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A Stable Isotope Dilution Method for a Highly Accurate Analysis of Karrikins

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A Stable Isotope Dilution Method for a Highly Accurate Analysis of Karrikins Hrdlička et al. Plant Methods (2021) 17:37 https://doi.org/10.1186/s13007-021-00738-1 Plant Methods RESEARCH Open Access A stable isotope dilution method for a highly accurate analysis of karrikins Jakub Hrdlička1,2, Tomáš Gucký3, Johannes van Staden4, Ondřej Novák1* and Karel Doležal1,2 Abstract Background: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory efects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profling of KARs in plant tissues has not been reported. Results: We present a sensitive method for the determination of two highly biologically active karrikins (KAR 1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efcient single-step sample purifcation with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the valida- tion of KAR quantifcation 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 confrmed by the suc- cessful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with diferent concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). Conclusions: Our 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. Keywords: Karrikins, Smoke water, Stable isotope labelled standard, Stable isotope dilution method, Ultra-high performance liquid chromatography (UHPLC), Tandem mass spectrometry (MS/MS) Background prepared saturated extracts (so-called smoke water, Karrikins (KARs) are small organic compounds derived SW). Te SW prepared from smoke produced by con- from butenolide molecules (Fig. 1a) with their principal trolled combustion devices can be used as an aford- efect on seed germination [1, 2]. Details of their origin able biostimulant in agriculture and horticulture [2, 5, is still unknown, but their formation requires oxygen and 6]. Te stimulatory efect of KARs is independent from a pyran ring derived from the heating of polysaccharides plant reproductive strategy, seed size and/or plant mor- and sugars [3, 4]. KARs are produced during wild fres phology or ecology. Tey are efective not only on plants and play a key role in the restoration of destroyed areas. from fre-prone areas, but also on various species from Moreover, they can also be components of artifcially diferent families and environments [5]. Tere are six known KARs (Fig. 1a), of which KAR 1 is most active on seed germination of fre-following plants, but the model *Correspondence: [email protected] plant Arabidopsis responds more strongly to KAR 2 [7]. 1 Laboratory of Growth Regulators, Institute of Experimental Botany, The In parallel, a structurally and functionally similar sub- Czech Academy of Sciences and Faculty of Science, Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic stance, named strigolactone (SL, Fig. 1b), has been shown Full list of author information is available at the end of the article to trigger germination of parasitic plants [8, 9]. KARs © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hrdlička et al. Plant Methods (2021) 17:37 Page 2 of 13 spectrometry (MS/MS) has been widely used to deter- mine levels of various plant hormones [reviewed in 12– 17]. Te complexity of plant matrix, where minor PGRs are present in the background of more abundant primary and secondary metabolites, requires a combination of proper sample preparation and high instrument perfor- mance (robustness and sensitivity). For assessment of the accuracy and precision of MS-based methods, the con- centration of each analyte should be calculated using the stable isotope dilution method (SIDM; [16]). In general, SIDM is used to determine the quantity of a chemical substance in a sample based on internal standard (IS) to analyte ratio. Moreover, IS labelled with stable isotopes such as deuterium (2H), 13C, 15 N, and/or 18O atoms bal- Fig. 1 The structures of six naturally occurring karrikins (a) and strigolactones (b). The structural similarity of KARs and SLs, naturally ances the inefciencies and/or losses within the process occurring strigol and synthetic analogue GR24, is due to the presence of sample preparation as well as the ion suppression of a 2-furanone moiety (red part) efects during the MS analysis [18]. In the case of using deuterium-labelled IS, three or more 2H are used. One reason is the stronger binding of 2H isotopes to car- bons than 1H isotopes, which can lead to small physico- are perceived by KARRIKIN-INSENSITIVE2 (KAI2), chemical diferences between the analyte and IS and a homolog of SLs receptor. In addition, both signaling thus potentially better chromatographic separation. In pathways share the same or similar components, such addition, a higher number of 2H prevents interferences as the F-box protein MORE AXILARY GROWTH2 of naturally occurring analyte isotopes with IS [19]. In (MAX2) and the transcriptional corepressors SUPPRES- plant hormone profling methods, the use of IS for tar- SOR OF MAX2 1 (SMAX1)-LIKE proteins (SMXL) [10, geted quantifcation analysis has become the primarily 11]. performed technique [showed in 20–23]. A better understanding of plant growth regulators’ Very recently, ultra-high performance liquid chroma- (PGRs) biosynthesis, metabolism and mode of action tography combined with tandem mass spectrometry requires analysis of changes in their levels in various plant (UHPLC–MS/MS) was used for KAR quantifcation [24]. organs and tissues in diferent physiological processes. A standard dilution method (SDM) using a KAR struc- Moreover, the biological activity of PGR compounds tural analogue and a standard addition method (SAM) is dependent on their concentration levels both in the were compared. Te SAM was successfully validated and whole plant and in individual organs. It is therefore of applied in the determination of KARs in eight smoke great importance to develop highly sensitive and robust water samples of various origins and ages. However, the analytical methods for monitoring the endogenous lev- SIDM was not tested due to the absence of an isotopically els of PGRs in various plant tissues. Such analyses still labelled standard. In this study, we have developed a com- remain challenging, since PGRs are present at very low plex analytical protocol suitable for the SPE-based isola- concentrations (pmol/g fresh weight) in a complex bio- tion of KARs supplemented by sensitive and selective logical matrix [12]. Typical analysis of plant hormones quantifcation using the UHPLC–MS/MS method. After usually includes a multi-step sample pre-treatment, synthesizing a new deuterium-labelled internal stand- such as solid phase extraction (SPE) and/or immunoaf- ard, we were able to apply the SIDM as a quantifcation fnity extraction followed by subsequent instrumental approach for KAR analyses. Finally, we demonstrated measurements of individual metabolites [13]. SPE is the utility of our MS-based method for KARs profling in most frequently used method to isolate compounds from samples of ten-day-old seedlings of Arabidopsis thaliana complex matrices. By removing interfering compounds treated with KAR1 and KAR2, which are commercially such as salts, pigments, polysaccharides, lipids and pro- available and commonly used in plant research. teins from the sample, the chemical noise is reduced and potential co-eluting analytes are eliminated. In addition, Results and discussion less complex samples are more gentle on highly sensitive 2 Preparation of stable isotope labelled standard [ H3]KAR1 instrumentation such as mass spectrometers. A previously published study showed the difculties During the last decade, liquid chromatography (LC) of KAR quantifcation due to the complexity of smoke or gas chromatography (GC) coupled to tandem mass water matrices [24]. Although the SAM method has Hrdlička et al. Plant Methods (2021) 17:37 Page 3 of 13 been validated, its application for the analysis of bio- Development of extraction and purifcation protocol logical samples is time consuming [25]. Moreover, the Due to the expected extremely low concentration of methodology can be less suitable for tissue specifc KARs in plants, it has been necessary to develop an ef- experiments due to the requirement for a large amount cient extraction step with a suitable solvent under opti- of plant material. Te targeted profling of KARs in mized conditions. Te intriguing similarity between two minute plant tissues using a new deuterium-labelled germination stimulants, karrikins and strigolactones internal standard was an essential requirement in the (Fig. 1) inspired us to develop extraction approaches feld of karrikin research.
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