Os10g0397400

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Annotated Information

Function

The rice (Oryza sativa) brassinosteroid (BR)-deficient dwarf2 (brd2) is a loss-of-function allele of Dim/dwf1, a gene homologous to the Arabidopsis DIM/DWF1.The gene brd2 mutation causes a semidwarf phenotype in the vegetative stage,with severe defects in internode elongation and panicle and seed development. Quantitative analysis of the endogenous sterol levels showed that brd2 is defective in the conversion of 24-MC to CR, as are the Arabidopsis dim/dwf1 and pea lkb mutants.

Expression

The brd2 mutatant possess a semidwarf phenotype in the vegetative stage.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.

Figure 1. Phenotype of the brd2 Mutant

Recently, we identified a dwarf mutant, brd2,which displays the typical BR-deficient phenotype but also has characteristics not observed in the other mutants.Figure 1A shows the gross morphology of the mutant in an early vegetative stage. At this stage, the mutant plant (right) displays a moderate dwarf phenotype with a height 70% of the wild-type plant (left) but no other obvious abnormalities. However, as development progresses, the phenotype becomes more severe with characteristics typical of BR-related mutants: dark-green, erect leaves and shortened leaf sheaths. After flowering, the height of the plant reaches only 40% of the wild type (Figure 1B). We compared the internode elongation patterns of brd2, the wild type, and other BR-deficient mutants (Figure 1C). In the wild type, the five uppermost internodes elongate; the internodes are numbered from top to bottom such that the uppermost, just below the panicle, is designated as the first internode (Figure 1C).By contrast, the three BR-deficient mutants show different elongation patterns:d2-1 has a specific inhibition in the second internode,brd1-1 is completely defective in internode elongation, and the first internode of brd2 elongates, but the lower internodes do not (Figure 1C).d61-2, which is defective in the rice Bri1 gene, also exhibits dwarfism with a lack of internode elongation except for the first internode.The brd2 mutant flowers and produces malformed panicles, but the numbers of spikelets and rachis branches, as well as fertility, are severely reduced (Figure 1D). The lengths of the first and second rachis branches in the panicle are also severely reduced. In contrast with these severe defects, the neck internode of the mutant is longer than that of the wild type (Figure 1D).This phenomenon is a common feature in rice BR-related mutants because long neck internodes are also observed in d2 and d61-1. In addition, the brd2 mutant also exhibits shortened grains (Figure 1E) and defective root elongation and development (Figure 1F). Although one seminal root develops normally during embryogenesis, after germination the seminal root elongates to only two-thirds of the wild-type root and formation of the crown root is inhibited, but lateral root formation is not as severely affected as crown root formation.

To isolate Brd2 by positional cloning, we first performed rough mapping of brd2 using 30 F2 progeny with the dwarf phenotype.Using 62 molecular markers, the brd2 locus was mapped to rice chromosome 10 between 21.8 and 30.2 centimorgan. Because a gene homologous to Arabidopsis DIM1/DWF1 is present in this area, we suspected that brd2 was identical to Dim/dwf1 (Figure 2A).The predicted Dim/dwf1 protein consists of 561 amino acids with 80% identity to the Arabidopsis DIM/DWF1 sequence, and comparison of the genomic and full-length cDNA sequences showed that Dim/dwf1 contains three predicted exons (Figure 2B). Direct sequencing of the brd2 mutant allele revealed a single base deletion(guanine) in exon 2, which leads to a frameshift and produces a premature stop codon near the mutation site (Figure 2B). The mutation causes the loss of the majority of a conserved FAD binding domain and the C-terminal region in Dim/dwf1 (Figure 2C).

We used gel blot analysis to examine the level of Dim/dwf1transcripts in the mutant. RNA from 10-d-old light-grown wild-type and mutant seedlings was isolated and probed with the 800-bp 39 fragment of the Dim/dwf1 cDNA. The Dim/dwf1transcript was detected in wild-type seedlings but not in the mutant (Figure 2D). A wild-type genomic DNA fragment containing the entire Dim/dwf1 was transformed into brd2, which restored the wild-type phenotype in all plants that were resistant to the selection marker, hygromycin (Figure 2E, right). Transformation with the empty vector had no effect on the dwarf phenotype(Figure 2E, left).Based on these results, we predict that brd2 is a knockout allele of Dim/dwf1.

Figure 2. Molecular Characterization of the brd2 Mutation

The Dim/dwf1 expression pattern in various organs was studied using RNA gel blot analysis. Dim/dwf1 transcripts were detected in all organs examined, albeit at different levels. The highest expression was in stems; intermediate expression was seen in roots, the shoot apex, and flowers; and expression was low in leaves and panicles (Figure 2F). RNA gel blot analysis was also used to examine the effect of exogenously applied BL on Dim/dwf1 expression (Figure 2G). Similar Dim/dwf1 expression levels were observed in the wild type, the BR-deficient mutant brd1, and d61-2, which is partially defective in the BR signaling pathway, and expression was not affected by treatment with BL. These results indicate that Dim/dwf1 expression is not regulated by BL, in contrast with D2 and BRD1, which are downregulated by BL in a feedback manner.

Evolution

The brd2 locus contains a single base deletion in the coding region of Dim/dwf1,a homolog of Arabidopsis thaliana DIMINUTO/DWARF1(DIM/DWF1).It is a knockout allele of Dim/dwf1. A knockout mutation, brd2, of the rice gene encoding a protein similar to the Arabidopsis DIM/DWF1 causes a semidwarf phenotype in the vegetative stage, with severe defects in internode elongation and panicle and seed development. Quantitative analysis of the endogenous sterol levels showed that brd2 is defective in the conversion of 24-MC to CR, as are the Arabidopsis dim/dwf1 and pea lkb mutants.

Labs working on this gene

• BioScience and Biotechnology Center,Nagoya University,Chikusa,Nagoya 464-8601,Japan
• RIKEN,Institute of Physical and Chemical Research,Wako-shi,Saitama 351-0198,Japan
• Department of Chemistry,Joetsu University of Education,Joetsu-shi,Niigata 943-8512,Japan

References

• 1.Zhi Hong,Miyako Ueguchi-Tanaka,Shozo Fujioka et al.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.The Plant Cell,2005,17(8):2243-2254
• 2.Tanaka,T., Antonio,B.A., Kikuchi,S. et al. The Rice Annotation Project Database (RAP-DB): 2008 update.Nucleic Acids Res.36 (DATABASE ISSUE),D1028-D1033(2008)
• 3.Itoh,T., Tanaka,T., Barrero,R.A. et al. Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana.Genome Res.17(2),175-183(2007)