Biosynthetic Pathways of Brassinolide in Arabidopsis

Abstract

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone 3 3-dehydro-6-deoxoteasterone 3 6-deoxotyphasterol 3 6-deoxocastasterone 3 6␣-hydroxycastasterone 3 castasterone 3 BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone 3 3-dehydroteasterone 3 typhasterol 3 castasterone 3 BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.

New Alleles of Rice <i>ebisu dwarf</i> (<i>d</i>2) Mutant Show both Brassinosteroid-Deficient and -Insensitive Phenotypes

“…Although the major pathway for brassinosteroid biosynthesis has been established [17][18][19], recent studies demonstrated the complexity of the pathway by revealing new branches and shortcut routes [20,21]. To clarify the function of the other biosynthetic enzymes involved in brassinosteroid biosynthesis for which mutants have not been identified, we screened rice mutant libraries and isolated three new candidate lines.…”

Brassinosteroid-6-Oxidases from Arabidopsis and Tomato Catalyze Multiple C-6 Oxidations in Brassinosteroid Biosynthesis

“…For example, det2 Noguchi et al, 1999b), cpd , dwf4 (Choe et al, 1998), dwf1/dim (Klahre et al, 1998;Choe et al, 1999a), ste1/dwf7 (Choe et al, 1999b), sax1 (Ephritikhine et al, 1999), dwf5 (Choe et al, 2000), fackel (Jang et al, 2000;Schrick et al, 2000), and bri1 (Clouse et al, 1996;Li and Chory, 1997). From the analysis of these mutants, we identified BL, castasterone (CS), typhasterol (TY), teasterone (TE), 6-deoxocastasterone (6-DeoxoCS), 6-deoxotyphasterol (6-DeoxoTY), 6-deoxoteasterone (6-DeoxoTE), and 6-deoxocathasterone (6-DeoxoCT) as endogenous BRs in various Arabidopsis tissues, such as shoots, siliques, and seeds (Fujioka et al, 1996Noguchi et al, 1999aNoguchi et al, , 2000. All BRs identified in Arabidopsis are important components of either the early or late C-6-oxidation pathways, indicating that both pathways are functional in this species.…”

Reversible Conversion In The Brassinosteroid Quartet Castasterone, Brassinolide And Their 3β-Epimers

“…The previously reported reversibility of the epimerization of the 3-hydroxyl group of brassinosteroids [20,21,35,36] showed a peak corresponding to labelled precursor 3 (RR t 1.100) and a peak corresponding to labelled castasterone 1 (RR t 1.419) in the seedlings of A. thaliana and S. cereale. In addition a peak corresponding to triply deuterated 3-epibrassinolide (4) (m/z 818; [M+H] + ; RR t 0.856) in the seedlings of S. cereale has been detected (Fig.…”

“…Inversion from 3β to 3α configuration has been demonstrated in the course of early and late C-6 oxidation [18 -19], and 3-epi-6-deoxocathasterone (3α-configuration) was identified as a metabolite of 6-deoxocathasterone (3β -configuration) [9]. Formation of teasterone (3β -epimer) from typhasterol (3α-epimer) through the 3-dehydro intermediate showed the reversibility of this reaction [20]. Brassinosteroids of the 24β series have also been used to show that inversion of configuration at C-3 is influenced by regio-and stereospecific formation of carbohydrate conjugates of the 3β -epimer [21].…”