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Plant Physiol. Apr 2004; 134(4): 1642–1653.
PMCID: PMC419838

An Overview of Gibberellin Metabolism Enzyme Genes and Their Related Mutants in Rice1,[w]

Abstract

To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment.

The GAs form a large family of tetracyclic diterpenoid phytohormones that are involved in the regulation of various growth and developmental processes in higher plants. Bioactive GAs, such as GA1 and GA4, are synthesized from trans-geranylgeranyl diphosphate (GGDP) as shown in Figure 1 (Hedden and Kamiya, 1997; Hedden and Phillips, 2000). GGDP is converted to the tetracyclic hydrocarbon ent-kaurene via ent-copalyl diphosphate (CDP) by two kinds of diterpene cyclases in plastids, CDP synthase (CPS) and ent-kaurene synthase (KS). ent-Kaurene is then modified by sequential oxidations to produce GA12 via ent-kaurenoic acid. These steps are catalyzed by two membrane-associated Cyt P450 monooxygenases, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO). The final stage of bioactive GA synthesis, from GA53/GA12 to GA1/GA4, is catalyzed through two parallel pathways (i.e. early-13-hydroxylation and non-13-hydroxylation pathways) by two soluble 2-oxoglutarate-dependent dioxygenases (2ODDs) in the cytosol, GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox). The bioactive GA1/GA4 and their immediate precursors GA20/GA9 are inactivated by a third 2ODD, GA 2-oxidase (GA2ox). In recent years, almost all of the genes encoding these seven GA metabolic enzymes (CPS, KS, KO, KAO, GA20ox, GA3ox, and GA2ox) and their related mutants have been isolated from various plants (Hedden and Phillips, 2000). A typical phenotype of these mutants, except the mutants with GA2ox, is dwarfism, a state that is restored by the application of exogenous GA.

Figure 1.
Principal pathway of GA metabolism in plants. GA13ox, GA 13-hydroxylase.

Dwarfism is one of the most valuable traits in crop breeding because semidwarf cultivars are more resistant to damage by wind and rain (lodging resistant) and are associated with stable increased yields (Evans, 1993). Because of their agronomic importance, a large number of dwarf mutants of rice (Oryza sativa) have been isolated, and several of them have been characterized as GA-deficient (Murakami, 1972; Kobayashi et al., 1989; Itoh et al., 2001; Sasaki et al., 2002a). In this study, we attempted to identify the genes encoding the seven GA metabolic enzymes and their related mutants to enhance our current understanding of the GA metabolic pathway in rice. Although there is only one KAO-like gene in the rice genome, two to nine candidate genes were identified for the other six enzymes. Based on the mutant and expression analyses, we have revealed that the enzymes catalyzing early steps in GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by small gene families. We have also found that some CPS-like and KS-like genes are likely to be involved in diterpene phytoalexin biosynthesis.

RESULTS

Isolation of Putative GA Metabolic Genes in Rice

The predicted amino acid sequences of GA metabolic enzyme genes from various plants (including Arabidopsis, lettuce [Lactuca sativa], pea [Pisum sativum], tomato [Lycopersicon esculentum], and rice) were used as probes to screen in silico all available rice DNA databases (Feng et al., 2002; Goff et al., 2002; Sasaki et al., 2002b; Yu et al., 2002; Kikuchi et al., 2003). Candidate GA sequences detected during this process were also used reiteratively as search probes. Through this exhaustive sequence search, we found four CPS-like genes (OsCPS1-4), nine KS-like genes (OsKS1-9), five KO-like genes (OsKO1-5), four GA20ox-like genes (OsGA20ox1-4), two GA3ox-like genes (OsGA3ox1 and 2), and four GA2ox-like genes (OsGA2ox1-4), whereas there was only one KAO-like gene (OsKAO; Table I). Localization of the 29 candidate genes on the rice genome revealed that they are distributed across nine of the 12 rice chromosomes (Fig. 2A). There are five small gene clusters that contain 16 of the 29 candidate genes (shaded in red in Fig. 2A; see below).

Figure 2.
Chromosomal location of the candidate genes for GA metabolism and contiguous arrangement of CPS-like and KS-like genes. A, Map position of the candidate genes for GA metabolism on the rice genome. B to E, Contiguous arrangement of CPS-like and KS-like ...
Table I.
GA biosynthetic genes and their homologs in rice

CPS-Like Genes

Four CPS-like genes were identified in the rice genome, including a pseudogene (OsCPS3), the transcript of which lacks the coding sequence below exon 8, whereas CPS is encoded by the single gene, AtCPS/GA1, in Arabidopsis. Mapping analysis revealed that OsCPS1 and OsCPS2 are located on chromosome 2 and that OsCPS3 and OsCPS4 are located on chromosomes 9 and 4, respectively (Table I; Fig. 2A). Interestingly, OsCPS2 on chromosome 2 was contiguously clustered with three KS-like genes, OsKS5, OsKS6, and OsKS7 (Table I; Fig. 2, A and B). Similarly, OsCPS4 was located near to another KS-like gene, OsKS4, on chromosome 4 (Table I; Fig. 2, A and C), whereas OsCPS3 was mapped onto chromosome 9 without linkage to any KS-like genes (Table I; Fig. 2A).

The deduced amino acid sequence of Arabidopsis AtCPS/GA1 showed highest homology with OsCPS1 (41% identity) and relatively lower identities with OsCPS2 to OsCPS4 (ranging between 35% and 37%). An Asp-rich box, DXDDTA, which is shared in many terpene cyclases, catalyzing cyclization without removal of the diphosphate group, was conserved in OsCPS2 (at residues 374–379) and OsCPS4 (at residues 319–324); however, the first Asp was replaced with Glu in OsCPS1 (at residue 418), and the C-terminal half (DTA) was exchanged to TGS in OsCPSL2 (at residues 364–366). A SAYDTAWVA motif that is conserved among CPS and KS proteins was also conserved in OsCPS1 (at residues 155–163) and OsCPS3 (at residues 97–105), whereas amino acid substitutions were observed in OsCPS2 (at residues 109–117) and OsCPS4 (at residues 56–64). Phylogenetic analysis of the cyclases (CPS and KS; Fig. 3A) revealed that OsCPS1 was most closely related to maize CPS, ZmCPS/AN1 (63% identity; Fig. 3A), whereas OsCPS2 to OsCPS4 are grouped as a different clade from the OsCPS1 clade and the CPS proteins in dicot plants. This suggests that cyclases OsCPS2 to OsCPS4 might have different functions from those of OsCPS1 and ZmCPS/AN1 (see below).

Figure 3.
Phylogenetic relationships among GA metabolic diterpene cyclases, CPS and KS (A), GA metabolic Cyt P450-dependent monooxygenases KO and KAO (B), and GA metabolic 2ODDs GA20ox, GA3ox, and GA2ox (C). At, Arabidopsis; Cm, pumpkin; Cs, Cucumis sativus; Hv, ...

KS-Like Genes

Nine KS-like genes were identified in the rice genome, including a pseudogene (OsKS9), which contained a premature stop codon in exon 3, whereas KS is encoded by the single-copy gene, AtKS/GA2, in Arabidopsis. OsKS1, OsKS2, and OsKS3 were arranged as tandem direct repeats at 104 centimorgan (cM) of chromosome 4 (Table I; Fig. 2, A and D). OsKS4 was located near to the position of OsCPS4 on chromosome 4 as mentioned above (Table I; Fig. 2, A and C). OsKS5, OsKS6, and OsKS7 were also arranged within 150 kb at 86 cM of chromosome 2, where OsCPS2 is also located (Table I; Fig. 2, A and B). OsKS8 was located on chromosome 11 contiguously to the position of OsKS9 in the same direction (Table I; Fig. 2, A and E).

OsKS1 showed highest similarity to Arabidopsis AtKS/GA2 (43% identity), whereas the other OsKS proteins showed between 37% and 41% identities with the Arabidopsis protein. The conserved Asp-rich sequence, DDXXD, which is considered to be involved in binding to the diphosphate group of the substrate, was conserved in all OsKS proteins (e.g. at residues 552–556 of OsKS1). In the SAYDTAWVA motif, the first Ala was replaced with Leu in OsKS1 (at residue 80), with Pro in OsKS2, OsKS3, and OsKS7, and with Ser in the other OsKS proteins. The second Ala in the SAYDTAWVA motif was conserved in all OsKS proteins (e.g. at residue 84 of OsKS1), although it was replaced with Ser in AtKS/GA2. Phylogenetic analysis revealed that KS-like proteins are divided into monocot and dicot groups, and the monocot group is subdivided into two further groups. OsKS1-4 and OsKS7 were more closely related to maize (Zea mays) ZmKS (49%–54% identities), whereas OsKS5, OsKS6, and OsKS8 constituted another group (39%–43% identities with ZmKS; Fig. 3A).

KO-Like Genes

Five KO-like genes were identified in the rice genome, whereas KO is encoded by the single copy gene, AtKO/GA3, in Arabidopsis. Mapping analysis of the five KO-like genes revealed that they are all mapped to chromosome 6, arranging in tandem as a gene cluster (Table I; Fig. 2A; Itoh et al., 2004). The deduced amino acid sequence of Arabidopsis AtKO/GA3 showed higher identities with OsKO1 to OsKO3 (about 53%) and lower identities with OsKO4 and OsKO5 (47% and 46%). Phylogenetic analysis of GA-related Cyt P450-dependent monooxygenases (Fig. 3B) revealed that OsKO proteins are more similar to each other (64%–93% identities) than to any other reported KO proteins (46%–53% identities) and are subdivided into two groups (i.e. one group contains OsKO1, OsKO2, and OsKO3, and another group involves OsKO4 and OsKO5).

KAO-Like Gene

One KAO-like gene was identified in the rice genome, whereas KAO is encoded redundantly by two genes, AtKAO1 and AtKAO2, in Arabidopsis. We searched the databases using several regions of KAO proteins from various plant species, including Arabidopsis, garden pea, pumpkin (Cucurbita maxima), barley (Hordeum vulgare), and maize, but we could not identify any other genes except OsKAO. Mapping analysis revealed that OsKAO is located on chromosome 6 (Table I; Fig. 2A). The deduced amino acid sequence of OsKAO showed approximately 51% identities with Arabidopsis AtKAO1 and AtKAO2. Phylogenetic analysis revealed that KAO-like proteins from monocot plants share higher amino acid identities with each other (76%–77% identities) and lower identities with those of dicot plants (51%–54% identities; Fig. 3B). OsKAO is categorized into the monocot group, which includes maize ZmKAO/Dwarf3 (77% identity) and barley HvKAO1/Grd5 (76% identity).

GA20ox-Like Genes

Four GA20ox-like genes were identified in the rice genome, two of which (OsGA20ox1 and OsGA20ox2) were identical to the genes reported previously (Toyomasu et al., 1997; Sasaki et al., 2002a); in comparison, five copies of GA20ox genes have been reported in Arabidopsis (Hedden et al., 2002). Mapping analysis revealed that OsGA20ox1 is mapped to chromosome 3 (Table I; Fig. 2A). OsGA20ox2 is located on the long arm of chromosome 1, which is identical to the rice Green Revolution gene, Semi-Dwarf1 (SD1; Table I; Fig. 2A; Sasaki et al., 2002a). OsGA20ox3 and OsGA20ox4 were mapped to chromosomes 7 and 5, respectively (Table I; Fig. 2A).

The deduced amino acid sequence of OsGA20ox1 showed highest homology with Arabidopsis AtGA20ox1/GA5 (51% identity), whereas OsGA20ox2 to OsGA20ox4 showed 45% to 48% identities to it, respectively. The consensus sequence NYYPXCXXP of 2ODDs for binding the common cosubstrate, 2-oxoglutarate (e.g. at residues 216–224 of OsGA20ox1) and the three His residues (e.g. at residues 79, 233, and 289 of OsGA20ox1) for binding Fe2+ were conserved in all OsGA20ox proteins. A sequence LPWKET, which is considered to be involved in the binding of the GA substrates, was also conserved in all OsGA20ox proteins (e.g. at residues 128–133 of OsGA20ox1). Phylogenetic analysis of 2ODDs (GA20ox, GA3ox, and GA2ox; Fig. 3C) revealed that the GA20ox proteins from dicot plants shared higher amino acid identity each other (49%–80% identities) and formed a single group. OsGA20ox2 showed higher similarity (61% identity) to OsGA20ox4 than the other OsGA20ox proteins, and OsGA20ox2 and OsGA20ox4 formed one subgroup (36%–48% identities with dicot proteins), whereas OsGA20ox1 and OsGA20ox3 were separately located from the OsGA20ox2/OsGA20ox4 subgroup (39%–54% and 39%–49% identities with dicot proteins, respectively).

GA3ox-Like Genes

Two GA3ox-like genes were identified in the rice genome, both of which have been already reported by Itoh et al. (2001), whereas four copies of GA3ox genes were reported in Arabidopsis (Hedden et al., 2002). Mapping analysis revealed that OsGA3ox1 is located on the short arm of chromosome 5 (Table I; Fig. 2A), and OsGA3ox2 is located at the short arm of chromosome 1, which is identical to the rice dwarf gene, D18 (Table I; Fig. 2A; Itoh et al., 2001).

The deduced amino acid sequence of OsGA3ox1 and OsGA3ox2 showed 29% and 33% identities with Arabidopsis AtGA3ox1/GA4, respectively. Two His residues (e.g. at residues 245 and 303 of OsGA3ox1) and one Asp residue (e.g. at residue 247 of OsGA3ox1) at cofactor binding site were conserved in OsGA3ox1 and OsGA3ox2. The phylogenetic analysis revealed that OsGA3ox1 and OsGA3ox2 are more similar to each other (51% identity) than to any other reported GA3ox proteins from dicot plants (27%–35% identities) and constituted a separate group (Fig. 3C). As there are no reports describing the structure of GA3ox genes in monocot plants except rice at present, we have to wait to conclude that the rice GA3ox genes show higher similarity to other genes from monocot plants than those from dicots.

GA2ox-Like Genes

Four GA2ox-like genes were identified in the rice genome, three of which (OsGA2ox1, OsGA2ox2, and OsGA2ox3) were identical to the genes reported previously (Sakamoto et al., 2001; Sakai et al., 2003), whereas seven copies have been previously reported in Arabidopsis (Schomburg et al., 2003). Mapping analysis revealed that OsGA2ox1 and OsGA2ox4 are located on chromosome 5, and OsGA2ox2 and OsGA2ox3 are mapped on chromosome 1 (Table I; Fig. 2A). In contrast to the genes encoding the early steps in GA biosynthesis, such as the CPS-like, KS-like, and KO-like genes, OsGA2ox genes located on the same chromosomes do not form gene clusters but instead are separated by large distances.

Two His residues (e.g. at residues 241 and 302 of OsGA2ox1) and one Asp residue (e.g. at residue 243 of OsGA2ox1), which are supposed to associate at the catalytic site and to bind with Fe2+, respectively, were conserved in all the rice GA2ox proteins that we found. Phylogenetic analysis revealed that GA2ox proteins are subdivided into three groups. Two Arabidopsis GA2ox proteins, AtGA2ox7 and AtGA2ox8, constituted a separate branch that showed less similarity to the other GA2ox proteins (16%–23% identities with the others). The remaining two groups contained both monocot and dicot plant proteins (i.e. OsGA2ox1 and OsGA2ox2 were grouped with AtGA2ox4, AtGA2ox5, and PsGA2ox2, whereas OsGA2ox3 and OsGA2ox4 were grouped with AtGA2ox1, AtGA2ox2, AtGA2ox3, and the others; Fig. 3C). The arrangement of these three subgroups suggests that these related subgroups of GA2ox proteins might have homologous functions crossing between monocot and dicot plants (Sakai et al., 2003; Schomburg et al., 2003).

Isolation of Rice GA-Deficient Mutants

In silico screening of GA metabolic enzyme genes in rice DNA databases revealed that rice contains 29 candidate genes related to GA metabolism. However, this does not mean that all of these candidate genes are involved in GA metabolism, and it is highly likely that some of them are not related to GA but to other metabolisms. To elucidate which candidate genes are involved in GA biosynthesis, we took a genetic approach; that is, when the genes involved in GA biosynthesis are deficient in their function, mutant plants should exhibit a GA-deficient phenotype. Using this method, we performed a large-scale screening of rice GA-deficient mutants to identify the genes involved in GA biosynthesis.

Figure 4A shows a typical phenotype of a rice GA-deficient mutant at the young seedling stage. The GA-related mutants showed dwarfism without the induction of additionally aberrant morphology. The final plant height of GA-deficient mutants ranged widely between <5% and 90% of the wild-type plants. The leaf blades of the mutant plants became dark green, shorter, and wider than those of the wild-type plants. In contrast to GA-insensitive mutants, exogenously applied bioactive GA3 rescued the dwarfism of GA-deficient mutants to restore final plant heights similar to those of the wild type (Fig. 4B).

Figure 4.
Typical phenotype of GA-deficient rice dwarf mutants (A) and rescue of the dwarf phenotype by external GA3 treatment (B). We used three criteria for screening of the GA-deficient mutants, that is, dwarfism without aberrant morphology, dark green leaves, ...

Based on these phenotypic features, we performed a large-scale screening of rice mutant collections, which were produced by a chemical mutagen, a retrotransposon (Tos17), and γ-ray irradiation. Through the first screening of these mutant collections, we selected more than several hundred dwarf mutants as candidates. We analyzed the detailed phenotype and GA responsibility of these candidates and finally identified 18 lines as GA-deficient mutants (Table II; Supplemental Fig. 1). Among them, nine lines were derived from the traditional GA-deficient mutants (four alleles of sd1, four alleles of d18, and one allele of d35; Ogawa et al., 1996; Itoh et al., 2001; Ashikari et al., 2002; Sasaki et al., 2002a), and the remaining nine mutant lines with recessive alleles were novel mutants. All 18 mutants could be categorized into three groups by their phenotypes; namely, severe dwarf without flower or seed development (category I; Fig. 5, B–E; Supplemental Fig. 2), severe dwarf with seed development (category II; Fig. 5F), and semidwarf with seed development (category III; Fig. 5A, plants 5, 9, and 11). Seeds of category II and category III mutants were normally germinated (data not shown).

Figure 5.
Phenotypes of rice GA-deficient dwarf mutants. A, Comparison of gross morphology between mutant plants and their original cultivars. 1, Nipponbare (original strain for oscps-1, osks1-1, osko2-1, and oskao-1); 2, oscps-1 (null allele); 3, osks1-1 (null ...
Table II.
The original mutant names, their wild-type parental lines, and the mutagen used

Mapping analysis revealed that each of the nine novel dwarf mutants could be assigned to mutation sites at one of four loci. The mutation sites of two lines, oscps1-1 (Fig. 5, A, plant 2, and B) and oscps1-2, both of which showed severe dwarf phenotype without flower or seed development (category I), were mapped onto chromosome 2, tightly linked to OsCPS1. The sequence of OsCPS1 in these two lines revealed that oscps1-1 had an insertion of Tos17 in exon 7, whereas OsCPS1 in oscps1-2 had nine nucleotides deleted in exon 7, which encode the well-conserved three amino acid sequence, DLF (Fig. 6). No bioactive GA1 was detected in oscps1-1 (Table III). In addition, the level of ent-kaurene was severely decreased in oscps1-1 compared to its original strain, Nipponbare (Table III). This result supports the hypothesis postulated above that oscps1 had a defect in the activity of CPS, which catalyzes the formation of CDP, the immediate precursors of ent-kaurene.

Figure 6.
Genomic structure of the GA metabolic genes. Boxes and lines indicate exons and introns, respectively. The mutation sites are indicated below each line. Arrowheads indicate the insertion sites of a rice retrotransposon, Tos17. Nucleotide substitutions ...
Table III.
Endogenous levels (ng g−1 fresh weight) of various GAs in wild-type and mutant seedlings

The mutation sites of three other lines, osks1-1 (Fig. 5, A, plant 3, and C), osks1-2, and osks1-3, all of which showed severe dwarf phenotype without flower or seed development (category I), were mapped onto chromosome 4, tightly linked to the gene cluster of OsKS1 to OsKS3. Sequence analysis of the OsKS genes in these three mutant lines revealed that OsKS1 was deleted approximately 4.5 kb nucleotides, including the C-terminal half of OsKS1 protein in osks1-3, whereas osks1-1 and osks1-2 had a Tos17 insertion in exons 3 and 14 of OsKS1, respectively (Fig. 6). No mutation was observed in the OsKS2 or OsKS3 gene from the three mutant lines. Again, no bioactive GA1 was detected in osks1-1 (Table III). Similar to the case of oscps-1, the level of ent-kaurene was severely decreased in osks1-1 compared to its original strain, Nipponbare (Table III). The result is consistent with the hypothesis that osks1 had a defect in the activity of KS, which catalyzes the formation of ent-kaurene from CDP.

The mutation site of osko2-1 (Fig. 5, A, plant 6, and D), which showed severe dwarf phenotype without flower or seed development (category I), was mapped onto chromosome 6, tightly linked to the cluster of OsKO1 to OsKO5. Sequence analysis of the five OsKO genes in osko2-1 revealed that Tos17 was inserted into exon 4 of OsKO2 (Fig. 6). Recently, we have also demonstrated that the d35 mutant (Fig. 5A, plant 5), which showed semidwarf phenotype with seed development (category II), is due to a loss of function of the OsKO2 gene (T. Tatsumi, H. Itoh, and M. Matsuoka, unpublished data). The OsKO2 sequence in d35 had a single nucleotide substitution in exon 5, which induced an amino acid change (Fig. 6); therefore, we designated d35/Tanginbozu as osko2-2, a weak allele of OsKO2. The observed levels of GA53, GA44, GA19, GA20, and GA1 were lower in osko2-2 than in the original strain, Ginbozu, whereas ent-kaurene was highly accumulated (approximately 10-fold) in osko2-2 (Table III). This result supports the hypothesis that osko2 is deficient in the KO activity, which catalyzes the formation of ent-kaurenoic acid from ent-kaurene, as has already been suggested (Ogawa et al., 1996).

The mutation sites of the final three lines, oskao-1 (Fig. 5, A, plant 7, and E), oskao-2, and oskao-3, which showed severe dwarf phenotype without flower or seed development (category I), were mapped onto chromosome 6, tightly linked to OsKAO. Sequence analysis of OsKAO from these three lines revealed that oskao-2 had a single nucleotide substitution in exon 1, which induced an exchange of highly conserved amino acid residue among this kind of Cyt P450 monooxygenases, whereas the other two lines (oskao-1 and oskao-3) had a Tos17 insertion in exons 5 and 6 of OsKAO, respectively (Fig. 6). The levels of GA53, GA44, GA19, GA20, and GA1 in oskao-1 were severely decreased relative to those in its original strain, Nipponbare (Table III). The level of ent-kaurene in oskao-1 was less decreased than that in oscps-1 and osks1-1. The result implies that oskao had a severe defect not in the formation of ent-kaurene but in the activity of KAO, which catalyzes the formation of GA12, the precursors of GA53.

Figure 5 also shows the traditional GA-deficient mutants such as sd1 and d18. All four sd1 mutant alleles showed a semidwarf phenotype, which is favorable for the breeding of high-yielding cultivars (category III; Fig. 5A, plant 9; Ashikari et al., 2002). In d18 mutants, two lines, d18-dy (Fig. 5A, plant 11) and d18k, exhibited a semidwarf phenotype (category III) caused by weak alleles, whereas the remaining two lines, d18-AD (Fig. 5, A, plant 12, and F) and d18h, were severely dwarfed (category II) by the null alleles (Itoh et al., 2001). It is noteworthy that reproductive development was not abolished even in the null alleles of d18 (arrows in Fig. 5F), although the mutant's vegetative development was severely affected. In contrast to these traditional mutant lines, both vegetative and reproductive developments were severely restricted in the novel GA-deficient mutants as mentioned above (Fig. 5, B–E), probably because rice geneticists and breeders historically screened only self-fertile mutants.

As already reported (Ashikari et al., 2002; Sasaki et al., 2002a), sd1-1 was caused by a 383-bp deletion, which induced a frameshift and created a premature stop codon (Fig. 5, plant 9), whereas the other three sd1 alleles had single nucleotide substitutions, which induced amino acid changes (sd1-2, sd1-3, and sd1-4; Fig. 6). The levels of GA44, GA19, GA20, GA1, GA29, and GA8 in sd1-1 were lower than in the original strain, Usen, whereas the amount of GA53 in sd1-1 was slightly higher (Table III). This result confirms that the activity of GA20ox, which catalyzes the steps from GA53 to GA20 via GA44 and GA19, was weaker in sd1 than in the wild-type plants.

In the strong alleles of the d18 mutants, d18-AD (Fig. 5F) showed an 8-kb deletion that includes the entire coding sequence of OsGA3ox2 (Fig. 6). In another strong allele, d18h, a 1-bp deletion in exon 2 shifts the reading frame, as reported by Itoh et al. (2001; Fig. 6). In the weak alleles, d18-dy showed a 9-bp deletion in exon 1, which induced a 3-amino acid deletion, and d18k had a single nucleotide substitution in exon 1, which induced an amino acid change (Fig. 6). Although no bioactive GA1 was detected in d18-AD, GA20 accumulated at an approximately 10-fold increase over its original strain, Akibare, at the vegetative stage (Table III). This result strongly suggests that d18-AD is defective in the activity of GA3ox, which catalyzes the step from GA20 to GA1.

Expression of GA Metabolic Genes in Wild-Type Rice

Semiquantitative reverse transcription (RT)-PCR analysis revealed that the isolated GA metabolic genes were expressed at different levels in various organs of wild-type rice (Fig. 7). OsCPS1, OsKS1, OsKO2/D35, and OsKAO were broadly expressed in all the organs of wild-type rice that we tested, including vegetative shoot apices, leaf sheaths, leaf blades, elongating stems, roots, immature panicles, and panicles at flowering time, whereas the levels of OsKS1 in roots and of OsKAO in leaf sheathes and stems were lower than those in other organs.

Figure 7.
Expression of the GA metabolic genes in various organs of the wild-type rice. Total RNAs were isolated from vegetative shoot apices (1), leaf sheaths (2), leaf blades (3), stems (4), roots (5), immature panicles (6), and panicles at flowering time (7), ...

All OsGA20ox genes were expressed in immature and mature panicles at different levels. In addition, OsGA20ox1 was expressed in all vegetative organs that we tested. OsGA20ox2/SD1 transcript was highly accumulated in stems and also in other vegetative organs. OsGA20ox3 expression was not observed in any vegetative organs, whereas OsGA20ox4 was moderately expressed in leaf sheaths, leaf blades, and stems and weakly in vegetative shoot apices and roots. The expression profile suggests that OsGA20ox2/SD1 is the dominant GA20ox in stems and that OsGA20ox1 and OsGA20ox4 could be also involved in GA biosynthesis in vegetative organs. This hypothesis corresponds well to the weak phenotype of the sd1 mutant relative to other GA-deficient rice mutants. Similarly to the case of OsGA20ox3, OsGA3ox1 was preferentially expressed in the panicles. In contrast, OsGA3ox2 was broadly expressed in all of the organs that we tested, corresponding to the severe phenotype of d18-AD at the vegetative stage but the mild phenotype at the reproductive stage.

OsGA2ox1 was preferentially expressed in roots, immature panicles, and mature panicles and also in vegetative shoot apices at low level. Transcripts were not observed in leaf sheaths, leaf blades, or stems, consistent with earlier studies (Sakamoto et al., 2001), although faint signals were detected in these organs when the cycle number of the PCR was increased (data not shown). Both OsGA2ox3 and OsGA2ox4 were broadly expressed in all organs of the wild-type rice we tested, whereas OsGA2ox2 expression was not detected in any organs (data not shown).

Although CPS-like, KS-like, and KO-like genes constitute small gene families, loss-of-function mutants of OsCPS1, OsKS1, and OsKO2 showed severe defects in both vegetative and reproductive development (category I). These results strongly suggest that only one gene for each enzyme functions for bioactive GA synthesis in rice. Because rice produces diterpene phytoalexins that are considered to be synthesized from GGDP (Wickham and West, 1992; Mohan et al., 1996), it is possible that some CPS-like and KS-like genes are involved in the biosynthesis of phytoalexins rather than GAs. Phytoalexin production is stimulated by various kinds of inducers, such as UV irradiation and the elicitors produced by pathogenic microorganisms (Wickham and West, 1992; Kato et al., 1995). We therefore examined CPS-like and KS-like gene expression in UV-irradiated rice seedlings and elicitor-treated rice cell cultures.

The expression of OsCPS1 and OsKS1, which function for GA biosynthesis, was unaltered in UV irradiated rice seedlings (Fig. 8). Similarly, OsKS3, OsKS5, and OsKS6 expression was not affected by the UV irradiation. On the other hand, the expression of OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8 was greater in UV irradiated seedlings in comparison to the nonirradiated seedlings (Fig. 8). OsKS2 expression was not detected either in the nontreated or treated seedlings (data not shown).

Figure 8.
Effect of UV irradiation and elicitor treatment on the levels of CPS-like and KS-like gene expression. Left, Comparison between UV irradiated for 30 min (+) and nonirradiated (−) wild-type seedlings. Right, Cultured cells of wild-type ...

In the cultured cells, transcript of OsCPS1, OsKS1, OsKS2, OsKS6, or OsKS8 was not detected in either the elicitor-treated or nontreated conditions (data not shown). Similarly to the case of UV-irradiated seedlings, OsKS3 or OsKS5 expression rate was not affected by the elicitor treatment. On the other hand, the expression of OsCPS2, OsCPS4, OsKS4, and OsKS7 was increased in the elicitor-treated cells compared to the nontreated cells (Fig. 8). The level of OsCPS2, OsCPS4, and OsKS7 transcripts gradually increased for 12 h after the elicitor treatment. The OsKS4 transcript increased until 6 h after the treatment, following which the expression level decreased.

DISCUSSION

In silico screening of the rice genomic and cDNA databases identified 29 candidate genes for seven GA metabolic enzymes. Four CPS-like, nine KS-like, and five KO-like genes were identified in the rice genome (Table I), whereas the Arabidopsis genome contains only one gene encoding each of CPS, KS, or KO (Hedden and Phillips, 2000). Interestingly, 11 genes out of 13 CPS-like and KS-like genes are arranged into four clusters on the rice chromosomes (Fig. 2, B–E). Similarly, the rice genome contains several GA20ox-like, GA3ox-like, and GA2ox-like genes, but their arrangement differs from that of the OsCPS, OsKS, and OsKO genes, and each gene is arranged separately on the genome. We could not find any sequences homologous to the reported KAO genes except OsKAO, and the mutation of OsKAO induced the severely dwarfed phenotype, indicating that KAO is encoded by a single gene, in contrast to other GA-metabolic enzymes.

To identify which gene(s) is actually involved in GA biosynthesis, we have collected and characterized 18 GA-deficient dwarf mutants. We categorized these mutants into three groups according to their phenotypes, that is, severe dwarf without flower or seed development (category I), severe dwarf with seed development (category II), and semidwarf suitable for breeding of high-yielding cultivars (category III). In the mutants grouped into the category I, bioactive GA production at the vegetative and reproductive stages was severely restricted, suggesting that the mutated gene encodes major enzymes involved in GA biosynthesis at both the vegetative and reproductive stages. In this context, the major enzymes involved in the early steps of GA biosynthesis (CPS, KS, KO, and KAO) are likely to be encoded by single genes (OsCPS1, OsKS1, OsKO2, and OsKAO, respectively) that are broadly expressed in both the vegetative and reproductive organs of the wild-type rice because the loss of function of these genes (three alleles of oscps1, two alleles of osks1, one allele of osko2, and three alleles of oskao) caused the severe phenotype characterized in the category I.

The null alleles of the loss-of-function mutants of OsGA3ox2, d18-AD, and d18h, were grouped into the category II. The phenotype suggests that bioactive GA production for shoot elongation is severely defective but that for reproductive development is less affected. This indicates that one gene (OsGA3ox2) contributes to the GA biosynthesis in shoot elongation, whereas another gene (OsGA3ox1) functions redundantly in the reproductive organ development. Supporting this hypothesis, both OsGA3ox1 and OsGA3ox2 produced in Escherichia coli showed GA3ox activity in vitro (Itoh et al., 2001), and OsGA3ox2 was broadly expressed in both vegetative and reproductive organs, whereas OsGA3ox1 was specifically expressed in the reproductive organs of wild-type rice (Fig. 7).

Functional redundancy in GA20ox proteins explains why the null mutation in OsGA20ox2, sd1, exhibited a semidwarf phenotype (category III). In addition to OsGA20ox2, other GA20ox genes, OsGA20ox1 and OsGA20ox4, were simultaneously expressed in all vegetative organs of rice, and all OsGA20ox genes were expressed in the reproductive organs. This overlap expression pattern, accompanied with the feedback up-regulation of other GA biosynthetic enzymes by the homeostatic system (Hedden and Phillips, 2000), compensate the defect in OsGA20ox2 function in shoot elongation, and consequently the defect in OsGA20ox2/SD1 induces suitable semidwarfism of the rice height for useful breeding.

GA2ox genes were also encoded by a small gene family in rice, and two of the four OsGA2ox genes (OsGA2ox3 and OsGA2ox4) were broadly expressed in all organs that we tested. When the GA2ox genes involved in GA catabolism are deficient in their function, mutant plants should show an elongated slender phenotype, as observed in the sln mutant of garden pea (Lester et al., 1999; Martin et al., 1999). We performed a large-scale screening of rice slender mutants and characterized more than 10 lines. However, all these mutants were allelic to the constitutive GA-responsive mutant, slender rice1 (Ikeda et al., 2001), and we could not identify the loss-of-function mutants for any of the OsGA2ox genes (data not shown). These results suggest that at least two GA2ox proteins, OsGA2ox3 and OsGA2ox4, which were simultaneously expressed in all organs of wild-type rice, function redundantly in the control of bioactive GA levels.

Although several CPS-like and KS-like genes were identified in rice, GA biosynthesis depends on only OsCPS1 and OsKS1. This begs the question of what functions do the other genes homologous to CPS and KS genes possess? The two successive type B and type A cyclizations, which are catalyzed by CPS and KS, respectively, are shared by the two pathways for GA biosynthesis and for the biosynthesis of polycyclic diterpenes, including rice diterpene phytoalexins, such as oryzalexins and momilactones (Mohan et al., 1996). Therefore, we suspected that some of the CPS-like and KS-like genes might encode enzymes involved in phytoalexin biosynthesis. As expected, the expression of two CPS-like (OsCPS2 and OsCPS4) and three KS-like (OsKS4, OsKS7, and OsKS8) genes were increased by UV irradiation, and four of these genes, OsCPS2, OsCPS4, OsKS4, and OsKS7, were also induced by the elicitor treatment. Therefore, these CPS-like and KS-like genes are considered likely to be involved in diterpene phytoalexin biosynthesis in response to pathogen infection and UV irradiation. This hypothesis is supported by the recent characterization of a rice diterpene cyclase gene, OsDTC1, which is identical to OsKS7. Biochemical studies revealed that OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of diterpenoid phytoalexins, (-)-phytocassanes (Cho et al., 2004). The other group has also determined the in vitro activity of OsCPSs and found that some OsCPSs are likely to be involved in the phytoalexin biosynthesis (T. Toyomasu, personal communication).

It is noteworthy that these UV and elicitor inducible CPS-like and KS-like genes are contiguously arranged at the same loci of chromosomes 2, 4, and 11 (Fig. 2, B–E). Furthermore, the regions at 86 cM of chromosome 2 (one CPS-like gene, three KS-like genes, and one KS-homologous sequence) and at 14.3 cM of chromosome 4 (one CPS-like gene and one KS-like gene) also contain genes encoding putative Cyt P450s, which are similar to geraniol 10-hydroxylase (Fig. 2, B and C). Such a contiguous arrangement of genes involved in consecutive steps in terpenoid biosynthesis also occurs in the Arabidopsis genome (Aubourg et al., 2002). Aubourg et al. (2002) predicted that the biological meaning of the clustered organization of terpenoid synthase (TPS) genes for consecutive steps is for coordinated gene expression in the multiply stepped modules of terpenoid biosynthesis. According to their prediction, the CPS-like and KS-like genes clustered in these loci might be coordinately regulated by the UV irradiation and elicitor treatments; however, in the Arabidopsis genome, the gene clusters do not consist of TPS-c class (AtCPS/GA1) or TPS-e class (AtKS/GA2) group of TPS genes but instead consist of the other TPS classes (TPS-a and TPS-b). The presence of the gene cluster consisting of TPS-c and TPS-e groups in the rice but not in the Arabidopsis genome could be related to the fact that rice actively produces specific kinds of diterpenes, oryzalexins and momilactones (both of which are derivatives of CDP) as dominant components of phytoalexins, whereas Arabidopsis does not.

The increased copy number of CPS-like and KS-like genes and their contiguous arrangement suggests that these genes evolved rapidly through gene duplication. In fact, the frequency of nucleotide polymorphism in these regions between the O. sativa subsp. japonica and O. sativa subsp. indica genomes is higher than in other regions (data not shown). The frequency of nucleotide polymorphism in these regions could be related to the presence of transposon-like sequences (Fig. 2, B and C). The two gene clusters (OsCPS2-OsKS5-OsKS6-OsKS7 and OsCPS4-OsKS4) contain two and seven copies of transposon-like sequences, respectively. It has been pointed out that retroelements generate instability in genomic regions by promoting recombination and translocation events (Aubourg et al., 2002). Such genomic instability also explains the frequent presence of small pieces of CPS-like and KS-like sequences in the rice genome (e.g. represented as KS-similar in Fig. 2B).

Based on the contrasting situation between the large copy number of the TPS-a and TPS-b genes and the single copy of GA biosynthetic genes in the Arabidopsis genome, Aubourg et al. (2002) suggested the possibility that the duplication of GA biosynthetic genes might be deleterious for growth and development controlled by GA. However, the rice genome has succeeded in increasing the copy number of CPS-like, KS-like, and KO-like genes. The original of the multiple copies of these genes should be the GA biosynthetic genes because the GA biosynthetic genes are widely shared by monocot and dicot plants, but the genes involved in phytoalexin synthesis are specific in the rice plant. Why did the duplicated genes derived from the GA biosynthetic genes rapidly loose their original GA biosynthesis activities even though the original genes retained them? One possible explanation is that the multiple copies of CPS-like, KS-like, and KO-like genes are deleterious for growth and development, as discussed by Aubourg et al. (2002). Further biochemical studies on these CPS-like, KS-like, and KO-like enzymes are necessary to confirm this proposition.

MATERIALS AND METHODS

Plant Materials

Seeds of wild-type and mutant rice (Oryza sativa) were immersed in water for 2 d, grown for 1 month in a greenhouse, and then transplanted to the field.

Sequence Analysis

BLAST search was performed against the rice DNA databases of the Rice Genome Research Program (http://rgp.dna.affrc.go.jp/), the Knowledge-based Oryza Molecular biological Encyclopedia (KOME; http://cdna01.dna.affrc.go.jp/cDNA/), Gramene (http://www.gramene.org/), the Torrey Mesa Research Institute (http://www.tmri.org/index.html), and the Beijing Genomics Institute (http://btn.genomics.org.cn/rice). The deduced amino acid sequences were aligned using the ClustalW program (http://www.ddbj.nig.ac.jp/E-mail/homology.html) with standard parameters, and the phylogenetic tree was obtained with the neighbor-joining method. To map each candidate gene onto the rice genome, linkage analysis was performed using a population of backcrossed inbred lines derived from the cross between Nipponbare (japonica) and Kasalath (indica) varieties. The linkage was calculated using the MAPMAKER program (Lander et al., 1987). The accession numbers of the sequences used in this paper are indicated in Supplemental Table II.

Mutant Analysis

The effect of GA treatment on the shoot elongation of mutant seedlings was evaluated by microdrop assay (Murakami, 1972). For the mapping of each mutant, linkage analysis was performed using an F2 population derived from the cross between the mutant (japonica) and Kasalath (indica) varieties.

GA Analysis

Rice leaves (1 g) were homogenized and extracted with 10 mL of ethyl acetate:methanol (1:1) twice. The extracts were combined, added with 50 ng or 100 ng (for Tanginbozu) of [17,17-2H2]ent-kaurene (purchased from Prof. L. Mander, Australian National University, Canberra, Australia), methylated with excess amount of diazomethane, and concentrated in vacuo. The concentrated extract was then dissolved with 10 mL of ethyl acetate:n-hexane (5:95), dried with Na2SO4, and half of the solution was applied to Bond Elut SI column (gel size 1 g). The column was flushed with air, and the eluate was concentrated and applied to HPLC analysis with a column of Docosil (250 mm length, 10 mm i.d.; Senshu Scientific, Tokyo). The column was eluted with CHCl3:methanol (5:95) at a flow rate of 2 mL/min, and the eluate was collected as ent-kaurene fraction from 45 to 50 min after injection. Gas chromatography-mass spectrometry was performed with an AUTO MASS mass spectrometer (JEOL, Akishima, Japan) connected to a Hewlett-Packard 5890 series II gas chromatograph (Palo Alto, CA) equipped with a column of DB-5 (30 m in length, 0.25 mm i.d., 0.25 μm film thickness; J & W Scientific, Folsom, CA). After the injection, the column oven temperature was maintained at 80°C for 1 min and then increased to 180°C at a rate of 20°C min−1, followed by further increment to 210°C at a rate of 2.5°C min−1. Then it was maintained at 210°C for 10 min and finally increased to 270°C at a rate of 20°C min−1. Authentic ent-kaurene was eluted approximately 18.6 min after the injection. The level of ent-kaurene was calculated by the area ratio between the peaks at mass-to-charge ratio of 274 (internal standard) and 272 (endogenous). Endogenous GA levels in wild-type and mutant seedlings were analyzed by gas chromatography-selected ion monitoring as described previously (Sakamoto et al., 2001).

Expression Analysis

RT-PCR was performed with DNase-treated total RNAs separately prepared from various organs of rice by using the Advantage RT-for-PCR kit (CLONTECH, Palo Alto, CA). The PCR cycles, in which PCR products were in exponential increase, were determined essentially as described by the supplier. The primer sequences used in this paper are indicated in Supplemental Table III. These primers specifically amplified the target gene sequences (data not shown). Seedlings of wild-type rice (cv Nipponbare) were UV irradiated for 30 min using a germicidal lamp (15 W) at a distance of 15 cm. After irradiation, these seedlings were incubated at 30°C in the dark for 24 h, and then under light for another 24 h. Total RNAs extracted from elicitor-treated rice cultured cells were kindly provided by Dr. Eiichi Minami (National Institute of Agrobiological Sciences, Tsukuba, Japan). Suspension cultured cells of wild-type rice (cv Nipponbare) were prepared as described previously (Yamada et al., 1993). Purified N-acetylchitoheptaose (Kuchitsu et al., 1993) was applied to the medium at a final concentration of 1 mg L−1.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank the National Institute of Agrobiological Sciences for providing seed materials. We also thank Dr. Eiichi Minami for providing the RNA samples extracted from elicitor-treated rice cultured cells and Dr. Tomonobu Toyomasu for sharing data before publication.

Notes

1This work was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project; grant no. IP–1010 to T.S.), the Program for Promotion of Basic Research Activities for Innovation of Biosciences (grant to M.U.-T. and H.K.), and the Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for the Center of Excellence to M.M.).

[w]The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.033696.

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