Gibberellin Regulates Pollen Viability and Pollen
Tube Growth in Rice
Tory Chhun,a,1Koichiro Aya,a,b,1Kenji Asano,a,bEiji Yamamoto,aYoichi Morinaka,cMasao Watanabe,d
Hidemi Kitano,aMotoyuki Ashikari,aMakoto Matsuoka,a,2and Miyako Ueguchi-Tanakaa
aBioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan
bJapan Society for the Promotion of Science, Chiyoda, Tokyo 102-8472, Japan
cHonda Research Institute Japan, Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan
dGraduate School of Life Science, Tohoku University, Aoba, Sendai 980-8577, Japan
Gibberellins (GAs) play many biological roles in higher plants. We collected and performed genetic analysis on rice (Oryza
sativa) GA-related mutants, including GA-deficient and GA-insensitive mutants. Genetic analysis of the mutants revealed
that rice GA-deficient mutations are not transmitted as Mendelian traits to the next generation following self-pollination of
F1 heterozygous plants, although GA-insensitive mutations are transmitted normally. To understand these differences in
transmission, we examined the effect of GA on microsporogenesis and pollen tube elongation in rice using new GA-
deficient and GA-insensitive mutants that produce semifertile flowers. Phenotypic analysis revealed that the GA-deficient
mutant reduced pollen elongation1 is defective in pollen tube elongation, resulting in a low fertilization frequency, whereas
the GA-insensitive semidominant mutant Slr1-d3 is mainly defective in viable pollen production. Quantitative RT-PCR re-
vealed that GA biosynthesis genes tested whose mutations are transmitted to the next generation at a lower frequency are
preferentially expressed after meiosis during pollen development, but expression is absent or very low before the meiosis
stage, whereas GA signal-related genes are actively expressed before meiosis. Based on these observations, we predict
that the transmission of GA-signaling genes occurs in a sporophytic manner, since the protein products and/or mRNA
transcripts of these genes may be introduced into pollen-carrying mutant alleles, whereas GA synthesis genes are trans-
mitted in a gametophytic manner, since these genes are preferentially expressed after meiosis.
Gibberellins (GAs) are tetracyclic diterpenoid compounds, some
of which function as endogenous plant growth regulators. Phe-
notypic analyses of mutants with reduced GA production and
responses to GA have revealed that active GAs play an essential
role in many aspects of plant growth and development, including
seedgermination, leafandstemelongation, flowerinduction and
development, and fruit and seed development (Davies, 2005;
Fleet and Sun, 2005). Reproductive development is one of the
most important physiological events in which GAs are involved
(Pharis and King, 1985; King and Evans, 2003). For example,
Arabidopsis thaliana GA-deficient and GA-insensitive mutants
have demonstrated that GA synthesis and signaling are impor-
tant for flower induction and the development of flower organs,
1999; Dill and Sun, 2001; Cheng et al., 2004). There is also evi-
dence that active GA synthesis and signaling are essential for
anther development. For example, the Arabidopsis mutant ga1
(a loss-of-function mutant in copalyl diphosphate synthase) has
the anthers of this mutant, microsporogenesis occurs but the
pollen grains are not viable (Goto and Pharis, 1999). Loss-of-
function mutants in DELLA proteins, which are negative regula-
floral organs in ga1, demonstrating that GA promotes these
events by countering the function of DELLA proteins (Cheng
et al., 2004; Tyler et al., 2004). Pollen development is also defec-
tive in the tomato (Solanum lycopersicum) GA-deficient mutants
and gib-2 (a loss-of-function mutant in kaurenoic acid oxidase)
(Nester and Zeevaart, 1988; Jacobsen and Olszewski, 1991). By
contrast with At ga1, the development of the anthers in the
Similarly, a rice (Oryza sativa) loss-of-function mutant in GAMYB,
which functions asa positive transacting factor in GA signaling in
cereal aleurone cells, shows some defects in anther and pollen
development (Kaneko et al., 2004). Murray et al. (2003) also
reported that the overproduction of barley (Hordeum vulgare)
GAMYB in transgenic barley causes abnormal anther develop-
ment (decreased anther length, lighter anther color, lack of
anther dehiscence, and male sterility). These observations dem-
onstrate that defects in GA synthesis and GA signaling block
anther and pollen development.
In addition, there have been some reports of the enhancement
of pollen tube growth in vitro following GA application (Bhandal
1These authors contributed equally to this work.
2Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the find-
ings presented in this article in accordance with the policy described in
the Instructions for Authors (www.plantcell.org) is: Makoto Matsuoka
WOnline version contains Web-only data.
The Plant Cell, Vol. 19: 3876–3888, December 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
and Malik, 1979; Viti et al., 1990). Using transgenic Arabidopsis
overexpressing a pea (Pisum sativum) cDNA encoding the GA-
inactivating enzyme Ps GA2ox2, Swain and colleagues investi-
gated the role of GAs in pollen tube growth (Singh et al., 2002;
Swain et al., 2004). The growth of pollen tubes carrying the 35S-
PsGA2ox2 transgene was lower than that of nontransformed
pollen tubes. This reduced pollen tube growth in the 35S-
in vitro; by GA hypersensitive mutations, such as spy-5, sly1gar2,
and rga; or by an RNA interference silencing construct targeting
PsGA2ox2. Furthermore, the in vitro treatment of wild-type
pollen tubes with the GA biosynthesis inhibitor uniconazole
retarded pollen tube elongation. These observations strongly
suggest that GAs are important for normal pollen tube growth,
although the known GA biosynthesis and GA response mutants
in various species have not been reported to exhibit an obvious
pollen tube elongation phenotype (Singh et al., 2002).
Riceisa monocotmodel plant for whichmuchinformation and
materialisavailable,including thewhole-genome sequence,full-
length cDNA clones, transformation systems, and many mutant
collections. Taking advantage of these materials, we have per-
formed extensive screens and characterization of GA-deficient
and GA-insensitive mutants. In a search for GA-deficient mu-
tants, we identified 18 mutants at six different loci that encode
the GA metabolic enzymes ent-copalyl diphosphate synthase
(CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO),
ent-kaurenoic acid oxidase (KAO), GA 20-oxidase, and GA
3-oxidase (Sakamoto et al., 2004). Based on these mutants and
the expression patterns of the corresponding genes in the wild
type, we demonstrated that the enzymes that catalyze early
steps in the GA biosynthetic pathway (CPS, KS, KO, and KAO)
are encoded by single genes (Os CPS1, KS1, KO2, and KAO,
respectively). Knockout mutations of these genes induce severe
dwarf phenotypes and are severely defected in flower and seed
development (Sakamoto etal.,2004). Wealsoisolated threeGA-
insensitive mutants: gid1, which is defective in a soluble GA
receptor (Ueguchi-Tanaka et al., 2005); gid2, which is defective
in the degradation of a rice DELLA protein (SLR1) that is involved
in GA signaling (Sasaki et al., 2003); and gamyb, which is de-
fective in a positive transcription factor in GA signaling (Kaneko
et al., 2004). We further isolated a mutant that constitutively
responds to GA, slender rice1 (slr1; Ikeda et al., 2001; Itoh et al.,
In the process of the genetic analysis of these rice GA-related
mutants, we noticed a difference in the genetic frequency of
transmission of mutant alleles (transmission frequency) of GA-
deficient and GA-insensitive genes. For example, the frequency
of a homozygous mutation of Os CPS1 (Os cps1) was less than a
few percent in F2 segregants derived from self-pollinated F1
plants heterozygous in the allele, whereas that of the gid1 mu-
tation almost fit Mendelian rules (see below). The differences in
the transmission frequencies of GA-deficient and GA-insensitive
mutations led us to speculate that a novel mechanism regulates
GA signaling during the rice fertilization process. In this study,
we attempted to determine why such differences between GA-
deficient and GA-insensitive mutations occur. We isolated two
new GA-deficient and GA-insensitive mutants of intermediate
severity; plants of each line produce a few seeds following self-
mission frequency of the GA-deficient mutation is caused by
GA-insensitive mutant is mainly defective in pollen developmen-
tal processes. Based on these observations, we discuss two
different critical steps involved in GA signaling and GA biosyn-
thesis in the process of pollen development.
and GA-Signaling Mutants
Table 1 shows the segregation ratios of a GA-deficient mutant
and a GA-insensitive mutant at the F2 generation stage of self-
pollinated F1 plants carrying heterozygous alleles. The GA-
deficient mutant tested was Os cps1-1, which contains a severe
was gid1-3,a severemutationin theGAreceptor, because these
mutants show the typical severe phenotypes of GA-related mu-
tants (Sakamoto et al., 2004; Ueguchi-Tanaka et al., 2005). The
F2 segregation of the GID1 locus almost fit the expected ratio,
Table 1. Segregation of F2 Plants Derived from Self-Pollinated F1 Plants Heterozygous at the GID1 (GA Receptor) Locus or the Os CPS1
(GA Synthesis Enzyme) Locus
GID1/gid1-3 3 GID1/gid1-3
CPS1/cps1-1 3 CPS1/cps1-1
aSignificant at the 0.5% level.
Numbers in parentheses indicate the percentage relative to the total number of plants. NS, not significant.
GA and Pollen Activities 3877
1 (GID1/GID1):2 (GID1/gid1):1 (gid1/gid1), indicating that the gid1
By contrast, the segregation of the cps1 locus was apparently
distorted, with the frequency of the cps/cps genotype;3% and
that of the CPS1/CPS1 genotype >40% (Table 1). Such low
transmission of the mutant allele in the next generation was ob-
served not only in cps1-1 but also in other GA-deficient mutants
Isolation and Characterization of the GA-Deficient
To study the mechanism of the distorted transmission of GA
deficiency mutations, we isolated a new GA-deficient mutant of
intermediate severity, since previously isolated GA-deficient
mutants barely develop floral organs. From >100 semidwarf mu-
tants, we selected one mutant that was produced using ethylene
imine treatment. The frequency of the dwarf phenotype in the F2
progenies of heterozygous plants did not agree with the ex-
pected 3:1 ratio (P < 0.005): the phenotype was found in;6% of
the F2plants(Table 2).Toinvestigate whetherthemale orfemale
gametophyte is defective in this mutant, we artificially crossed
the mutant withthe cultivar from which itwas derived, Fujiminori,
using emasculated flowers. When the mutant was used as the
female parent (pollen receiver), we found that 70 to 80% of the
seeds were fertile, and fertile seeds were produced at a similar
frequency following artificial self-pollination of the original culti-
var (Table 3). However, when the mutant was used as a male
mutation caused a defect in the male gametophyte. The mutant
exhibited reduced pollen germination and elongation (see be-
low). We designated this mutant reduced pollen elongation1
Next, we confirmed that the abnormal phenotypes of rpe1 are
caused by a defect in GA biosynthesis. Using 30 dwarf F2 plants
of a cross between rpe1 (japonica) and Kasalath (indica), we
analyzed the linkage between the RPE1 locus and molecular
markers corresponding to four GA synthesis genes, Os CPS1,
KS1, KO2, and KAO, all of which have already been mapped and
not examine the genes encoding two enzymes that catalyze later
GA biosynthesis steps, GA20 oxidase and GA3 oxidase, be-
cause previous studies had revealed that defects in these en-
zymes do not affect fertility (Sakamoto et al., 2004). The Os KAO
molecular marker was completely linked with the rpe1 mutation,
suggesting that RPE1 is identical to Os KAO. We then examined
the sequence of Os KAO in the rpe1 mutant and found one
To confirm that KAO corresponds to the rpe1 locus, we per-
formed a complementation test. A 9.4-kb DNA fragment con-
taining the entire KAO sequence, including;3.0 and;1.0 kb of
the 59- and 39-flanking regions, respectively, was introduced into
rpe1 by Agrobacterium tumefaciens–mediated transformation.
The dwarf phenotype of all of the plants that were resistant to
hygromycin, the selection marker used for transformation, was
rescued (see Supplemental Figure 1B online). We had previously
isolated knockout alleles of KAO that exhibit a severe dwarf
phenotype and barely develop floral organs (Sakamoto et al.,
2004). These observations indicate that the rpe1 mutant pheno-
type is caused by a partial defect in the functioning of KAO.
rpe1 Develops Normal Flowers but Shows Impaired Pollen
Germination and Elongation
rpe1 exhibits a typical GA-related dwarf phenotype (Figure 1A).
The mutant develops normal flowers with normal pistils and
stamens (Figures 1B to 1D). Although the pollen viability and the
number of mature pollen grains in rpe1 are similar to those of the
wild type (Figure 1E, Table 4), the seed viability is ;40% com-
pared with the viability of the wild type of >90% (Table 4). We
examined the germination and elongation of pollen by staining
had germinated and elongated (Figures 2B and 2E). By contrast,
;20% of the rpe1 pollen grains had germinated, and <10% had
elongated (Figures 2A and 2E). Impaired pollen germination and
elongation were also observed following pollination of wild-type
stigma with rpe1 pollen (Figure 2C), whereas wild-type pollen
germinated normally and elongated on the rpe1 stigma (Figure
2D), indicating that the impaired pollen germination and elonga-
tion is due to a defect in rpe1 pollen. At 2 h after pollination,
several elongated wild-type pollen tubes were observed in the
ovary, and some had reached the wild-type ovule (arrowhead in
Table 2. Segregation of F2 Plants Derived from Self-Pollinated F1
Plants Heterozygous at the RPE1 Locus
RPE1/rpe1 3 RPE1/rpe1
Wild Type rpe1Total x2(3:1)
182 (93.8%) 12 (6.2%) 194
190 (93.5%) 13 (6.4%) 203
258 (93.8%) 17 (6.2%) 275
aSignificant at the 0.5% level.
Numbers in parentheses indicate the percentage relative to the total
number of plants.
Table 3. Reciprocal Crossing of rpe1 and Wild-Type Plants
WT(\) 3 WT(_) Fertilea
15 (78.9%)4 (21.1%) 19
aFlowers that set seed were judged as fertile, and those that failed were
judged as infertile.
Numbers in parentheses indicate the percentage relative to the total
number of spikelets. The percentage of success of artificial crossing of
rice is ;80%. WT, wild type.
3878 The Plant Cell
Figure 2G), but no elongated rpe1 pollen tubes were seen in the
mutant ovary (Figure 2F). However, after 4 h, elongating pollen
tubes were sometimes observed to reachthe ovule in the mutant
ovary (arrowhead in Figure 2H). This indicates that the rpe1 mu-
tation does not completely abolish pollen germination or elonga-
tion but does severely disturb these processes. This observation
is also consistent with the semifertility of rpe1 (Table 2). We con-
is due to the rpe1 mutation (Figures 2I and 2J).
GA Is Essential for the Germination and Elongation of
We confirmed that the impaired germination and elongation of
tubes had reached the ovule by 2 h after pollination, as observed
with wild-type pollen (cf. Figures 3D with 3C and 2G).
We also investigated the GA dose dependency of pollen ger-
mination and elongation. Both events were increased by an in-
crease in the GA4concentration to 10?7M and then decreased
by treatment with 10?6M GA4(Figure 4). With 10?7M GA4, the
frequencies of germinating and elongating pollen in the mutant
were similar to those in the wild type, indicating that the impaired
on a defect in GA synthesis. Interestingly, in both wild-type and
mutant flowers treated with 10?6M GA4, the frequencies of both
events were lower than those observed following treatment with
10?7M GA4. This result shows that a higher GA4concentration
has an inhibitory effect on these events (see Discussion). Similar
observations of high GA concentrations inhibiting wild-type
pollen tube elongation have been reported (Singh et al., 2002).
Isolation and Characterization of a New GA-Insensitive
The above results with rpe1 clearly demonstrate that a defect in
GA synthesis causes impaired pollen germination and elonga-
GA-insensitive mutant. One mild GA-insensitive dwarf mutant
Figure 1. Phenotype of the rpe1 Mutant.
(A) Gross morphology of wild-type Fijiminori (left) and the rpe1 mutant (right) at the ripening stage. Bar ¼ 30 cm.
(B) Flowers of the wild type (left) and rpe1 (right). Cp, carpel; Le, lemma; Lo, lodicle; Pl, palea; St, stamen. Bar ¼ 2 mm.
(C) Mature stamens of the wild type (left) and rpe1 (right). An, anther; Fl, filament. Bar ¼ 1 mm.
(D) Pistils of the wild type (left) and rpe1 (right). Bar ¼ 1 mm.
(E) Pollen grains stained with I2-KI solution. Wild type (left) and rpe1 (right). Pollen grains staining black were judged as viable, and those staining yellow
or light red were judged as sterile. Bars ¼ 100 mm.
Table 4. Pollen and Seed Viability in the rpe1 Mutant at the Mature Stage
Wild Type (Fujiminori)rpe1 Wild Type (T65)Slr1-d3
Viable pollen (%)a
Number of pollen grains
Seed viability (%)b
91.61 6 7.04
4317 6 257
92.30 6 0.97
88.57 6 6.52
4300 6 427
42.39 6 2.38*
90.5 6 0.84
4875 6 730
82.0 6 1.27
29.20 6 4.51*
4592 6 474
56.0 6 3.03*
aPollen grains staining black with I2-KI solution were judged as viable, and those staining yellow or light red were considered sterile. The percentage of
viable pollen was calculated relative to the total pollen counted. *Significantly different from the wild type (P < 0.01).
bSeed viability represents the percentage of spikelet number that set seed per total number.
GA and Pollen Activities 3879
developed floral organs (Figure 5). However, as this mutant has
normal fertility, it was unsuitable for the above purpose. Another
GA-insensitive mutant, gid1-7, has a more severe phenotype
than gid1-8, as it is completely defective in pollen development
(Figure 5); therefore, this mutant was also unsuitable for our ex-
periment. Thus, we performed a screen for a new GA-insensitive
mutant with semidwarfism and semifertility and found one can-
didate that fulfilled these conditions.
This new mutant has a semidwarf phenotype with a height
similar to that of rpe1 at the heading stage (Figure 5). The mutant
develops flowers with normal stamens and pistils (Figure 5). Mo-
lecular characterization of this mutant revealed that the mutation
is located on the SLR1 locus and results in an amino acid substi-
Figure 2 online). The SLR1 locus encodes a DELLA protein, a
suppressor protein in GA signaling, and its GA-dependent
Figure 2. Germination and Elongation of rpe1 Pollen.
(A) to (D) and (F) to (J) Growth of pollen of rpe1 ([A], [C], [F], [H], and [I]) and the wild type ([B], [D], [G], and [J]) on rpe1 ([A], [D], [F], [H], and [J]) or
wild-type stigmas ([B], [C], [G], and [I]). Pollen grains were stained with aniline blue at 30 min ([A] to [D]), 2 h ([F], [G], [I], and [J]), or 4 h (H) after artificial
pollination. Arrowheads indicate pollen tube elongation. Bars ¼ 200 mm.
(E) Germination and elongation frequencies of wild-type and rpe1 pollen. Open and closed bars represent the frequencies of wild-type and rpe1 pollen
germination or elongation, respectively. Germination and elongation frequencies were estimated by staining with aniline blue at 30 min and 2 hafter self-
3880The Plant Cell
degradation is essential for GA action in rice (Ikeda et al., 2001;
(Zea mays), and rice, some lines with mutations in DELLA genes
that cause in-frame deletions or amino acid exchanges in con-
served domains, such as the DELLA and TVHYNP domains,
produce mutant proteins that constitutively suppress GA action,
even under high GA conditions (Peng et al., 1997, 1999; Itoh
et al., 2002). Similarly, the semidwarf phenotype of the new mu-
tant was inherited in a semidominant fashion. These results indi-
cate that the mutant carries a gain-of-function allele of the SLR1
locus whose product functions constitutively to partially suppress
plants for further studies.
Slr1-d3 is semifertile, even though it develops normal flowers
with morphologically normal stamens and pistils (Figure 5). The
anthers of Slr1-d3 appear normal and produce a similar number
of pollen grains per spikelet as those of the cultivar from which it
was derived, T65 (Table 4). However, the viability of the mutant
pollen is lower than that of wild-type pollen (Figures 6A and 6D,
Table 4). Interestingly, however, when we examined the germi-
nation and elongation of Slr1-d3 pollen at 30 min after pollination
by the same way as rpe1, the frequencies were much lower than
those of the wild type, while the elongation was almost similar to
those of the wild type and much faster than those of rpe1 (cf.
Figures 6E with 2A and 6B). Two hours after artificial self-
pollination, an elongated pollen tube was sometimes observed
to reach the ovule in self-pollinated Slr1-d3 ovary, but elongated
pollen tubes were observed more frequently in the wild-type
ovary (cf. Figures 6C and 6F). These findings suggest that the
semifertility of the Slr1-d3 mutant is mainly caused by a high
Figure 3. Effect of GA on Pollen Tube Elongation.
Growth of pollen from rpe1 ([A] to [D]) on its own stigmas that were left
treated with mock ([A] and [C]) or with GA4([B] and [D]). Pollen grains
were stained with aniline blue at 30 min ([A] and [B]) or 2 h ([C] and [D])
after artificial pollination. Emasculated wild-type flowers with stamens
removed were pretreated with GA4for 30 min before pollination. Arrow-
head indicates pollen elongation. Bars ¼ 200 mm.
Figure 4. Dose-Dependent Effect of GA4on Pollen Germination and
Frequencies of pollen germination (A) and elongation (B) were estimated
by counting the germinated and elongated pollen grains stained with
aniline blue after 30 min or 2 h, respectively, on artificially pollinated wild-
type stigmas treated with various concentrations of GA4. The GA4
treatment procedure was the same as in Figure 3. Open and closed
bars represent the frequencies for wild type and rpe1 pollen, respec-
tively. * and ** indicate significant differences at the 5 and 1% levels,
respectively, as judged using the Student’s t test.
GA and Pollen Activities3881
Why Do rpe1 and Slr1-d3 Show Different Phenotypes in the
We next examined the expression profiles of GA-signaling and
GA synthesis genes at various anther developmental stages. As
mentioned above, rice knockout mutants in genes encoding
enzymes that catalyze early steps in the GA synthesis pathway
(Os cps1, ks1, ko2, and kao) show severe defects in flower and
seed development, whereas those catalyzing later steps (sd1
and d18) show normal flower and seed development (Itoh et al.,
2001; Ashikari et al., 2002; Sasaki et al., 2002, Sakamoto et al.,
2004). Thus, we focused on expression analysis of four of the
genes that encode early enzymes in the pathway: CPS1, KS1,
KO2, and KAO. We also analyzed the expression of four GA
signaling–related genes: GID1, GID2, SLR1, and GAMYB. Based
on previous observations by Itoh et al. (2005), we divided the
process of rice anther development into five stages: establish-
ment of pollen mother cells (stage 1), meiosis (stage 2), tetrad
(stage 3), microspore (stage 4), and mature pollen (stage 5)
(Figure 7A). We performed real-time RT-PCR analysis using RNA
isolated from whole anthers at various stages but not from pollen
or pollen mother cells because of difficulty of isolation of some
Figure 5. Phenotypes of GA-Insensitive Mutants with Different Levels of Impairment.
Gross morphology at the ripening stage. Flower, stamen, and pistil of the wild type (T65), gid1-8, Slr1-d3, and gid1-7 (left to right). Wild-type and gid1-8
produce fertile flowers, Slr1-d3 produces semifertile flowers, and gid1-7 does not produce fertile flowers.
Figure 6. Viability, Germination, and Elongation of Slr1-d3 Pollen.
(A) and (D) Viability of wild-type T65 (A) and Slr1-d3 (D) pollen. Pollen grains were stained with I2-KI solution. Pollen grains staining black were judged as
viable, and those staining yellow or light red were considered sterile.
(B),(C),(E), and (F) Growth of pollen of thewild type ([B] and [C]) and Slr1-d3 ([E]and [F])on their own stigmas. Pollen was stained with aniline blue after
30 min ([B] and [E]) or 2 h ([C] and [F]). Artificial pollination was performed by hand. Arrowheads indicate pollen elongation.
Bars ¼ 200 mm.
3882 The Plant Cell
amounts of RNAs. The results of real-time RT-PCR revealed that
all of the GA synthesis–related genes examined were predom-
inantly expressed at stage 3 or later, which corresponds to the
KAO was seen in the mature pollen stage (stage 5), whereas ex-
pression level of KS1 was lower at stage 5 than that at stage 4.
High level expression of CPS1 in mature pollens has also been
the premeiotic stage, however, no (CPS1, KS1, and KO2) or low
(KAO) expression was observed (Figure 7B). By contrast, all of
premeiotic stage, and their expression was rapidly decreased
after stage 3 (Figure 7B).
We also examined the segregation ratio in progenies of het-
erozygous plants of GA-deficient (cps1, ks1, ko2, and kao) and
GA-insensitive (gid1-1, gid2-2, slr1-1, Slr1-d3, and gamyb-2)
of the GA-insensitive mutants almost fit the expected ratio with
Figure 7. Expression Pattern of GA Synthesis and GA-Signaling Genes in the Process of Anther Development.
(A) Anther development of wild-type rice at various stages. Each developmental stage was scored according to the length of the lemma: pollen mother
cell stage (stage 1, lemma 2 mm), meiosis stage (stage 2, 2 to 3 mm), tetrad stage (stage 3, 3 to 5 mm), microspore stage (stage 4, 5 to 8 mm), and
mature pollen stage (stage 5, ;8 mm). Arrow indicates the timing of cytokinesis of meiosis. PMC, pollen mother cell; MC, meiocyte; Tds, tetrads; MS,
microspore; MP, mature pollen. Bars ¼ 25 mm.
(B) Quantitative RT-PCR analysis of GA biosynthesis and GA-signaling genes at various anther developmental stages. The numbers 1 to 5 correspond
to the anther developmental stages. Relative mRNA level was determined by normalizing the PCR threshold cycle number of each gene with that of the
Actin1 reference gene, and data were the average of three replicates. The top panel represents the relative expression levels of all genes tested at
stages 1 to 5. The bottom panel shows the enlarged view of the expression levels of genes with lower expression at stages 1 to 4. The striped and open
boxes represent the GA synthesis and GA-signaling genes, respectively. Arrows indicate the timing of cytokinesis of meiosis. Error bars represent SE of
three biological replicates.
GA and Pollen Activities 3883
one exception of Slr1-d3 (see Discussion). However, the segre-
gation of all of the GA-deficient mutants examined was appar-
ently distorted. Interestingly, the segregation frequencies of the
;8%, whereas that of cps1 was 2 to 3% and that of ko2-1 was
<1%. We alsoconfirmed the statistical significance of these seg-
cps1-2, and ko2 mutations and that of ko2-1 was significantly
lower than that of kaos and ks1-2.
There is a correlation between the transmission frequency of
these mutations and their expression patterns during the pro-
cess of pollen development. For example, KAO, whose mutation
showed the highest transmission among these mutants, was ex-
pressed most rapidly during the developmental process in these
genes, whereas CPS1 and KO2, whose mutations showed lower
transmission, were expressed onlyatlate stages (Figure7B).The
correlation between the expression pattern and the transmission
is critical in determining the transmission frequencies of mutant
alleles of GA synthesis genes (see Discussion). Furthermore, it is
noteworthy that the transmission frequency of a mild allele of
kao, rpe1 (Table 2), is similar to those of corresponding null al-
in the KAO enzymatic activities of the mild allele (rpe1) and the
strong alleles (kao-1 and -3) is irrelevant in the process of pollen
germination and elongation, even though such differences clearly
reflect a difference in the severities of their dwarfism. Similarly, a
mild allele of ko2, d35Tan-Ginbozu(ko2-2), which is a well-known
semidwarf mutant of a high-yielding variety and therefore exhibits
very good fertility (Itoh et al., 2004), showed a lower segregation
ratio (;15%) in its F2 progeny. This suggests that even a mild
mutation that is thought to induce no abnormality except dwarf-
ism also causes a defect in pollen germination and elongation. In
other words, pollen germination and elongation might be very
sensitive to the GA level, as are leaf and stem elongation (see
In this study, we attempted to reveal the molecular mechanisms
of differences in the transmission frequencies of GA-deficient
and GA-insensitivemutationsin the F2segregation stage.For this
GA-insensitive Slr1-d3. Phenotypic analyses of these mutants,
as well as genetic analyses of previously isolated GA-related
mutants, demonstrated that even though both GA-deficient and
GA-insensitive mutations affect pollen activity and consequently
induce a male-sterilephenotype,GA-deficient mutantsare mainly
defective in pollen germination and elongation and GA-insensitive
mutants are mainly defective in pollen development. Expression
analysis revealed that the expression of genes involved in GA
signaling, such as GID1, GID2, SLR1, and GAMYB, actively oc-
curs at the premeiosis stage in the pollen developmental pro-
cess, whereas the expression of genes involved in GA synthesis,
such as CPS1, KS1, KO2, and KAO, occurs preferentially after
meiosis (Figure 7B). Taking these observations together, we pre-
dict that differences in the transmission frequencies of GA-
deficient and GA-insensitive mutations depend on differences in
the expression levels of these genes in the pollen developmental
process (Figure 8). The GA-signaling genes are actively ex-
pressed in anthers, specifically in pollen mother cells and the
tapetum (Kaneko et al., 2003, 2004; Tsuji et al., 2006), before
meiosis, and the mRNA and/or protein products of these genes
might be introduced into pollen carrying mutant alleles. Conse-
quently, the transmission of the GA-signaling genes occurs in a
sporophytic manner. By contrast, the GA synthesis genes are
preferentially expressed after meiosis; therefore their transmis-
sion depends on the genotypes of each pollen grain in a game-
GA synthetic genes in the pollen developmental process and
their transmission frequencies supports this hypothesis.
De Novo Synthesis of GA Is Essential for Pollen
Germination and Elongation
Examination of the rpe1 phenotype demonstrated that pollen
germination and elongation essentially depend on the de novo
synthesis of GA in rice (Figure 2). The dependence of pollen
germination and elongation on GA was also supported by ex-
periments with exogenous treatments of GA4(Figure 3). Swain
and colleagues reported that GA plays a physiological role in
Arabidopsis pollen tube growth, based on experiments using a
transgeniclinecarryinga cDNA encodingthe pea GA-inactivating
enzyme GA2 oxidase2 under the control of the 35S promoter
(Singh etal.,2002; Swain etal.,2004). The authorsobserved that
the growth of pollen tubes carrying the transgene was lower than
that of normal pollen, and the impaired pollen tube growth was
partially reversed by GA application in vitro or by combination
with mutations that cause an increased GA response, such as
spy-5, sly1gar2, or rga (Singh et al., 2002; Swain et al., 2004). The
inhibitory effect ofuniconazole onpollen tubegrowthinvitro also
Table 5. Genetic Analysis of GA-Deficient and GA-Signaling Mutants
The letters a to d denote statistically significant differences (P < 0.05)
according to the x2goodness-of-fit test followed by the Tukey’s wholly
significant difference test. NS, not significant; *, significant at the 0.5%
level; **, included wild (SLR1/SLR1) and mild dwarf (Slr1-d3/SLR1)
3884 The Plant Cell
supported their hypothesis (Singh et al., 2002). Although some
studies of the role of GA in pollen tube growth were discussed
based on results of the application of GA on pistils or the in vitro
observations by Swain and colleagues were the first report that
directly suggested the importance of GA in pollen tube elonga-
tion. Curiously, however, no GA mutants previously identified in
pea, Arabidopsis, or other species have been reported to show
impaired pollen development (Swain et al., 1997). In this regard,
Singh et al. (2002) discussed the possibility that many of these
mutants may have relatively mild changes in pollen tube elon-
gation that cannot be easily identified. This is not the case with
mutants with defects in early steps of the GA biosynthetic path-
way show distorted pollen transmission frequencies, even with
a mild mutation, d35Tan-Ginbozu. As d35Tan-Ginbozuwas used in the
production of a high-yielding variety, Tan-Ginbozu, this single
recessive mutation was thought to cause no abnormal pheno-
ever, this study shows that the d35Tan-Ginbozumutation causes
not only a semidwarf phenotype but also impaired fertility. This
elongation, is one of the most sensitive biological events and
reflects the endogenous GA level. This idea is supported by the
observation that the transmission frequency of an intermediate
kao-1 and kao-3. This result indicates that the remaining activity
of the KAO enzyme produced by rpe1, which can cause partial
shoot and stem elongation and also supports almost complete
flower organ development (Figure 1), does not function in pollen
tube elongation. In other words, the acceptable value of GA syn-
thetic activity should be high enough for normal pollen elonga-
tion, and even mild defects in GA synthesis severely affect GA
activity. The reason why GA-deficient mutants of other species
do not exhibit an obvious impaired pollen tube elongation phe-
notype has not yet been elucidated. One possibility is that these
GA-deficient mutations are inherited in a sporophytic manner
because of the expression of these genes in the premeiotic
stage, as in the rice genes involved in GA signaling.
Function of the GID1/DELLA-Mediated GA Perception
System in Pollen Tube Elongation
The semifertility of the Slr1-d3 homozygous mutant was mainly
caused by impaired pollen development instead of impaired
pollen tube elongation (Figure 6). However, the requirement for
GA synthesis in rice pollen for pollen tube elongation directly
indicates that the GA-signaling pathway should work in pollen.
Why then is the critical step in the fertilization process different
between the GA-signaling and GA-deficient mutants? One pos-
pathway is not essential for pollen tube elongation and that an
cess. However, this possibility is unlikely, at least in Arabidopsis,
because of the following observations. The impaired pollen elon-
gation of transgenic Arabidopsis ectopically expressing a gene
tant of the GA signaling–specific F-box protein gene (SLY1), and
(Singh et al., 2002), both of which cause the GA-hypersensitive
phenotype as a result of changes in the sensitivity of the GID1/
DELLA-mediated GA-signaling pathway. The lack of functional
versions of three DELLA proteins, RGL1, RGL2, and RGA, com-
pletely restoredpetal and stamendevelopment in ga1-3, a severe
GA-deficient Arabidopsismutant,andpermitted normalseedset
(Cheng et al., 2004), supporting the above idea. Furthermore, in
Figure 8. Model to Explain Differences in the Transmission Frequencies of GA-Deficient and GA-Insensitive Mutations.
Pollen carrying GA-deficient mutations, which are poorly transmitted, does not synthesize GA, which is essential for pollen tube germination and
elongation, because the expression of these genes occurs after meiosis. By contrast, GA signaling functions normally in pollen carrying GA-insensitive
mutations because the GA-signaling genes are actively expressed just before meiosis; therefore, their products should be transported into pollen
carrying mutant alleles. þ/? indicates a heterozygous genotype of pollen mother cells, whereas þ and – indicate the wild-type and mutant alleles,
respectively, of pollen cells. Gray shading indicates the expression of GA synthesis or GA-signaling genes in pollen mother cells or pollen.
GA and Pollen Activities 3885
rice, we also observed that the transmission of Slr1-d3, the
partially dominant allele of slr1, was significantly lower than the
theoretical value (<20%, Table 5). A dominant-negative form of
to affect pollens carrying the Slr1-d3 allele rather than those
carrying the wild allele. The relatively mild effect of the Slr1-d3
allele onthesegregationratioratherthanGA-deficient mutations
can be discussed as follows. All pollen grains from the hetero-
zygous plants (þ/Slr1-d3) will have some carryover Slr1-d3
mRNA. Consequently, the additional copies of Slr1-d3 mRNA
in pollens may only have a minor additional inhibitory effect on
pollen germination and growth. These observations and discus-
sion support the idea that the GID1/DELLA-dependent GA-
signaling pathwayalsofunctionsinpollentubeelongationin rice.
In addition, the inhibitory effect of a high concentration of GA4
on pollen germination and elongation (Figure 4) suggests that an
alternative GA-signaling pathway might be involved in these
events because other GA-responding events mediated by the
GID1/DELLA perception system do not show such an inhibitory
effect at high concentrations. Actually, when we consider the
molecular mechanism of the GID1/DELLA system, in which GA
induces the degradation of DELLA proteins by collaboration with
the GA signal-specific SCF complex, it is difficult to believe that
overdosing with GA can cause an inhibitory effect in this system.
Such an inhibitory effect of high concentrations of GA on pollen
Taking all these observations together, we predict a possible
scenario for the involvement of GA in rice pollen germination and
the synthesized GA is perceived by the GID1/DELLA-mediated
an alternative pathway might function to suppress GA signaling
at higher GA levels. Further study is required to confirm an
alternative pathway of GA signaling in pollen.
Isolation of the rpe1 and Slr1-d3 Mutants
The rice (Oryza sativa) mutants rpe1 and Slr1-d3 were initially isolated in a
screen for dwarf rice by treatment with ethylene imine and N-methyl-
for 3 d, sown in artificial soil, and then grown for 20 to 30 d. The seedlings
were then transplanted into a rice field and transferred to a glasshouse at
the heading stage to examine pollen growth.
To examine the segregation between the homozygous GID1/GID1, the
heterozygous GID1/gid1-3, and the homozygous gid1-3/gid1-3 and be-
tween the homozygous CPS1/CPS1, the heterozygous CPS1/cps1-1,
and the homozygous cps1-1/cps1-1, DNA was extracted from leaves of
14-d-old seedlings derived from GID1/gid1-3 and CPS1/cps1-1 plants
(see below for the DNA extraction procedure). The primers used for PCR
amplification are listed in Supplemental Table 1 online. PCR amplification
conditions were an initial incubation at 948C for 5 min followed by 35
cycles of 948C for 30 s, 608C for 30 s, and 728C for 45 s, with a final exten-
sion for 7 min at 728C. To observe the band patterns that revealed the
homozygous and heterozygous genotypes, 10 mL of each PCR product
was separated by electrophoresis in agarose gels (2.5% agarose in TBE
Treatment with GA and a GA Inhibitor
Three microliters of solutions containing 10?9, 10?8, 10?7, or 10?6M GA
were applied directly to the insides of emasculated spikelets.
Viable Pollen Assays
To evaluate pollen viability, six anthers before flowering were removed
a fine powder and stained with 10 mL of 1% (v/v) of I2in 3% (v/v) KI, and
scope. Pollen grains that were round in shape and stained black were
judged as viable or living pollen, and sterile or dead pollen was stained
yellow or light red.
Examination of Pollen Tube Growth
Aniline blue staining was performed as described by Ryan et al. (1998)
and Singh et al. (2002) with modifications.
Rice flowers were emasculated and artificially pollinated by hand. After
30 min, the pistils were removed and directly stained with aniline blue
on a glass slide for a few minutes before observation by UV micro-
scopy (Olympus; U-TV 0.5 XC). Germinated pollen refers to pollen grains
attached to the stigma whose pollen tube growth can be detected by
in 3:1 ethanol:acetic acid for 30 min and then softened in 1 N KOH for 30
for a few minutes and stained with 0.1% aniline blue in K3PO4buffer, pH
8.5, for 2 h at room temperature. The pistils were rinsed briefly in distilled
water and mounted in 50% glycerol. The samples were visualized by UV
microscopy. Elongated pollen refersto germinatedpollen whose filament
tubes elongate in a time-dependent manner.
DNA Sequence Analysis
To examine the nucleotide sequence in the rice GA biosynthetic gene Os
KAO (accession number AK069429), DNA was extracted from leaves of
2-week-old seedlings of the rpe1 mutant and the wild type from which it
was derived (cv Fujiminori). DNA extraction was performed using an
ISOPLANT extraction kit (Nippon Gene). PCR was then performed using
the primers listed in Supplemental Table 1 online. The PCR conditions
were 948C for 5 min followed by 40 cycles of 948C for 30 s, 558C for 30 s,
and 728C for 30 s. The amplified DNA fragments were then separated by
electrophoresis on a 0.7% (w/v) low-melting-point agarose gel, and the
fragments were sequenced directly with appropriate primers.
RNA Isolation and RT-PCR Analysis
Total RNA was extracted using the RNeasy plant mini kit (Qiagen) from
wild-type rice anthers at the various stages as described in Figure 7A.
First-strand cDNA was synthesized from ;1 mg of total RNA with an
oligo(dT) primer and Omniscript RT kit (Qiagen). Each transcript for GA-
related genes was quantified by a real-time PCR analysis using 10% of
3886The Plant Cell
the resulting cDNA as a template. Real-time PCR was performed with the
LightCycler system (Roche) and the QuantiTect SYBR Green PCR kit
(Qiagen). For this analysis, a linear standard curve and threshold cycle
number versus log (designated transcript level) were constructed using a
series of dilutions of each PCR product (10?17, 10?18, 10?19, and 10?20
M); the levels of the transcript in all unknown samples were determined
accordingto the standard curve. The Actin1 gene was usedas aninternal
standard for normalizing cDNA concentration variations. Data were the
average of three replicates. The sequences of primer pairs are listed in
Supplemental Table 1 online.
Plant materials fixed in formalin:acetic acid:70% ethanol (1:1:18) were
dehydrated through a graded ethanol series and embedded in Paraplast
Plus (Sherwood Medical). Microtome sections (10 mm thick) were stained
with 0.2% hematoxylin.
A 9.4-kb genomic DNA fragment containing the full-length rice KAO gene
was separately isolatedbydigestingaBAC clonewith SmaIand XhoIand
with XhoI and DraI. The two isolated DNA fragments were each inserted
into the pBluescript II SKþ vector between the SmaI and XhoI sites or the
XhoI and DraI sites, respectively. The vectors containing the inserts were
redigested with SmaI and XhoI or with XhoI and XbaI, respectively. The
two insert fragments were then ligated and fused into pBluescript II SKþ
between the SmaI and XbaI sites. Finally, the DNA fragment (9.4 kb) was
subcloned between the SmaI and XbaI sites in a binary vector containing
kanamycin and hygromycin resistance genes, pBI-Hm12 (provided by
H.Hirano).Theinsertcontaining thefull-length KAOwastransformedinto
the rpe1 mutant using Agrobacterium tumefaciens (Hiei et al., 1994).
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL data libraries under the following accession
At RGA (At2g01570), At RGL1 (At1g66350), At RGL2 (At3g03450), At
RGL3 (At5g17490), Br RGA1 (AY928549), Hv D3 (Q9AXH9), Hv SLN1
Os GAMYB (AK102841), Os KAO (AK069429), Os KO2 (AK066285), Os
KS1 (AY347878), Os SLR1 (BAE96289), Zm D3 (AAC49067), Zm D8
(Q9ST48), and Te Rht-D1b (Q9ST59).
The following materials are available in the online version of this article.
Supplemental Figure 1. Mutation Site of the rpe1 Allele.
Supplemental Figure 2. Diagram of the Structure of the DELLA
Proteins and the Mutation Site of Slr1-d3 and Alignment of SLR1 and
Other DELLA Proteins at the TVHYNP Domain.
Supplemental Table 1. List of Primer Sequences.
We thank Kazuhiro Kobayashi and Yoshiaki Harushima for suggestions
regarding the examination of pollen tube elongation and Hitomi Kihara,
Mayuko Kawamura, and Hiroko Ohmiya for technical assistance. This
study was supported by a Grant-in-Aid from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (19570037 to M.U.-T.,
18075003 to M.W., and 18107001 and 18075006 to M.M.), the Ministry
of Agriculture, Forest, and Fisheries of Japan (Green Technology Project
IP1003; M.M. and M.A.), the Target Protein Project (M.M.), and research
fellowships from the Japan Society for the Promotion of Science for
Young Scientists (K.A. and K.A.).
Received August 3, 2007; revised November 10, 2007; accepted No-
vember 18, 2007; published December 14, 2007.
Ashikari, M., Sasaki, A., Ueguchi-Tanaka, M., Itoh, H., Nishimura, A.,
Datta, S., Ishiyama, K., Saito, T., Kobayashi, M., Khush, G.S.,
Kitano, H., and Matsuoka, M. (2002). Loss-of-function of a rice
gibberellin biosynthetic gene, GA20 oxidase (GA20ox-2), led to the
rice ‘Green Revolution’. Breed. Sci. 52: 143–150.
Bhandal, I.S., and Malik, C.P. (1979). Effect of gibberellic acid,
(2-chloroethyl)phosphoric acid, actinomycin-D and cycloheximide
on the activity and leaching of some hydrolases in pollen suspension
cultures of Crotalaria juncea. Physiol. Plant. 45: 297–300.
Cheng, H., Qin, L., Lee, S., Fu, X., Richards, D.E., Cao, D., Luo, D.,
Harberd, N.P., and Peng, J. (2004). Gibberellin regulates Arabidopsis
floral development via suppression of DELLA protein function. Devel-
opment 131: 1055–1064.
Davies, P.J. (2005). Plant Hormones. (Dordrecht, The Netherlands:
Kluwer Academic Publishers).
Dill, A., and Sun, T.-P. (2001). Synergistic derepression of gibberellin
signaling by removing RGA and GAI function in Arabidopsis thaliana.
Genetics 159: 777–785.
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.
Fukuda, A., Nemoto, K., Chono, M., Yamaguchi, S., Nakajima, M.,
Yamagishi, J., Maekawa, M., and Yamaguchi, I. (2004). Expression
pattern of the coparyl diphosphate synthase gene in developing rice
anthers. Biosci. Biotechnol. Biochem. 68: 1814–1816.
Goto, N., and Pharis, R.P. (1999). Role of gibberellin in the develop-
ment of floral organs of the gibberellin-deficient mutant, ga1-1, of
Arabidopsis thaliana. Can. J. Bot. 77: 944–954.
Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient trans-
formation of rice (Oryza sativa L.) mediated by Agrobacterium and se-
quence analysis of the boundaries of the T-DNA. Plant J. 6: 271–282.
Ikeda, A., Ueguchi-Tanaka, M., Sonoda, Y., Kitano, H., Koshioka, M.,
Futsuhara, Y., Matsuoka, M., and Yamaguchi, J. (2001). slender
rice, a constitutive gibberellin response mutant, is caused by a null
mutation of the SLR1 gene, an ortholog of the height-regulating gene
GAI/RGA/RHT/D8. Plant Cell 13: 999–1010.
Itoh, H., Tatsumi, T., Sakamoto, T., Otomo, K., Toyomasu, T.,
Kitano, H., Ashikari, M., Ichihara, S., and Matsuoka, M. (2004). A
rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin bio-
synthesis enzyme, ent-kaurene oxidase. Plant Mol. Biol. 54: 533–547.
Itoh, H., Ueguchi-Tanaka, M., Sato, Y., Ashikari, M., and Matsuoka,
M. (2002). The gibberellin signaling pathway is regulated by the
appearance and disappearance of SLENDER RICE1 in nuclei. Plant
Cell 14: 57–70.
Itoh, H., Ueguchi-Tanaka, M., Sentoku, N., Kitano, H., Matsuoka, M.,
and Kobayashi, M. (2001). Cloning and functional analysis of two
gibberellin 3b-hydoroxylase genes that are differently expressed
during the growth of rice. Proc. Natl. Acad. Sci. USA 98: 8909–
Itoh, J., Nonomura, K., Ikeda, K., Yamaki, S., Inukai, Y., Yamagishi,
H., Kitano, H., and Nagato, Y. (2005). Rice plant development: from
zygote to spikelet. Plant Cell Physiol. 46: 23–47.
GA and Pollen Activities3887
Jacobsen, S.E., and Olszewski, N.E. (1991). Characterization of the
arrest in anther development associated with gibberellin deficiency of
the gib-1 mutant of tomato. Plant Physiol. 97: 409–414.
Kaneko, M., Inukai, Y., Ueguchi-Tanaka, M., Itoh, H., Izawa, T.,
Kobayashi, Y., Hattori, T., Miyao, A., Hirochika, H., Ashikari, M.,
and Matsuoka, M. (2004). Loss-of-function mutations of the rice
GAMYB gene impair a-amylase expression in aleurone and flower
development. Plant Cell 16: 33–44.
Kaneko, M., Itoh, H., Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M.,
Ashikari, M., and Matsuoka, M. (2003). Where do gibberellin biosyn-
thesis and gibberellin signaling occur in rice plants? Plant J. 35: 104–115.
King, R.W., and Evans, L.T. (2003). Gibberellins and flowering of
grasses and cereals: prizing open the lid of the ‘‘florigen’’ black box.
Annu. Rev. Plant Biol. 54: 307–328.
Koornneef, M., and van der Veen, J.H. (1980). Induction and analysis
of gibberellin sensitive mutants in Arabidopsis thaliana (L.). Theor.
Appl. Genet. 58: 257–263.
Murray, F., Kalla, R., Jacobsen, J., and Gubler, F. (2003). A role for
HvGAMYB in anther development. Plant J. 33: 481–491.
Nester, J.E., and Zeevaart, J.A.D. (1988). Flower development in normal
tomato and a gibberellin-deficient (ga-2) mutant. Am. J. Bot. 75: 45–55.
Peng, J., Carol, P., Richards, D.E., King, K.E., Cowling, R.J., Murphy,
G.P., and Harberd, N.P. (1997). The Arabidopsis GAI gene defines a
signaling pathway that negatively regulates gibberellin responses.
Genes Dev. 11: 3194–3205.
Peng, J., et al. (1999). Green revolution genes encode mutant gibber-
ellin response modulators. Nature 400: 256–261.
Pharis, R.P., and King, R.W. (1985). Gibberellins and reproductive
development in seed plants. Annu. Rev. Plant Physiol. 36: 517–568.
Ryan, E., Grierson, I.C., Cavell, A., Steer, M., and Dolan, L. (1998).
TIP1 is required for both tip growth and non-tip growth in Arabidopsis.
New Phytol. 138: 49–58.
Sakamoto, T., et al. (2004). An overview of gibberellin metabolism
enzyme genes and their related mutants in rice. Plant Physiol. 134:
Sasaki, A., Ashikari, M., Ueguchi-Tanaka, M., Itoh, H., Nishimura, A.,
Swapan, D., Ishiyama, K., Saito, T., Kobayashi, M., Khush, G.S.,
Kitano, H., and Matsuoka, M. (2002). A mutant gibberellin-synthesis
gene in rice. Nature 416: 701–702.
Sasaki, A., Itoh, H., Gomi, K., Ueguchi-Tanaka, M., Ishiyama, K.,
Kobayashi, M., Jeong, D.H., An, G., Kitano, H., Ashikari, M., and
Matsuoka, M. (2003). Accumulation of phosphorylated repressor for
gibberellin signaling in an F-box mutant. Science 299: 1896–1898.
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.
Swain, S.M., Muller, A.J., and Singh, D.P. (2004). The gar2 and rga
alleles increase the growth of gibberellin-deficient pollen tubes in
Arabidopsis. Plant Physiol. 134: 694–705.
Swain, S.M., Reid, J.B., and Kamiya, Y. (1997). Gibberellins are
required for embryo and seed development in pea. Plant J. 12:
Tsuji, H., Aya, K., Ueguchi-Tanaka, M., Shimada, Y., Nakazono, M.,
Watanabe, R., Nishizawa, N.K., Gomi, K., Shimada, A., Kitano, H.,
Ashikari, M., and Matsuoka, M. (2006). GAMYB controls different
sets of genes and is differentially regulated by microRNA in aleurone
cells and anthers. Plant J. 47: 427–444.
Tyler, L., Thomas, S.G., Hu, J., Dill, A., Alonso, J.M., Ecker, J.R., and
Sun, T.-P. (2004). DELLA proteins and gibberellin-regulated seed
germination and floral development in Arabidopsis. Plant Physiol. 135:
Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M., Itoh, H., Katoh, E.,
Kobayashi, M., Chow, T.-Y., Hsing, Y.C., Kitano, H., Yamaguchi, I.,
and Matsuoka, M. (2005). GIBBERELLIN INSENSITIVE DWARF1
encodes a soluble receptor for gibberellin. Nature 437: 693–698.
Viti, R., Bartolini, S., and Vitagliano, C. (1990). Growth regulators on
pollen germination in olive. Acta Hortic. 286: 227–230.
Wilson, R., Heckman, J.W., and Somerville, C. (1992). Gibberellin is
required for flowering in Arabidopsis thaliana under short days. Plant
Physiol. 100: 403–408.
3888 The Plant Cell