Acidic Phytohormones Research Articles

Highly sensitive analysis of photoregulation and dynamic distribution of phytohormones based on nanoconfined liquid phase nanoextraction

BackgroundAs a vital energy source, light is one of the most significant environmental signals for plants' growth and development. The crosstalk amongst phytohormones regulated by light exhibits quantitative dynamic changes, but methodologies to analyze their distribution during plant growth are still limited. Rapid, highly sensitive, low-invasive detection and simultaneous assessment of the levels of multiple classes of phytohormones have important phytology applications, however the existing sample pretreatment strategies remain intricate, laborious, and far from being developed for in vivo high-sensitivity testing. (81) ResultsWe applied a nanoconfined liquid phase nanoextraction (NLPNE) technique based on acidified carbon nanofibers (ACNFs) in combination with LC-ESI-MS/MS for highly sensitive analysis of acidic phytohormones’ photoregulation and dynamic distribution. In this system, the mass transfer ability of analytes entering the nanoconfined space is significantly improved given the nanoconfined effect. In particular, the accelerated and strong adsorption of alkaline compounds to the ACNFs surface provide minimum interference for acidic compounds (photosensitive phytohormones), which facilitates their simple, fast, and selective quantification with improved sensitivity. The ACNFs-NLPNE strategy achieved quantitative enrichment of multi-class phytohormones in less than 5 min, and detection limits down to 0.49 fg mL−1. Moreover, we monitored the phytohormone changes under red and blue monochromatic light with relative standard deviations <13.4 %. The results further indicated that short-time red light regulation promoted Lepidium sativum L. growth while blue light inhibited it. (141) SignificanceA nanoconfinement effect-based sample pretreatment platform was developed for monitoring photoregulation phytohormones dynamic distribution with higher sensitivity and stability. Our findings highlighted the importance of the NLPNE approach in providing an accurate plant crosstalk information at the molecular level, which opens a promising avenue for investigating internal hormonal responses to external stimuli. (52).

Fabrication of magnetic ordered mesoporous carbon for quantitative analysis of acidic phytohormones in mushroom samples prior to their determination by ultra-high-performance liquid chromatography–tandem mass spectrometry

AbstractA novel method was established for analysing trace four acidic phytohormones, namely, indole-3-acetic acid, 3-indolebutyric acid, abscisic acid, and 1-naphthylacetic acid, using magnetic ordered mesoporous carbon (MOMC). MOMC was facilely synthesised via self-assembly strategy with a direct carbonisation process. The properties of MOMC were characterised using various instruments. MOMC exhibited excellent adsorption capacity towards the analytes. Various critical parameters which may influence the enrichment efficiency were evaluated, including amount of MOMC, extraction conditions, and desorption conditions. An efficient method based on MOMC magnetic solid-phase extraction coupled with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was developed to analyse the trace four acidic phytohormones, with good correlation coefficients (R2 = 0.9965–0.9998) and low limits of detection (0.13–9.7 ng L−1, S/N = 3). Trace acidic phytohormones in Agaricus bisporus and Hypsizygus marmoreus samples were determined with satisfactory recoveries (91.8–108%) and reproducibility (2.6–6.3%). The features indicated that MOMC provides an efficient platform for mushroom sampling; the developed method is convenient, promising, and sensitive for the detection of trace phytohormones in complicated mushroom samples.

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing

BackgroundAcidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants.ResultsA high-throughput sample preparation method for liquid chromatography–tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports.ConclusionThe method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Determination of thirteen acidic phytohormones and their analogues in tea (Camellia sinensis) leaves using ultra high performance liquid chromatography tandem mass spectrometry

Trace plant hormones play an important role in tea growth, development and quick response to biotic and abiotic stresses. However, lack of a sensitive method limits the research on plant hormone regulation for tea quality and yields. Herein, a highly sensitive method was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for profiling and quantification of 13 acidic phytohormones and their analogues, including auxins, abscisic acid and gibberellins in fresh tea leaves. After optimizing the different C18 columns and mobile phase systematically, an Agilent Eclipse Plus C18 column combined with the mobile phase A (acetonitrile) and B (water) was employed. Target acidic phytohormones were extracted using acidified methanol, and tea matrices were cleaned up by dispersive solid phase adsorbents of polyvinylpolypyrrolidone (PVPP) and graphitized carbon black (GCB) followed by polymer-based mixed-mode cation-exchange solid phase extraction. The method showed good linearity for all 13 analytes with regression coefficients (R2) > 0.998. Satisfactory recoveries of 12 analytes spiked with three levels ranged from 71.8% to 109.9%, while intra-day and inter-day precisions were below 20%. Limits of detection (LODs) and limits of quantitation (LODs) for 12 acidic phytohormones were 0.1–4.2 μg kg−1 and 0.3–13.9 μg kg−1, respectively. Finally, this method was firstly employed to analyze 13 analytes in fresh tea leaves (with the treatment of dormancy, light qualities, exogenous hormones and infestation of pests), highlighting its sufficient capability for rapid analysis of multiclass phytohormones in agriculture field.

Simultaneous determination of acidic phytohormones in cucumbers and green bean sprouts by ion-pair stir bar sorptive extraction-high performance liquid chromatography

A simple, sensitive and inexpensive method of ion-pair stir bar sorptive extraction (IP-SBSE) combined with high performance liquid chromatography -ultraviolet (HPLC-UV) detection was developed for the simultaneous determination of three acidic phytohormones (salicylic acid (SA), indole-3-acetic acid (IAA), and abscisic acid (ABA)) in vegetables. With the addition of cationic surfactant-cetyltrimethylammonium bromide (CTAB) in sample solutions, ion pair between CTAB and target phytohormones were formed, leading to an improvement of the extraction efficiency for target phytohormones by C18 silica coated SBSE. The factors affecting the extraction efficiency of three target phytohormones by IP-C18 coated SBSE were optimized, and the optimal experimental conditions were established. Under the optimal conditions, the limits of detection for SA, IAA and ABA obtained by IP-C18-SBSE-HPLC-UV were 2.7, 2.1 and 0.3μgL−1, with the relative standard deviations of 6.0%, 5.0% and 8.3%, respectively, and the enrichment factor was 32–49-fold. The developed method was applied to the determination of SA, IAA and ABA in cucumbers and green bean sprouts, and the recoveries of SA, IAA and ABA for the spiked samples were between 72.8% and 109.5%.

Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry.

In the current study, a novel method for high-throughput and sensitive determination of 12 phytohormones in plants was developed by using sequential solvent induced phase transition extraction (SIPTE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In sequential SIPTE, 0.1% formic acid (v/v) and 50mM NaHCO3 aqueous solution were used for enrichment and purification of alkaline and acidic phytohormones from the acetonitrile extract of plant tissues in sequence, in which hydrophobic solvent (toluene) was added to the acetonitrile aqueous mixture for driving the phase separation. Under optimized sequential SIPTE conditions, the phytohormones in acetonitrile extract of plant tissues could be effectively enriched and purified, which was in favor of the following UPLC-MS/MS analysis with less matrix effect. The phytohormones could be detected using the developed sequential SIPTE-UPLC-MS/MS method with the limits of the detection (LODs) ranging from 0.56 to 438.60pgmL(-1) and linear range over 2 orders of magnitude with correlation coefficients (r)>0.9970. The relative recoveries of the detected phytohormones were in the range of 85.1-114.6%. Finally, the proposed method was applied to simultaneous determination of endogenous phytohormones in different tissues of model plants (Oryza sativa and Arabidopsis thaliana) with small amount of sample size (5mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants.

Magnetic solid phase extraction coupled with in situ derivatization for the highly sensitive determination of acidic phytohormones in rice leaves by UPLC-MS/MS.

A simple, rapid and sensitive method based on magnetic solid phase extraction (MSPE) coupled with in situ derivatization (ISD) was developed for the determination of endogenous acidic phytohormones in rice leaves by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. With this method, acidic phytohormones were extracted onto the surface of a TiO2/magnetic hollow mesoporous silica sphere (MHMSS) through hydrophilic interaction, and then in situ derivatization was performed by the subsequent addition of 3-bromoactonyltrimethylammonium bromide (BTA). Thus, the process integrated extraction, purification, and derivatization into one step. Additionally, the permanent positively charged moiety from BTA significantly improved the ionization efficiencies of the acidic phytohormones. Several parameters affecting the efficiencies of the extraction, derivatization, and desorption were evaluated. The signal intensities of acidic phytohormones increased by 2 to 481 fold after treatment with MSPE-ISD. Under the optimized conditions, several endogenous acidic phytohormones, including GA4, GA9, GA20, JA, IAA, and ABA, were identified and quantified in rice leaves by the MSPE-ISD method. The limits of detection (LODs) were in the range of 1.03-91.21 pg mL(-1). The relative recoveries ranged from 71.6-112.8%, with the intra- and interday relative standard deviations (RSDs) being less than 14.9% and 16.2%, respectively. Taken together, the proposed method provides a novel approach of combining magnetic solid phase extraction and in situ derivatization for the highly sensitive determination of endogenous acidic phytohormones.

Polyaniline sheathed electrospun nanofiber bar for in vivo extraction of trace acidic phytohormones in plant tissue

For in vivo extraction of trace and polar components in tissues, extraction materials should be biocompatible, highly permeable to matrix and have high extraction capacity for polar and ionic compounds. In this study, nanofibers (∼200nm) with rough polyaniline (PAn) sheath were prepared successfully for the first time by coaxial electrospinning and coelectrospun polymer sacrificing method. The nanofibers (0.5mg) were then compressed into tiny bar (Φ1mm×2mm) and inserted directly into the aloe leaf for in vivo extraction of trace phytohormones. Due to the large surface area (13.97m2/g) of nanofibers and large through pores formed between fibers, high extraction capacity (up to 10μg/bar) and permeability to tissue matrix of the tiny bar can be obtained. Moreover, PAn sheath has high adsorption equilibrium constants (107–108mL/mol) for acidic phytohormones in aloe leaf. Four phytohormones were detected by in vivo extraction with a spatial resolution of 3–8mm3, temporal resolution of 20min, and limit of detection down to 60pg/g. The quantification results were well met with that by traditional in vitro organic solvent extraction.

Study of field mobilities dependence and direct separation of acidic phytohormones by differential mobility spectrometry–mass spectrometry

The direct separation of five acidic phytohormones including auxin (indole-3-acetic acid, indole-3-propionic acid, and indole-3-butyric acid), jasmonic acid, and abscisic acid by differential mobility spectrometry–mass spectrometry (DMS–MS) with the use of the polar gas-phase chemical modifier 2-propanol was demonstrated. The method was rapid and simple to operate with selectivity and repeatability. The influence of experimental conditions on the separation, such as modifier types and concentration, separation voltage, and temperature, was systematically investigated. The field mobilities dependence was measured as the normalized α function using the developed DMS–MS method. All the analytes behaved as type A ions and the clustering separation mechanism dominated. These results not only gave insights of ion properties but also contributed to the prediction of DMS separation by choosing targeted and appropriate conditions for the study of acidic phytohormones.

Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis

In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid–liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.

Simultaneous determination of several phytohormones in natural coconut juice by hollow fiber-based liquid–liquid–liquid microextraction-high performance liquid chromatography

A simple, selective, sensitive and inexpensive method of hollow fiber-based liquid–liquid–liquid microextraction (HF-LLLME) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV) detection was developed for the determination of four acidic phytohormones (salicylic acid (SA), indole-3-acetic acid (IAA), (±) abscisic acid (ABA) and (±) jasmonic acid (JA)) in natural coconut juice. To the best of our knowledge, this is the first report on the use of liquid phase microextraction (LPME) as a sample pretreatment technique for the simultaneous analysis of several phytohormones. Using phenetole to fill the pores of hollow fiber as the organic phase, 0.1 mol L −1 NaOH solution in the lumen of hollow fiber as the acceptor phase and 1 mol L −1 HCl as the donor phase, a simultaneous preconcentration of four target phytohormones was realized. The acceptor phase was finally withdrawn into the microsyringe and directly injected into HPLC for the separation and quantification of the target phytohormones. The factors affecting the extraction efficiency of four phytohormones by HF-LLLME were optimized with orthogonal design experiment, and the data was analyzed by Statistical Product and Service Solutions (SPSS) software. Under the optimized conditions, the enrichment factors for SA, IAA, ABA and JA were 243, 215, 52 and 48, with the detection limits (S/N = 3) of 4.6, 1.3, 0.9 ng mL −1 and 8.8 μg mL −1, respectively. The relative standard deviations (RSDs, n = 7) were 7.9, 4.9, 6.8% at 50 ng mL −1 level for SA, IAA, ABA and 8.4% at 500 μg mL −1 for JA, respectively. To evaluate the accuracy of the method, the developed method was applied for the simultaneous analysis of several phytohormones in five natural coconut juice samples, and the recoveries for the spiked samples were in the range of 88.3–119.1%.

Antagonism between salicylic and abscisic acid reflects early host–pathogen conflict and moulds plant defence responses

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana. We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.

The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites.

Through complex networks of signaling interactions, phytohormones regulate growth, development, reproduction and responses to biotic and abiotic stress. Comprehensive metabolomic approaches, seeking to quantify changes in vast numbers of plant metabolites, may ultimately clarify these complex signaling interactions and consequently explain pleiotropic effects on plant metabolism. Synergistic and antagonistic phytohormone signaling interactions, referred to as crosstalk, are often considered at the level of transduction without proper consideration of synthesis or accumulation of phytohormones because of the limitation and difficulty in quantifying numerous signals. Significant progress has recently been made in the expansion of metabolic profiling and analysis of multiple phytohormones [Birkemeyer et al. (J. Chromatogr. A, 2003, 993, 89); Chiwocha et al. (Plant J., 2003, 35, 405); Müller et al. (Planta, 2002, 216, 44); Schmelz et al. (Proc. Natl Acad. Sci. USA, 2003, 100, 10552)]. We recently presented a novel metabolic profiling approach to the analysis of acidic phytohormones and other metabolites based on a simplistic preparation scheme and analysis by chemical ionization-gas chromatography/mass spectrometry. We now provide a detailed description of this vapor phase extraction technique and use pathogen infection of Arabidopsis with Pseudomonas syringae DC3000 to illustrate metabolic changes in salicylic acid, cinnamic acid, jasmonic acid, indole-3-acetic acid, abscisic acid, unsaturated C(18) fatty acids, 12-oxo-phytodienoic acid, and phytotoxin coronatine. Directions for further method expansion are provided and include issues of recovery, derivatization, range of accessible analytes, optimization, reproducibility and future directions.

A multiplex GC-MS/MS technique for the sensitive and quantitative single-run analysis of acidic phytohormones and related compounds, and its application to Arabidopsis thaliana.

A highly sensitive and accurate multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique is reported for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid. The optimized setup allows the routine processing and analysis of up to 60 plant samples of between 20 and 200 mg of fresh weight per day. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signalling molecules. Whole-plant organ-distribution maps for indole-3-acetic acid, abscisic acid, jasmonic acid, 12-oxo-phytodienoic acid and salicylic acid were generated for Arabidopsis thaliana (L.) Heynh. For leaves of A. thaliana, a spatial resolution of hormone quantitation down to approximately 2 mm(2) was achieved.