller
growth throughout the rice life cycle was examined. Tiller number
increased from 30 to 50 DAI (active tillering), remained constant
until 75 DAI, and then increased again until 90 DAI (late tillering)
when the experiment was terminated (Figure 3C). Expression of
each group of GA2oxs paralleled the active and late tillering
stages (cf. Figures 3B with 3C). Except for a slight reduction in the
reproductive phase, the expression of GA3ox2, which encodes a
GA3ox involved in GA biosynthesis, was not significantly altered
in leaves throughout the rice life cycle.
Bioactive GAs are well known for promoting germination, so
the role of GA2oxs during germination was studied. Seeds were
imbibed for various lengths of time. Germination was observed
from 1 DAI and reached almost 100% at 2 DAI (Figure 4A). Total
RNA was isolated from embryos after imbibition of seeds, and
temporal expression profiles of six GA2oxs were analyzed by
RT-PCR. The accumulation of most GA2ox mRNAs was detect-
able starting from 0 to 1 DAI and maintained at similar levels
afterward, except that of GA2ox5 and GA2ox9 was moderately
reduced at 2 DAI (Figure 4B). GA2ox6 had a distinct expression
pattern, as its mRNA quickly accumulated from 0.5 to 1 DAI and
then decreased significantly from 2 to 4 DAI. Low-level accumu-
lation of GA3ox2 mRNA was detected at 0 DAI and then at
similarly high levels after 0.5 DAI. This study demonstrated that
reduced expression of three C
20
GA2oxs seems to correlate with
the rapid seed germination at 2 DAI.
Functional Analysis of GA2oxs with T-DNA
Activation–Tagged Rice Mutants
To study the functions of GA2oxs in rice, we screened for
mutants in a T-DNA–tagged rice mutant library, the Taiwan Rice
Insertional Mutant (TRIM) library (Hsing et al., 2007). The T-DNA
tag used for generating the TRIM library contained multiple
cauliflower mosaic virus 35S promoter (CaMV35S) enhancers
adjacent to the left border, which activate promoters located
near T-DNA insertion sites (Hsing et al., 2007). Two GA2ox-
activated dwarf mutants, M77777 and M47191, were identified
by a forward genetics screen, and another two mutants, M27337
and M58817, were identified by a reverse genetics screen of the
library (Figure 5).
The severe dwarf mutant M77777, designated as GA2ox3
ACT
,
carries a T-DNA insertion at a position 587 bp upstream of the
translation start codon of GA2ox3 (Figure 5A). Accumulation of
GA2ox3 mRNA was significantly enhanced in the heterozygous
mutant. The GA2ox3
ACT
mutant did not produce seeds and was
therefore maintained and propagated vegetatively.
The semidwarf mutant M27337, designated as GA2ox5D335-
341
ACT
, carries a T-DNA insertion in the coding region, at a
position 23 bp upstream of the translation stop codon of GA2ox5
(Figure 5B). Truncation of GA2ox5 by T-DNA resulted in a loss of
seven amino acids at the C terminus of the putative GA2ox5
polypeptide. Accumulation of the truncated GA2ox5 mRNA was
significantly enhanced by T-DNA activation tagging in both
homozygous and heterozygous mutants, but the semidwarf
phenotype was observed only in the homozygous mutant. The
GA2ox5D335-341
ACT
homozygous mutant had an average plant
height of 90% and produced seeds with an average fertility of
88% of the wild type (Table 1).
The severe dwarf mutant M47191, designated as GA2ox6
ACT
,
carries a T-DNA insertion at a position 2.1 kb upstream of the
translation start codon of GA2ox6 (Figure 5C). Accumulation of
GA2ox6 mRNA was significantly enhanced by T-DNA activation
tagging, and a severe dwarf phenotype was observed in both
heterozygous and homozygous mutants. The GA2ox6
ACT
heter-
ozygous mutant produced seeds with an average fertility of only
43% of the wild type after >5 months of cultivation (Table 1).
The semidwarf mutant M58817, designated as GA2ox9
ACT
,
carries a T-DNA insertion at a position 2.4 kb upstream of the
translation start codon of GA2ox9 (Figure 5D). Accumulation of
GA2ox9 mRNA was significantly enhanced by T-DNA activation
Figure 4. C
20
GA2oxs Could Be Responsible for Regulating Seed
Germination.
(A) Germination rate of rice seeds reached 100% at 2 DAI.
(B) Expression patterns of GA2oxs in rice seedlings between 0 and ;5
DAI. Total RNA was isolated from embryos at each time point and
analyzed by RT-PCR. The 18S rRNA gene (rRNA) was used as a control.
2606 The Plant Cell
tagging, and the semidwarf phenotype was observed in both
the homozygous and heterozygous mutants. The GA2ox9
ACT
homozygous mutant had an average plant height of 76% and
produced seeds with an average fertility of 92% of the wild type
(Table 1).
The three activation-tagged mutants, GA2ox5D335-341
ACT
,
GA2ox6
ACT
, and GA2ox9
ACT
, were further characterized. Prog-
enies displayed the same phenotypes as their parents, with
GA2ox5D335-341
ACT
and GA2ox9
ACT
growing slightly shorter
than the wild type, while GA2ox6
ACT
remained severely dwarfed
throughout all growth stages (Figures 6A and 6B). GA2ox5D335-
341
ACT
and GA2ox9
ACT
displayed a normal height but had longer
roots and higher tiller numbers than the wild type (Table 1). Other
traits significantly altered in the severe dwarf GA2ox6
ACT
mutant
included shorter leaves, later heading date, reduced panicle
length, higher tiller numbers, lower grain weight, and lower seed
fertility compared with the wild type (Table 1). Germination of
GA2ox6
ACT
seeds was also significantly delayed, as it took
20 d to reach 90% germination rate, while the wild-type and
GA2ox9
ACT
mutant seeds took only 2 d to reach a germination
rate of 97 and 98%, respectively (Figure 6C). Germination of
GA2ox5D335-341
ACT
seeds was delayed for 4 d to reach a final
88% germination rate (Figure 6C).
Differential Activity of C
20
GA2oxs in Inactivation of GA in
Transgenic Rice and Tobacco
To verify the function of GA2ox5 and GA2ox6 in dwarfism of rice
plants, full-length cDNAs of GA2ox5 and GA2ox6 were isolated
from rice and fused downstream of the maize ubiquitin (Ubi)
(Sun and Gubler, 2004) promoter, generating Ubi:GA2ox5 and
Ubi:GA2ox6 constructs for rice transformation. More than
30 independent transgenic rice lines were obtained for each
construct and all showed dwarf phenotypes with slight varia-
tions in final height. The overall phenotypes of Ubi:GA2ox5 and
Ubi:GA2ox6 T1 plants were similar to the GA2ox6
ACT
mutant,
except that the seed fertility of Ubi:GA2ox6 transgenic rice
(average 64%) was higher than that of Ubi:GA2ox5 transgenic
rice (average 30%) (Figures 7A and 7B, Table 1). These results
demonstrated that ectopic overexpression of GA2ox5 and
GA2ox6 was able to recapitulate the dwarf phenotype in trans-
genic rice.
To examine whether rice GA2oxs are functional in dicots,
Ubi:GA2ox5 and Ubi:GA2ox6 constructs were used for tobacco
transformation. Transgenic tobacco showed the same retarda-
tion of plant growth but to different extents. While Ubi:GA2ox5
reduced plant height to 32% and seed production to 62% and
Ubi:GA2ox6 reduced plant height to 67% of the wild-type
tobacco, Ubi:GA2ox6 had no effect on seed production (Figures
7C and 7D, Table 2). The flowering time was delayed ;2to4
weeks for all transgenic tobacco. Growth of hypocotyls and roots
of 18-d-old T1 transgenic tobacco seedlings was slightly re-
tarded by overexpression of GA2ox6 but significantly retarded
by overexpression of GA2ox5, compared with the wild type
(Figures 7E and 7F, Table 2). These studies demonstrated that
the two rice GA2oxs have similar functions in monocots and
dicots, with GA2ox5 being more potent in inactivation of GA than
GA2ox6 in both transgenic rice and tobacco.
Figure 5. Severely Dwarfed and Semidwarfed Rice Mutants Obtained
by T-DNA Activation Tagging.
(A) The severe dwarf mutant GA2ox3
ACT
(M77777).
(B) The semidwarf mutant GA2ox5D335-341
ACT
(M27337).
(C) The severe dwarf mutant GA2ox6
ACT
(M47191).
(D) The semidwarf mutant GA2ox9
ACT
(M58817).
Accumulationof mRNA was analyzed by RT-PCR, with the 18S rRNA gene
(rRNA) as a control. T/T and T/W, homozygous and heterozygous mutant,
respectively. In the diagram, an asterisk indicates translation start codon,
filled box indicates exon, triangle indicates T-DNA, arrowheads indicate
position of primers used for RT-PCR analysis, and scale bar represents
DNA length for each gene. The box in the triangle indicates the position of
the CaMV35S enhancers (next to the left border of T-DNA).
Rice GA 2-Oxidase Family 2607
Only Shoot, but Not Root, Elongation Is Inhibited by
GA Deficiency
To determine whether the dwarfism of rice mutants overex-
pressing GA2ox was a result of a reduction of bioactive GAs,
GA2ox6
ACT
mutant seeds were germinated on Murashige and
Skoog medium with or without a supplement of 5 mMGA
3
.
Addition of GA
3
promoted germination of GA2ox6
ACT
seeds
(Figure 8A), indicating that GA deficiency inhibited GA2ox6
ACT
mutant seed germination. Plant height of 18-d-old wild-type
seedlings was only slightly enhanced by GA
3
treatment; by
contrast, height of the dwarf GA2ox6
ACT
mutant seedlings was
significantly enhanced by GA
3
treatment, with recovery of up to
84% of the wild-type height (Figure 8B). Root lengths of the wild-
type and GA2ox6
ACT
mutant seedlings were similar, and both
were effectively enhanced by GA
3
treatment (Figure 8B). We
noticed that root elongation of GA2ox6
ACT
was slower initially
after germination (Figure 8A, top panel), but it sped up after 6 DAI
and became similar to the wild type at 18 DAI (Figure 8B). A
similar phenomenon was also observed for root elongation of
Ubi:GA2ox5 and Ubi:GA2ox6 transgenic rice. GA2ox6 mRNA in
leaves and roots accumulated to a higher level in the GA2ox6
ACT
mutant than in the wild type, but both were unaffected by GA
3
treatment (Figure 8C), indicating that shoot and root elongation
was promoted by exogenous GA
3
despite high level of GA2ox6
expression. These results show that GA deficiency only inhibited
stem, but not root, elongation.
GA Deficiency Promotes Early Tillering and Adventitious
Root Growth
We found that rice mutants with activated GA2oxs or transgenic
rice overexpressing GA2oxs formed tillers earlier and in higher
number than the wild type. In the GA2ox6
ACT
mutant and
Ubi:GA2ox5 and Ubi:GA2ox6 transgenic seedlings, after growth
of the main stem from the embryo at 2 DAI, a subsequent swelling
on the embryo surface adjacent to the base of the main stem was
observed at 3 DAI (Figure 9A, panels 2 to 4). Then a first and even
a second tiller grew out from the swollen embryo surface from
9 to 15 DAI (Figures 9B and 9C). Each tiller grew out from its own
coleoptile (Figure 9D), suggesting that these tillers developed
independently in the embryo. Both mutant and transgenic seed-
lings showed early tillering (Figure 9E). The swollen embryo
surface, where a tiller was about to emerge, was not observed in
the wild type (Figure 9A, panel 1). All new tillers in the mutant and
transgenic rice had their own adventitious roots (Figure 9D), a
feature similar to tillers from the wild-type plant around 30 DAI.
Despite retardation of shoot elongation, total root length of
mutant and transgenic seedlings was similar to the wild type at
15 DAI. Quantification of the data also revealed that stem
elongation was inhibited, while tiller and root numbers of mutant
and transgenic rice at 18 DAI were enhanced, compared with the
wild type (Figure 10).
C
20
GA2oxs Specifically Inactivate C
20
-GA Precursors
To examine if GA metabolism in the rice GA2ox6
ACT
mutant was
altered, GAs were purified from leaves of 18-d-old seedlings
and mature plants and subjected to gas chromatography–mass
spectrometry (GC-MS)–selected ion monitoring for quantifica-
tion (Lee and Zeevaart, 2002). In seedlings and mature leaves,
the level of active GA
1
was much lower in mutants (0.1 and 0 ng/
g, respectively) than in the wild type (0.6 and 0.7 ng/g, respec-
tively); by contrast, the level of GA
97
was much higher in mutants
(28.7 and 10.8 ng/g, respectively) than in the wild type (3.6 and
0.5 ng/g, respectively) (Table 3). Due to small amounts of
material, few GAs could be quantified for comparison in both
the mutant and wild-type leaves. In a second experiment, large
decreases in GA
53
and GA
19
and an increase in GA
110
were also
observed in the mutant.
GA
12
and GA
53
were converted to GA
110
and GA
97
, re-
spectively, in vitro by the Arabidopsis C
20
GA2ox7 through
2b-hydroxylation (Schomburg et al., 2003). In this study, the in
Table 1. Characterization of Rice Mutants and Transgenic Rice Overexpressing GA2oxs
Traits Wild Type
GA2ox5D335-341
ACT
(T3)
a
GA2ox9
ACT
(T3)
GA2ox6
ACT
(T2)
Ubi:GA2ox5
(T1)
Ubi:GA2ox6
(T1)
Tiller number of seedling
(18 DAI)
1.0 60.0
b
(100)
c
1.8 60.8 (180) 1.0 60.0 (100) 2.6 60.5 (260) 2.7 60.6 (270) 2.5 60.7 (250)
Root length (cm) at 18 DAI 6.3 60.9 (100) 15.7 63.2 (249) 11.0 61.9 (175) 5.8 61.8 (92) 6.3 62.1 (100) 6.6 60.4 (105)
Plant height (cm) at 120 DAI 109.5 62.5 (100) 98.0 67.1 (90) 83.2 64.1 (76) 16.6 61.7 (15) 16.7 62.8 (15) 12.1 62.7 (11)
Length of leaf bellow
flag leaf (cm) at 120 DAI
49.9 66.0 (100) 49.6 65.1 (100) 49.3 63.3 (99) 12.2 60.9 (24) 10.6 61.2 (21) 8.1 60.8 (16)
Width of leaf bellow flag
leaf (cm) at 120 DAI
1.64 60.1 (100) 1.66 60.1 (101) 1.75 60.2 (107) 1.51 60.1 (92) 1.2 60.1 (73) 1.04 60.1 (63)
Heading day (DAI) 108.7 61.5 107.6 61.3 107.9 61.0 > 150 >150 >150
Panicle length (cm) 21.6 62.0 (100) 20.3 61.5 (94) 19.7 61.8 (91) 7.7 61.6 (36) 5.9 60.8 (27) 7.5 60.9 (35)
Total Tiller number 11.0 61.8 (100) 20.3 64.1 (185) 13.4 62.9 (122) 17.6 63.7 (160) NA 18.8 64.5 (171)
Grain weight (g/100 grains) 2.44 60.1 (100) 2.04 60.1 (84) 2.34 60.2 (96) 1.54 (63) 1.43 (59) 1.98 (81)
Fertility (%) 92.6 64.2 (100) 81.1 65.4 (88) 85.4 68.9 (92) 39.5 618 (43) 27.7 612.0 (30) 59.4 64.4 (64)
a
T1, T2, and T3 in parenthesis indicate generation of mutants.
b
SE;n¼20 for GA2ox5D335-341
ACT
, GA2ox9
ACT
,andGA2ox6
ACT
;n¼10 for Ubi:GA2ox5 and Ubi:GA2ox6.
c
Values in parentheses indicate % of the wild type. NA: not available. DAI: days after imbibition.
2608 The Plant Cell
vitro activity of the rice GA2ox5 and GA2ox6 was also investi-
gated by overexpression as fusion proteins with glutathione
S-transferase in Escherichia coli. Although these fusion proteins
formed protein bodies, they were partially purified (see Supple-
mental Figure 5 online) and enzyme activities analyzed. In assays
with
14
C-labeled GAs and analysis by reverse-phase HPLC with
online radioactivity monitoring (Lee and Zeevaart, 2005), no
metabolism of GA
44
,GA
19
,GA
20
, and GA
1
was observed, but
GA
12
and GA
53
were converted to radioactive products with
Figure 6. Overexpression of GA2oxs Has Different Effects on Rice Seed
Germination and Seedling Growth.
(A) Morphology of T1 seedlings at 18 DAI.
(B) Seedling heights of GA2ox5D335-341
ACT
and GA2ox9
ACT
mutants
were slightly shorter, while seedlings of GA2ox6
ACT
were much shorter
than the wild type. Heights of eight plants in each line were measured,
and error bars indicate the SE of the mean at each time point.
(C) Germination rate was normal for the GA2ox9
ACT
mutant, slightly
delayed for the GA2ox5D335-341
ACT
mutant, and significantly delayed
for the GA2ox6
ACT
mutant compared with the wild type. Numbers of
seeds for determining germination rates were 154, 50, 156, and 49 for the
wild type, GA2ox5D335-341
ACT
,GA2ox6
ACT
,andGA2ox9
ACT
, respec-
tively.
Figure 7. Overexpression of GA2ox5 in Transgenic Rice and Tobacco
Causes More Severe Dwarfism Than Overexpression of GA2ox6.
(A) and (B) Rice transformed with Ubi:GA2ox5 and Ubi:GA2ox6.
(C) to (F) Tobacco transformed with Ubi:GA2ox5 and Ubi:GA2ox6.
Transgenic plants showed different degrees of dwarfism compared
with the control rice or tobacco transformed with vector only (MS).
Photographs of transgenic tobacco were taken at the heading stage ([C]
and [D])and18d([E] and [F]) after sowing of seeds.
Rice GA 2-Oxidase Family 2609
retention times similar to those of GA
110
and GA
97
, respectively.
17,17-[
2
H
2
]GAs were used to identify the products by GC-MS.
Results showed that GA2ox6 could convert GA
12
to GA
110
and
GA
53
to GA
97
(Table 4). GA2ox5 had the same catalytic activity as
GA2ox6 but activity was weaker, perhaps due to less successful
expression in E. coli or lower inherent activity. Nevertheless,
these studies provide evidence that overexpression of GA2ox5
and GA2ox6 reduced in vivo synthesis of bioactive GA
4
and GA
1
from GA
12
and GA
53
, respectively.
Motif III Is Necessary for Activity of C
20
GA2oxs
C
20
GA2oxs, including Arabidopsis GA2ox7 and GA2ox8 and
spinach GA2ox3, contain three unique conserved motifs that are
absent in other GA2oxs (Lee and Zeevaart, 2005). These con-
served motifs are also present in rice GA2ox5, GA2ox6, and
GA2ox9 (see Supplemental Figure 2 online). No function of these
conserved motifs has yet been determined in plants. The rice
GA2ox5D335-341
ACT
mutant, which overexpressed GA2ox5
with four amino acids in motif III deleted, exhibited a less severe
mutant phenotype. This observation prompted us to investigate
the function of motif III in C
20
GA2oxs. Truncated cDNAs of
GA2ox5 and GA2ox6, with deletion of nucleotides encoding
motif III (amino acid residues 325 to 341 and 338 to 358,
respectively), were fused downstream of the Ubi promoter to
generate constructs Ubi:GA2ox5-IIIDand Ubi:GA2ox6-IIID(Fig-
ure 11A) for rice transformation. More than 30 independent
transgenic plants were obtained for each construct. As shown in
Figure 11B, these transgenic plants exhibited the same normal
phenotype as the control transformed with the empty vector (cf.
panels 2 and 5 with panel 3), which was in contrast with the dwarf
phenotype of rice plants transformed with Ubi:GA2ox5 and
Ubi:GA2ox6 (panels 1 and 4). Plant height, panicle number,
and seed germination were normal in all transgenic plants
overexpressing GA2oxs without motif III. These results indicate
that deletion of motif III reduces or eliminates the activity of
GA2ox5 and GA2ox6.
In vitro activity of GA2ox6-IIIDprotein was examined by
overexpression in E. coli as a fusion protein (see Supplemental
Figure 6 online). Both the GA2ox6 and GA2ox6-IIIDrecombinant
proteins partially converted GA
12
to a product that cochromato-
graphed with the standard GA
110
(see Supplemental Figure 7
online). Deletion of motif III appeared to reduce the catalytic
activity of the recombinant protein, as the relative amount of
GA
110
to GA
12
was significantly decreased in GA2ox6-IIID. This
reduced activity could be the result of an inherent change in
catalytic activity, but it could also be the result of unequal enzyme
concentrations in the assays.
GA Deficiency Promotes Expression of OSH1 and TB1
Our preliminary studies demonstrated that tiller and adventitious
root growth was promoted by GA deficiency and inhibited by GA
3
(see Supplemental Figure 3A online). Additionally, the inhibition
of tillering by GA
3
was independent of rice growth stage (see
Supplemental Figure 3B online). To determine whether GA
deficiency induced expression of OSH1 and TB1 that in turn
promotes tillering and root development, GA2ox5D335-341
ACT
,
GA2ox6
ACT
, and wild-type seedlings were grown in media with or
without 5 mMGA
3
after germination. Rice embryos containing
tiller buds were collected at 12 DAI. RT-PCR and real-time
quantitative RT-PCR analyses revealed that levels of both OSH1
and TB1 mRNAs were significantly higher in mutants than in the
wild type, whereas GA
3
significantly reduced levels of both
mRNAs in mutants (Figure 12A; RT-PCR data are shown in
Supplemental Figure 4 online). The increase in OSH1 and TB1
mRNA levels correlated well with early tillering and adventitious
root development in both mutants, whereas GA
3
coordinately
suppressed OSH1 and TB1 mRNA accumulation and tillering
and adventitious root development in both mutants (cf. Figures
12A with 12B). It is not clear why the accumulation of OSH1 and
TB1 mRNAs in the wild type was enhanced by GA
3
(Figure 12A;
see Supplemental Figure 4 online); nevertheless, its stem be-
came more slender and adventitious root growth was inhibited in
the mutants (Figure 12B).
DISCUSSION
The GA2ox Family Is Differentially Regulated and Acts in
Concert or Individually to Control GA Levels
Studies of the effects of GAs on plant growth and development
have been hindered by their low abundance and variation in time
and place. Examination of expression of genes encoding en-
zymes involved in GA biosynthesis and catabolism provides an
alternative approach for such studies. In this study, we showed
that GA2oxs are differentially regulated during the rice life cycle,
indicating that they act in concert or individually to control GA
levels during rice development. For example, seven GA2oxs,
excluding GA2ox2 and GA2ox6, were downregulated in
Table 2. Characterization of Transgenic Tobacco Overexpressing Rice GA2ox5 and GA2ox6
Traits Wild Type Ubi:GA2ox5 Ubi:GA2ox6
Root length (mm) at 18 DAI 20.8 62.7
a
(100)
b
9.6 64.6 (46) 17.7 63.6 (85)
Hypocotyl length (mm) at 18 DAI 6.5 60.8 (100) 3.2 60.6 (49) 4.6 61.0 (71)
Final plant height (cm) 127.7 64.7 (100) 41.2 618.9 (32) 85.3 69.4 (67)
Number of leaves to inflorescence 18.3 60.6 (100) 20.8 63.3 (114) 19.0 60.8 (104)
Seeds yield (g)/plant 31.1 60.0 (100) 19.3 63.4 (62) 31.3 64.1 (100)
a
SE with n¼40.
b
Values in parentheses indicate percentage of the wild type.
2610 The Plant Cell
correlation with the phase transition from vegetative to repro-
ductive growth (Figure 3B). This result is consistent with the
role of GA in promoting flowering in maize and Arabidopsis
(Evans and Poethig, 1995; Blazquez et al., 1998). In another
example, GA2ox6 was significantly downregulated and GA2ox5
and GA2ox9 were moderately downregulated at 2 DAI, which
correlated with rapid seed germination (Figure 4). It indicates that
at least the class C
20
GA2oxs might act coordinately in regulating
GA levels necessary for germination. It is unclear how GA2oxs
are differentially regulated. Due to the complicated feedback
regulatory network and temporal and spatial expression of
GA2oxs and other enzymes involved in GA biosynthesis and
catabolism, and the interaction between GA metabolism and
response pathways (Olszewski et al., 2002; Yamauchi et al.,
2007), deciphering the regulatory mechanism of GA2ox expres-
sion by more extensive biochemical and genetic studies is
required to better understand their exact functions during rice
growth and development.
C
20
GA2oxs Cause Less Severe GA-Defective Phenotypes
Than C
19
GA2oxs
In this study, we observed that overexpression of C
20
GA2oxs
caused less severe GA-defective phenotypes in rice than C
19
GA2oxs. For example, rice overexpressing C
19
GA2oxs, includ-
ing GA2ox3
ACT
mutant and Act1:GA2ox1 and Act1:GA2ox3
transgenic rice, exhibited severe dwarfism and bore no seeds
despite long cultivation periods (Figure 5A; Sakamoto et al.,
2001; Sakai et al., 2003). However, rice overexpressing C
20
GA2oxs, including GA2ox6
ACT
mutant and Ubi:GA2ox5 and
Ubi:GA2ox6 transgenic rice, although they also exhibited severe
dwarfism, did produce seeds after prolonged cultivation. The
GA2ox9
ACT
mutant, which also overexpressed a C
20
GA2ox,
exhibited a semidwarf phenotype and produced a close to
normal amount of seeds with normal germination rate. Similarly,
heterologous overexpression of C
20
GA2ox, including the spin-
ach GA2ox3 and rice GA2ox5 and GA2ox6, in transgenic to-
bacco resulted in typically GA deficient dwarfism and late
flowering, but normal flowers and seed production were ob-
tained (Figures 7C and 7D; Lee and Zeevaart, 2005).
In transgenic tobacco overexpressing spinach GA2ox3, al-
though GA precursors were deactivated, GA 20-oxidase could
still convert a small amount of the precursor to GA
1
(Lee and
Zeevaart, 2005). This observation could be explained by two
possibilities. First, the total amount of C
20
-GA precursors is too
high to be completely deactivated by C
20
GA2oxs, compared
with the total amount of C
19
-GA precursors and active C
19
-GAs
that are deactivated by C
19
GA2oxs. This notion is supported by
studies in rice, Arabidopsis, and tobacco showing that the total
amount of C
20
-GA precursors in these plants is significantly
higher than the total amount of C
19
-GA precursors and active
C
19
-GAs (Sakamoto et al., 2001; Schomburg et al., 2003; Lee
and Zeevaart, 2005). Alternatively, C
20
-GA precursors are so
diverse that it allows some of them to escape from inactivation by
C
20
GA2oxs, so that they can be converted by GA 20-oxidase
and GA 3-oxidase to active C
19
-GAs. These studies may also
explain why C
20
GA2oxs cause less severe GA-defective phe-
notype than C
19
GA2oxs.
Activity of C
20
GA2oxs seems to be conserved in monocots
and dicots. For example, ectopic overexpression of GA2ox5
caused more severe effects than GA2ox6 on plant growth and
seed development in both transgenic rice and tobacco. How-
ever, differences in effects of GA deficiency on plant growth were
observed between monocots and dicots. For example, GA
Figure 8. Only Shoot, but Not Root, Growth Is Inhibited by GA Deficiency.
(A) Treatment with GA
3
(5 mM) promoted germination and seedling
growth of the GA2ox6
ACT
mutant (photo taken at 6 DAI).
(B) Overexpression of GA2ox6 in rice mutants reduces shoot, but not
root, growth. Treatment with GA
3
(5 mM) recovered plant height of the
GA2ox6
ACT
mutant and root growth of both the wild type and mutant. A
total of eight plants at 18 DAI were used for measuring plant height and
root length, and error bars indicate the SE of the mean.
(C) Accumulation of GA2ox6 mRNA in leaves and roots of wild-type and
mutant seedlings (at 18 DAI) was not altered by GA
3
treatment. The 18S
rRNA gene (rRNA) was used as a control. þand , presence and
absence, respectively.
Rice GA 2-Oxidase Family 2611
deficiency inhibits root elongation only in transgenic tobacco
(Figures 7E and 7F; Lee and Zeevaart, 2005) but not in transgenic
rice (Figure 8B, Table 1). Furthermore, tillering was promoted in
transgenic rice (Figure 10), but branching in transgenic tobacco
was not (Lee and Zeevaart, 2005).
C
20
GA2oxs contain three unique and conserved motifs, and
among them, the recombinant GA2ox6 was shown to be capable
of catalyzing 2b-hydroxylation of C
20
-GAs, instead of C
19
-GAs
catalysis by C
19
GA2oxs; this is similar to C
20
GA2oxs from
other plant species (e.g., the Arabidopsis GA2ox7 and GA2ox8)
(Schomburg et al., 2003) and spinach GA2ox3 (Lee and Zeevaart,
2005). These findings suggest that C
20
GA2oxs have a substrate
specificity distinct from that of C
19
GA2oxs (Figure 1). In this study,
motif III was found to play a role in activity of C
20
GA2oxs.
Overexpression of GA2ox5 missing four amino acids in motif III in
the GA2ox5D335-341
ACT
mutant reduced GA concentration to a
Figure 9. GA Deficiency Promotes Early Tillering and Adventitious Root Growth.
(A) Swelling on the embryo surface adjacent to the base of the main stem (MS) (positions indicated by arrows) was observed in the GA2ox6
ACT
mutant
and Ubi:GA2ox5 and Ubi:GA2ox6 transgenic rice (panels 2 to 4) but not in the wild type (panel 1) (photos taken at 3 DAI).
(B) First tiller (1T) grew out from the swollen embryo surface of mutant (photo taken at 9 DAI).
(C) First and second tillers (1T and 2T) formed in some seedlings of mutant (photo taken at 15 DAI).
(D) Each tiller grew out of its own coleoptile, and all new tillers in the mutant had their own adventitious roots (photo taken at 21 DAI). Panel 2 is a higher
magnification of the boxed area in panel 1 that reveals coleoptiles (1C and 2C, respectively) and adventitious roots (1R and 2R, respectively) of the main
stem and first tiller.
(E) Dwarfism and early tillering of seedlings of mutant and transgenic rice compared with the wild type (photo taken at 12 DAI). Panel 2 is a higher
magnification of the boxed area in panel 1 that reveals main stem and first tiller.
2612 The Plant Cell
level that promoted tillering, but only slightly inhibited stem elon-
gation and had no significant effect on seed production (Table 1),
indicating that the truncated enzyme is at least partially functional.
This notionis further supported by thelikely reduced in vitro activity
of recombinant GA2ox6-IIID(see Supplemental Figure 7 online). It
also suggests that tillering, stem elongation, and seed production
are regulated by different GA concentrations.
T-DNA Activation–Tagged Rice Mutants Facilitate Study of
Physiological Functions of Genes Involved in GA
Metabolism and Regulation
More than 18 GA-deficient mutants have been identified by
screening rice mutant populations that were generated by
chemical mutation, retrotransposon (Tos17)insertion,and
g-irradiation (Sakamoto et al., 2004). Despite extensive efforts,
loss-of-function mutations in GA2ox that caused an elongated
slender phenotype were not found in these mutant populations,
probably due to functional redundancy of the GA2ox multigene
family; however, gain-of-function mutations in a GA2ox that
caused a dwarf phenotype were also not found in these mutant
populations (Sakamoto et al., 2004). In this study, severe dwarf
and semidwarf rice mutants were identified by forward and
reverse genetics screens, respectively, of the TRIM mutant library
(Hsing et al., 2007). All these mutants displayed specific pheno-
types due to activation of individual GA2oxs.
An additional advantage of the T-DNA activation approach is
that it allows the overexpression of GA2oxs under the control of
their native promoters. The T-DNA activation approach has been
shown to mainly elevate the expression level of nearby genes
without altering the original expression pattern in general (Jeong
et al., 2002). The controlled expression of GA2oxs in the right
time and right place may give rise to phenotypes that facilitate
not only identification of mutants with altered GA2ox functions
but also study of functions of other genes involved in growth and
development through control of GA metabolism and signaling
pathways in rice.
GA Signaling Represses OSH1 and TB1 Expression That in
Turn Inhibits Tillering
Despite the important contribution of tillering and root system to
grain yield, the mechanisms that control these two developmen-
tal processes in rice are mostly unclear. It is interesting to note
that high tillering often accompanies dwarfism in rice (Ishikawa
et al., 2005). A recent study showed that overexpression of the
YABBY1 gene, a feedback regulator of GA biosynthesis, in
transgenic rice leads to reduced GA level, increased tiller num-
ber, and a semidwarf phenotype (Dai et al., 2007), which provides
a clue that GA might coordinately control the two opposite
developmental processes.
In this study, using GA-deficient mutants, we demonstrated
that stem elongation was inhibited but tillering was promoted by
GA deficiency; by contrast, stem elongation was promoted but
tillering was inhibited by GA
3
(Figure 12). Consequently, we
conclude that GA concomitantly promotes shoot elongation and
inhibits tillering (Figure 1). However, an increase in tillering
indicates a loss of apical dominance. Studies mostly with dicots
Figure 10. GA Deficiency Increases Rice Tiller and Root Numbers.
Mutant (GA2ox6
ACT
) or transgenic (Ubi:GA2ox5 and Ubi: GA2ox6) seeds
germinated on Murashige and Skoog agar medium for 18 DAI. Plant
height and tiller and root numbers of 10 plants in each line were
determined, and error bars indicate the SE.
Table 3. GA Content in the GA2ox6
ACT
Mutant and the Wild Type
Content
a
Sample GA
1
GA
97
Leaves from seedlings at 18 DAI
GA2ox6
ACT
0.1 28.7
Wild type 0.6 3.6
Leaves from mature plants
GA2ox6
ACT
0.0 10.8
Wild type 0.7 0.5
a
GA content in ng g
1
dry weight.
Rice GA 2-Oxidase Family 2613
indicate that this process is mediated by a network of hormonal
signals: apically produced auxin inhibits axillary meristems, while
cytokinin promotes meristem growth (Busov et al., 2008). A
HIGH-TILLERING DWARF1 (HTD1) gene, encoding a carotenoid
cleavage dioxygenase, negatively regulates tiller bud outgrowth
in rice (Zou et al., 2006). As HTD1 expression is induced by auxin
(1-naphthalene acetic acid),it has been suggested that auxin may
suppress rice tillering partly through upregulation of HTD1 tran-
scription (Zou et al., 2006). Further studies are required to under-
stand whether auxin, cytokinin, and GA signaling interact and
control tillering in rice.
In this study, we also showed that growth of adventitious roots
was induced by GA deficiency and suppressed by GA
3
(Figure
12). This observation is supported by a recent study that shows
that GA
3
inhibits adventitious root formation in Populus (Busov
et al., 2006). Additionally, overexpression of DELLA-less versions
of GA insensitive (GAI) and repressor of GAI-like 1, which
conferred GA insensitivity in transgenic Populus trees, led to
dwarfism and an increase in adventitious root growth (Busov
et al., 2006). Crown rootless (Crl1) promotes crown and lateral
root formation, and Crl1 itself is upregulated by auxin in rice
(Inukai et al., 2005). However, aboveground organs are normal in
the crl1 mutant (Inukai et al., 2005), indicating that adventitious/
crown root growth might be regulated by a root-specific auxin
signaling pathway. Again, further studies are required to deter-
mine whether auxin and GA signaling interact to regulate root
growth in rice.
MOC1 is an essential regulator of rice tiller bud formation and
development (Li et al., 2003). Overexpression of MOC1 also
promotes tiller growth and inhibits stem elongation in transgenic
rice, and OSH1 and TB1 are downstream positive regulators
themselves positively regulated by MOC1 in tiller development
(Li et al., 2003; Wang and Li, 2005). In addition to tillering, seed
germination and fertilization are also impaired in the moc1
mutant, which indicates that MOC1 might be involved in GA
signaling pathways by serving as both a positive and negative
regulator (Wang and Li, 2005). However, it is unclear how MOC1
interacts with the GA signaling pathway for regulation of OSH1
and TB1 expression and, thus, tiller development.
We were unable to detect MOC1 mRNA in GA2ox6
ACT
mutant
and wild-type seedlings by the RT-PCR method in a quantitative
manner, probably due to its low abundance in the axillary buds
(Li et al., 2003). Nevertheless, we showed that expression of
OSH1 and TB1 in tiller buds was induced by GA deficiency and
suppressed by GA
3
(Figure 12A). Meanwhile, development of
tiller and adventitious roots was promoted by GA deficiency and
inhibited by GA
3
(Figure 12B). Consequently, our study provides
evidence that GA negatively regulates OSH1 and TB1 expression
that in turn inhibits tiller development (Figure 1). However,
whether GA inhibition of adventitious root development is also
mediated through OSH1 and TB1 cannot be decided from our
results.
It is unclear why exogenous GA confers opposite effects, by
repressing OSH1 and TB1 mRNA accumulations in the mutants,
but inducing their expression in the wild type (Figure 12A).
Nevertheless, the role of OSH1 in positive regulation of tiller
development is also supported by some earlier studies showing
that overexpression of the rice OSH1 led to formation of multiple
shoot and floral apices in transgenic Arabidopsis (Matsuoka
et al., 1993) and multiple shoots in transgenic tobacco and rice
(Kano-Murakami et al., 1993; Sentoku et al., 2000). The role of
TB1 in tiller development is more intriguing, as overexpression of
Figure 11. Motif III Is Necessary for Activity of GA2ox5 and GA2ox6.
(A) Design of constructs encoding the full-length and motif III–truncated
GA2ox5 and GA2ox6. Boxes indicate positions of three highly conserved
amino acid motifs. The last amino acid residue was shown at the C
terminus of deduced polypeptides.
(B) Comparison of morphology among transgenic rice overexpressing
full-length and motif III–truncated GA2ox5 and GA2ox6 and vector
pCAMBIA1301 only (CK).
Table 4. Identification of Products Formed after Incubation of
Recombinant GA2ox6 from Rice with GA
12
or GA
53
Mass Spectra of Products
a
Substrate Product m/z (% Relative Abundance)
17, 17-[
2
H
2
]GA
12
17, 17[
2
H
2
]GA
110
M
þ
450 (5), 435 (7), 418 (39),
390 (60), 375 (6), 328 (6),
318 (13), 300 (97), 285 (81),
274 (53), 260 (34), 259 (46),
241 (100), 225 (34),
201 (23), 197 (12), 145 (37)
17, 17-[
2
H
2
]GA
53
17, 17[
2
H
2
]GA
97
M
þ
538 (35), 523 (9), 506 (8),
479 (13), 448 (2), 389 (9),
373 (7), 329 (14), 299 (3),
239 (38), 210 (61), 209
(100), 195 (17), 179 (16),
147 (14), 119 (14)
a
As the methyl ester trimethylsilyl ethers.
2614 The Plant Cell
the rice TB1 alone inhibits lateral branching but not the propa-
gation of axillary buds in transgenic rice (Takeda et al., 2003).
Whether co-overexpression of OSH1 and TB1 promotes tillering
remains for further studies.
Use of C
20
GA2ox in Plant Breeding
Semidwarfism is one of the most valuable traits in crop breeding
because it results in plants that are more resistant to damage by
wind and rain (lodging resistant) and that have stable yield
increases. It is a major factor in the increasing yield of the green
revolution varieties (Peng et al., 1999; Spielmeyer et al., 2002).
However, the creation of such varieties has relied on limited
natural genetic variation within crop species. Overexpression of
GA2oxs is an easy way to reduce GA levels in transgenic plants.
However, constitutive ectopic overexpression of most GA2oxs
caused severe dwarfism and low seed production in various
plant species because active GAs were probably deactivated as
soon as they were produced (Sakamoto et al., 2001; Singh et al.,
2002; Schomburg et al., 2003; Biemelt et al., 2004; Lee and
Zeevaart, 2005; Dijkstra et al., 2008). Expression of rice GA2ox1
under the control of the rice GA3ox2 promoter, at the site (shoot
apex) of active GA biosynthesis, led to a semidwarf phenotype
with normal flowering and grain development (Sakamoto et al.,
2003).
In this study, our discoveries offer three different approaches
for breeding plants with reduced height, increased root biomass,
and normal flowering and seed production by overexpression of
C
20
GA2oxs. First, overexpression of GA2ox9 generated a semi-
dwarf rice variety. The average grain weight and fertility of the
GA2ox9
ACT
mutant were only slightly reduced (by 8 and 4%,
respectively), but tiller number increased 22% compared with
the wild type (Table 1), which suggests a potential yield increase.
Second, overexpression of C
20
GA2oxs with defective motif III
could also generate a semidwarf rice variety. The average grain
weight and fertility of the GA2ox5D335-341
ACT
mutant was
reduced by 16 and 12%, respectively, but tiller number increased
almost twofold (Table 1), which also suggests a potential for
overall yield increase. Third, overexpression of a selected C
20
GA2ox gene, such as GA2ox6, which has less effect on plant
growth under the control of a weak promoter or its native
promoter, could be beneficial for breeding a semidwarf plant
without sacrificing seed production. It is interesting to note that
both number and length of adventitious roots of both
GA2ox5D335-341
ACT
and GA2ox9
ACT
increased (Table 1), a trait
that could be beneficial for increased nutrient and water uptake
from soil and carbon sequestration from aerial tissues and for
bioremediation. Our studies have provided important information
for the future application of C
20
GA2oxs to improve yields in a
wide range of plant species.
Figure 12. GA
3
Suppresses OSH1 and TB1 Expression and Inhibits Tiller
and Root Development.
(A) Wild-type and GA2ox6
ACT
and GA2ox5D335-341
ACT
mutant seeds
were germinated in Murashige and Skoog agar medium with (þ)or
without ()5mMGA
3
. Total RNA was isolated from embryos containing
tiller buds at 12 DAI and analyzed by quantitative RT-PCR analysis using
primers that specifically amplified rice OSH1 and TB1 cDNAs. RNA levels
were quantified and normalized to the level of rRNA. The highest mRNA
level was assigned a value of 100, and mRNA levels of other samples
were calculated relative to this value. Error bars indicate the SE for three
replicate experiments.
(B) Seedlings used in (A) were photographed prior to RNA isolation.
Panels 1 and 2 are higher magnifications of boxed areas for
GA2ox5D335-341
ACT
and GA2ox6
ACT
mutants without GA
3
treatment
to reveal the main stem (MS) and first tiller (1T).
Rice GA 2-Oxidase Family 2615
METHODS
Plant Materials
The rice cultivar Oryza sativa cv Tainung 67 was used in this study. Mutant
and wild-type seeds were surface sterilized in 2.5% NaClO, placed on
Murashige and Skoog agar medium (Murashige and Skoog Basal Me-
dium; Sigma-Aldrich), and incubated at 288C with 16 h light and 8 h dark
for ;15 to 20 d. Plants were transplanted to pot soil and grown in a net
house. Transgenic tobacco (Nicotiana tabacum) seeds were surface-
sterilized in 2.0% NaClO, placed on half-strength Murashige and Skoog
agar medium, incubated at 228C with 16 h light and 8 h dark for 15 to 20 d,
and then transferred to pot soil and grown in a net house.
Database Searching and Phylogenetic Analysis of Rice GA2oxs
GA2oxs were identified by BLAST search of the NCBI database (http://
www.ncbi.nlm.nih.gov/BLAST/), TIGR database (http://www.tigr.org/
tdb/e2k1/osa1/irgsp.shtml), and Rice Genome Annotation (RiceGAAS)
database (http://ricegaas.dna.affrc.go.jp) with the conserved domain in
the 2-oxoglutarate–dependent oxygenase family and nucleotide se-
quences of four previously identified rice GA2oxs(GA2ox1 to GA2ox4)
(Sakamoto et al., 2001, 2004; Sakai et al., 2003) and two uncharacterized
GA2oxs (GA2ox5 and GA2ox6) (Lee and Zeevaart, 2005). Deduced amino
acid sequences of GA2oxs were aligned with the ClustalW2 (version
2.0.8) and AlignX (Vector NTI, version 9.0.0; Informax) programs, and
conserved residue shading was performed using the BioEdit Sequence
Alignment Editor (version 7.0.7.0) for generation of the PHYLIP file (see
Supplemental Data Sets 1 and 2 online). Evolutionary relationships were
deduced using the neighbor-joining algorithm (Saitou and Nei, 1987).
Bootstrapping was performed using the PHYLIP program (version 3.6.7)
with 1000 replicates. The unrooted phylogenetic tree was constructed
using the MEGA 4 phylogenetic analysis program (Tamura et al., 2007).
The Knowledge-based Oryza Molecular Biological Encyclopedia data-
base (http://cdna01.dna.affrc.go.jp/cDNA) was used for cDNA searching.
T-DNA Flanking Sequence Analysis
Genomic DNA was extracted with a CTAB extraction buffer as described
(Doyle and Doyle, 1987). T-DNA flanking sequences were rescued using a
built-in plasmid rescue system (Upadhyaya et al., 2002) and analyzed
with an ABI Prism 3100 DNA sequencer (Applied Biosystems) using DNA
sequences 100 bp upstream of the T-DNA right border (Hsing et al., 2007)
as primer. T-DNA flanking sequences were searched using BLASTN
against the NCBI database for assignment in the rice BAC/PAC site, and
gene dispersions were annotated by the RiceGAAS database.
RT-PCR and Real-Time Quantitative RT-PCR Analyses
Total RNA was purified from rice tissues using Trizol reagent (Invitrogen)
and treated with RNase-free DNase I (Promega). The DNase-digested
RNA sample was used for reverse transcription by Superscript III reverse
transcriptase (Invitrogen). Samples, which served as cDNA stocks for
PCR analysis, were stored at 708C.
RT-PCR analysis was performed in a 15-mL solution containing 0.9 mL
cDNA stock using GoTaq DNA polymerase (Promega). For GA2ox2,
GA2ox7, and GA2ox8 that had very low mRNA abundance, RT-PCR
analysis was performed using KOD Hot Start DNA Polymerase (Novagen).
All PCR reactions were performed in a 15-mL reaction solution containing
0.9 mL cDNA, using a programmable thermal cycler (PTC-200; MJ
Research). Concentrations of all GA2ox mRNAs were very low; therefore,
higher PCR cycles in the linear quantitative range were performed. The
numbers of PCR cycles were 33 for GA2ox1 and GA2ox3, 34 for the rest of
GA2oxs, and 24 for rRNA. RT-PCR products were fractionated in a 1.5%
agarose gel and visualized by ethidium bromide staining. All RT-PCR
analyses were performed more than three times from at least two batches
of RNA samples with similar results.
The same RNA samples were further used to analyze OSH1 and TB1
expression profiles using quantitative RT-PCR analysis by the ABI 7300
system as described (Dai et al., 2007; Lu et al., 2007). SYBR green was
used to monitor the kinetics of PCR product in real-time RT-PCR. 18S
rRNA was used as an internal control to quantify the relative transcript
level of OSH1 and TB1 in 15-DAI shoots.
Rice and Tobacco Transformation
Full-length GA2ox5 and GA2ox6 cDNAs were PCR amplified from rice
mRNA based on their putative open reading frames annotated with the
RiceGAAS database. A BamHI restriction site was designed at the 59end
of DNA primers used for PCR amplification (see Supplemental Table 4
online). The PCR products of 1043 and 1094 bp were ligated into the
pGEM-T Easy cloning vector (Promega), and their sequences were
confirmed by DNA sequencing. Plasmid pAHC18 (Bruce et al., 1989)
was derived from plasmid pUC18 that contains the Ubi promoter and
nopaline synthase (Nos) terminator. GA2ox5 and GA2ox6 cDNAs were
then excised with BamHI from the pGEM-T Easy vector and ligated into
the same site between the Ubi promoter and Nos terminator in plasmid
pAHC18. Plasmids containing Ubi:GA2ox5 and Ubi:GA2ox6 were linear-
ized with HindIII and inserted into the same site in pCAMBIA1301
(Hajdukiewicz et al., 1994). The resulting binary vectors were transferred
into Agrobacterium tumefaciens strain EHA105 and used for rice and
tobacco transformation as described (Krugel et al., 2002).
DNA fragments of GA2ox5-IIID325-341 and GA2ox6-IIID338-358 were
PCR amplified. PCR products of 992 and 1031 bp were cloned into the
pCAMBIA1301 binary vector, following procedures described above, for
generation of binary vectors containing Ubi:GA2ox5-IIID325-341 and
Ubi:GA2ox6-IIID338-358 for rice transformation.
Expression and Activity Assay of Recombinant GA2ox5 and GA2ox6
Full-length cDNAs of GA2ox5 and GA2ox6 in the pGEM-T Easy cloning
vector were digested with BamHI and subcloned into the same site in
pGEX-5X expression vector (Amersham Biosciences). The resulting
expression vectors were used to transform Escherichia coli strain BL21-
CodonPlus (DE3) RIPL (Stratagene). A volume 500 mL culture in Luria-
Bertani broth (Difco) with 100 mg L
1
ampicillin was incubated at 3 78Cuntil
cell densityreached an OD
600
around 0.6 to ;0.8.The recombinant protein
induction was performed by adding0.3 mM isopropyl-b-D-thiogalactopyr-
anosideand incubated at 288C for another3 h. Bacteria were collectedfrom
the culture medium by centrifugation, resuspended in a BugBuster Protein
Extraction Reagent (a buffer containing DTT, rBenzonase Nuclease, and
rLysozyme, as indicated in the manufacturer’s instruction manual; Nova-
gen), and centrifuged again. The supernatant protein extracts were partially
purified by elution through a GST-Bind resin (Novagen) with 50 mM Tris
buffer, pH 8.0, containing 10 mM reduced glutathione and stored at 808C.
The frozen extracts were lyophilized, shipped to J.A.D.Z.’s lab at Michigan
State University, and reconstituted with cold distilled water for enzyme
activity assays as described (Lee and Zeevaart, 2002, 2005).
Analysis of Endogenous GA Levels
The procedures for extraction, purification, and quantification of endog-
enous GAs have been described elsewhere (Talon et al., 1990; Zeevaart
et al., 1993; Schomburg et al., 2003).
Primers
Nucleotides for all primers used PCR and RT-PCR analyses are provided
in Supplemental Table 4 online.
2616 The Plant Cell
Accession Numbers
Sequence data from this article can be found in the NCBI or TIGR
database under the following accession numbers: AtGA2ox1, AJ132435;
AtGA2ox2, AJ132436; AtGA2ox3, AJ132437; AtGA2ox4, AY859740;
AtGA2ox6, AY859741; AtGA2ox7, NM109746; AtGA2ox8, NM118239;
CmGA2ox, AJ302041; LsGA2ox1, AB031206; NtGA2ox1, AB125232;
NtGA2ox3, EF471117; NtGA2ox5, EF471118; PcGA2ox1, AJ132438;
poplar GA2ox1, AY392094; PsGA2ox1, AF056935; PsGA2ox2,
AF100954; SoGA2ox1, AF506281; SoGA2ox2, AF206282; SoGA2ox3,
AY935713; 18S rRNA, AH001794; OSH1, D16507; and TB1, AY286002.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The Rice GA2ox Family.
Supplemental Figure 2. Amino Acid Sequence Alignment of
Rice GA2oxs (OsGA2ox1, OsGA2ox3, OsGA2ox5, OsGA2ox6, and
OsGA2ox9), Arabidopsis GA2oxs (AtGA2ox7 and AtGA2ox8), and
spinach GA2ox (SoGA2ox3).
Supplemental Figure 3. GA
3
Represses Tiller Growth Independent of
Growth Stages.
Supplemental Figure 4. GA
3
Suppresses OSH1 and TB1 Expression.
Supplemental Figure 5. Production, Purification, and SDS-PAGE
Analyses of GST-Fused Recombinant GA2ox5 and GA2ox6.
Supplemental Figure 6. Conversion of [
14
C]GA
12
to [
14
C]GA
110
by
Recombinant GA2ox6 and GA2ox6-IIIDProteins.
Supplemental Table 1. Putative GA2ox Gene Family in Rice (Oryza
sativa).
Supplemental Table 2. Comparison of Deduced Amino Acids among
Rice GA2oxs.
Supplemental Table 3. Gene Names and Accession Numbers of 19
GA2oxs from Different Plant Species.
Supplemental Table 4. Primers Used for T-DNA Flanking Sequence,
PCR, and RT-PCR Analyses and Plasmid Construction.
Supplemental Data Set 1. Text File Corresponding to the Phyloge-
netic Tree in Figure 2.
Supplemental Data Set 2. Text File Corresponding to the Phyloge-
netic Tree in Supplemental Figure 1B.
ACKNOWLEDGMENTS
We thank Tuan-hua David Ho and Harry Wilson for their critical review of
this manuscript and, Mei-Chu Chung, Chyr-Guan Chern, Chang-Sheng
Wang, Ming-Jen Fan, Lin-Chih Yu, Lin-yun Kuang, Tung-Hi Tseng,
Ming-Jier Jiang, and Wen-Bin Tseng for their technical assistance. We
also thank Bev Chamberlin of the Michigan State University Mass
Spectrometry Facility for assistance with GC-MS. This work was
supported by grants from Academia Sinica (AS92-AB-IMB-03 and
AS93-AB-IMB-02 to S.-M.Y.), the Council of Agriculture (94-AS-5.2.1-
S-a1-8 to L.-J.C.), and the National Science Council (NSC92-2317-B-
001-036 to S.-M.Y. and NSC96-2317-B-005-007 to L.-J.C.) of the
Republic of China.
Received May 22, 2008; revised September 19, 2008; accepted Sep-
tember 30, 2008; published October 24, 2008.
REFERENCES
Biemelt, S., Tschiersch, H., and Sonnewald, U. (2004). Impact of
altered gibberellin metabolism on biomass accumulation, lignin bio-
synthesis, and photosynthesis in transgenic tobacco plants. Plant
Physiol. 135: 254–265.
Blazquez, M.A., Green, R., Nilsson, O., Sussman, M.R., and Weigel,
D. (1998). Gibberellins promote flowering of Arabidopsis by activating
the LEAFY promoter. Plant Cell 10: 791–800.
Bruce, W.B., Christensen, A.H., Klein, T., Fromm, M., and Quail, P.H.
(1989). Photoregulation of a phytochrome gene promoter from oat
transferred into rice by particle bombardment. Proc. Natl. Acad. Sci.
USA 86: 9692–9696.
Busov, V., Meilan, R., Pearce, D., Rood, S., Ma, C., Tschaplinski, T.,
and Strauss, S. (2006). Transgenic modification of gai or rgl1 causes
dwarfing and alters gibberellins, root growth, and metabolite profiles
in Populus. Planta 224: 288–299.
Busov, V.B., Brunner, A.M., and Strauss, S.H. (2008). Genes for
control of plant stature and form. New Phytol. 177: 589–607.
Choi, Y.-H., Kobayashi, M., Fujioka, S., Matsuno, T., Hirosawa, T.,
and Sakurai, A. (1995). Fluctuation of endogenous gibberellin levels
in the early development of rice. Biosci. Biotechnol. Biochem. 59:
285–288.
Dai, M., Zhao, Y., Ma, Q., Hu, Y., Hedden, P., Zhang, Q., and Zhou,
D.X. (2007). The rice YABBY1 gene is involved in the feedback
regulation of gibberellin metabolism. Plant Physiol. 144: 121–133.
Dijkstra, C., Adams, E., Bhattacharya, A., Page, A., Anthony, P.,
Kourmpetli, S., Power, J., Lowe, K., Thomas, S., Hedden, P.,
Phillips, A., and Davey, M. (2008). Over-expression of a gibberellin
2-oxidase gene from Phaseolus coccineus L. enhances gibberellin
inactivation and induces dwarfism in Solanum species. Plant Cell Rep.
27: 463–470.
Doyle, J.J., and Doyle, J.L. (1987). A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15.
Evans, M.M., and Poethig, R.S. (1995). Gibberellins promote vegeta-
tive phase change and reproductive maturity in maize. Plant Physiol.
108: 475–487.
Fleet, C.M., and Sun, T.P. (2005). A DELLAcate balance: the role of
gibberellin in plant morphogenesis. Curr. Opin. Plant Biol. 8: 77–85.
Graebe, J.E. (1987). Gibberellin biosynthesis and control. Annu. Rev.
Plant Physiol. 38: 419–465.
Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994). The small, versatile
pPZP family of Agrobacterium binary vectors for plant transformation.
Plant Mol. Biol. 25: 989–994.
Hedden, P., and Phillips, A.L. (2000). Gibberellin metabolism: New
insights revealed by the genes. Trends Plant Sci. 5: 523–530.
Hsing, Y.-I., et al. (2007). A rice gene activation/knockout mutant
resource for high throughput functional genomics. Plant Mol. Biol. 63:
351–364.
Hubbard, L., McSteen, P., Doebley, J., and Hake, S. (2002). Expres-
sion patterns and mutant phenotype of teosinte branched1 correlate
with growth suppression in maize and teosinte. Genetics 162: 1927–
1935.
Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M., Shibata, Y., Gomi,
K., Umemura, I., Hasegawa, Y., Ashikari, M., Kitano, H., and
Matsuoka, M. (2005). Crown rootless1, which is essential for crown
root formation in rice, is a target of an AUXIN RESPONSE FACTOR in
auxin signaling. Plant Cell 17: 1387–1396.
Ishikawa, S., Maekawa, M., Arite, T., Onishi, K., Takamure, I., and
Kyozuka, J. (2005). Suppression of tiller bud activity in tillering dwarf
mutants of rice. Plant Cell Physiol. 46: 79–86.
Jeong, D.H., An, S., Kang, H.G., Moon, S., Han, J.J., Park, S., Lee,
Rice GA 2-Oxidase Family 2617
H.S., An, K., and An, G. (2002). T-DNA insertional mutagenesis for
activation tagging in rice. Plant Physiol. 130: 1636–1644.
Kano-Murakami, Y., Yanai, T., Tagiri, A., and Matsuoka, M. (1993). A
rice homeotic gene, OSH1, causes unusual phenotypes in transgenic
tobacco. FEBS Lett. 334: 365–368.
King, R.W., and Evans, L.T. (2003). Gibberellins and flowering of
grasses and cereals. Annu. Rev. Plant Biol. 54: 307–328.
Krugel, T., Lim, M., Gase, K., Halitschke, R., and Baldwin, I.T. (2002).
Agrobacterium-mediated transformation of Nicotiana attenuata,a
model ecological expression system. Chemoecology 12: 177–183.
Lee, D.J., and Zeevaart, J.A.D. (2002). Differential regulation of RNA
levels of gibberellin dioxygenases by photoperiod in spinach. Plant
Physiol. 130: 2085–2094.
Lee, D.J., and Zeevaart, J.A.D. (2005). Molecular cloning of GA
2-oxidase3 from spinach and its ectopic expression in Nicotiana
sylvestris. Plant Physiol. 138: 243–254.
Li, X., et al. (2003). Control of tillering in rice. Nature 422: 618–621.
Lu, C.-A., Lin, C.-C., Lee, K.-W., Chen, J.-L., Huang, L.-F., Ho, S.-L.,
Liu, H.-J., Hsing, Y.-I., and Yu, S.-M. (2007). The SnRK1A protein
kinase plays a key role in sugar signaling during germination and
seedling growth of rice. Plant Cell 19: 2484–2499.
Martin, D.N., Proebsting, W.M., and Hedden, P. (1999). The SLENDER
gene of pea encodes a gibberellin 2-oxidase. PlantPhysiol. 121: 775–781.
Matsuoka, M., Ichikawa, H., Saito, A., Tada, Y., Fujimura, T., and Kano-
Murakami, Y. (1993). Expression of a rice homeobox gene causes
altered morphology of transgenic plants. Plant Cell 5: 1039–1048.
Olszewski, N., Sun, T.-p., and Gubler, F. (2002). Gibberellin signaling:
Biosynthesis, catabolism, and response pathways. Plant Cell 14:
S61–S80.
Peng, J., et al. (1999). ‘Green revolution’ genes encode mutant gibber-
ellin response modulators. Nature 400: 256–261.
Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol.Biol. Evol. 4: 406–425.
Sakai, M., Sakamoto, T., Saito, T., Matsuoka, M., Tanaka, H., and
Kobayashi, M. (2003). Expression of novel rice gibberellin 2-oxidase
gene is under homeostatic regulation by biologically active gibber-
ellins. J. Plant Res. 116: 161–164.
Sakamoto, T., Kobayashi, M., Itoh, H., Tagiri, A., Kayano, T., Tanaka,
H., Iwahori, S., and Matsuoka, M. (2001). Expression of a gibberellin
2-oxidase gene around the shoot apex is related to phase transition in
rice. Plant Physiol. 125: 1508–1516.
Sakamoto, T., Morinaka, Y., Ishiyama, K., Kobayashi, M., Itoh, H.,
Kayano, T., Iwahori, S., Matsuoka, M., and Tanaka, H. (2003).
Genetic manipulation of gibberellin metabolism in transgenic rice. Nat.
Biotechnol. 21: 909–913.
Sakamoto, T., et al. (2004). An overview of gibberellin metabolism
enzyme genes and their related mutants in rice. Plant Physiol. 134:
1642–1653.
Sato, Y., Hong, S.-K., Tagiri, A., Kitano, H., Yamamoto, N., Nagato,
Y., and Matsuoka, M. (1996). A rice homeobox gene, OSH1,is
expressed before organ differentiation in a specific region during early
embryogenesis. Proc. Natl. Acad. Sci. USA 93: 8117–8122.
Schomburg, F.M., Bizzell, C.M., Lee, D.J., Zeevaart, J.A.D., and
Amasino, R.M. (2003). Overexpression of a novel class of gibberellin
2-oxidases decreases gibberellin levels and creates dwarf plants.
Plant Cell 15: 151–163.
Sentoku, N., Sato, Y., and Matsuoka, M. (2000). Overexpression of
rice OSH genes induces ectopic shoots on leaf sheaths of transgenic
rice plants. Dev. Biol. 220: 358–364.
Singh, D.P., Jermakow, A.M., and Swain, S.M. (2002). Gibberellins are
required for seed development and pollen tube growth in Arabidopsis.
Plant Cell 14: 3133–3147.
Spielmeyer, W., Ellis, M.H., and Chandler, P.M. (2002). Semidwarf
(sd-1), ‘‘green revolution’’ rice, contains a defective gibberellin
20-oxidase gene. Proc. Natl. Acad. Sci. USA 99: 9043–9048.
Sun, T.P., and Gubler, F. (2004). Molecular mechanism of gibberellin
signaling in plants. Annu. Rev. Plant Biol. 55: 197–223.
Takeda, T., Suwa, Y., Suzuki, M., Kitano, H., Ueguchi-Tanaka, M.,
Ashikari, M., Matsuoka, M., and Ueguchi, C. (2003). The OsTB1
gene negatively regulates lateral branching in rice. Plant J. 33:
513–520.
Talon, M., Koornneef, M., and Zeevaart, J.A.D. (1990). Endogenous
gibberellins in Arabidopsis thaliana and possible steps blocked in the
biosynthetic pathways of the semidwarf ga4 and ga5 mutants. Proc.
Natl. Acad. Sci. USA 87: 7983–7987.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA) Software Version
4.0. Mol. Biol. Evol. 24: 1596–1599.
Tanimoto, E. (2005). Regulation of root growth by plant hormones:
Roles for auxin and gibberellin. Crit. Rev. Plant Sci. 24: 249–265.
Thomas, S.G., Phillips, A.L., and Hedden, P. (1999). Molecular cloning
and functional expression of gibberellin 2- oxidases, multifunctional
enzymes involved in gibberellin deactivation. Proc. Natl. Acad. Sci.
USA 96: 4698–4703.
Upadhyaya, N.M., et al. (2002). An iAc/Ds gene and enhancer trapping
system for insertional mutagenesis in rice. Funct. Plant Biol. 29:
547–559.
Wang, Y., and Li, J. (2005). The plant architecture of rice (Oryza sativa).
Plant Mol. Biol. 59: 75–84.
Yamaguchi, S. (2008). Gibberellin metabolism and its regulation. Annu.
Rev. Plant Biol. 59: 225–251.
Yamauchi, Y., Takeda-Kamiya, N., Hanada, A., Ogawa, M., Kuwahara,
A., Seo, M., Kamiya, Y., and Yamaguchi, S. (2007). Contribution
of gibberellin deactivation by AtGA2ox2 to the suppression of germi-
nation of dark-imbibed Arabidopsis thaliana seeds. Plant Cell Physiol.
48: 555–561.
Zeevaart, J.A.D., Gage, D.A., and Talon, M. (1993). Gibberellin A1 is
required for stem elongation in spinach. Proc. Natl. Acad. Sci. USA
90: 7401–7405.
Zou, J., Zhang, S., Zhang, W., Li, G., Chen, Z., Zhai, W., Zhao, X.,
Pan, X., Xie, Q., and Zhu, L. (2006). The rice HIGH-TILLERING
DWARF1 encoding an ortholog of Arabidopsis MAX3 is required
for negative regulation of the outgrowth of axillary buds. Plant J. 48:
687–698.
2618 The Plant Cell
