Analytical Methods For Determination Of Phytohormones In Plant

Phytohormones are important class of compounds, essential for plant growth and development. As secondary metabolites, even at trace level, they are of particular interest because they regulate numerous plant processes such as germination, growth, reproduction and protective response against stress. Typically, they are divided into several classes, including auxins, abscisic acid (ABA), gibberellins (GAs), cytokinins (CKs), jasmonates, salicylates, brassinosteroids (BRs) and ethylene (ET) on the basis of their structures and physiological functions. Although, each class of phytohormones is distinguished for their specific biological functions, but all of these are involved to regulate the plant growth and development, and plant response against biotic and abiotic stresses by means of synergistic or antagonistic effect. These phenomena are usually referred as cross-talk of phytohormones. The mechanisms of phytohormonescross-talk are complex and refined, and some of them still remain unclear. Analytical methods for simultaneous determination of multi-class phytohormones in plant tissues play a crucial role in elucidating the cross-talk mechanisms of the phytohormones..In current work, we have developed rapid and effective sample pretreatment methods, including magnetic solid-phase extraction (MSPE) and solvent induced phase transition extraction (SIPTE) for simultaneous determination of multi-class endogenous phytohormones by LC-MS.1. A simple and effective sample preparation method was established for CKs purification from crude plant extraction by HILIC. Despite the simple strategy for sample pretreatment, relative recoveries were quite high (88.0%-118.1%). Finally, this developed method was successfully applied for the quantification of endogenous CKs from only 20 mg plant sample and the limits of detection (LODs) ranged from 8.0 to 4.0 pg g-1FW.2. We developed a rapid and high-throughput method by combination the UPLC-MS/MS and HILIC-MSPE for the determination of CKs in rice samples. To obtain good separation of CKs isomers and avoid the mutual interference in MS detection of CKs, UPLC was employed for CKs separation prior to MS analysis. Finally, out of 16 identified CKs,10 endogenous CKs were successfully quantified in 50 mg Oryza sativa sample using the developed MSPE-UPLC-MS/MS method. Due to high throughput of the strategy,40 sample could be analyzed within one day.3. Based on the work described above, we further developed a simple and reliable UPLC-MS/MS method for the analysis of acidic phytohormones and CKs by employing magnetic TiO2 nanoparticles as sorbent. Adding one step further, we used the MSPE-UPLC-MS/MS method to explore the change of phytohormones in rice under Cadmium (Cd) stress.4. Boronate affinity MSPE was employed to improve the purification ability of sample pretreatment method. By combining, TiO2-based MSPE and boronate affinity MSPE, we developed UPLC-MS/MS method for the profiling of phytohormones including IAA, ABA, JA,4GAs,3 BRs and 6 CKs in 100 mg rape flower samples.5. In order to avoid the drying step of crude plant extract, we proposed a successive SIPTE method for rapid and sensitive detection of phytohormones in plant. During the sample pretreatment procedure, toluene was added to the ACN:water mixture to drive the phase separation. The proposed method is simple but effectively removed matrix effect during ESI-MS analysis. Using this protocol, quantification of endogenous phytohormones in tiny plant tissues (5 mg, fresh weight) was achieved successfully.