To transposon tag the tomato Dwarf (D) gene, a tomato line that carries a T-DNA containing a maize Activator (Ac) transposable element closely linked to D was pollinated with a stock homozygous for the d mutation. Hybrid seedlings were screened for dwarf progeny, and three independent dwarf lines were obtained. Two of these lines showed inheritance of a recessive phenotype similar to that conferred by the extreme dwarf (dx) allele. Variegation for the dwarf phenotype in one of these lines suggested that D had been tagged by Ac. Genomic DNA adjacent to Ac in these two lines was isolated by use of the inverse polymerase chain reaction, and the two insertions mapped approximately 2 kb apart. Partial complementation of d was observed when the corresponding wild-type sequence was used in transformation experiments. A cDNA clone of D was sequenced, and the predicted amino acid sequence has homology to cytochrome P450 enzymes.

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.

Based on its compact habit, Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.), has been proposed as a preferred variety to carry out molecular research in tomato. This cultivar, however, is poorly characterized. It is shown here that Micro-Tom has mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. The presence of the self-pruning mutation in Micro-Tom, that generates a determinate phenotype, was confirmed by crossing and sequence analysis. It was also found that Micro-Tom has a mutation in the DWARF gene (d) that leads to mis-splicing and production of at least two shorter mRNAs. The d mutation is predicted to generate truncated DWARF protein. The d sequence defect co-segregates with dark-green and rugose leaves, characteristics of brassinosteroid biosynthesis mutants. Micro-Tom also carries at least another mutation producing internode length reduction that affects plant height but not active gibberellin (GA) levels, which were similar in dwarf and tall Micro-TomxSeverianin segregants. GAs and brassinosteroids act synergistically in Micro-Tom, and the response to GA depends on brassinosteroids because the elongation of internodes was at least six times higher when GA(3) was applied simultaneously with brassinolide. A novel variety, Micro-0 that is fully susceptible to C. fulvum and almost as dwarf as Micro-Tom, has been generated from the cross of Cf0xMicro-Tom. This line represents a valuable resource for future analysis of Cf resistance genes through breeding or transformation.