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, 17 (8), 2243-54

The Rice Brassinosteroid-Deficient dwarf2 Mutant, Defective in the Rice Homolog of Arabidopsis DIMINUTO/DWARF1, Is Rescued by the Endogenously Accumulated Alternative Bioactive Brassinosteroid, Dolichosterone

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The Rice Brassinosteroid-Deficient dwarf2 Mutant, Defective in the Rice Homolog of Arabidopsis DIMINUTO/DWARF1, Is Rescued by the Endogenously Accumulated Alternative Bioactive Brassinosteroid, Dolichosterone

Zhi Hong et al. Plant Cell.

Abstract

We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective.

Figures

Figure 1.
Figure 1.
Phenotype of the brd2 Mutant. (A) Gross morphology of wild-type (left) and brd2 (right) seedlings grown for 40 d in soil. Bar = 10 cm. (B) Gross morphology of field-grown wild type (left) and brd2 (right) at the flowering stage. Bar = 15 cm. (C) Comparison of the internode elongation pattern of brd2 with previously characterized rice BR-deficient mutants. The five uppermost internodes (I to V) of the wild type were elongated. Bar = 10 cm. (D) Panicle structure. The wild-type plant (left) developed ∼120 seeds per panicle, and the brd2 mutant panicle developed ∼30 seeds with poor germination rates. Arrows indicate the positions of the nodes. Bar = 10 cm. (E) Morphology of unhulled grains (top) and brown rice (bottom). The mutant (right) produced shortened grains. Bar = 1 cm. (F) Root morphology. Seedlings were grown at 30°C in water for 1 week in the light. Bar = 1 cm.
Figure 2.
Figure 2.
Molecular Characterization of the brd2 Mutation. (A) The brd2 mutation was mapped at ∼25 centimorgan on chromosome 10, near the map position of Dim/dwf1. (B) Schematic diagram of the structure of Dim/dwf1 and the site of the mutation in the brd2 allele. Closed boxes represent exons, and open boxes indicate introns. The first Met (ATG) and the stop codon (TAA) are located in exons 2 and 3, respectively. The single base deletion in the brd2 allele is indicated by an arrow, and the premature stop codon is marked with an asterisk. (C) The deduced amino acid sequence of Dim/dwf1. The mutation site is marked with a closed triangle, and the FAD binding domain is underlined. (D) Levels of Dim/dwf1 transcripts in wild-type (left) and brd2 (right) plants. Ten micrograms of total RNA was loaded per lane. Ethidium bromide–stained rRNA bands were monitored as a loading control. (E) Molecular complementation. Transgenic plants derived from mutant callus transformed with the Dim/dwf1 gene had the wild-type phenotype, in contrast with the dwarf phenotype of the mutant (right), and transformants containing the empty vector had the dwarf phenotype (left). (F) Organ-specific expression of the Dim/dwf1 gene in the wild type. RT, root; SA, shoot apex; LB, leaf blade; LS, leaf sheath; ST, stem; PA, panicle; FL, flower. Five micrograms of total RNA was loaded per lane, and ethidium bromide–stained rRNA bands were monitored as a loading control. (G) The effect of BL on Dim/dwf1 expression. Total RNA was isolated from wild-type, brd1-1, and d61-2 seedlings either treated for 10 d with a final concentration of 1 μM BL (+) or untreated (−); 7.5 μg was loaded per lane.
Figure 3.
Figure 3.
Phenotypes of Dim/dwf1 Antisense and Sense Transgenic Plants. (A) Gross morphology of an antisense plant (left), a control plant containing the empty vector (middle), and a sense plant (right) 3 months after transplantation. Bar = 20 cm. (B) Leaf morphology: a wild-type leaf (center) bends away from the vertical axis of the leaf toward the abaxial side, an antisense leaf (left) is more erect, and that of an overproducer (right) is more bent. (C) Panicle structure: the antisense plant (left) generates poor panicles with lower spikelet numbers than the wild type (center), whereas the overproducer (right) generates superior panicles with increased spikelet numbers. (D) Internode elongation pattern. The antisense plant (left) is severely inhibited in the elongation of internodes II through V, whereas the overproducer has increased numbers and lengths of elongated internodes in comparison with the wild type (center). Bar = 20 cm. (E) RNA gel blot analysis of the endogenous and transformed Dim/dwf1 transcripts. Two micrograms of total RNA was loaded per lane. An 800-bp cDNA fragment containing the coding sequence was used as a probe to detect both sense and antisense Dim/dwf1 transcripts (top), and a 200-bp probe containing only the 3′-noncoding sequence of the Dim/dwf1 transcript was used to detect endogenous Dim/dwf1 expression (middle). Ethidium bromide–stained rRNA bands were monitored as a loading control (bottom).
Figure 4.
Figure 4.
Quantitative Analysis of Endogenous BR Intermediates in Wild-Type and brd2 Plants. Shoots of wild-type and mutant plants were harvested after growth in a greenhouse for 2 months. BR levels (ng/g FW) in wild-type (left) and brd2 (right) plants are shown below each product. BR levels were measured in two independent experiments. na, not analyzed; nd, not detected.
Figure 5.
Figure 5.
Comparison of Dwarfism and Level of BRs between brd2 and Other Rice BR-Deficient Mutants. (A) Two-week-old seedlings are shown. The mutants were germinated and grown in MS agar. The background of d2-1, d2-2, and d11-1 is T65 (Taichung 65); brd2, brd1-1, brd1-2, and brd1-3 are Ni (Nipponbare) background; and that of d11-2 is Shi (Shiokari). Bar = 5 cm. (B) Comparison of the level of TY, 6-DeoxoCS, and CS in the BR-deficient mutants. The background plant of each mutant is written in parentheses. The level of BRs in d2, d11, brd1-1, and brd1-2 are adopted from the results previously reported (Hong et al., 2002, 2003; Tanabe et al., 2005). CS level in brd1-3 was not determined because of contamination with unknown compound(s).
Figure 6.
Figure 6.
The Sterol Biosynthetic Pathway and Sterol Contents in the Wild Type and brd2. Proposed biosynthetic pathways of the wild type (left) and brd2 (right) are shown. The end products of the Dim/dwf1-requiring pathways are enclosed in boxes with solid lines, and those of the Dim/dwf1-free pathway are enclosed in boxes with dashed lines. Sterol levels were measured by two independent experiments. The level of DS was not corrected by an internal standard. The number of arrows does not represent the number of chemical reaction steps. nd, not detected.
Figure 7.
Figure 7.
BR Biological Activity of DS. (A) Phenotypic rescue of the abnormal morphology of brd1-1 by treatment with DS and CS. The malformed phenotype and shortened leaf sheaths of a brd1-1 plant grown for 4 weeks were not rescued by the mock treatment (cont.), whereas 1 μM DS or CS restored the sheath elongation and morphology of newly expanded leaves. White brackets indicate the relative length of the uppermost leaf sheaths. (B) Lamina joint bending test. DS increased the lamina joint bending of the wild type and brd2 in a dose-responsive manner but not of the BR-perception mutant d61-2. CS treatment had an effect similar to DS treatment, although the efficiency of CS was slightly higher than that of DS in the wild type and lower in brd2. Shown at right are typical lamina joint bending results after treatment with 1 μg CS or DS. (C) Inhibition of root elongation by treatment with DS or CS. Plants were germinated on agar lacking (−BR) or containing 10−7 M CS or DS. Seedlings were examined 4 d after germination. (D) Effect of CS and DS on root elongation in wild-type, brd2, and d61-2 seedlings. The plants were germinated as in (A) with the indicated concentrations of CS or DS. The data presented are the means of results from six plants. Error bars = sd.

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