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

Abstract

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 (cLC-MS) method for the sensitive assay of in-vitro 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-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-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 (K m) of recombinant GA3-oxidase in Escherichia coli (E. coli) 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.

The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis

“…As GIM2 was predicted to belong to the 2OG‐Fe(II) oxygenase family (Figure S3), we explored its enzymatic activity using a method designed to measure GA3ox1 activity (Chen et al ). To ensure the efficiency of our enzyme assay system, we first determined the catalytic efficiency of GA3ox1 against its substrate, GA 9 ; as expected, the product GA 4 was produced (Figure S7A).…”

“…The catalytic activity of GIM2 protein for the substrate ABA was analyzed using an HPLC‐UV instrument (260 nm) (High Performance Liquid Chromatography, Shimadzu, Japan). The catalytic activity of GIM2 for GA intermediates was analyzed by LC‐MS as described by Chen et al () with minor modifications.…”

“…Cell lysates extracted from the E. coli expressing empty vector GST and GST-GA3ox1 were used as the controls. The enzymatic assays were performed as described previously with minor modification (Williams et al 1998;Yamaguchi et al 1998;Chen et al 2013;Zhao et al 2013). The substrate, such as ABA (Sigma, USA), GA 9 , GA 12 , GA 20 , or GA 53 (OlChemIm, Czech Republic), was incubated with cell lysates in a total reaction volume of 100 mL.…”

Translating Flowering Time from Arabidopsis thaliana to Brassicaceae and Asteraceae Crop Species

“…The GA pathway is well described in A. thaliana (reviewed in [192]). In brief, the last steps of the GA pathway involves the conversion of GA 12 into GA 9 and GA 53 into GA 20 , by GA 20-oxidases (GA20ox1-5), the conversion of GA 9 and GA 20 into bioactive GA 4 and GA 1 , respectively, by GA 3-oxidases (GA3ox1-3) and the deactivation of GA 4 and GA 1 by GA 2-oxidases (GA2ox1-5) [193,194,195,196,197,198]. Bioactive GAs binds to GIBBERELLIN INSENSITIVE DWARF1 ( GID1a , - b and - c ) to promote degradation of DELLA proteins [199,200,201], negative regulators of gibberellin signaling that act immediately downstream of the GA receptor.…”

Monoliths in capillary electrochromatography and capillary liquid chromatography in conjunction with mass spectrometry

“…The obtained detection limits of the GAs were in the range of 0.62–0.90 fmol (0.18–0.26 ng/mL). The developed method was further applied to the evaluation of endogenous GA3‐oxidase activity in different types of plant tissues, including rice embryos, rice seedlings, Arabidopsis thaliana ( A. thaliana) seedlings, and A. thaliana flowers .…”