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 intermediates 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 quantification method for
BR analysis is still a bottleneck technique for in-depth studying of the role of
BRs during the life cycle of plants.In the past 20 years, most of the detection and identification processes of
BRs could be described briefly as the following steps. The harvested plant materials were lyophilized and then ground to a fine powder, followed by the CH
3OH/CHCl
3 extraction. The concentrate was then partitioned with the CHCl
3/H
2O system three times. After that, the CHCl
3 fraction was subjected to a silica gel column for
BR enrichment, and the collected
BRs-containing fraction was purified with Sephadex LH-20 column and Sep-Pak Plus C18 cartridge in sequence. At last, the collected fractions were purified with preparative HPLC and then derivatized for analysis with gas chromatography-mass spectrometry (
MS) under selected ion monitoring mode (
Hong et al., 2005;
Nomura et al., 2005;
Kim et al., 2006). So far, this protocol has been proven to be workable in most cases and provided a great quantity of valuable data for plant physiological research (
Hwang et al., 2006;
Lee et al., 2010). However, at least more than 20 g of plant materials were consumed for quantifying/identifying
BRs in one plant sample without replicates (
Hong et al., 2005;
Bancos et al., 2006;
Kim et al., 2006), and it is difficult to collect sufficient plant tissues for
BR measurement in some rare model plant mutants. In addition, the method involved multiple tedious and labor-intensive steps, which might result in poor recovery and low sensitivity, especially for some labile
BR intermediates. The traditional method took one person about 3 to 4 d to treat one batch of samples. Most of the
BR measurement experiments were performed without biological replicates using traditional methods due to the disadvantages mentioned above, which discounted the reliability of the results. Therefore, a simple, rapid, and sensitive analysis method for
BRs is in urgent need, along with the development of
BR research.Recently, several efforts were made to improve the
BR determination (
Svatos et al., 2004;
Huo et al., 2012). The consumption of plant material was reduced to 2 g after modifying the
BRs with a new derivatization reagent for further ultra-performance liquid chromatography (
UPLC)-multiple reaction monitoring (
MRM)-
MS detection. However, the purification process consisting of deproteinization and multiple solid-phase extraction (
SPE) steps was still quite tedious and couldn’t guarantee covering the four most important bioactive
BRs, including brassinolide (
BL), castasterone (
CS), teasterone (
TE), and typhasterol (
TY; ). In our previous study (
Xin et al., 2013), we reported a simple, convenient, and high-sensitivity method for detection of endogenous
BRs from real plant materials based on the dual role of specific boronate affinity. Although it was the first time to measure multiple
BRs in subgram plant materials and the time duration of the method decreased to one-third of that previously reported, the synthesis of boronate affinity-functionalized magnetic nanoparticles made the method difficult to follow in biological laboratories for routine analysis.
Open in a separate windowChemical structure of four major bioactive
BRs.
BRs are neutral steroid compounds with a common four-ring cholestane skeleton and hydroxyl groups on A ring and/or the side chain linked to D ring. Especially, the vicinal diol moieties on C
22 and C
23 sites of
BL,
CS,
TY, and
TE allow these bioactive
BRs to be derivatized with ionizable reagents for
MS response enhancement. Considering the unique physicochemical properties of
BRs, we herein developed a simplified high-sensitivity analytical method based on mixed-mode anion exchange (MAX)-cation exchange (MCX) solid phase extraction (SPE) purification, vicinal diol derivatization combined with
UPLC-
MRM3-
MS detection for quantification of
BL,
CS,
TE, and
TY in model plants (). The performance of the method was demonstrated by determination of
BRs in different tissues of both wild-type and mutant Arabidopsis (
Arabidopsis thaliana) and rice.
Open in a separate windowSimplified high-sensitivity protocol for quantitative analysis of
BRs. IS, Internal standards. [See online article for color version of this figure.]
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