Simultaneous analysis of defense-related phytohormones in Arabidopsis thaliana responding to fungal infection.
OBJECTIVE
Simultaneous analysis of defense-related phytohormones can provide insights into underlying biochemical interactions. Ultra-high-performance liquid chromatographic (UHPLC) techniques hyphenated to electrospray ionization mass spectrometry (ESI-MS) are powerful analytical platforms, suitable for quantitative profiling of multiple classes of metabolites.


METHODS
An efficient and simplified extraction method was designed followed by reverse-phase UHPLC for separation of seven phytohormones: salicylic acid, methyl salicylate, jasmonic acid, methyl jasmonate, absiscic acid, indole acetic acid, and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. A triple quadrupole multiple reaction monitoring (MRM) method was developed for MS quantification. The methods were applied to analyze phytohormones in Arabidopsis leaf tissue responding to biotic stresses.


RESULTS
Under the optimized conditions, the phytohormones were separated within 15 min, with good linearities and high sensitivity. Repeatable results were obtained, with the limits of detection and quantification around 0.01 ng/μL (∼9 ng/g tissue). The method was validated and applied to monitor, quantify, and compare the temporal changes of the phytohormones under biotic stress.


CONCLUSIONS
Quantitative changes indicate increased production of defense phytohormones from the various classes. The analytical method was useful and suitable to distinguish distinctive variations in the phytohormonal profiles and balance in A. thaliana leaves resulting from pathogen attack.
Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa.
Katlego B
KB
Riet
Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa.
Nombuso
N
Ndlovu
Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa.
Lizelle A
LA
Piater
Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa.
Ian A
IA
Dubery
Arabidopsis thaliana; pathogen infection; phytohormones; quantitative analysis