Novel sterols are produced in fk-J79 mutants. (A) fk-J79 mutation causes both the reduction of BRS and the accumulation of three abnormal 8,14-diene sterols. (B) A hypothetical model depicts the effects of fk-J79 mutation on plant development.

The fk-J79 mutation affects sterol biosynthesis and an application of BRs does not rescue the fk-J79 mutant's defect in hypocotyl elongation. (A) A simplified schematic sterol biosynthetic pathway in Arabidopsis. Values below each compound represent endogenous levels (per gram fresh weight) in fk-J79 (top) and wild type (bottom). (nd) Not detected. (B) The wild-type and fk-J79 or det2 mutant seedlings 7 DAG, treated with (+) or without (−) 1 μm of brassinolide (BL).

fk-J79 mutant is defective in embryogenesis. (A,B) Wild-type (A) and fk-J79 (B) embryos at the early heart stage. Arrow in B denotes oblique cell division in the epidermal layer. (Asterisk) Cotyledon primordium; (arrowhead) O′ line; (pv) provascular cells. (C,D) Wild-type (C) and fk-J79 (D) embryos at the late heart stage. Arrowheads in D denote small protrusions on the embryo surface. (Asterisk) Cotyledon primordium. (E–G) Wild-type (E) and fk-J79 (F,G) embryos at the bending cotyledon stage. (H,J) Magnification of E, F, and G, respectively. (Arrows) The region at the cotyledon base. Bars, 50 μm in A–D; 100 μm in E–J.

Morphological phenotypes of the fk-J79 mutant. (A,B) Scanning electron micrograph (SEM) of a light-grown wild-type (A) or fk-J79 (B) seedling 4 days after germination (DAG). Bar, 0.5 mm. (C,D) Top view of a light-grown wild-type (C) or fk-J79 (D) seedling 7 DAG. Note that leaf axis in the center are perpendicular to the cotyledon in wild type but not in fk-J79. (Arrow) A fused cotyledon. Bar, 1 mm in C and 0.5 mm in D. (E,F) Transverse section of a wild-type (E) or fk-J79 (F) cotyledon. (Arrows) Vascular bundles. (Asterisks) A region missing the epidermal cell layer in fk-J79. Bar, 100 μm. (G) SEM of a light-grown fk-J79 seedling (7 DAG). (Arrows) Three adventitious shoot meristems on a large, flat shoot tip region. Bar, 0.25 mm; a wild-type seedling is shown for comparison (inset). (Arrowhead) The single shoot apical meristem. Bar, 2 mm. (H) Four-week-old greenhouse grown wild-type and fk-J79 plants. Bar, 0.5 cm. (I) Six-week-old wild-type and twelve-week-old fk-J79 plants grown in a greenhouse. Bar, 2.5 cm.

FK expression. (A) RNA gel blot analysis of FK expression in different stages. RNA samples were prepared from dark-grown 7 DAG, or light-grown 4-, 7-, 14-, or 21-DAG wild type on MS medium. For each sample, 5 μg of total RNA was loaded. Equal loading was determined by the ethidium bromide staining of rRNA bands. A 1.3-kb fragment (Δ13) containing full-length FK cDNA was labeled and used as a probe for the hybridization. (B) RNA blot analysis of FK expression in different tissues. RNA samples were prepared from the wild-type rosette leaves (4-week old), inflorescence stems, flowers, siliques, and root. RNA gel electrophoresis, probe preparation, and blot hybridization are as described in A. (C–G) The expression of GUS (indicated by blue stain) in FK::GUS transgenic plants. (C) A flower showing high levels of GUS expression in anthers (white arrow) and ovules (black arrow); (D) a seedling 24 hr after germination; (E) shoot tip; (F) leaf of a seedling 7 DAG showing blue stain in vascular tissue and trichomes; (G) root-tips. Bars, 500 μm in C and F, 200 μm in D and E, and 300 μm in G.

fk-J79 is allelic to fk-X224 and is defective in cell division, cell expansion, and vascular development. (A) RNA blot analysis of FK in BE (WT), fk-J79, Ler (WT), and fk-X224 plants. RNA samples were prepared from 2-week-old light-grown plants on MS medium. For each sample, 5 μg of total RNA was loaded. Equal loading was determined by the ethidium bromide staining of rRNA bands. A 1.3-kb fragment (Δ13) containing full-length FK cDNA was labeled and used as a probe for the hybridization. (B) Protein gel blot analysis of FK in wild-type, fk-J79/+, and fk-J79 plants. Proteins were prepared from 2-week-old plants, separated on SDS–polyacrylamide gel, blotted to the membrane, and probed with antibody against FK. (C–E) Vasculature pattern in wild-type (C) and fk-J79 (D,E) seedlings. Seedlings were cleared using Hoyer's solution (Berleth and Jürgens 1993). As denoted by arrows, the vascular bundles from cotyledons meet at the top of the hypocotyl in wild type, but at the top of the root in fk-J79. Bars, 0.5 mm in (C–E). (F) Longitudinal section of an fk-J79 seedling (4 DAG). (Arrows) A leaf on the left and a fused organ on the right. (Asterisk) A depressed shoot apical region. Note that the large cortical cells expand in the lateral direction. (V) Vascular bundle; bar, 200 μm. (G–J) Transverse section of the hypocotyl in the wild-type (G) and fk-J79 mutant (H) and root in the wild type (I) and fk-J79 mutant (J). The vascular stele is denoted by a single arrow in the wild type (G). A large and disorganized vascular region is denoted by three arrows in the fk-J79 mutant (H). Bar, 100 μm.

Cloning, sequence analysis, and mutant complementation. (A) DNA blot analysis of FK in wild-type, fk-J79/+, and fk-J79 plants. Genomic DNA were digested with BamHI and separated by gel electrophoresis and blotted to a nylon membrane. A 1.1-kb EcoRI–HindIII fragment (λ4-1.2-2) that contained the left border of T-DNA and a flanking Arabidopsis genomic sequence was labeled and used as a probe for the hybridization. (B) Predicted protein sequence of FK. The signature motif of sterol reductase is underlined. (FK sequence has been deposited in GenBank, accession no. AF257178 and AF263244.) (C) FK sequence is similar to human LBR (38%, LBR, GenBank accession no. L25941), yeast C-14 sterol reductase (34%, ERG24, GenBank accession no. P32462), human sterol reductases (39%, SR-1, GenBank accession no. AF096304; 34%, SR-2 GenBank accession no. AF034544 or AF096305), Arabidopsis Δ⁷ sterol reductase (ST7R, GenBank accession no. U49398) and yeast C-24 sterol reductase (ERG4, GenBank accession no. P25340). Numbers at right represent length of the predicted proteins. Shaded cylinders denote transmembrane helices predicted by the SOSUI Program (http://www.tuat.ac.jp/∼mitaku/adv_sosui). Areas denoted by the arrow bar are the homologous regions aligned on the basis of the result of the BLAST search of the GenBank (Altschul et al. 1997). (D) A T-DNA is inserted 79 bp upstream of the predicted start codon (ATG) of FK in fk-J79 mutant. An ORF (D61) of 1671 bp is transcribed in an opposite direction to FK. The 1786-bp region between FK and D61 genes is a putative shared promoter. A comparison between cDNA and genomic sequences of FK revealed 13 exons (shaded boxes) and 12 introns (lines between hatched boxes). (E) Complementation of fk-J79 mutant. A NotI–EcoO1091 genomic fragment containing a 1786-bp shared promoter and 2734 bp of FK or 1671 bp of D61 was inserted into a binary vector. Each construct was transfected into Agrobacterium tumefaciens and the strain was used to transform fk-J79/+ plants by the vacuum infiltration method. Ratios are the number of fk-J79 to the wild-type plants.