| Acidic plant hormones are a family of small molecular compounds with carboxyl groups, including abscisic acid (ABA), auxins, gibberellins (GAs), jasmonic acid (JA), and salicylic acid (SA). These hormones regulate almost all of plant physiological processes, including seed germination, stem elongation, flowering and fruiting, response to biotic and abiotic stress, and so forth. In addition, acidic plant hormones also play a critical role in the agricultural production. By up or down regulating the expression of genes involving the biosynthesis and transduction of acidic plant hormones, crop varieties with high yield, drought tolerance and pest resistance can be achieved. It provides a feasible solution to the global food crisis. And fully understanding the molecular mechanism of acidic plant hormones is essential for the agricultural application. While, the molecular mechanism study relies on the analysis of endogenous acidic plant hormones.Up to date, several routine analytical methods can quantify acidic plant hormones in plant tissues crude extracts and cell lysates, such as gas-chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). However, it remains a challenge that quantitative profiling of acidic plant hormones in plant tissues with a micro amount. It is mainly attributed to several reasons:1. The trace concentration (ng/g or even pg/g) of endogenous acidic plant hormones requires an ultra-sensitive analytical methods. But in negative mode, the electrospray ionization (ESI) efficiency of acidic analytes remains unsatisfactory to the sensitive analysis;2. There is a variety of acidic plant hormones. For instance, the number of identified GAs is up to144, even10GAs involves the biosynthesis of bioactive GAs. Obviously, it frequently happens that analytes are co-eluted during chromatographic separation, which influences the identification and quantification of target analytes.3. In the higher plant species, there are many kinds of impurities with high concentration and similar chemical/physical properties to analytes. It leads to tedious sample preparation, inefficient hormone purification, and restricts the trace analysis of acidic plant hormones. Based on the issues above, in this dissertation, the author employed derivatization approach to improve the ESI-MS response of acidic analytes, used organic polymer monolithic columns to baseline separate multiple acidic plant hormones with, established online trapping system to enhance the detection sensitivity of analytes, and realized the quantitative profiling of acidic plant hormones in rice leaves with a micro amount. Special contents of the dissertation include the following aspects.1. We employed3-bromoactonyltrimethylammonium bromide (BTA) to derive15acidic plant hormones of5groups, including abscisic acid, auxins, GAs, jasmonic acid, salicylic acid. Compared to the intensity of ion signal detected with negative ion mode, the intensity of ion signal of BTA-derivatives detected in the positive ion mode dramatically increased by5?10folds. Additionally, the positively charged BTA-derivatives were separated by capillary electrophoresis (CE) using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. With the proposed method,14acidic plant hormones can be determined in rice leaves.2. For the baseline separation of15derived acidic plant hormones, poly(methacrylic acid-co-ethylene glycol dimethacrylate)(MAA-cY;-EDMA) monolith was prepared. We investigated the influence of the hydrophilicity of porogens on the monolithic porous structure and the exposure of carboxyl groups. The characterization indicated that monolithic column prepared with the "hydrophilic" progenic system possessed homogeneous column bed, highly exposure of carboxyl groups. We also investigated the chromatographic retention mechanism of target analytes, it indicated that derived plant hormones were retained on the prepared poly(MAA-co-EDMA) monolithic column by the weak cation exchange/reversed-phase (WCX/RP) mixed-mode chromatographic retention. Finally,15derived acidic plant hormones can be baseline separated with a salt concentration gradient.3. We established the online enrichment system to enhance the detection capacity for derived acidic plant hormones. We investigated the extraction recovery of different types of polymer monolithic capillaries and optimized the online trapping parameters. Under optimal conditions, the signal intensities of the analytes were significantly improved by at least100folds. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from1.05to122.4pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. To improve the efficiency of sample preparation, tandem solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was employed. With the proposed method here, we were able to simultaneously determine11endogenous acidic phytohormones from only5mg of rice leave sample, which dramatically decreased the required sample amount for the profiling of low abundant acidic plant hormones.4. 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 (GAi, GA4, GA9, GA20). An anion exchange/hydrophobic polymer monolithic column was employed for the separation of all target GAs. Using a large injection volume of1200nL, the detection sensitivity was apparently improved. Furthermore, we optimized the ESI-MS parameter to suppress the in-source collision activated dissociation (CAD) occurrence of GAs. Taken together, the developed method was sensitive and robust. The developed method was further applied in the evaluation of endogenous GA3-oxidase activity in different types of plant tissues, including rice embryos, rice seedlings, A. lhaliana seedlings, and A. thaliana flowers. |
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