The Arabidopsis \u3cem\u3edwf/ste1\u3c/em\u3e Mutant is Defective in the Δ\u3csup\u3e7\u3c/sup\u3e 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 Δ7 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 Arabidopsis \u3cem\u3edwarf1\u3c/em\u3e Mutant is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis

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

The Arabidopsis leucine-rich repeat receptor kinase BIR3 negatively regulates BAK1 receptor complex formation and stabilizes BAK1

BAK1 is a co-receptor and positive regulator of multiple ligand-binding leucine-rich-repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana. BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand-binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand-binding receptors to prevent BAK1 receptor complex formation

The Control of Cell Expansion, Cell Division, and Vascular Development by Brassinosteroids: A Historical Perspective

Steroid hormones are important signaling molecules in plants and animals. The plant steroid hormone brassinosteroids were first isolated and characterized in the 1970s and have been studied since then for their functions in plant growth. Treatment of plants or plant cells with brassinosteroids revealed they play important roles during diverse developmental processes, including control of cell expansion, cell division, and vascular differentiation. Molecular genetic studies, primarily in Arabidopsis thaliana, but increasingly in many other plants, have identified many genes involved in brassinosteroid biosynthesis and responses. Here we review the roles of brassinosteroids in cell expansion, cell division, and vascular differentiation, comparing the early physiological studies with more recent results of the analysis of mutants in brassinosteroid biosynthesis and signaling genes. A few representative examples of other molecular pathways that share developmental roles with brassinosteroids are described, including pathways that share functional overlap or response components with the brassinosteroid pathway. We conclude by briefly discussing the origin and conservation of brassinosteroid signaling

BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana

Brassinosteroid-insensitive 1 (BRI1) of Arabidopsis thaliana encodes a cell surface receptor for brassinosteroids. Mutations in BRI1 severely affect plant growth and development. Activation tagging of a weak bri1 allele (bri1-5) resulted in the identification of a new locus, brs1-1D. BRS1 is predicted to encode a secreted carboxypeptidase. Whereas a brs1 loss-of-function allele has no obvious mutant phenotype, overexpression of BRS1 can suppress bri1 extracellular domain mutants. Genetic analyses showed that brassinosteroids and a functional BRI1 protein kinase domain are required for suppression. In addition, overexpressed BRS1 missense mutants, predicted to abolish BRS1 protease activity, failed to suppress bri1-5. Finally, the effects of BRS1 are selective: overexpression in either wild-type or two other receptor kinase mutants resulted in no phenotypic alterations. These results strongly suggest that BRS1 processes a protein involved in an early event in the BRI1 signaling

CYP83B1, a Cytochrome P450 at the Metabolic Branch Point in Auxin and Indole Glucosinolate Biosynthesis in Arabidopsis

Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci-nitro compound, 1-aci-nitro-2-indolyl-ethane, with a K(m) of 3 μM and a turnover number of 53 min(−1). The aci-nitro compound formed reacts non-enzymatically with thiol compounds to produce an N-alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis