The Arabidopsis dwarf 1 Mutant Is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis 1

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2),constitutive photomorphogenesis and dwarfism(cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22α-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism. Disciplines Amino Acids, Peptides, and Proteins | Biology | Nucleic Acids, Nucleotides, and Nucleosides | Plant Sciences Comments At the time of this publication, Dr. Gregory was affiliated with the University of Arizona, but he is now a faculty member at the University of Pennsylvania. Author(s) Sunghwa Choe, Brian P. Dilkes, Brian D. Gregory, Amanda S. Ross, Heng Yuan, Takahiro Noguchi, Shozo Fujioka, Suguru Takatsuto, Atsushi Tanaka, Shigeo Yoshida, Frans E. Tax, and Kenneth A. Feldmann This technical report is available at ScholarlyCommons: http://repository.upenn.edu/biology_papers/19 The Arabidopsis dwarf1 Mutant Is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis Sunghwa Choe, Brian P. Dilkes, Brian D. Gregory, Amanda S. Ross, Heng Yuan, Takahiro Noguchi, Shozo Fujioka, Suguru Takatsuto, Atsushi Tanaka, Shigeo Yoshida, Frans E. Tax, and Kenneth A. Feldmann* Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (S.C., B.P.D., B.D.G., A.S.R., H.Y., A.T., F.E.T., K.A.F.); Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351–0198, Japan (T.N., S.F., S.Y.); Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943–8512, Japan (S.T.); and Department of Environment and Resources, Japan Atomic Energy Research Institute, 1233 Watanuki-machi, Takasaki-shi, Gunma 370–1292, Japan (A.T.) Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd ), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22a-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism. T-DNA-insertion mutagenesis has proven to be useful for the isolation of many important genes controlling plant growth and development (Choe and Feldmann, 1998). The Arabidopsis dwarf1 (dwf1) mutant was originally isolated from a T-DNA mutant population, and was the first mutant shown to cosegregate with the selectable marker in the T-DNA (Feldmann et al., 1989). The dwf1 mutant was identified because of its short stature, dark-green leaves, reduced fertility, and robust stems when grown in the light. Physiologically, dwf1 was not rescued by any of the known growth-promoting phytohormones such as GA3 or auxin (Feldmann et al., 1989). Using the plant DNA flanking the T-DNA as a probe, DWF1 was cloned and sequenced (accession no. U12400). Independently, Takahashi et al. (1995) isolated a morphologically similar mutant, diminuto (dim), from a different T-DNA mutant collection. Cloning and sequencing revealed that dim is disrupted in the DWF1 sequence, indicating that it is an allele of dwf1. One year later, Kauschmann et al. (1996) isolated another allele of DWF1 from a transposon-tagged population. They identified three tiny mutants named cabbage1, cabbage2, and cabbage3 (cbb1, cbb2, and cbb3). Altmann et al. (1995) found the sequence of genomic DNA flanking the transposon in cbb1 to be identical to that of DWF1. Kauschmann et al. (1996) originally found that cbb1 (dwf1-6) could be rescued by exogenous application of brassinosteroids, suggesting that cbb1 (dwf1-6) is defective in brassinosteroid biosynthesis. They also analyzed the expression of genes known to be involved in cell elongation, such as g-tonoplast intrinsic protein (g-TIP) (Höfte et al., 1992; Phillips and Huttly, 1994), and cell wall-modifying enzymes such as xyloglucan endotransglycosylases, including TOUCH4 (TCH4) (Xu et al., 1995) and MERI5 (Medford et al., 1991). The steady-state mRNA levels of TCH4 and MERI5 were lowered, whereas the expression of g-TIP was increased in the cbb mutants. Based on this, they proposed that a defect in brassinosteroid biosynthesis in cbb1 (dwf1-6) leads to failure in cell elongation, which requires partial activity of the cell wallmodifying enzymes TCH4 and MERI5. Brassinolide, the proposed end product of the brassinosteroid-biosynthetic pathway, is synthesized from sterol substrates. Therefore, plants defective in the biosynthetic steps leading from mevalonic acid to sterol, as well as in steps modifying sterols to brassinolide, could display the characteristic dwf phenotype. Currently, dwf7 is reported to be defective in a step of sterol biosynthesis (Choe et al., 1999), and three mutants, deetiolated2 (det2) (Li et al., 1 This research was supported by the National Science Foundation (grant no. 9604439 to K.A.F.) and by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports, and Culture of Japan (grant no. 10460050 to S.F.). * Corresponding author; e-mail feldmann@ag.arizona.edu; fax 1–520 – 621–7186. Abbreviations: EMS, ethyl methanesulfate; EST, expressed sequence tag; SIM, selective ion monitoring; SSLP, simple sequence length polymorphism. Plant Physiology, March 1999, Vol. 119, pp. 897–907, www.plantphysiol.org © 1999 American Society of Plant Physiologists 897 www.plantphysiol.org on July 12, 2017 Published by Downloaded from Copyright © 1999 American Society of Plant Biologists. All rights reserved. 1996), dwf4 (Choe et al., 1998), and constitutive photomorphogenesis and dwarfism (cpd) (Szekeres et al., 1996), are defective in the brassinosteroid-specific biosynthetic steps, from campesterol to brassinolide. Specifically, dwf7 is blocked in the sterol C-5 desaturation step, which is the most upstream step identified in dwf mutants thus far (Choe et al., 1999). Fujioka et al. (1997) have shown det2 to be blocked in the 5a-reduction step converting campesterol to campestanol. Choe et al. (1998) have proposed that dwf4 is disrupted in the 22a-hydroxylation step, which is hypothesized to be the rate-limiting step in brassinosteroid biosynthesis. Finally, Szekeres et al. (1996) have found cpd to be defective in the 23a-hydroxylation step following dwf4. Both DWF4 and CPD have been assigned to the same group of Cyt P450 proteins (CYP90) because they share more than 40% identity. In addition to these biosynthetic mutants, Clouse et al. (1996) have also identified brassinosteroid insensitive1 (bri1), a mutant insensitive to brassinosteroids. Recently, BRI1 was cloned and was shown to encode a Leu-rich repeat receptor kinase, suggesting a role in brassinosteroid signal perception and transduction (Li and Chory, 1997). All of the brassinosteroid dwarf mutants share characteristic phenotypes in the light, as described above, as well as abnormal skotomorphogenesis in the dark, including short hypocotyls and expanded cotyledons. Recent characterization of these dwf mutants provides compelling evidence that brassinosteroids are essential modulators for proper growth and development in plants. To understand all of the roles assigned to brassinosteroids in plants, the identification of the components of the brassinosteroid pathway and the regulation of endogenous brassinosteroid biosynthesis is critical. The proposed brassinosteroid-biosynthetic pathway predicts that there are at least 20 genes involved in brassinolide synthesis, which begins with squalene (Choe et al., 1999). To identify mutants in each biosynthetic step, we are characterizing a large collection of Arabidopsis dwarfs with the characteristic brassinosteroid dwarf phenotype. Currently, we have identified 12 different brassinosteroid loci. Six of these mutants, bri1 (dwf2) (Clouse et al., 1996; Li and Chory, 1997), cpd (dwf3) (Szeker