Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants

Abstract

Rationale The plant hormone auxin, indole‐3‐acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques. Methods In this study, we detailed optimized conditions for high‐performance liquid chromatography coupled with triple‐quadrupole mass spectrometry (LC/MS). We compared LC/MS conditions, such as columns, mobile phases, parameters of acquisition methods (selective or multiple ion monitoring), dwell times (DTs), and channel numbers, using differentially mixed authentic auxin and its related compounds. We further investigated pretreatment methods through the optimization of auxin recovery and irrelative compound removal from plant tissues prior to the LC/MS analysis. Results Our LC/MS analysis demonstrated the particular importance of the column, DTs, and channel numbers on detection sensitivity. Our comparative analysis developed optimal pretreatment methods, including the pulverization of plants, concentration of extract through centrifugal evaporation, and removal of irrelative metabolites using liquid–liquid extraction and a spin filter. We injected plant samples into our LC/MS system, quantified auxin and eight related compounds in a single measurement, and determined the auxin increase in an auxin over‐producing mutant. Conclusions Our practical optimization of LC/MS conditions and pretreatment methods provides detailed experimental processes toward the sensitive quantification of auxin from 10 mg of plant tissue. These processes have not always been clearly documented; therefore, our protocol could broadly contribute to technical advances in plant growth and development research.

AtIAR1 is a Zn transporter that regulates auxin metabolism in Arabidopsis thaliana

“…Precise quantification of metabolite levels is essential to understanding changes in IAA metabolism. LC-MS was used to quantify IAA and IAA-related metabolites using D 2 -IAA as an internal standard based on previous approaches ( Sugahara et al ., 2020 ). A methodology adapted for the current study ( Sugahara et al ., 2020 ), as shown in the Materials and methods, produced an appropriate linear response range for metabolite analysis in Arabidopsis whole seedlings for a variety of IAA-related metabolites including IAA precursors (Trp and IAN), IAA, IAA conjugates (IAA–Ala, IAA–Asp, and IAA–Glc), and degradation pathway intermediates (oxIAA) ( Supplementary Table S7 ).…”

“…LC-MS was used to quantify IAA and IAA-related metabolites using D 2 -IAA as an internal standard based on previous approaches ( Sugahara et al ., 2020 ). A methodology adapted for the current study ( Sugahara et al ., 2020 ), as shown in the Materials and methods, produced an appropriate linear response range for metabolite analysis in Arabidopsis whole seedlings for a variety of IAA-related metabolites including IAA precursors (Trp and IAN), IAA, IAA conjugates (IAA–Ala, IAA–Asp, and IAA–Glc), and degradation pathway intermediates (oxIAA) ( Supplementary Table S7 ). Levels of IAA–Ala were too low to detect in this analysis, a result that aligns with previous work suggesting that the content of IAA–Ala is below that of other conjugates including IAA–Glc and IAA–Asp ( Sugahara et al ., 2020 ).…”

“…A methodology adapted for the current study ( Sugahara et al ., 2020 ), as shown in the Materials and methods, produced an appropriate linear response range for metabolite analysis in Arabidopsis whole seedlings for a variety of IAA-related metabolites including IAA precursors (Trp and IAN), IAA, IAA conjugates (IAA–Ala, IAA–Asp, and IAA–Glc), and degradation pathway intermediates (oxIAA) ( Supplementary Table S7 ). Levels of IAA–Ala were too low to detect in this analysis, a result that aligns with previous work suggesting that the content of IAA–Ala is below that of other conjugates including IAA–Glc and IAA–Asp ( Sugahara et al ., 2020 ). This differing abundance suggests some degree of selection between conjugates as an IAA store, although the mechanisms and significance of this selection remain unclear.…”

“…Sterilized and vernalized seeds were sown on Hoagland solution-based EDTA-washed agar medium with 0, 1, or 150 µM ZnSO 4 , and grown vertically for 16 d. In a protocol adapted from Sugahara et al . (2020) , 100 mg pools of 16-day-old whole seedlings were snap-frozen in liquid nitrogen and homogenized with 3 mm tungsten carbide beads with a TissueLyser LT homogenizer.…”

Development and Comprehensive SPE-UHPLC-MS/MS Analysis Optimization, Comparison, and Evaluation of 2,4-Epibrassinolide in Different Plant Tissues

“…In recent years, there have been lots of discoveries relating to the anabolism of BR [13], its physiological function and applications [14], and the signal transduction regulatory network [15]. However, the content of EBL is lower than other plant hormones (generally at the concentration level of ng/g to pg/g), which makes the accurate qualitative and quantitative determination of EBL content difficult, and which greatly restricts the research on this plant hormone [16,17]. Additionally, the quantitative analysis of BR content in plants is of great significance when studying the mechanisms of BR.…”