Analytical Methods for Plant Hormones

What are plant hormones?

Plant hormones are trace organic compounds synthesized in plants that have significant effects on plant growth and development. They can be transferred from the site of production to the site of action. They can regulate plant growth and development at low concentrations, and are involved in almost every process of plant growth and development, from influencing cell division, elongation and differentiation to germination, rooting, flowering, fruiting, dormancy and abscission. Plant hormones mainly include ethylene, auxin, abscisic acid, gibberellins, cytokinins, jasmonates, brassinosteroids and salicylates, etc. In addition, some secondary metabolites, such as nitric oxide (NO) and recently discovered strigolactones, have also been suggested to act in a hormone-like manner in regulating plant growth and development.

Plant hormones also play a significant role in initiating defense responses when plants are subjected to biotic and abiotic damage such as pathogen infestation, gnawing by pests and animals, drought and salt stress.

Plant hormones can both directly affect crop yield by regulating growth and development, and reduce yield loss by participating in the regulation of crop adaptation to unfavorable conditions. The research results of plant hormones have revolutionized the improvement of crop yield and are of great value in agricultural applications. Therefore, the accurate determination of plant hormones in plants has attracted increasing attention.

The hormone content in plants is extremely low, unstable in nature, and easily disturbed by other secondary metabolites. In addition, some of the plant mutant materials used in plant physiology research are very precious and few in quantity. Therefore, the method of detection must be very sensitive and specific.

Extraction and purification of plant hormones

As a special group of plant secondary metabolites, the content of plant hormones in plants is very low, usually only one ten-thousandth or even lower than that of common plant secondary metabolites, in the range of 0.1~50 ng/g fresh weight.

In addition, plant extracts are complex multi-component mixtures, and the secondary metabolites they contain can seriously interfere with the qualitative and quantitative analysis of plant hormones. In order to identify and quantify phytohormones, it is necessary to establish and optimize the extraction and purification process of phytohormones, remove impurities that affect the analysis of phytohormones in crude plant extracts as much as possible, and enrich the target compounds to obtain fully purified samples for final analysis and determination.

Plant materials are usually processed by selecting fresh or lyophilized treated plant materials under liquid nitrogen freezing conditions for adequate grinding. Then a suitable extraction method is selected to extract the plant hormones from these materials, mainly using solvent extraction. The crude extracts of plants extracted by organic solvents contain a large amount of plant secondary metabolites, while the content of plant hormones in the crude extracts is very low. Purification techniques such as liquid-phase extraction, solid-phase extraction and solid-phase microextraction can purify and enrich the target plant hormones of interest, reduce or eliminate the large number of complex background interferences in plant tissues, and thus make the samples suitable for quantitative analysis.

Detection of plant hormones

The selectivity of the target can be greatly enhanced by using mass spectrometry. Tandem mass spectrometry (MS/MS) is achieved using time-tandem ion trap mass spectrometry or spatially tandem triple quadrupole (QQQ) mass spectrometry. Multiple reaction monitor (MRM) mode is commonly used in phytohormone quantification.

The high sensitivity of mass spectrometry greatly reduces the amount of sample to be measured, which makes the determination of endogenous hormones in some valuable plant mutant materials simple and easy, and enables the simultaneous detection of multiple plant hormones.

Ultra-performance liquid chromatography (UPLC) uses columns with small particle size (1.7 μm) packing for separation and can withstand pressures of up to 1000 bar (1 bar = 105 Pa), providing high separation capacity and fast analysis speed. The combination of UPLC and MS has greatly improved the efficiency of the instrument. The powerful separation efficiency and speed of the UPLC-MS provides a high throughput of a wide range of plant hormones.