Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.     

The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.     

The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis.

dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22alpha-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22alpha-hydroxylase.     

BIN2/DWF12 Antagonistically Transduces Brassinosteroid and Auxin Signals in the Roots of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Plant growth-stimulating hormones brassinosteroids (BRs) function via interactions with other hormones. However, the mechanism of these interactions remains to be elucidated. The unique phenotypes of brassinosteroid insensitive2/dwarf12-D (bin2/dwf12-D) mutants, such as twisted inflorescences and leaves, suggested that BIN2, a negative regulator of BR signaling, may be involved in auxin signaling. Furthermore, previously, we showed that auxin stimulates DWF4 expression. To determine the possible role of BIN2/DWF12 in Auxin signaling, we measured DWARF4pro:GUS activity through both GUS histochemical staining and in vivo GUS assay. We found that the GUS activity in the bin2/dwarf12-1D background dramatically increased relative to control. In addition, the number of lateral roots (LR) in bin2/dwf12-1D was greater than wild type, and the optimal concentration for auxin-mediated lateral root induction was lower in bin2/dwf12-1D; these findings suggest that BIN2 plays a positive role in auxin signaling. In contrast, ABA repressed both DWF4pro:GUS expression and lateral root development. However, the degree of repression was lower in bin2/dwf12-1D background, suggesting that BIN2 plays a role in ABA-mediated DWF4pro:GUS expression and subsequently in lateral root development, too. Therefore, it is likely that BIN2 plays a role of signal integrator for multiple hormones, such as BRs, auxin, and ABA.     

The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone

We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective.     

An Arabidopsis brassinosteroid-dependent mutant is blocked in cell elongation.

Cell elongation is a developmental process that is regulated by light and phytohormones and is of critical importance for plant growth. Mutants defective in their response to light and various hormones are often dwarfs. The dwarfed phenotype results because of a failure in normal cell elongation. Little is known, however, about the basis of dwarfism as a common element in these diverse signaling pathways and the nature of the cellular functions responsible for cell elongation. Here, we describe an Arabidopsis mutant, dwarf4 (dwf4), whose phenotype can be rescued with exogenously supplied brassinolide. dwf4 mutants display features of light-regulatory mutants, but the dwarfed phenotype is entirely and specifically brassinosteroid dependent; no other hormone can rescue dwf4 to a wild-type phenotype. Therefore, an intact brassinosteroid system is an absolute requirement for cell elongation.     

Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.     

Genetic evidence for an essential role of brassinosteroids in plant development

Brassinosteroids which show high structural similarity to animal steroid hormones elicit a variety of growth responses when exogeneously applied to plant tissues. Thus far however, the function of endogeneous brassinosteroids in higher plants has been unclear. This paper describes three extremely dwarfed Arabidopsis thaliana mutants, cbb1 (dwf1-6), cbb2 and cbb3, which are impared in cell elongation controlled by brassinosteroids. While cbb1 (dwf1-6) and cbb3 can be phenotypically normalized to wild-type by feeding with brassinosteroids indicating deficiencies of brassinosteroid biosynthesis, cbb2 is brassinosteroid-insensitive and defines a function required for further metabolic conversion necessary for biological activity or for perception/signal transduction of these growth-regulating plant steroid hormones. Expression of the meri5 and TCH4 genes is low in all three cbb mutants and can be restored to wild-type levels by brassinosteroid treatment in the cbb1 (dwf1-6) and cbb3 mutants but are unaffected in the cbb2 mutant. These data indicate that brassinosteroids are essential for proper plant development and play an important role in the control of cell elongation.     

Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Delta7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C-labeled precursors in dwf5 verified a deficiency in a sterol Delta7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol Delta7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3' and 5' splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith-Lemli-Opitz syndrome are caused by loss-of-function mutations in a sterol Delta7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.     

'Rinrei', a brassinosteroid-deficient dwarf mutant of faba bean (Vicia faba L.)

A dwarf mutant of broad bean ( Vicia faba L.), the variety Rinrei, has been created by γ -ray irradiation. Rinrei is characterized by dark green leaves and by reduced plant length, internode and petiole length, shoot weight, and number of branches. Genetic analysis of hybrids between Rinrei and two wild-type lines indicated that these characteristics are controlled by a single recessive gene. The phenotype of Rinrei was restored to that of the wild type by application of brassinolide, but not by GA₃. Qualitative and quantitative analysis by gas chromatography–mass spectrometry indicated that 24-methylenecholesterol and isofucosterol accumulated in Rinrei to levels more than 30 times higher than in the wild type. In contrast, Rinrei had lower than wild-type levels of campesterol, sitosterol and brassinosteroids. Therefore, Rinrei is a brassinosteroid-deficient mutant defective in sterol C-24 reduction. The gene was tentatively designated as brassinosteroid deficient dwarf 1 , bdd1 , which seems to be a homologue of Arabidopsis dwf1 ( dim , cbb1 ) and pea lkb .     

Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutation or a brassinosteroid biosynthesis inhibitor rescues defects in tropic responses of hypocotyls in the arabidopsis mutant nonphototropic hypocotyl 4

The nonphototropic hypocotyl 4 (nph4)/auxin response factor 7 (arf7) mutant of Arabidopsis (Arabidopsis thaliana) is insensitive to auxin and has defects in hypocotyl tropism, hook formation, differential leaf growth, and lateral root formation. To understand an auxin-signaling pathway through NPH4, we carried out screening of suppressor mutants of nph4-103 and obtained a dwarf suppressor mutant, suppressor of nph4 (snp2). snp2 had short hypocotyls in the dark condition and dark green and round leaves, short petioles, and more lateral shoots than the wild type in the light condition. The snp2 phenotypes were rescued by adding brassinolide to the growth medium in both light and dark conditions. Genetic mapping, sequence analysis, and a complementation test indicated that snp2 was a weak allele of DWARF4 (DWF4), which functions in brassinosteroid (BR) biosynthesis. snp2, which was renamed dwf4-101, exhibited photo- and gravitropisms of hypocotyls similar to those of the wild type with a slightly faster response in gravitropism. dwf4-101 almost completely suppressed defects in both tropisms of nph4-103 hypocotyls and completely suppressed hyponastic growth of nph4-103 leaves. Treatment with brassinazole, an inhibitor of BR biosynthesis, also partially rescued the tropic defects in nph4-103. Hypocotyls of nph4-103 were auxin insensitive, whereas hypocotyls of dwf4-101 were more sensitive than those of the wild type. dwf4-101 nph4-103 hypocotyls were as sensitive as those of dwf4-101. Auxin inducibility of massugu 2 (MSG2)/IAA19 gene expression was reduced in nph4-103. mRNA level of MSG2 was reduced in dwf4-101 and dwf4-101 nph4-103, but both mutants exhibited greater auxin inducibility of MSG2 than the wild type. Taken together, dwf4-101 was epistatic to nph4-103. These results strongly suggest that BR deficiency suppresses nph4-103 defects in tropic responses of hypocotyls and differential growth of leaves and that BR negatively regulates tropic responses.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. I. Molecular, cellular and physiological characterization of the Arabidopsis bul1 mutant, defective in the Δ7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis

Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bul1-1 mutant indicated that bul1-1 is defective in the Δ⁷-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes. Received: 28 April 2000 / Accepted: 6 October 2000     

Rice CYP734As function as multisubstrate and multifunctional enzymes in brassinosteroid catabolism

Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors.     

BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana

The ben1-1D (bri1-5 enhanced 1-1dominant) mutant was identified via an activation-tagging screen for bri1-5 extragenic modifiers. bri1-5 is a weak mutant allele of the brassinosteroid receptor gene, BRI1. Overexpression of BEN1 greatly enhances the defective phenotypes of bri1-5 plants. Removal of BEN1 by gene disruption in a Col-0 wild-type background, on the other hand, promotes the elongation of organs. Because BEN1 encodes a novel protein homologous to dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (BAN), BEN1 is probably involved in a brassinosteroid metabolic pathway. Analyses of brassinosteroid profiles demonstrated that BEN1 is indeed responsible for regulating the levels of several brassinosteroids, including typhasterol, castasterone and brassinolide. In vivo feeding and in vitro biochemical assays suggest that BEN1 is probably involved in a new mechanism to regulate brassinosteroid levels. These results provide additional insight into the regulatory mechanisms of bioactive brassinosteroids.