Use of chemical labeling-assisted liquid chromatography-mass spectrometry for discovering derivatives of brassinosteroids.

Brassinosteroids (BRs) are plant steroid hormones that are involved in the regulation of plant growth and development as well as environmental adaptation. The discovery of new BR derivatives is beneficial to their biosynthesis and regulation mechanisms research. However, there are few reports on the methods for exploring new BRs, and the existing methods tend to lack coverage. In this work, we established a comprehensive, highly sensitive and selective structure-oriented method for the screening and structural identification of potential BRs in plants using chemical isotope labeling-assisted liquid chromatography-high resolution mass spectrometry (CL-LC-HRMS). The potential BRs were speculated according to the structural features of the reported BRs, and those speculated BRs containing cis-diol groups were labeled by isotope reagents of 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) and 2-methyl-4-PAMBA-d5 to improve the sensitivity and selectivity of MS detection. In addition, the fragmentation of 2-methyl-4-PAMBA-labeled BRs via collision-induced dissociation (CID) led to the generation of reporter ions, which contributed to specific screening of potential BRs. In-depth structure of potential BRs was elucidated by analyzing multistage MS (MSn) fragmentation patterns. Using our proposed method, a total of 16 potential BRs were detected from plant samples including 5 new ones, of which one new BR derivative was identified as 2-deoxy-3-epi-6-deoxy-dolichosterone, and this new BR may be involved in the biosynthesis of BRs as precursor of 6-deoxo-dolichosterone. The method developed in this work is promising for screening and identifying new BR derivatives in plants, thereby supplementing the biosynthesis pathway of BRs.

Sensors for the quantification, localization and analysis of the dynamics of plant hormones.

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.

Therapeutic Potential of Brassinosteroids in Biomedical and Clinical Research.

Steroids are a pivotal class of hormones with a key role in growth modulation and signal transduction in multicellular organisms. Synthetic steroids are widely used to cure large array of viral, fungal, bacterial, and cancerous infections. Brassinosteroids (BRs) are a natural collection of phytosterols, which have structural similarity with animal steroids. BRs are dispersed universally throughout the plant kingdom. These plant steroids are well known to modulate a plethora of physiological responses in plants leading to improvement in quality as well as yield of food crops. Moreover, they have been found to play imperative role in stress-fortification against various stresses in plants. Over a decade, BRs have conquered worldwide interest due to their diverse biological activities in animal systems. Recent studies have indicated anticancerous, antiangiogenic, antiviral, antigenotoxic, antifungal, and antibacterial bioactivities of BRs in the animal test systems. BRs inhibit replication of viruses and induce cytotoxic effects on cancerous cell lines. Keeping in view the biological activities of BRs, this review is an attempt to update the information about prospects of BRs in biomedical and clinical application.

A Mass Spectrometry-Based Study Shows that Volatiles Emitted by Arthrobacter agilis UMCV2 Increase the Content of Brassinosteroids in Medicago truncatula in Response to Iron Deficiency Stress.

Iron is an essential plant micronutrient. It is a component of numerous proteins and participates in cell redox reactions; iron deficiency results in a reduction in nutritional quality and crop yields. Volatiles from the rhizobacterium Arthrobacter agilis UMCV2 induce iron acquisition mechanisms in plants. However, it is not known whether microbial volatiles modulate other metabolic plant stress responses to reduce the negative effect of iron deficiency. Mass spectrometry has great potential to analyze metabolite alterations in plants exposed to biotic and abiotic factors. Direct liquid introduction-electrospray-mass spectrometry was used to study the metabolite profile in Medicago truncatula due to iron deficiency, and in response to microbial volatiles. The putatively identified compounds belonged to different classes, including pigments, terpenes, flavonoids, and brassinosteroids, which have been associated with defense responses against abiotic stress. Notably, the levels of these compounds increased in the presence of the rhizobacterium. In particular, the analysis of brassinolide by gas chromatography in tandem with mass spectrometry showed that the phytohormone increased ten times in plants grown under iron-deficient growth conditions and exposed to microbial volatiles. In this mass spectrometry-based study, we provide new evidence on the role of A. agilis UMCV2 in the modulation of certain compounds involved in stress tolerance in M. truncatula.

Simultaneous Determination of Multiclass Phytohormones in Submilligram Plant Samples by One-Pot Multifunctional Derivatization-Assisted Liquid Chromatography-Tandem Mass Spectrometry.

Phytohormones play crucial roles in every aspect of plant life, and their regulatory functions rely on complex crosstalk networks among the different classes of phytohormones in an antagonistic or synergistic manner. Therefore, the simultaneous determination of multiclass phytohormones is important for studies of phytohormone functions and networks. However, due to the heterogeneity of the sensitivity resulting from structural diversity and the low content in the plant samples, simultaneous determination of multiclass phytohormones is challenging, especially in very tiny plant tissues or organs. Here, we describe a novel method for the simultaneous determination of 31 phytohormones from different classes in a single run. This uses a one-pot multifunctional derivatization coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). N, N-Diethyl ethylenediamine (DEED) and 2-methyl-4-phenylaminomethyl-benzeneboronic acid (2-methy-4-PAMBA) were used for derivatization of carboxylated phytohormones and brassinosteroids (BRs), respectively, to simultaneously improve detection sensitivities of carboxylated phytohormones and BRs. The method was fully validated for the 31 targeted phytohormones and could quantify multiclass endogenous phytohormones in small amounts of fresh plant samples (0.02-5 mg, FW). Finally, we used this method to investigate the spatial-temporal distribution of multiple phytohormones in reproductive organs of a single flower of Arabidopsis thaliana for the first time. This method could be a powerful auxiliary tool for studies of phytohormone functions and regulatory networks.

A new boronic acid reagent for the simultaneous determination of C27-, C28-, and C29-brassinosteroids in plant tissues by chemical labeling-assisted liquid chromatography-mass spectrometry.

Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C27-, C28-, or C29-BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C27-BRs exhibit similar bioactivities to C28- and C29-BRs, the biosynthetic pathways of C27-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C27-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C27-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C27-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C27-, C28-, and C29-BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C27-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C27-BRs (28-norCS and 28-norBL), 3 C28-BRs (CS, BL, and TY), and a single C29-BR (28-homoBL), are found to be 0.10-1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d5 labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C27-BRs. Graphical abstract.

Profiling of potential brassinosteroids in different tissues of rape flower by stable isotope labeling - liquid chromatography/mass spectrometry analysis.

Brassinosteroids (BRs) play crucial roles in a variety of physiological processes in plants. The full elucidation of the functions of RBs relies on sensitive detection and accurate measurement of BRs in plants. However, the identification and quantification of BRs are challenging due to their low abundance as well as poor ionization efficiencies during mass spectrometry-based analysis. Herein, we developed a highly sensitive and selective strategy for profiling potential BRs in plants by stable isotope labeling liquid chromatography multiple reaction monitoring scan mass spectrometry (SIL-LC-MRM-MS) analysis. In the strategy, we used a pair of stable isotope labeling reagents 4-phenylaminomethyl-benzeneboronic acid (4-PAMBA) and d5-4-phenylaminomethyl-benzeneboronic acid (4-PAMBA-d5) that can react with C22-C23 cis-diol on BRs for profiling potential BRs in plant tissues. The 4-PAMBA and 4-PAMBA-d5 labeled BRs could generate two characteristic neutral loss under collision induced dissociation (CID), respectively, which is used to establish the MRM-based detection and screening. The precursor ions of BRs labeled with 4-PAMBA and 4-PAMBA-d5 were set according to the reported structures of BRs, and the corresponding product ions were predicted by subtracting the lost neutral loss. In this respect, corresponding precursor ions and product ions in MRM transitions are formed. The peak pairs with a fixed mass difference, similar retention times and intensities were assigned as potential BRs. Using the developed SIL-LC-MRM-MS strategy, we successfully found 13 potential BR in different tissues of rape flower. Taken together, the SIL-LC-MRM-MS analytical strategy is promising for profiling potential BRs as well as other compounds that have the same functional moiety from complex biological samples.

4-Mercaptophenylboronic acid-modified spirally-curved mesoporous silica nanofibers coupled with ultra performance liquid chromatography-mass spectrometry for determination of brassinosteroids in plants.

Herein, for the first time, thiol-functionalized mesoporous silica (mSiO2-SH) nanofibers with a spirally-curved twisted hexagonal morphology were synthesized via a simple one-pot protocol. 4-Mercaptophenylboronic acids (4-MPBA) were attached onto the mSiO2-SH nanofibers via disulfide bond, serving as boronate affinity sorbent to selectively capture brassinosteroids (BRs) from plant extract. The resulting BRs-MPBA derivatives were easily eluted from the sorbent by cleaving the disulfide bond, which was subsequently subjected to ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Thus, the in situ extraction/derivatization/desorption method coupled with UPLC-MS was established for the fast, sensitive and selective detection of BRs in plant tissues. Finally, based on the developed method, endogenous BRs were successfully detected in leaf of H. lupulus L., silique of A. thaliana, and panicle of O. sativa L.

Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry.

Brassinosteroids (BRs) are regarded as the sixth plant hormone that is widely distributed in the plant kingdom. Sensitive quantification of BRs will be greatly benefit to illuminate the detail mechanisms about how BRs play crucial role in plant developmental processes such as cell division, cell expansion, cytodifferentiation, seed germination, vegetative growth and resisting biological or abiotic stress. In the current study, we developed a method for rapid and sensitive determination of endogenous BRs in plant tissues by combining LC-MS and a novel sample preparation strategy, in which the plant tissue extract was supplied to solid phase boronate affinity labeling and extraction, followed by desorption and salt-induced phase transition extraction for further purification. Under the optimized conditions, good linearity was obtained for 6 BR with correlation coefficients (r) ranging from 0.9988 to 0.9999. The limits of detection (LODs, S/N = 3) ranged from 1.4 to 2.8 pg mL-1. The recoveries were between 93.4% and 116.2% with the relative standard deviations (RSDs) ranging from 2.8% to 15.8%. Finally, the developed method was successfully applied to the analysis of 6 endogenous BR in various plant tissues including 20 mg FW Oryza sativa shoot, 10 mg FW Oryza sativa root, 20 mg FW Arabidopsis thaliana shoot, 4 Arabidopsis thaliana flowers (2.8 mg) and one Brassica napus stamen (3.0 mg) with concentration ranging from 0.26 to 157.28 ng g-1 FW.

Pursuing extreme sensitivity for determination of endogenous brassinosteroids through direct fishing from plant matrices and eliminating most interferences with boronate affinity magnetic nanoparticles.

Brassinosteroids (BRs) are important plant hormones regulating plant growth and development. High-performance analytical methods for quantifying endogenous BRs are important for studying the molecular mechanisms of BR action. Herein we developed a high-performance sample pretreatment method based on boronate affinity magnetic nanoparticles (BAMNPs). The high specificity of boronate affinity enables direct fishing of BRs from plant matrices. The strong binding energy makes it possible to remove most contaminants in plant matrices with a small loss of target BRs. Besides these advantages, the novel two-step oxidation-hydrolysis elution system raised BR recoveries to 70.5%-98.2%, which was much higher than other boronate affinity applications. The high cleanliness of the final eluents lowered the matrix effects to 85.2%-92.4%. As a result, this method enables simultaneously good recoveries of endogenous BRs and thorough removal of matrix interferences, which greatly improves the sensitivity of BR analysis and reduces the use of plant materials for routine analysis to <10 mg. In addition, the sample handling time can be shortened to <3 h due to the operating convenience of BAMNPs and their easy separation from plant powders. Based on these advantages of BAMNP solid phase extraction, the organ-specific BR distribution analysis in Arabidopsis and rice tissues demonstrates excellent sensitivity, good reproducibility and high throughput of the method. Graphical abstract A high-sensitivity and time-saving UPLC-MS/MS-based quantification method for brassinosteroids (BRs) was developed through directly fishing BRs from plant matrices and eliminating most matrix interferences with as-prepared boronate affinity magnetic nanoparticles (BAMNPs).

Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

In situ fabrication of label-free optical sensing paper strips for the rapid surface-enhanced Raman scattering (SERS) detection of brassinosteroids in plant tissues.

A surface-enhanced Raman scattering (SERS)-functionalized, gold nanoparticle (AuNPs)-immobilized paper strip was developed to realize label-free rapid detection of brassinosteroids (BRs) in plant tissues. The AuNPs paper strip was fabricated through utilizing poly(γ-glutamic acid) (γ-PGA) as the linker and concentrator for the directly immobilized AuNPs on paper, which can circumvent many drawbacks of the existing SERS substrates. An enhancement factor of 1.13×108, high reproducibility (relative standard deviation of 4.78%), highly sensitive detection limit with a correlation coefficient of 0.99 (1×10-11M 4-mercaptophenylboronic acid), and long-term stability for the responses of the SERS-functionalized AuNPs paper strip to Raman intensities were achieved by optimizing reaction conditions including the concentrations of γ-PGA and HAuCl4/NaBH4. Ultimately, this developed paper strip was applied to the determination of BRs in plant tissues, obtaining precisions with RSD values in a range of 3.0-13.8%. The SERS-functionalized AuNPs paper strip featuring label-free rapid analysis, and high sensitivity could serve as an excellent alternative for on-site rapid detections.

The determination of 22 natural brassinosteroids in a minute sample of plant tissue by UHPLC-ESI-MS/MS.

The triterpenoid plant hormones brassinosteroids (BRs) are believed to influence almost every aspect of plant growth and development. We have developed a sensitive mass spectrometry-based method for the simultaneous profiling of twenty-two naturally occurring brassinosteroids including biosynthetic precursors and the majority of biologically active metabolites. Using ultra-high performance liquid chromatographic (UHPLC) analysis, the run time was reduced up to three times (to 9 min) in comparison to standard HPLC BRs analyses, the retention time stability was improved to 0.1-0.2 % RSD and the injection accuracy was increased to 1.1-4.9 % RSD. The procedures for extraction and for two-step purification based on solid-phase extraction (SPE) were optimised in combination with subsequent UHPLC analysis coupled to electrospray ionisation tandem mass spectrometry (ESI-MS/MS) using Brassica flowers and Arabidopsis plant tissue extracts. In multiple reaction monitoring (MRM) mode, the average detection limit for BRs analysed was close to 7 pg, and the linear range covered up to 3 orders of magnitude. The low detection limits for this broad range of BR metabolites enabled as little as 50 mg of plant tissue to be used for quantitative analyses. The results of determinations exploiting internal standards showed that this approach provides a high level of practicality, reproducibility and recovery. The method we have established will enable researchers to gain a better understanding of the dynamics of the biosynthesis and metabolism of brassinosteroids and their modes of action in plant growth and development.

Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry.

Determination of endogenous brassinosteroids (BRs) in limited sample amount is vital to elucidating their tissue- and even local tissue-specific signaling pathway and physiological effects on plant growth and development. In this work, an ultra-sensitive quantification method was established for endogenous BRs in milligram fresh plant by using pipette-tip solid-phase extraction coupled with ultra-performance liquid chromatography tandem mass spectrometry (PT-SPE-UPLC-MS/MS), in which a quaternary ammonium phenyl boronic acid, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was first developed for chemical derivatization of BRs. Due to the cationic quaternary ammonium group of BTBA, the ionization efficiencies of the BRs chelates with BTBA (BTBA-BRs) were enhanced by 1190-448785 times, which is the highest response enhancement factor among all derivatization reagents reported for BRs. In addition, PT-SPE packed with C18 sorbent was first used for purifying BRs from plant extracts, so the required sample amount was minimized, and recoveries higher than 91% were achieved. Under the optimized conditions, the minimal detectable amounts (MDA) of five target BRs were in the range of 27-94 amol, and the correlation coefficients (R(2)) were >0.9985 over four orders of magnitude. The relative recoveries of 75.8-104.9% were obtained with the intra- and inter-day relative standard deviations (RSDs) less than 18.7% and 19.6%, respectively. Finally, three BRs were successfully quantified in only 5mg fresh rice plant samples, and 24-epiBL can even be detected in only 0.5mg FW rice leaf segments. It is the first time that the BRs content in sub-milligram fresh plant sample has been quantified.

Determination of endogenous brassinosteroids using sequential magnetic solid phase extraction followed by in situ derivatization/desorption method coupled with liquid chromatography-tandem mass spectrometry.

In this study, a sequential magnetic solid phase extraction followed by in situ derivatization/desorption method was proposed for the fast, selective and sensitive determination of brassinosteroids (BRs) in plant tissues. Magnetic sorbent for quick, easy, cheap, effective, rugged and safe method (mQuEChERS) and polymer(4-vinylphenylboronic acid-co-ethylene glycol dimethacrylate) coated Fe3O4@SiO2 (p(4-VPBA-co-EGDMA) coated Fe3O4@SiO2) were prepared and characterized. Using them as sorbents, pigments and hydrophilic interferents were firstly removed from plant extract by mQuEChERS, and then endogenous BRs were selectively enriched by p(4-VPBA-co-EGDMA) coated Fe3O4@SiO2 through boronate affinity interaction. After loading BRs on p(4-VPBA-co-EGDMA) coated Fe3O4@SiO2, instead of directly eluting free BRs, the adsorbed BRs were released by adding 4-(N,N-dimethyamino)phenylboronic acid (4-DMAPBA) solution for in situ derivatizaiton/desorption of BRs based on a transesterification reaction between the boronate moieties of p(4-VPBA-co-EGDMA) coated Fe3O4@SiO2 and 4-DMAPBA, finally the resultant solution was submitted to LC-MS/MS for quantification. The whole procedure of the sequential MSPE could be accomplished within 1h, and the matrix effect to MS signal after the sample pretreatment was estimated to be in the range of 93.0-97.4%. The established method provided broad linear dynamics ranges (1.0-100.0pg/mL) with correlation coefficients (R) >0.9978, substantial sensitivity (limits of detection ranged from 0.27 to 1.29pg/mL), high reproducibility (intra-day and inter-day relative standard deviations (RSDs) less than 14.8%) and satisfactory accuracy (recoveries ranged from 74.0%-116.6%). Furthermore, endogenous BRs were successfully detected in one flower of Brassica napus L. (22.5-542.7pg/g fresh weight) and other plant tissues (13.7-289.8pg/g fresh weight).

GC-MS studies reveal stimulated pesticide detoxification by brassinolide application in Brassica juncea L. plants.

Imidacloprid (IMI) is a commonly used pesticide against aphids and accumulates in plant parts, maximum in leaves. Present study was conducted to check the efficiency of seed pre-soaking with 24-epibrassinolide (24-EBL) for reduction of this pesticide in the leaves of Brassica juncea L. plants raised from 24-EBL pre-soaked seeds and grown in soils supplemented with IMI. Leaves were analyzed for IMI residues using gas chromatography-mass spectrometry (GC-MS). Activities of guaiacol peroxidase (POD), glutathione reductase (GR), and glutathione S-transferase (GST), and glutathione (GSH) content were determined by spectrophotometry. Soil containing 350 mg IMI/kilogram soil resulted in 88.66 µg/g fresh weight (FW) of IMI residue in the leaves, which was maximum decrease to 35.31 µg/g FW (60.17 %), when seeds were pre-soaked in 100 nM 24-EBL. In this treatment (350 mg IMI/kilogram soil + 100 nM 24-EBL), GSH content, GR, POD, and GST activities were increased by 42.30, 34.5, 20.5, and 13.4 %, respectively, as compared to plants grown in soils amended with 350 mg IMI/kilogram soil.

4-Phenylaminomethyl-Benzeneboric Acid Modified Tip Extraction for Determination of Brassinosteroids in Plant Tissues by Stable Isotope Labeling-Liquid Chromatography-Mass Spectrometry.

Monitoring brassinosteroids (BRs) has been of major interest of researchers as these substances play a crucial role in a variety of phytological processes in plants. However, the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues. In this study, a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis. In the proposed strategy, a mixed mode sorbent (C8-SO3H) in tip was modified with 4-phenylaminomethyl-benzeneboric acid (4-PAMBA) through cation exchange and hydrophobic interactions, and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boric acid groups of 4-PAMBA and cis-diol on BRs. The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO3H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile, followed by LC-MS/MS analysis. BR standards, isotopically labeled with d5-4-phenylaminomethyl-benzeneboric acid (4-PAMBA-d5) were introduced to improve the assay precision of LC-MS/MS. Under the optimized conditions, the overall process could be completed within 1 h, which is greatly improved in speed compared with previously reported protocols. In addition, the detection sensitivities of labeled BRs were improved by over 2000-fold compared with unlabeled BRs, thus the consumption of plant materials was reduced to 50 mg. Finally, the proposed method was applied for the investigation of BRs response in rice toward Cd stress.

[Effect of exogenous brassinolide on morphological characters and contents of seven chemical constituents of Glycyrrhiza uralensis].

The transplants of the two-year-old Glycyrrhiza uralensis were subjected to four concentration of brassinolide (BR 0.1, 0.4, 0.7, 1.0 mg•L⁻¹) in July. The morphological characters ( plant height, stem diameter, nodes number, internode length and root length , root thick, root fresh weight and root dry weight ) were measured and seven kinds of chemical constituents (glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, liquiritin apioside, isoliquiritin apioside) were determined by HPLC with the aim of increasing sinter output and improving quality of G. uralensis. Then the long-term dynamic changes of these morphological characters and chemical compositions' content were analyzed. The results showed that morphological characters of plant height, stem diameter, root length , root thick, root fresh weight and root dry weight increased remarkably with the 0.7 mg•L⁻¹ BR stimulating 2 months later,the increase rates were： 15.09%,6.15%,16.52%,8.46%,21.90%,29.41%, respectively. The content of glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, liquiritin apioside, isoliquiritin apioside were increased 20.16%,45.31%,53.56%,27.66%,23.54%,8.46% with the 0.7 mg•L⁻¹ BR stimulating 2 months later. The best effects were achieved in 2 months after brassinolide stimulating. The conclusions prove that morphological characters and the main chemical constituents accumulation of G. uralensis could be effected by exogenous BR stimulation in certain case.

A bioassay for brassinosteroid activity based on the in vitro fluorimetric detection of nitric oxide production.

Recent studies have shown that low concentrations of brassinolide induce a rapid generation of nitric oxide in mesophyll cells of maize leaves, which can be easily detected by fluorimetric methods. In this work we describe a series of natural and synthetic brassinosteroids that are able to trigger in vitro NO production in tomato cells that exhibits dose-response behavior. We propose that this effect can be used to develop a new rapid and very sensitive bioassay for brassinosteroid activity that offers several advantages when compared to the current methodologies.

Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings.

A crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC-MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone↔28-homotyphasterol→28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone↔typhasterol→castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice.