Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 15 (12), 2900-10

A Rice Brassinosteroid-Deficient Mutant, Ebisu Dwarf (d2), Is Caused by a Loss of Function of a New Member of Cytochrome P450

Affiliations

A Rice Brassinosteroid-Deficient Mutant, Ebisu Dwarf (d2), Is Caused by a Loss of Function of a New Member of Cytochrome P450

Zhi Hong et al. Plant Cell.

Abstract

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.

Figures

Figure 1.
Figure 1.
d2 Mutants Displaying Pleiotropic Abnormalities. (A) Gross morphology at the heading stage of wild-type (left), mild allele (d2-2; center), and strong allele (d2-1; right) plants grown in the field. Bar = 20 cm. (B) Elongation pattern of the second internode. The second internode from the top was shortened completely in d2-1 and partially in d2-2. From left to right, wild type, d2-2, and d2-1. Arrows indicate the positions of the nodes. Bar = 5 cm. (C) Leaf morphology. The leaf blade of the wild type (left) bends away from the vertical axis of the leaf sheath toward the abaxial side. The leaf of d2-2 (right) is erect. Arrows show the lamina joint. Bar = 5 cm. (D) Panicle morphology of the wild type (left) and d2-2 (right). Arrows indicate the positions of the nodes. Bar = 5 cm. (E) Grain morphology. The mutant (right) has slightly shortened grains. Bar = 1 cm.
Figure 2.
Figure 2.
Photomorphogenic Phenotype of d2 Grown in the Dark. Wild-type (left), d18 (GA-deficient mutant; center), and d2-2 (right) plants were grown in complete darkness. Arrows indicate the nodes, and arrowheads indicate the mesocotyls. Bars = 1 cm.
Figure 3.
Figure 3.
Elongation of the Second Leaf Sheath in Wild-Type and d2-2 Plants after Treatment with BL. The length of the second leaf sheath was measured 2 weeks after wild-type (WT; closed squares) and d2 (open circles) seeds were germinated on MS medium (Murashige and Skoog, 1962) containing various concentrations of BL. n = 25.
Figure 4.
Figure 4.
Physical Map of the D2 Gene and Structure of the D2 Protein. (A) High-resolution linkage and physical map of the d2 locus. The vertical bars represent the molecular markers, and the numbers of recombinant plants are indicated above the linkage map. The d2 mutation was tightly linked with a marker, C52409. The physical distances between adjacent markers are shown in parentheses. The D2 gene consists of eight exons and seven introns. Closed and open rectangles indicate exons and introns, respectively. Mutations identified in d2-1 and d2-2 are indicated. (B) Comparison of amino acid sequences of D2/CYP90D2 and other CYP90C and CYP90D proteins. According to the nomenclature for the P450 superfamily (Nelson et al., 1996), rice D2 and its D2 homolog were named CYP90D2 and CYP90D3, respectively. Dashes indicate gaps introduced to maximize alignment. Identical amino acids are represented by white-on-black letters. Triangles indicate the positions of intron insertions. Multiple sequence alignment was performed using the CLUSTAL W analysis tool in DDBJ. (C) Phylogenetic relationship between D2 and the BR biosynthetic P450 protein. D2/CYP90D2 is highly similar to the Arabidopsis BR biosynthetic P450 proteins CYP90D1, ROT3/CYP90C1 (Kim et al., 1998), CYP90A1(Szekeres et al., 1996), CYP90B (Choe et al., 1998), CYP85 (Bishop et al., 1996), and CYP85A1 (Shimada et al., 2001). The structural relationship was calculated using CLUSTAL W and illustrated using Treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
Figure 5.
Figure 5.
Phenotypic Complementation by the Introduction of D2. d2-1 mutant plants containing the empty vector (left) and the DNA fragment encompassing the entire D2 gene (right) are shown. Bar = 20 cm.
Figure 6.
Figure 6.
Expression Pattern of D2/CYP90D2 and CYP90D3 in Various Organs, and the Negative Feedback Effect of BL on D2/CYP90D2 Expression. (A) Organ-specific expression of D2/CYP90D2 and CYP90D3 in a wild-type plant. Total RNA was isolated from root (RT), flower (FL), panicle (PA), shoot apex (SA), leaf blade (LB), leaf sheath (LS), elongating stem (ST1), and elongated stem (ST2), and RT-PCR was performed. Signals were detected with the 32P-labeled cDNA clone indicated at left. Expression of the Actin gene was used as a control. (B) Negative feedback regulation of D2/CYP90D2 by BL. Total RNA was prepared from 10-day-old seedlings of the wild type (WT), brd1-1, brd1-2, and d61-2 with or without exogenous application of 10−6 M BL. Expression of the Actin gene was used as a control.
Figure 7.
Figure 7.
Quantitative Analysis of the Endogenous BR Intermediates in Wild-Type, d2-1, and d2-2 Plants. Sterol and BR levels (ng/g fresh weight) in wild-type (left), d2-2 (middle), and d2-1 (right) plants are shown below each product. nd, not detected.
Figure 8.
Figure 8.
Effect of BR Intermediates on the Degree of Inclination of the Leaf Lamina in Wild-Type and d2 Plants. (A) Typical response of the second leaf lamina joint in wild-type plants to treatment with BR intermediates. (B) to (H) The dose response to BR intermediates (ng/plant) of the bending angle in wild-type (WT; closed squares) and d2-2 (open circles) plants. Data presented are means from 10 plants. Error bars indicate standard deviations. (I) Pathway of late BR biosynthesis. BR intermediates that effectively, partially, or slightly increased the bending angle in d2-2 plants are indicated by closed, gray, or open boxes, respectively. Abbreviations are as in Figure 7.

Similar articles

See all similar articles

Cited by 145 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback