An Improved Simplified High-Sensitivity Quantification Method for Determining Brassinosteroids in Different Tissues of Rice and Arabidopsis    

Abstract

Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work. Brassinosteroids (BRs) have been considered as the sixth class of endogenous plant hormones with wide occurrence across the plant kingdom (Bajguz and Tretyn, 2003). BRs play a key role in a variety of physiological processes, such as cell elongation, vascular differentiation , reproductive development, photomorphogenesis, stress tolerance, and so on (Hayat, 2010). Recently, it was found that BR deficiency could increase grain yield in rice (Oryza sativa) by more than 30%, which showed a food security-enhancing potential and guided new green revolution in the future (Sakamoto et al., 2006; Wu et al., 2008). Since BRs were first isolated and identified from rape (Brassica napus) pollen in 1970s (Mitchell et al., 1970; Grove et al., 1979), the natural occurrence of more than 60 BRs in a large quantity of plant species has been reported (Hayat, 2010). To date, research on the occurrence of BRs in different plants, physiological properties, and their action modes has made much progress (Fujioka and Yokota, 2003; Symons et al., 2008; Kim and Wang, 2010; Tang et al., 2010; Tong and Chu, 2012). However, so far, only limited information was obtained to understand the molecular mechanism of the physiological role of BRs. For example, although the biosynthetic pathway of C 28 BRs has been well established, the biosynthesis of C 27 and C 29 BRs remains unclear, and some intermedi-ates on their biosynthetic pathways still need to be elucidated (Noguchi et al., 2000; Fujita et al., 2006). The plant physiology research of BRs is speeded up by employing BR mutants on biosynthesis and signaling pathways (Yamamuro et al., 2000; Hong et al., 2003; Kwon and Choe, 2005; Tanabe et al., 2005); however, a simple, high-sensitivity screening, detection, and q...

MdWRKY9 overexpression confers intensive dwarfing in the M26 rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression

“…One month after transplanting the transgenic and control apple plants, the endogenous BRs (castasterone (CS) and BL), IAA, GA and CTK concentrations were determined. Quantification of endogenous BRs (BL and CS) concentrations was performed based on the method reported previously with some simplifications in sample pretreatment (Xin et al ., ). The harvested plant materials were first ground to a fine powder with a MM‐400 mixer milling (Retsch, Haan, Germany ).…”

“…MRM transitions and the related compound‐dependent parameters were set as described by Xin et al . (). Each experiment was independently repeated three times.…”

Response of the plant hormone network to boron deficiency

“…For gibberellins a separate sample of freeze‐dried material was extracted by acetonitrile/water (Extraction III). All protocols were adapted and modified procedures from literature (Kojima et al ., ; Seo et al ., ; Urbanova et al ., ; Xin et al ., ,b) and are described in detail in the Methods S1 and Tables S3 and S4.…”

A Comprehensive and Effective Mass Spectrometry-Based Screening Strategy for Discovery and Identification of New Brassinosteroids from Rice Tissues

“…The BR candidates were purified according to a protocol developed in our lab as previously reported, but with minor modifications (Xin et al, 2013b). The MAX cartridge was activated and equilibrated with MeOH, water, 1 M KOH, 10% (v/v) MeOH, and 95% (v/v) MeOH in turn, and the MCX cartridge with MeOH, water, 5% (v/v) formic acid (FA), and 10% (v/v) MeOH.…”

“…However, these screening methods are not suitable for BRs due to the lower sensitivity of these survey scan modes in triple quadrupole MS and the extremely low endogenous BRs concentrations in plant tissues. Therefore, we focused on developing simple, selective, and high-efficiency sample pretreatment methods, while improving the ionization efficiency of BRs via derivitization for high-sensitivity detection and quantification of known BRs in previous studies (Xin et al, 2013a,b). In the present study, we tailored a sensitive, structure-oriented, and high performance MS-based strategy to screen and identify novel BRs from plant tissues based on multiple reaction monitoring (MRM) data-dependent enhanced product ion (EPI) scan merged with high resolution MS survey-dependent MS/MS analysis.…”