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, 130 (3), 1152-61

Isolation and Characterization of a Rice Dwarf Mutant With a Defect in Brassinosteroid Biosynthesis

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Isolation and Characterization of a Rice Dwarf Mutant With a Defect in Brassinosteroid Biosynthesis

Masaki Mori et al. Plant Physiol.

Abstract

We have isolated a new recessive dwarf mutant of rice (Oryza sativa L. cv Nipponbare). Under normal growth conditions, the mutant has very short leaf sheaths; has short, curled, and frizzled leaf blades; has few tillers; and is sterile. Longitudinal sections of the leaf sheaths revealed that the cell length along the longitudinal axis is reduced, which explains the short leaf sheaths. Transverse sections of the leaf blades revealed enlargement of the motor cells along the dorsal-ventral axis, which explains the curled and frizzled leaf blades. In addition, the number of crown roots was smaller and the growth of branch roots was weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored the normal phenotypes, we designated the mutant brassinosteroid-dependent 1 (brd1). Further, under darkness, brd1 showed constitutive photomorphogenesis. Quantitative analyses of endogenous sterols and brassinosteroids (BRs) indicated that BR-6-oxidase, a BR biosynthesis enzyme, would be defective. In fact, a 0.2-kb deletion was detected in the genomic region of OsBR6ox (a rice BR-6-oxidase gene) in the brd1 mutant. These results indicate that BRs are involved in many morphological and physiological processes in rice, including the elongation and unrolling of leaves, development of tillers, skotomorphogenesis, root differentiation, and reproductive growth, and that the defect of BR-6-oxidase caused the brd1 phenotype.

Figures

Figure 1
Figure 1
Morphology of brd1 plant. A, Three-week-old brd1 seedling grown in the growth chamber. Bar = 1 cm. B, Eighty-day-old wild-type (WT) and brd1 plants grown in soil in the growth chamber. Bar = 10 cm. C, Close-up of brd1 in B. D, Six-month-old brd1 plant with a short panicle grown in the greenhouse in soil at 28°C. E, Wild-type and brd1 panicles. Bar = 1 cm. F, Seeds of wild-type and brd1 plants. Bar = 5 mm.
Figure 2
Figure 2
Phenotypic restoration of brd1 plant by BL. Seedlings were germinated and grown on one-half-strength Murashige and Skoog medium containing both 3% (w/v) Suc and 0.4% (w/v) Gelrite. When the fourth leaf emerged (about 10 d after sowing), plants were transplanted into Kimura's B solution (Sato et al., 1996) with or without BL. The photograph was taken after an additional 30 d of growth.
Figure 3
Figure 3
Length of the eighth leaf of wild-type (WT) and brd1 plants grown with or without BL. A, Length of the eighth leaf sheath grown with or without BL. B, Length of the eighth leaf blade grown with or without BL. Seedlings were germinated and grown on one-half-strength Murashige and Skoog medium containing both 3% (w/v) Suc and 0.4% (w/v) Gelrite. When the fourth leaf emerged (about 10 d after sowing), plants were transplanted to Kimura's B solution culture medium with or without 40 nm BL. The lengths of the leaves were measured after an additional 30 d of growth. The results are presented as mean values ± sd from five to seven plants.
Figure 4
Figure 4
Light microscopy of wild-type and brd1 leaves sectioned longitudinally and transversely. A through C, Longitudinal sections of the central region of the eighth leaf sheath of wild-type (A), brd1 (B), and brd1 in the presence of 40 nm BL (C). D through F, Transverse sections of the central region of the eighth leaf blade of wild-type (D), brd1 (E), and brd1 in the presence of 40 nm BL (F). Arrows indicate the cutting point shown in G to I. Broken arrows indicate the cutting point shown in J to L. Arrowheads indicate motor cells. G through L, Longitudinal sections of the central region of the eighth leaf blade of wild-type (G and J), brd1 (H and K), and brd1 in the presence of 40 nm BL (I and L). Seedlings were germinated and grown on one-half-strength Murashige and Skoog medium containing both 3% (w/v) Suc and 0.4% (w/v) Gelrite. When the fourth leaf emerged (about 10 d after sowing), plants were transplanted to Kimura's B solution culture medium with or without 40 nm BL. When the 11th leaf blade emerged, the eighth leaf was examined. Bar = 50 μm.
Figure 5
Figure 5
Photomorphogenic reaction. A, Two-week-old wild-type (WT) and brd1 seedlings grown in the dark. Black bar indicates the mesocotyl length; white bars indicate coleoptile lengths. B and C, WT and brd1 seedlings were grown under white light (L) or in the dark (D) for 2 weeks. The lengths of mesocotyls (B) and coleoptiles (C) of these seedlings were measured individually. WT and brd1 seedlings were germinated and grown on one-half-strength Murashige and Skoog medium containing 3% (w/v) Suc and 0.4% (w/v) Gelrite. The results are presented as mean values ± sd from seven to 10 plants. n.d., The corresponding tissue was not detected.
Figure 6
Figure 6
Number of crown roots at different leaf emergence stages. The number of crown roots which extended over 2 mm long was counted. Seedlings were grown in the light on one-half-strength Murashige and Skoog medium containing 3% (w/v) Suc and 0.4% (w/v) Gelrite. The results are presented as mean values ± sd from 10 plants. WT, Wild-type. n.d., Crown roots were not detected.
Figure 7
Figure 7
Root morphology. A through C, Photographs of roots. D through F, Roots observed under stereomicroscope. A and D, Wild type; B and E, brd1; C and F, brd1 in the presence of 1 nm BL. Seedlings were germinated and grown on one-half-strength Murashige and Skoog medium containing 3% (w/v) Suc and 0.4% (w/v) Gelrite. When the fourth leaf emerged (about 10 d after sowing), plants were transplanted into Kimura's B solution with or without BL. The photograph was taken after an additional 60 d of growth. Bar = 1 cm in A through C and 1 mm in D through F. Circles indicate secondary branched roots.
Figure 8
Figure 8
BL biosynthetic pathway and BR content. BR amounts, in nanograms per gram fresh weight, of wild-type and brd1 plants are shown in white and red boxes, respectively. ND, Not detected. BR6ox, BR-6-oxidase.
Figure 9
Figure 9
A, Schematic representation of OsBR6ox and the location of the deletion in the brd1 mutant. The closed boxes indicate exons. The approximate locations of primer sequences are shown by arrows. AU100843 is an EST clone of rice. B, PCR analysis of OsBR6ox in brd1, brd1/+, and wild-type plants. An ethidium bromide-stained agarose gel shows PCR products generated by using primers D4 and D6R, which amplify the genomic DNA containing exons 4 to 9.
Figure 10
Figure 10
Multiple sequence alignment of OsBR6ox with known sequences for BR-6-oxidases. The GenBank/EMBL/DNA data bank of Japan accession numbers are AB035868 for the Arabidopsis BR6ox gene and U54770 for the tomato Dwarf gene. The amino acid sequence of OsBR6ox was deduced from the DNA sequence of contig 18,223 from the Web site at http://btn.genomics.org.cn/rice (Yu et al., 2002). The heme-binding signature sequence of cytochrome P450 is underlined. As a result of the frameshiftings deletion in OsBR6ox in brd1, the carboxy-terminal region of the ORF (from Asp-300 to Tyr-470) is disrupted. Reverse contrast characters highlight identical amino acid residues. Gaps introduced to improve alignment are shown by hyphens.

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