A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones

Abstract

One of the hottest topics in plant hormone biology is the crosstalk mechanisms, whereby multiple classes of phytohormones interplay with each other through signaling networks. To better understand the roles of hormonal crosstalks in their complex regulatory networks, it is of high significance to investigate the spatial and temporal distributions of multiple -phytohormones simultaneously from one plant tissue sample. In this study, we develop a high-sensitivity and high-throughput method for the simultaneous quantitative analysis of 44 phytohormone compounds, covering currently known 10 major classes of phytohormones (strigolactones, brassinosteroids, gibberellins, auxin, abscisic acid, jasmonic acid, salicylic acid, cytokinins, ethylene, and polypeptide hormones [e.g., phytosulfokine]) from only 100 mg of plant sample. These compounds were grouped and purified separately with a tailored solid-phase extraction procedure based on their physicochemical properties and then analyzed by LC-MS/MS. The recoveries of our method ranged from 49.6% to 99.9% and the matrix effects from 61.8% to 102.5%, indicating that the overall sample pretreatment design resulted in good purification. The limits of quantitation (LOQs) of our method ranged from 0.06 to 1.29 pg/100 mg fresh weight and its precision was less than 13.4%, indicating high sensitivity and good reproducibility of the method. Tests of our method in different plant matrices demonstrated its wide applicability. Collectively, these advantages will make our method helpful in clarifying the crosstalk networks of phytohormones.

Keywords: LC–MS/MS; phytohormones; plant tissue; quantitative analysis; solid-phase extraction.

Figure 1

Overall Design of the MCX–WAX SPE of All Phytohormone Classes in a Single Plant Sample. MeOH, methanol; FA, formic acid; MCX, mixed-mode strong cation exchange cartridge; WAX, mixed-mode weak anion exchange cartridge.

Figure 2

Investigation of SPE Conditions for Neutral and Alkaline Phytohormones. (A) Effect of the flow-through volume on the recoveries of neutral phytohormones. Three replicates were measured for each condition. Error bars represent means ± SD (n = 3). (B) Influence of the elution volume on the recoveries of CKs and ACC. Error bars represent means ± SD (n = 3).

Figure 3

Optimization of SPE Conditions for Acidic Phytohormones. (A) Influence of different acid types on the recoveries of acidic phytohormones. The volume of washing solvent applied is 3 ml. (B) Influence of FA concentration on the recoveries of acidic phytohormones. The volume of washing solvent applied is 3 ml. (C) Effect of elution volume on the recoveries of weaker acidic phytohormones (GAs, IAA, ABA, and JA). (D) Effect of elution volume on the recoveries of stronger acidic phytohormones (SA and PSK). Error bars represent means ± SD (n = 3).

Figure 4

Fold Changes of Endogenous Levels of Multiple Phytohormones in d14 and Wild-Type Shiokari Rice Roots. Rice was grown in −Pi culture medium for 3 weeks. C_d14 and C_WT represent the endogenous levels of phytohormones in d14 and wild-type rice, respectively. Y values were calculated from three replicates of the measured endogenous levels.

Figure 5

Temporal Changes in the Contents of Nine Classes of Phytohormones during the Reproductive Growth Stages of Wild-Type Nipponbare Rice. (A) GA4, gibberellin GA₄; CS, castasterone; tZ, trans-zeatin; iP, N⁶-(Δ²-isopentenyl)adenine; PSK, phytosulfokine. (B) ACC, 1-aminocyclopropane-l-carboxylic acid; SA, salicylic acid; JA, jasmonic acid; ABA, abscisic acid; IAA, indole-3-acetic acid. The grain-filling sample was harvested about 20 days after flowering. Error bars represent means ± SD (n = 3).