Analysis of Underivatized Brassinosteroids by HPLC/APCI-MS. Occurrence of 3-Epibrassinolide in Arabidopsis thaliana

Abstract

In the course of investigations on brassinosteroid (BS) biosynthetic and metabolic pathways in plants, an approach using HPLC/APCI-MS analysis of underivatized BS for their identification in plant material has been developed. Its application to root-callus suspension cultures of Arabidopsis thaliana led to the first identification of 3-epibrassinolide as natural brassinosteroid.

Labeled brassinosteroids for biochemical studies

“…The obtained compounds were used for biosynthetic studies. Three new brassinosteroids were identified in these experiments, including 3‐epibrassinolide in Arabidopsis thaliana ,27 secasterol and 2,3‐diepisecasterone in Secale cereale 28. A new biosynthetic subpathway from typhasterol/teasterone via 2,3‐epoxybrassinosteroids to castasterone was found in seedlings of S. cereale 29.…”

Determination of brassinosteroids in the sub‐femtomolar range using dansyl‐3‐aminophenylboronate derivatization and electrospray mass spectrometry

“…An analytical method previously developed in our laboratory10 used the atmospheric pressure chemical ionization (APCI) inlet for the ionization. The lowest LOD achieved was only 10 ng μL −1 , far below the sensitivity needed for biosynthetic studies of plant‐derived BS using deuterium‐labeled precursors.…”

“…traditionally play an important role in this research field as well, but are not suitable for biosynthetic studies. More recently, high‐performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) analyses of free10 or derivatized BS11,12 were introduced. A fluorescent tagging of BS with dansyl‐3‐aminophenylboronic acid improved the limit of detection (LOD) down to the femtomolar (fmol) range 13…”

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

“…The reverse conversion of 3-epicastasterone and 2-epicastasterone to castasterone was detected recently in seedlings of Secale cereale [10]. The conversion of brassinolide to 3-epibrassinolide, a naturally occurring brassinosteroid in Arabidopsis [24], and the reverse conversion are reported here for the first time and, together with the corresponding conversions between castasterone and 3-epicastasterone, are discussed in the context of hormone homeostasis in plants.…”

“…In A. thaliana, brassinosteroids [25 -26] have been reported several times [27 -29]. Castasterone (1) and brassinolide (2) are native also to Arabidopsis shoots and seeds, and 3-epibrassinolide (4) was recently found in root-callus suspension cultures of ecotype C24 [24]. Schmidt et al [30] reported castasterone (1) together with other brassinosteroids from seeds of S. cereale.…”