Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, Department of Chemistry, Wuhan University, Wuhan, China.
PLoS One. 2013 Jul 26;8(7):e69629. doi: 10.1371/journal.pone.0069629. Print 2013.
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.
生物活性赤霉素（GAs）在植物生长和发育中起着关键的调节作用。在 GAs 的生物合成中，GA3-氧化酶催化最后一步生成生物活性 GAs。因此，评估 GA3-氧化酶的活性对于阐明 GAs 控制的植物生长调控机制至关重要。然而，评估内源性 GA3-氧化酶的催化活性仍然具有挑战性。在本研究中，我们通过分析 GA3-氧化酶（GA1、GA4、GA9、GA20）的催化底物和产物，开发了一种毛细管液相色谱-质谱（cLC-MS）方法，用于灵敏测定体外重组或内源性 GA3-氧化酶。成功制备了阴离子交换/疏水聚([2-(甲基丙烯酰氧)乙基]三甲基铵-co-二乙烯基苯-co-乙二醇二甲基丙烯酸酯)(META-co-DVB-co-EDMA)整体柱，用于所有目标 GA 的分离。GA 的检测限（LOD，Signal/Noise = 3）范围为 0.62-0.90 fmol。我们测定了大肠杆菌（E. coli）细胞裂解物中重组 GA3-氧化酶的动力学参数（K m），与以前的报道一致。此外，通过使用同位素标记的底物，我们成功评估了四种植物样品中内源性 GA3-氧化酶将 GA9 转化为 GA4 的活性，据我们所知，这是首次报道植物内源性 GA3-氧化酶活性的定量。综上所述，该方法为植物内源性 GA3-氧化酶活性的评估提供了一个很好的解决方案，这可能有助于深入研究 GAs 在植物生理学中控制的生长调控机制。
