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, 12 (9), 1591-606

Loss of Function of a Rice Brassinosteroid insensitive1 Homolog Prevents Internode Elongation and Bending of the Lamina Joint

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Loss of Function of a Rice Brassinosteroid insensitive1 Homolog Prevents Internode Elongation and Bending of the Lamina Joint

C Yamamuro et al. Plant Cell.

Abstract

Brassinosteroids (BRs) are plant growth-promoting natural products required for plant growth and development. Physiological studies have demonstrated that exogenous BR, alone or in combination with auxin, enhance bending of the lamina joint of rice. However, little is known about the function of endogenous BR in rice or other grass species. We report here the phenotypical and molecular characterization of a rice dwarf mutant, d61, that is less sensitive to BR compared to the wild type. We cloned a rice gene, OsBRI1, with extensive sequence similarity to that of the Arabidopsis BRI gene, which encodes a putative BR receptor kinase. Linkage analysis showed that the OsBRI1 gene is closely linked to the d61 locus. Single nucleotide substitutions found at different sites of the d61 alleles would give rise to amino acid changes in the corresponding polypeptides. Furthermore, introduction of the entire OsBRI1 coding region, including the 5' and 3' flanking sequences, into d61 plants complemented the mutation to display the wild-type phenotype. Transgenic plants carrying the antisense strand of the OsBRI1 transcript showed similar or even more severe phenotypes than those of the d61 mutants. Our results show that OsBRI1 functions in various growth and developmental processes in rice, including (1) internode elongation, by inducing the formation of the intercalary meristem and the longitudinal elongation of internode cells; (2) bending of the lamina joint; and (3) skotomorphogenesis.

Figures

Figure 1.
Figure 1.
Phenotype of the d61 Mutants. (A) Schematic representation of the various elongation patterns of internodes in the wild type (N) and various rice dwarf mutants (dn-, dm-, d6-, nl- and sh-types), adapted from Takeda (1977). (B) Gross morphology of a wild-type plant (left); d61-1 mutant (center), a weak allele; and d61-2 mutant (right), a strong allele. (C) Elongation pattern of internodes. The wild-type plant (left) shows the N-type of the elongation pattern, whereas the d61-1 (center) and d61-2 (right) mutants show typical dm- and d6-type patterns, respectively. The number of each internode is indicated. (D) Panicle structure. The wild-type plant (left) has a short panicle; the d61-1 (center) and d61-2 (right) mutants have longer panicles. The arrows indicate the nodes. (E) Leaf morphology. The leaf of the wild-type plant (left) is bent at the lamina joint indicated by the white arrow, whereas the leaves of d61-1 (center) and d61-2 (right) mutants are more erect. (F) Leaf sheath morphology. The leaf sheath in the d61-1 (center) and d61-2 (right) mutants is shorter than in the wild-type plant (left).
Figure 2.
Figure 2.
Structure of Well-Developed Internodes from Wild-Type and d61-2 Rice Plants and Orientation of Microtubules in Elongating Cells in the First Internode of Wild-Type and d61-2 Plants. (A) and (B) Longitudinal sections of the first internode from the wild type and d61-2, respectively. (C) and (D) Longitudinal sections of the second internode from the wild type and d61-2, respectively. (E) and (F) Longitudinal sections of the third internode from the wild type and d61-2, respectively. (G) and (H) Longitudinal sections of the fourth internode from the wild type and d61-2, respectively. (I) and (J) Immunofluorescence images of the microtubule arrangement in internodal parenchyma cells of the first internode from the wild type and d61-2, respectively. (K) and (L) Schematic presentation of the microtubule arrangement in internodal parenchyma cells of the first internode from the wild type and d61-2, respectively. Bars in (A) to (H) = 100 μm; bars in (I) to (L) = 50 μm.
Figure 3.
Figure 3.
Response of Seedlings to BL. (A) Seeds of the wild type (left) and the dwarf mutants d61-1 (center) and d61-2 (right) were germinated on agar plates in the presence (+) or absence (−) of 1 μM BL. Seedlings were examined 1 day after germination. (B) Effect of BL on the coleoptile and root elongation in wild-type and d61 seedlings. The plants were germinated in the same conditions as (A) with the indicated concentration of BL. Data presented are the means of results from five plants. Bars indicate sd.
Figure 4.
Figure 4.
Effect of BL on the Degree of Inclination of Etiolated Leaf Lamina in Wild-Type and d61 Plants. (A) Typical response of the second leaf lamina joint from wild-type, d61-1, and d61-2 plants to BL at 10−3 μg mL−1. (B) The dose response to BL of the bending angle in the wild type and d61 mutants. Data presented are the means of results from six plants. Bars indicate sd.
Figure 5.
Figure 5.
Endogenous BRs in Wild-Type and d61-2 Rice Plants. The amounts (ng g−1 fresh weight) of BL and its biosynthetic precursors in d61-2 (upper) and wild-type (lower) rice plants are shown. ND, not detected.
Figure 6.
Figure 6.
DET Phenotype of the Wild Type, d61, and Two Gibberellin-Deficient Rice Mutants (d18 and d35) in the Dark. The left- and right-hand seedlings of each pair were grown for 2 weeks in the light and the dark, respectively. Arrows indicate the nodes and arrowheads indicate the mesocotyls.
Figure 7.
Figure 7.
Length of the Second Lower Internodes of the Wild Type and Mutants Grown in the Dark. Data presented are the means of the results from five plants. Bars indicate sd.
Figure 8.
Figure 8.
Deduced Amino Acid Sequences of OsBRI1 and Arabidopsis BRI1. Identical residues are shaded. The underlined regions indicate (1) a putative signal peptide, (2) a leucine zipper motif, (3) N-side, and (4) C-side of a cysteine pair.
Figure 9.
Figure 9.
Complementation and Antisense Phenotype of OsBRI1. (A) d61 plant with the wild-type OsBRI1gene (left) and d61 plant introduced with a vector only (right). (B) Dwarf phenotype of OsBRI1 antisense plants with intermediate and severe phenotypes (right) in comparison with a wild-type plant (left). (C) Close-up view of OsBRI1 antisense plants with the severe phenotype. (D) Naked culm internodes of transgenic plant. From left to right, wild-type plant with normal elongation pattern of internodes, and OsBRI1 antisense plants with the dm, dm–d6, and d6 phenotypes, respectively. (E) Abnormal leaf morphology of an OsBRI1 antisense plant with a severe phenotype, showing lack of developed sheath organs. (F) Panicle morphology in wild-type (left) and OsBRI1 antisense plants with the mild (center) and intermediate (right) phenotypes. The arrows indicate the nodes. (G) Leaf morphology of wild-type (left) and OsBRI1 antisense plants with mild phenotype (right), showing the erect leaves in the latter. Bar in (C) = 5 cm; bar in (E) = 10 cm; bar in (F) = 2 cm.
Figure 10.
Figure 10.
Expression Pattern of OsBRI1 in Various Organs. Total RNA (10 μg) from various organs of wild-type plants was probed by hybridization with an OsBRI1 cDNA clone. (A) Organ-specific expression of OsBRI1. Lane 1, leaf blade; lane 2, leaf sheath; lane 3, developed flower; lane 4, rachis; lane 5, shoot apex; lane 6, root; and lane 7, seed. (B) Region-specific expression of OsBRI1 in developing first internodes. Lane 1, node; lane 2, divisional zone; lane 3, elongation zone; and lane 4, elongated zone. (C) Differential expression of OsBRI1 in each elongating internode. Lanes 1 to 4, the divisional and elongation zones of the first to fourth internodes, respectively, at the actively elongating stage for each internode; lane 5, the unelongated stem at the vegetative phase. (D) Light-dependent and BL-dependent expression of OsBRI1 (upper panel). Rice seedlings were grown for 10 days in the light (lanes 1 and 2) or dark (lanes 3 and 4) on agar plates in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 1 μM BL. The middle panel shows the amount of the actin mRNA probed with an expressed sequence tag clone, S14002, from Rice Genome Project as a control.

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