Brassinolide Biosynthesis in A. thaliana
Explore and order pathway-specific siRNAs, real-time PCR assays, and expression vectors. View pathway information and literature references for your pathway.
  • Click on your proteins of interest in the pathway image or review below
  • Select your genes of interest and click "add selection"
  • When you have finished your gene selection, click "Find Products" to find assays, arrays, or create custom products
Download Image Terms of Use Download PPT
Pathway Navigator
Brassinolide Biosynthesis in A. thaliana

BRs (Brassinosteroids) are Plant Steroid Hormones that influence a wide range of important Developmental and Physiological processes, including Regulation of Gene Expression, Cell Division and Expansion, Differentiation, Programmed Cell Death, and Homeostasis. The regulation of these processes by BRs, acting together with other plant hormones, leads to the promotion of Stem Elongation and Pollen tube Growth, Leaf Bending and Epinasty, Root Growth Inhibition, Proton-Pump Activation, and Xylem Differentiation. The pathways of BR biosynthesis have been elucidated by a series of detailed biochemical studies. The biosynthetic pathway to BL (Brassinolide), the biologically most active BR, can be divided into General Sterol Synthesis (Cycloartenol to Campesterol), and the BR-Specific Pathway from Campesterol to BL. BL is a polyhydroxylated derivative of 5-Alpha-Cholestan, namely (22R,23R,24S)-2-Alpha,3-Alpha,22,23-Tetrahydroxy-24-Methyl-B-Homo-7-Oxa-5Alpha-Cholestan-6-One.  The BR family consists of BL and about 40 other free BRs plus 4 conjugates. These differ from BL by variations at C-2 and C-3 in the A ring; the presence of a Lactone, Ketone, or De-Oxo function at C-6 in the B ring; the stereochemistry of the hydroxyl groups in the side chain, and the presence or absence of a methyl (methylene) or ethyl (ethylene) group at C-24. The conjugates are glycosylated, meristylated and laurylated derivatives of the hydroxyls in ring A or in the side chain. The optimal structure for highest BR activity normally is that found in BL, consisting of a Lactone function at C-6/C-7, cis-Vicinal hydroxyls at C-2 and C-3, R configuration of the hydroxyls at C-22/C-23 and a methyl substitution at C-24 (Ref.1 & 2).

The BR biosynthesis pathways are conserved between Catharanthus roseus, Arabidopsis, Pisum sativum, Lycopersicon esculentum and Oryza sativa. BL, the proposed end product of the BR-biosynthetic pathway in Arabidopsis, is synthesized from Sterol substrate, Campesterol. Besides their role as BR precursors, Campesterol are integral membrane components which serve to regulate the fluidity and permeability of membranes and directly affect the activity of membrane associated proteins, including enzymes and signal transduction components. The first reaction towards BL biosynthesis is the conversion of Campesterol to Campestanol, and then Campestanol is converted to Castasterone through either the Early C6-oxidation pathway or the Late C6-oxidation pathway. In the Early C-6 oxidation pathway, hydroxylation of the side chain occurs after C6 oxidation, whereas in the Late C-6 oxidation pathway the hydroxylation of the side chain occurs before position 6 of the B-ring is oxidized. Finally Castasterone is converted to BL, the most active BR (Ref.3).

The conversion of the membrane Sterol Campesterol to BL occurs via a series of reductions, hydroxylations, epimerizations and oxidations that have been extensively studied in several species. The conversion of Campesterol to Campestanol is not a single step, but composed of the biosynthetic sequence of Campesterol   4-en-3Beta-ol ---> 4-en-3-one ---> 3-one ---> Campestanol in Arabidopsis. In the first step,  Campesterol is converted to Campest-4-en-3Beta-ol in presence of enzyme Delta-5-3-Ketosteroid Isomerase. Enzymes that catalyze the conversion from 3-Beta-Hydroxy-Delta,5-6-Steroid to 3-oxo-Delta-4-5 Isomerase have been reported in Bacteria and Mammals. Campest-4-en-3 Beta-ol is converted to Campest-4-en-3-one in presence of enzyme 3-Beta-Hydroxysteroid Dehydrogenase. In the next step, Campest-4-en-3-one is converted to Campestanol via 5-Alpha-Campestan-3-one. 3-Oxo-5Alpha-Steroid 4-Dehydrogenase family members (encoded by Det2 in Arabidopsis) catalyze the formation of 5-Alpha-Campestan-3-one (Ref.2 & 4).

From Campestanol, the BL biosynthetic pathway diverges into the Early and Late C-6 oxidation branches. BRs lacking a Ketone or Lactone in the B ring occur widely, although they have low biological activity in bioassays. The endogenous occurrence of 6-Deoxocastasterone, 6-Deoxotyphasterol, 3-Dehyrdo-6-Deoxoteasterone and 6-Deoxoteasterone has been demonstrated in numerous plants where Castasterone and BL are also present including Bean, Wheat, Rice, Rye, Arizona cypress, C. roseus and Arabidopsis. The Co-occurrence of 6-Deoxo and 6-Oxo forms of BL precursors suggests that a Late C-6 oxidation pathway, in which Ketone formation at C-6 follows A ring Hydroxyl and side chain modification, operates simultaneously with Early C-6 oxidation in a wide array of plants (Ref.5).

Campestanol is first converted to 6-Deoxocathasterone and 6-Oxocampestanol. 6-Oxocampestanol formation occurs via 6Alpha-Hydroxy Campestanol. 6-Oxocampestanol once formed is converted to Cathasterone. The conversion of Campestanol to 6-Deoxocathasterone (Late C-6 oxidation) and of 6-Oxocampestanol to Cathasterone (Early C-6 oxidation) are both accomplished by the product of the Dwf4 gene, Steroid 22-Alpha-Hydroxylase, which encodes a Cytochrome P450 with sequence similarity to Mammalian Steroid Hydroxylases. The next step in both branches of the pathway also involves side chain hydroxylation and is catalyzed by the product of the Cpd gene, which encodes a Cytochrome P450, CYP90A1 (Cytochrome-P450-90A1) with 43% identity (66% similarity) to Dwf4. The Cpd mutant is an extreme dwarf which is rescued only by 23-Alpha-Hydroxylated BRs, indicating that Cpd acts as a C-23 Steroid. Cpd catalyzes conversion of Deoxocathasterone to 6-Deoxoteasterone as well as formation of Teasterone from Cathasterone. Both Teasterone and 6-Deoxoteasterone ultimately leads to formation of Typhasterol via intermediate steps. Teasterone forms Typhasterol via 3-Dehydroteasterone, whereas formation of Typhasterol from 6-Deoxoteasterone occurs via 3-Dehydro-6-Deoxoteasterone and 6-Deoxotyphasterol. Typhasterol is then converted to Castasterone. Rot3 gene, encoding the protein CYP90C1 (Cytochrome-P450-90C1), appears to be required for the conversion of Typhasterol to Castasterone. Castasterone can also be formed from 6-Deoxotyphasterol through the formation of 6-Deoxocastasterone and 6Alpha-Hydroxycastasterone, which is catalyzed by the enzyme BR6Ox (Brassinosteroid-6-Oxidase). Castasterone, once formed leads to formation of BL. Thus, BL biosynthesis in Arabidopsis appears to be more of a metabolic grid than two independent linear pathways of Early and Late C-6 oxidation. Double mutant studies showing additive phenotypes between mutants at different steps of the pathway supports the grid hypothesis vs. the independent linear pathways (Ref.6 & 7).