Abstract
BackgroundKarrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported.ResultsWe present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l).ConclusionsOur results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.
Highlights
Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity
Preparation of stable isotope labelled standard [2H3]KAR1 A previously published study showed the difficulties of KAR quantification due to the complexity of smoke water matrices [24]
We have developed a new sensitive and specific method for isolation and analysis of karrikin compounds in small amounts of plant tissue samples
Summary
Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. Karrikins (KARs) are small organic compounds derived from butenolide molecules (Fig. 1a) with their principal effect on seed germination [1, 2] Details of their origin is still unknown, but their formation requires oxygen and a pyran ring derived from the heating of polysaccharides and sugars [3, 4]. It is of great importance to develop highly sensitive and robust analytical methods for monitoring the endogenous levels of PGRs in various plant tissues. Such analyses still remain challenging, since PGRs are present at very low concentrations (pmol/g fresh weight) in a complex biological matrix [12]. Less complex samples are more gentle on highly sensitive instrumentation such as mass spectrometers
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