Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

<div><p>Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (<i>c</i>LC-MS) method for the sensitive assay of <i>in-vitro</i> recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-<i>co</i>-divinylbenzene-<i>co</i>-ethylene glycol dimethacrylate)(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (<i>K</i>_m) of recombinant GA3-oxidase in <i>Escherichia coli</i> (<i>E. coli</i>) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.</p></div

Characterizations of the META-silica hybrid monolithic column.

<p>(A) – (C) SEM images. (A) ×1,000 wide-view, (B) ×4,000 close-up-view, and (C) ×15,000 close-up-view. (D) The N₂ isothermal plot with the inset showing the pore-size distribution. (E) The effect of flow rate on the back pressure of the monolithic column. (F) Van Deemter plot of the height equivalent to a theoretical plate as a function of flow rate. Experimental conditions: column, poly(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column (30-cm long, 100 µm <i>i.d.</i>, 360 µm <i>o.d.</i>); UV detection wavelength, 254 nm for acrylamide, 214 nm for thiourea and benzene; mobile phase used in (E), ACN; mobile phase used in (F), ACN/H₂O (60/40, v/v).</p

Linearity, LODs and LOQs of GAs obtained by <i>c</i>LC-MS method.

<p>Linearity, LODs and LOQs of GAs obtained by <i>c</i>LC-MS method.</p

Extracted ion chromatogram of five GA standards.

<p>Shown in inset is the total ion chromatogram of five GA standards. Experimental conditions: column, poly(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column (30-cm long, 100 µm <i>i.d.</i>, 360 µm <i>o.d.</i>); flow rate, 800 nL/min; mobile phase, ACN/H₂O/FA (60/40/0.6, v/v/v). Order of peaks: 1. GA₁, 2. GA₂₀, 3. GA₅₃, 4. GA₄, 5. GA₉.</p

The reactions catalyzed by GA3-oxidase.

<p>In each metabolic reaction, the modification is highlighted in red.</p

Optimization of the mass spectrometry detection conditions of GA₁.

<p>(A) Full-scan spectrum of GA₁. (B) Full-scan spectrum of GA₁ with optimized ESI source conditions. Experimental conditions: 1 µg/mL GA₁ were infused in mobile phase (ACN/H₂O/FA, 60/40/0.6, v/v/v) at a flow rate of 3 µL/min.</p

Extracted ion chromatogram of the catalytic products and substrates of GA3-oxidase using 5 mg of rice embryos.

<p>Experimental conditions: column, poly(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column (30-cm long, 100 µm <i>i.d.</i>, 360 µm <i>o.d.</i>); flow rate, 800 nL/min; mobile phase, ACN/H₂O/FA (60/40/0.6, v/v/v). Order of peaks: 1. [²H₂]GA₂₀, 2. [²H₂]GA₅₃ (I.S.), 3. [²H₂]GA₄, 4. [²H₂]GA₉.</p