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Please input one-sentence summary here. The gene OsDWARF4 was found in young elongating tissues.

Annotated Information

Brassinosteroids influence both plant height and leaf erectness in rice[1]. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE1 (bri1) mutants, which are deficient in the brassinosteroid receptor, have a severe dwarf phenotype[2]. The rice mutants d61-1 and d61-2, which are weak mutant alleles of OsBRI1, a homolog of A. thaliana BRI1, have both a semi-dwarf phenotype and more erect leaves[3]. In rice, an erect leaf phenotype has also been associated with loss-of-function, brassinosteroid-deficient mutants such as brassinosteroid-deficient dwarf1 (brd1), which is defective in OsDWARF (the homolog of tomato CYP85A1/DWARF[4]), and ebisu dwarf (d2), which is defective in CYP90D2/D2 (ref.[5]). However, the grain yield of d61, brd1 and d2 mutants is decreased because of morphological alterations in their reproductive development.


New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields.The erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology[6].


The affected gene, OsDWARF4, was identified by characterizing the site of insertion of the Tos17 retrotransposon.

Molecular characterization of OsDWARF4 and OsDWARF4L1/D11

OsDWARF4 is a homolog of A. thaliana DWARF4, which encodes a cytochrome P450, CYP90B1 (Fig. 1a). CYP90B1/DWARF4 catalyzes C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis[7]. Only one DWARF4 homolog was identified in the rice genome. OsDWARF4 is located on the short arm of chromosome 3 (35 cM). Its predicted open reading frame consists of eight exons, and encodes a protein of 502 amino acids (Fig. 1b,c). OsDWARF4 is most closely related to DWARF4 (66.3% amino-acid identity; Fig. 1a). The structure of OsDWARF4 is similar to that of DWARF4 throughout its length: five domains found in cytochrome P450s—proline rich, A (also referred to as dioxygen binding), B (steroid binding), C and heme binding—are highly conserved (Fig. 1c). In wild-type rice, OsDWARF4 transcripts are most abundant in leaf blades and roots, but are also found in all other plant parts tested (Fig. 1d). Transcript levels were decreased by exogenous brassinolide treatment, and increased in brassinosteroid-insensitive d61-3 and brassinosteroid-deficient brd1-1 (Fig. 1e). These results suggest that OsDWARF4 expression is feedback regulated, as is that of A. thaliana brassinosteroid-biosynthesis genes[8]. OsDWARF4L1 was identified as an OsDWARF4-like gene by in silico screening of rice DNA databases. OsDWARF4L1 is located on the long arm of chromosome 4 (70.9 cM). A recent study demonstrated that a rice semi-dwarf and short-grain mutant, d11, has a loss-of-function mutation in the OsDWARF4L1 gene[9]. OsDWARF4L1/D11 shows the highest homology with A. thaliana CYP724A1, and is closely related to rice OsDWARF4 and A. thaliana DWARF4 (47.4%, 37.7% and 41.7% amino-acid identities, respectively; Fig. 1a). In wild-type rice, OsDWARF4L1/D11 transcripts were most abundant in vegetative shoot apices, leaf sheaths and roots, and found at more moderate levels in other organs (Fig. 1d). They were decreased by exogenous brassinolide treatment, and increased in d61-3 and brd1-1 (Fig. 1e). These results indicate that OsDWARF4L1/D11 expression is regulated by a feedback mechanism, as is that of OsDWARF4.

Figure 1. Molecular characterization of OsDWARF4 and OsDWARF4L1/D11 (a) Phylogenetic relationships among brassinosteroid-biosynthetic cytochrome P450s of rice, A. thaliana and tomato. Bar, 0.1 amino-acid substitutions per site. (b) Genomic organization of the OsDWARF4 gene. Boxes represent exons, and lines separating boxes represent introns. The arrowhead indicates the site of Tos17 insertion in osdwarf4-1. (c) Relative positions of the major domains in OsDWARF4. All of the major domains found in the cytochrome P450 superfamily are conserved in OsDWARF4. The arrowhead indicates the site of Tos17 insertion in osdwarf4-1. (d) Expression of OsDWARF4 and OsDWARF4L1/D11 in various organs of wild-type rice. The gene encoding Histone H3 was used as a control. (e) Feedback regulation of OsDWARF4 and OsDWARF4L1/D11 in brassinolide-treated wild-type plants (left) and brassinosteroid-related mutants (right). Values under each panel indicate relative amounts of each transcript. (f) Function of recombinant CYP90B2/OsDWARF4 and CYP724B1/D11 proteins. Campesterol (CR) was incubated with each recombinant protein and analyzed by GC-MS. Inset shows that the mass spectrum of the trimethylsilyl ester of each metabolite coincided with that of the trimethylsilyl ether of synthesized (22S)-22-hydroxycampesterol (22-OHCR).

Phenotypes of rice mutants defective in brassinosteroid biosynthesis

In osdwarf4-1, Tos17 insertion into exon 6 causes a premature termination in domain C (arrowhead in Fig. 1b,c). Because a premature stop codon before the heme-binding domain caused loss-of-function of A. thaliana DWARF4 (ref.[7]), we consider osdwarf4-1 to be a null allele. Unexpectedly, osdwarf4-1 plants show only weak morphological phenotypes, such as a slightly dwarfed stature and more erect leaves (Fig. 2a,b). Both of these phenotypes were complemented by the introduction of an entire OsDWARF4 gene (data not shown). These results seem inconsistent with the fact that rice d61-3 and brd1-1, which are loss-of-function mutants of OsBRI1 and OsDWARF respectively, display severe dwarfing and have malformed leaves (for example, see brd1-1 in Fig. 2a), and suggest functional redundancy in brassinosteroid biosynthesis in rice. Because null alleles of d11, such as d11-4, also show a weak phenotype[9](Fig. 2a),OsDWARF4L1/D11 and OsDWARF4 may function redundantly. To confirm this possibility, researcher produced double mutants by crossing homozygous osdwarf4-1 and d11-4 plants. Among the F2 population, 34 of 576 plants showed severe dwarfing and malformed leaves with tortuous leaf blades, indistinguishable from the brassinosteroid-deficient brd1-1 phenotype (Fig. 2a,c,d). This ratio fits the theoretical 9:3:3:1 (wild-type:osdwarf4-1:d11-4:osdwarf4-1/d11-4) segregation predicted by mendelian inheritance. PCR and sequence analyses confirmed that these plants were osdwarf4-1/d11-4 double mutants (data not shown). Endogenous levels of brassinosteroid intermediates in brd1 clearly indicate that OsDWARF catalyzes C-6 oxidation[4]. However, quantification in d2 could not determine the step(s) catalyzed by CYP90D2/D2, because CYP90D2/D2 functions redundantly with another gene, CYP90D3, and loss-of-function of CYP90D2/D2 have caused only limited defects in brassinosteroid biosynthesis[5]. Similarly, endogenous levels of brassinosteroid intermediates were obviously changed in the osdwarf4-1/d11-4 double mutant relative to osdwarf4-1 or d11 single mutants. Although brassinolide was not detected in either the wild-type or osdwarf4-1/d11-4, another bioactive brassinosteroid, castasterone (CS), was detected in the wild-type but not in osdwarf4-1/d11-4, confirming that osdwarf4-1/d11-4 is brassinosteroid-deficient (Fig. 2e). Levels of the other five intermediates downstream of C-22 hydroxylation—6-deoxocathasterone (6-DeoxoCT), 6-deoxoteasterone (6-DeoxoTE), 3-dehydro-6-deoxoteasterone (6-Deoxo3DT), 6-deoxotyphasterol (6-DeoxoTY) and 6-deoxocastasterone (6-DeoxoCS)—were also greatly reduced in osdwarf4-1/d11-4 (Fig. 2e). This indicates that both proteins are involved in C-22 hydroxylation. It is unclear why the upstream compound, campestanol, did not accumulate in osdwarf4-1/d11-4. Because A. thaliana CYP90B1/DWARF4 also catalyzes C-22 hydroxylation of other upstream compounds such as campesterol and (24R)-ergost-4-en-3-one[10], it is possible that campestanol is not the major substrate for CYP90B2/OsDWARF4 and CYP724B1/D11 in rice.

Figure 2. Phenotypes of rice mutants defective in brassinosteroid biosynthesis.(a) Comparison of gross morphology between the wild-type, single mutants (osdwarf4-1, d11-4, brd1-1), and a double mutant (osdwarf4-1/d11-4). Bar, 30 cm. (b) Erect leaf phenotype of osdwarf4-1. The degree of bending between leaf blade and sheath of osdwarf4-1 (right) is less than that of the wild-type (left). lb, leaf blade; ls, leaf sheath. Bar, 3 cm. (c,d) Close-up views of the osdwarf4-1/d11-4 double mutant (c) and brd1-1 (d). Bars, 5 cm. (e) Endogenous contents of brassinosteroid intermediates in the wild-type and osdwarf4-1/d11-4 double mutant. CN, campestanol; 6-DeoxoCT, 6-deoxocathasterone; 6-DeoxoTE, 6-deoxoteasterone; 6-Deoxo3DT, 3-dehydro-6-deoxoteasterone; 6-DeoxoTY, 6-deoxotyphasterol; 6-DeoxoCS, 6-deoxocastasterone; CS, castasterone; BL, brassinolide.

Characterization of a field-grown osdwarf4-1 mutant

Because the harvest index (grain/(grain plus straw)) of rice is about 0.5, further increases in yield potential will have to involve increases in crop biomass driven by more net photosynthesis[11]. In rice, the contribution of lower leaves to photosynthesis is significant, even though their photosynthetic capacity is considerably less than that of upper leaves[12]. Erect leaves allow greater penetration of light to lower leaves, thereby optimizing canopy photosynthesis[13]. Therefore, the most likely explanation for the increase in above-ground biomass in osdwarf4-1 plots under dense planting conditions (more than 1.2 times that in wild-type under conventional planting condition) is that the shade of the upper leaves is minimized, and the lower leaves receive more light to drive higher rates of photosynthesis (Fig. 3 and Supplementary Table 2 online). No difference was observed in the heading date (flowering time) and the grain-filling period (time to maturity) between wild-type and osdwarf4-1 plants (data not shown). In the wild-type, the above-ground biomass was increased in response to higher fertilizer application under conventional planting (22.2 plants m-2), but was significantly decreased under dense planting with doubled fertilizer application owing to reduced photosynthesis caused by lodging (Fig. 3a). In contrast, the above-ground biomass of osdwarf4-1 was significantly (P < 0.01) increased by dense planting at each fertilizer rate. Consequently, the above-ground biomass in osdwarf4-1 plots under dense planting with standard and a 1.5-fold increase in fertilizer application was approx1.34 and 1.52 times that in wild-type plots under conventional cultivation conditions. The increased biomass depended mainly on accelerated formation of tillers, which have the ability to generate a panicle (panicle number, Fig. 3b). Both total and fertile grain numbers tended to increase in densely planted osdwarf4-1 plots (Fig. 3c), resulting in significantly increased grain yield even when the fertilizer application was not increased (for example, the osdwarf4-1 yield was 6.19 t ha-1 under dense planting with standard fertilizer application whereas the wild-type yield was 4.69 t ha-1 under conventional conditions; Fig. 3d). However, the increase in grain yield was not as much as that in above-ground biomass, because the harvest index of osdwarf4-1 (0.41) was slightly lower than that of the wild-type (0.44). A similar field test gave comparable results.

Figure 3. Characterization of a field-grown osdwarf4-1 mutant.(a) Comparison of biomass production between the wild-type and osdwarf4-1. White and black bars indicate above-ground and panicle dry weight, respectively. (b) Comparison of panicle number (white bar) and panicle length (black bar). (c) Comparison of total (white bar) and fertile (black bar) grain number. (d) Comparison of estimated grain yields between wild-type (white bar) and osdwarf4-1 (black bar). W, wild-type, M, osdwarf4-1. Density indicates conventional planting (22.2 plants m-2) or dense planting (44.4 plants m-2). Fertilizer indicates the level of nitrogen fertilizer. Conventional condition is times 1 (6 g m-2), and two increased conditions are times 1.5 and times 2 (9 and 12 g m-2 respectively). Lowercase letters indicate significant differences at the level of P < 0.01 within a parameter (Tukey's Honest Significant Difference test).

Overexpression of OsDWARF4

Overexpression of DWARF in transgenic tomato increases plant height[14], and overexpression of DWARF4 in transgenic A. thaliana and tobacco increases vegetative growth and seed yield[15]. In rice, however, the role of brassinosteroid in regulating leaf angle has been known for over 40 years[16],[17]. Whereas brassinosteroid deficiency increased the erectness of leaves, the opposite phenotype (increased leaf angle) was observed in transgenic rice overexpressing OsDWARF4 (27.6° versus 10.0° in the wild-type, P < 0.01), and was inherited in their progeny (data not shown). Because inclined leaves are unfavorable for photosynthesis when plants are grown in a group, we have to carefully evaluate whether the overexpression of a brassinosteroid-biosynthesis gene in transgenic rice increases grain yield as is the case in transgenic plants overexpressing DWARF4.


More erect leaves improve not only light distribution but also the size and use of leaf nitrogen reservoirs for grain growth[13]. The erect leaf phenotype caused by brassinosteroid deficiency improved biomass production under field conditions, resulting in increased grain yield without the negative environmental effects associated with fertilizers. Recently identified rice homologs of A. thaliana BAS1 that encodes the brassinosteroid catabolic enzyme, CYP734A1 (ref.[18]). Overexpression of these rice homologs in transgenic rice successfully induced the brassinosteroid-deficient phenotypes (unpublished results). Although ideally, expression of these genes should be restricted to particular tissues and organs such as leaf laminae, it should be possible to generate erect leaf varieties by the introduction of a single transgene. In conclusion, engineering more erect leaves modulating brassinosteroid metabolism can be combined with dense planting to improve rice grain yields.

Labs working on this gene

Please input related labs here.


  1. Sakamoto, T. & Matsuoka, M. Generating high-yielding varieties by genetic manipulation of plant architecture. Curr. Opin. Biotech. 15, 144–147 (2004).
  2. Li, J. & Chory, J. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, 929–938 (1997).
  3. Yamamuro, C. et al. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12, 1591–1606 (2000).
  4. 4.0 4.1 Hong, Z. et al. Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. Plant J. 32, 495–508 (2002).
  5. 5.0 5.1 Hong, Z. et al. A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell 15, 2900–2910 (2003).
  6. Sakamoto T;Morinaka Y;Ohnishi T;Sunohara H;Fujioka S;Ueguchi Tanaka M;Mizutani M;Sakata K;Takatsuto S;Yoshida S;Tanaka H;Kitano H;Matsuoka M Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice Nature Biotechnology, 2006, 24(1): 105-109.
  7. 7.0 7.1 Choe, S. et al. The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10, 231–243 (1998).
  8. Bancos, S. et al. Regulation of transcript levels of the Arabidopsis cytochrome P450 genes involved in brassinosteroid biosynthesis. Plant Physiol. 130, 504–513 (2002).
  9. 9.0 9.1 Tanabe, S. et al. A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length. Plant Cell 17, 776–790 (2005).
  10. Fujioka, S., Takatsuto, S. & Yoshida, S. An early C-22 oxidation branch in brassinosteroid biosynthetic pathway. Plant Physiol. 130, 930–939 (2002).
  11. Mann, C.C. Crop scientists seek a new revolution. Science 283, 310–314 (1999).
  12. Horton, P. Prospects for crop improvement through the genetic manipulation of photosynthesis: morphological and biochemical aspects of light capture. J. Exp. Bot. 51, 475–485 (2000).
  13. 13.0 13.1 Sinclair, T.R. & Sheehy, J.E. Erect leaves and photosynthesis in rice. Science 283, 1455 (1999).
  14. Bishop, G.J. et al. The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis. Proc. Natl. Acad. Sci. USA 96, 1761–1766 (1999).
  15. Choe, S. et al. Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis. Plant J. 26, 573–582 (2001).
  16. Maeda, E. Rate of lamina inclination in excised rice leaves. Physiol. Plant. 18, 813–827 (1965).
  17. Wada, K. et al. Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol. 22, 323–325 (1981).
  18. Neff, M.M. et al. BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proc. Natl. Acad. Sci. USA 96, 15316–15323 (1999).

Structured Information