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, 17 (3), 776-90

A Novel Cytochrome P450 Is Implicated in Brassinosteroid Biosynthesis via the Characterization of a Rice Dwarf Mutant, dwarf11, With Reduced Seed Length

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A Novel Cytochrome P450 Is Implicated in Brassinosteroid Biosynthesis via the Characterization of a Rice Dwarf Mutant, dwarf11, With Reduced Seed Length

Sumiyo Tanabe et al. Plant Cell.

Abstract

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.

Figures

Figure 1.
Figure 1.
Pleiotropic Abnormalities Displayed by d11 Mutants. (A) Gross morphology. From left to right: T65, d11-1, Shiokari, d11-2, d61-1, and d2-1 plants grown in the fields at heading stages. T65 and Shiokari, the recurrent parents for d11-1 and d11-2, respectively, were used as wild-type plants. Bar = 20 cm. (B) Elongation patterns of the second internode. See (A) for rice plants. The second internode was extremely shortened in d11, d61-1, and d2-1 mutants. Thin and thick arrows indicate the upper and lower ends of the second internode, respectively. The second internodes of the wild-type plants were too long (see Table 1 for the length) to show their whole figures in this photograph. Bar = 1 cm. (C) Grain morphology. See (A) for rice plants. d11 mutants have shortened grains. Bar = 0.5 cm. (D) Panicle morphology. See (A) for rice plants. Brackets indicate the basal rachis internodes. Arrows show degenerated primary branches. The lengths of the basal rachis internodes of d11, d61-1, and d2-1 mutants are longer than those of wild-type plants. Bar = 5 cm.
Figure 2.
Figure 2.
Aberrant Skotomorphogenesis of d11 Mutants. Plants were germinated and grown in darkness. (A) From left to right: wild-type, d11-1, d61-1, and d2-1 plants. T65, the recurrent parent for d11-1, d61-1, and d2-1, was used as wild-type plants. Bar = 5 cm. (B) to (E) The node positions of the wild-type, d11-1, d61-1, and d2-1 plants are scaled up in (B) to (E), respectively, and indicated by arrows. The positions of the mesocotyls in these plants are indicated by arrowheads. Bar = 1 cm.
Figure 3.
Figure 3.
Elongation of the Second Leaf Sheath in Wild-Type Plants and d11 Mutants after Treatment with BL. The length of the second leaf sheath was measured 2 weeks after wild-type (closed squares) and d11-1 (open squares) seeds were germinated on half-strength MS medium containing various concentrations of BL. T65, the recurrent parent for d11-1, was used as wild-type plants. n = 30. Error bars indicate standard deviations.
Figure 4.
Figure 4.
Physical Map of the d11 Locus and Mutation Sites in d11 Alleles. (A) High-resolution linkage and physical map of the d11 locus. Vertical lines represent the positions of molecular markers (C559, G7008, W390, and L353), and the numbers of recombinants are indicated above the linkage map. Genetic distances (centimorgan [cM]) between adjacent markers are shown in parentheses. The physical map of the d11 locus was constructed using three PAC clones, and the candidate region of the d11 mutation was present between markers T and K. (B) Genomic structure of the D11 gene and positions of mutations in d11 alleles. Black boxes indicate exons. The D11 gene consists of nine exons and eight introns. The mutated DNA sequences of d11 alleles were shown in the bottom. d11-1 has a one-base deletion in the second exon, and d11-2 has a one-base insertion in the seventh exon. d11-3 has a one-base substitution in the fourth exon, and d11-4 has a one-base substitution of guanine for thymine at the last position of intron 3. A mutated mRNA with a five-base deletion in exon 4 (GCCAG) is produced in d11-4. The DNA sequences shown in the lowest part of the figure indicate those of mutated exons (d11-1, d11-2, and d11-3) and of the splicing site containing the mutated intron (d11-4).
Figure 5.
Figure 5.
Structure of the D11 Protein. Comparisons of amino acid sequences among D11, DWF4-OsH, and DWF4. In accordance with the nomenclature of the P450 superfamily (Nelson et al., 1996), the D11 protein was named CYP724B1. Because the amino acid sequence of the D11 protein showed similarity to the sequences of BR-biosynthetic P450 proteins belonging to the CYP90B subfamily, the sequences of D11 (this study), DWF4-OsH, and DWF4 (Choe et al., 1998) were compared. Multiple sequence alignment was performed using the ClustalW analysis tool in the DNA Data Bank of Japan. Dashes (-) indicate gaps introduced to maximize alignment. Identical amino acid residues are represented by white-on-black letters. The triangles (d11-1), open circles (d11-4), closed circles (d11-3), and closed squares (d11-2) indicate the positions of deletion, splicing, amino acid substitution, and insertion mutations, respectively. Various domain regions, such as the anchor region, the Pro-rich region, Domain A, Domain B, Domain C, and the heme binding domain, are labeled (Kalb and Loper, 1988).
Figure 6.
Figure 6.
Phylogenic Relationship in the Plant P450 Superfamily. BR biosynthesis: D11 (this study), DWARF (Bishop et al., 1996), AtBR6ox (Shimada et al., 2001), OsDWARF (Hong et al., 2002), CPD (Szekeres et al., 1996), AtCYP90D1 (Hong et al., 2003), OsCYP90D3 (Hong et al., 2003), D2 (Hong et al., 2003), DWF4 (Choe et al., 1998), and DWF4-OsH (registered in GenBank). GA biosynthesis: AtKAO1 and AtKAO2 (Helliwell et al., 2001). Auxin biosynthesis: CYP79B2 (Hull et al., 2000), CYP79B3 (Hull et al., 2000), and CYP83B1 (Bak and Feyereisen, 2001). Other: ROT3 (Kim et al., 1998). The structural relationship was calculated by ClustalW and illustrated by Treeview.
Figure 7.
Figure 7.
Phenotypic Complementation by Introduction of the D11 Gene. The entire D11 gene, excised at the restriction sites XbaI and EcoRI (Figure 4B), was subcloned into a binary vector, pPZP2H-lac, to generate pPZP2H-lac-D11. The pPZP2H-lac and pPZP2H-lac-D11 were introduced into the Agrobacterium, EHA101. Transformants were generated from d11-1 using the Agrobacterium-mediated transformation method. Transformant-D11 and transformant-control vector were obtained by the infections of Agrobacterium containing pPZP2H-lac-D11 and pPZP2H-lac as the control vector, respectively. T65 was the recurrent parent for d11-1 and used as the wild-type plant. (A) Gross morphology. Left, the wild type (T65); center, transformant-D11; right, transformant-control vector. Arrows and arrowheads indicate mature leaves and ears, respectively. Bar = 20 cm. (B) Seeds. See (A) for rice plants. Seed form of transformant-D11 was the same as that of the wild-type plant. Bar = 0.5 cm.
Figure 8.
Figure 8.
Comparison of D11 Gene Expression in Various Organs and the Negative Feedback Effect of BL on Gene Expression. T65, the recurrent parent for d11-1, d2-1, and d61-2, was used as wild-type plants. (A) Organ-specific expression of the D11 gene in the wild-type plants. Total RNAs were isolated from shoots, roots, internodes, and florets before flowering and florets after flowering, and RT-PCR was conducted to amplify a fragment of the D11 cDNA with specific primers. The expression of the Actin gene was used as a control. (B) Negative feedback regulation of the D11 and OsBRI1 genes by BL. Total RNA was prepared from 2-week-old wild-type, d11-1, d2-1, and d61-2 seedlings with (+) or without (−) exogenous supplement of 10−6 M BL. RT-PCR was conducted as described in (A). The expression of the Actin gene was used as a control.
Figure 9.
Figure 9.
Quantitative Analysis of Endogenous BR Intermediates in Wild-Type and d11 Plants. BR intermediates were isolated from shoots (A) and florets before flowering (B) as described in Methods. Sterol and BR levels (ng/g fresh weight) of two d11 alleles and their respective recurrent parents are shown below each product. The levels in d11-1 and its recurrent parent, T65, are indicated right and left, respectively, in the top parts marked with open circles. The levels in d11-2 and its recurrent parent, Shiokari, are indicated right and left, respectively, in the bottom parts marked with closed squares. The biosynthetic steps, in which D11 protein may participate, are indicated as “possible defect step 1” in (A) and “possible defect step 2” in (B). The steps catalyzed by D2 and OsDWARF (BRD1) proteins are shown as D2 and OsDWARF, respectively. nd, not detected; na, not analyzed.
Figure 9.
Figure 9.
Quantitative Analysis of Endogenous BR Intermediates in Wild-Type and d11 Plants. BR intermediates were isolated from shoots (A) and florets before flowering (B) as described in Methods. Sterol and BR levels (ng/g fresh weight) of two d11 alleles and their respective recurrent parents are shown below each product. The levels in d11-1 and its recurrent parent, T65, are indicated right and left, respectively, in the top parts marked with open circles. The levels in d11-2 and its recurrent parent, Shiokari, are indicated right and left, respectively, in the bottom parts marked with closed squares. The biosynthetic steps, in which D11 protein may participate, are indicated as “possible defect step 1” in (A) and “possible defect step 2” in (B). The steps catalyzed by D2 and OsDWARF (BRD1) proteins are shown as D2 and OsDWARF, respectively. nd, not detected; na, not analyzed.
Figure 10.
Figure 10.
Effects of BR Intermediates on the Degree of Inclination of the Leaf Lamina in Wild-Type and d11 Plants. (A) to (K) T65 and d11-1. (L) to (V) Shiokari and d11-2. The effect of BR intermediates (ng/plant) on the degree of inclination of the leaf lamina in wild-type plants (closed squares) and d11 mutants (open squares). Data presented are means from 10 plants. Error bars indicate standard deviations.

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