Hormonal profiles in dormant turions of 22 aquatic plant species: do they reflect functional or taxonomic traits?

BACKGROUND AND AIMS: Turions are vegetative, dormant overwintering organs formed in aquatic plants in response to unfavourable ecological conditions. Contents of cytokinin (CK) and auxin metabolites and ABA as main growth and development regulators were compared in innately dormant autumnal turions of 22 aquatic plant species of different functional ecological or taxonomic groups with those in non-dormant winter apices in three aquatic species and with those in spring turions of four species after their overwintering. METHODS: The hormones were analysed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: In innately dormant turions, the total contents of each of the four main CK types, biologically active forms and total CKs differed by two-three orders of magnitude across 22 species; the proportion of the active CK forms was 0.18-67 %. Similarly, the content of four auxin forms was extremely variable and the IAA proportion as the active form was 0.014-99 %. The ABA content varied from almost zero to 54 µmol kg-1 dry weight and after overwintering, it usually significantly decreased. Hormone profiles depended most of all functional traits studied on the place of turion sprouting (surface vs. bottom) and suggest that this trait is crucial for turion ecophysiology. CONCLUSIONS: The key role of ABA in regulating turion dormancy was confirmed. However, the highly variable pattern of the ABA content in innately dormant and in overwintered turions indicate that the hormonal mechanism regulating the innate dormancy and its breaking in turions are not united within aquatic plants.

Characteristics of turion development in two aquatic carnivorous plants: Hormonal profiles, gas exchange and mineral nutrient content.

Turions are vegetative, dormant, and storage overwintering organs formed in perennial aquatic plants in response to unfavorable ecological conditions and originate by extreme condensation of apical shoot segments. The contents of cytokinins, auxins, and abscisic acid were estimated in shoot apices of summer growing, rootless aquatic carnivorous plants, Aldrovanda vesiculosa and Utricularia australis, and in developing turions at three stages and full maturity to reveal hormonal patterns responsible for turion development. The hormones were analyzed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. Photosynthetic measurements in young leaves also confirmed relatively high photosynthetic rates at later turion stages. The content of active cytokinin forms was almost stable in A. vesiculosa during turion development but markedly decreased in U. australis. In both species, auxin content culminated in the middle of turion development and then decreased again. The content of abscisic acid as the main inhibitory hormone was very low in growing plants in both species but rose greatly at first developmental stages and stayed very high in mature turions. The hormonal data indicate a great strength of developing turions within sink-source relationships and confirm the central role of abscisic acid in regulating the turion development.

In situ separation and visualization of isomeric auxin derivatives in Arabidopsis by ion mobility mass spectrometry imaging.

In situ separation and visualization of synthetic and naturally occurring isomers from heterogeneous plant tissues, especially when they share similar molecular structures, are a challenging task. In this study, we combined the ion mobility separation with desorption electrospray ionization mass spectrometry imaging (DESI-IM-MSI) to achieve a direct separation and visualization of two synthetic auxin derivatives, auxinole and its structural isomer 4pTb-MeIAA, as well as endogenous auxins from Arabidopsis samples. Distinct distribution of these synthetic isomers and endogenous auxins in Arabidopsis primary roots and hypocotyls was achieved in the same imaging analysis from both individually treated and cotreated samples. We also observed putative metabolites of synthetic auxin derivatives, i.e. auxinole amino acid conjugates and hydrolysed 4pTb-MeIAA product - 4pTb-IAA, based on their unique drifting ion intensity patterns. Furthermore, DESI-IM-MSI-revealed abundance of endogenous auxins and synthetic isomers was validated by liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that DESI-IM-MSI could be used as a robust technique for detecting endogenous and exogenous isomers and provide a spatiotemporal evaluation of hormonomics profiles in plants.

Hormonal crosstalk controls cell death induced by kinetin in roots of Vicia faba ssp. minor seedlings.

Studies of vitality/mortality of cortex cells, as well as of the concentrations of ethylene (ETH), gibberellins (GAs), indolic compounds/auxins (ICs/AUXs) and cytokinins (CKs), were undertaken to explain the hormonal background of kinetin (Kin)-regulated cell death (RCD), which is induced in the cortex of the apical parts of roots of faba bean (Vicia faba ssp. minor) seedlings. Quantification was carried out with fluorescence microscopy, ETH sensors, spectrophotometry and ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS). The results indicated that Kin was metabolized to the transport form, i.e., kinetin-9-glucoside (Kin9G) and kinetin riboside (KinR). KinR was then converted to cis-zeatin (cZ) in apical parts of roots with meristems, to cis-zeatin riboside (cZR) in apical parts of roots without meristems and finally to cis-zeatin riboside 5'-monophosphate (cZR5'MP), which is indicated to be a ligand of cytokinin-dependent receptors inducing CD. The process may be enhanced by an increase in the amount of dihydrozeatin riboside (DHZR) as a byproduct of the pathway of zeatin metabolism. It seems that crosstalk of ETH, ICs/AUXs, GAs and CKs with the cZR5'MP, the cis-zeatin-dependent pathway, but not the trans-zeatin-dependent pathway, is responsible for Kin-RCD, indicating that the process is very specific and offers a useful model for studies of CD hallmarks in plants.

Cytokinins act synergistically with heat acclimation to enhance rice thermotolerance affecting hormonal dynamics, gene expression and volatile emission.

Heat stress is a frequent environmental constraint. Phytohormones can significantly affect plant thermotolerance. This study compares the effects of exogenous cytokinin meta-topolin-9-(tetrahydropyran-2-yl)purine (mT9THP) on rice (Oryza sativa) under control conditions, after acclimation by moderate temperature (A; 37 °C, 2h), heat stress (HS; 45 °C, 6h) and their combination (AHS). mT9THP is a stable cytokinin derivative that releases active meta-topolin gradually, preventing the rapid deactivation reported after exogenous cytokinin application. Under control conditions, mT9THP negatively affected jasmonic acid in leaves and abscisic and salicylic acids in crowns (meristematic tissue crucial for tillering). Exogenous cytokinin stimulated the emission of volatile organic compounds (VOC), especially 2,3-butanediol. Acclimation upregulated trans-zeatin, expression of stress- and hormone-related genes, and VOC emission. The combination of acclimation and mT9THP promoted the expression of stress markers and antioxidant enzymes and moderately increased VOC emission, including 2-ethylhexyl salicylate or furanones. AHS and HS responses shared some common features, namely, increase of ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), cis-zeatin and cytokinin methylthio derivatives, as well as the expression of heat shock proteins, alternative oxidases, and superoxide dismutases. AHS specifically induced jasmonic acid and auxin indole-3-acetic acid levels, diacylglycerolipids with fewer double bonds, and VOC emissions [e.g., acetamide, lipoxygenase (LOX)-derived volatiles]. Under direct HS, exogenous cytokinin mimicked some positive acclimation effects. The combination of mT9THP and AHS had the strongest thermo-protective effect, including a strong stimulation of VOC emissions (including LOX-derived ones). These results demonstrate for the first time the crucial contribution of volatiles to the beneficial effects of cytokinin and AHS on rice thermotolerance.

From synthesis to the biological effect of isoprenoid 2'-deoxyriboside and 2',3'-dideoxyriboside cytokinin analogues.

Isoprenoid cytokinins are a class of naturally occurring plant signaling molecules. A series of prepared compounds derived from isoprenoid cytokinins (isopentenyladenine, trans-zeatin and cis-zeatin) with attached 2'-deoxy-d-ribose or 2',3'-dideoxy-d-ribose at the N9 position of the purine were prepared and their biological activities were examined. Different synthetic approaches were employed. The final compounds were characterized with variety of physicochemical methods (TLC, HPLC-MS, and NMR) and their cytokinin activity was determined in classical bioassays such as Amaranthus, tobacco callus, detached wheat leaf senescence and Arabidopsis thaliana root elongation inhibition assay. In addition, compounds were screened for activation of the cytokinin signaling pathway (bacterial receptor, competitive ligand binding and ARR5::GUS assay) to provide a detailed assessment of CK structure-activity relationship. The prepared compounds were found to be non-toxic to human cells and the majority of assays exhibited the highest activity of free bases while 2',3'-dideoxyribosides had very weak or no activity. In contrast to the free bases, all 2'-deoxyriboside derivatives were not toxic to tobacco callus even at the highest tested concentration (10-4 moL/l) and compound 1 (iPdR) induced betacyanin synthesis at higher concentration even stronger than iP free base in the Amaranthus bioassay. The general cytokinin activity pattern base > riboside >2'-deoxyriboside > 2',3'-dideoxyriboside was distinguished.

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

Cytokinins and auxins in organs of aquatic carnivorous plants: what do they reflect?

BACKGROUND AND AIMS: Aquatic carnivorous plants have typical rootless linear shoots bearing traps and exhibit steep physiological polarity with rapid apical growth. The aim was to analyse auxin and cytokinin metabolites in traps, leaves/shoots and shoot apices in several species of genera Aldrovanda and Utricularia to elucidate how the hormonal profiles reflect the specific organ functions and polarity. METHODS: The main auxin and cytokinin metabolites were analysed in miniature samples (>2 mg dry weight) of different organs of Aldrovanda vesiculosa and six Utricularia species using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: Total contents of biologically active forms (free bases, ribosides) of all four main endogenously occurring cytokinin types were consistently higher in traps than in leaves in four Utricularia species with monomorphic shoots and/or higher than in shoots in two Utricularia species with dimorphic shoots. In Aldrovanda traps, the total content of different cytokinin forms was similar to or lower than that in shoots. In U. australis leaves, feeding on prey increased all cytokinin forms, while no consistent differences occurred in Aldrovanda. In four aquatic Utricularia species with monomorphic shoots, the content of four auxin forms was usually higher in traps than in leaves. Zero IAA content was determined in U. australis leaves from a meso-eutrophic site or when prey-fed. CONCLUSIONS: Different cytokinin and auxin profiles estimated in traps and leaves/shoots of aquatic carnivorous plants indicate an association with different dominant functions of these organs: nutrient uptake by traps versus photosynthetic function of traps. Interplay of cytokinins and auxins regulates apical dominance in these plants possessing strong polarity.

Glucose-6-P/phosphate translocator2 mediates the phosphoglucose-isomerase1-independent response to microbial volatiles.

In Arabidopsis (Arabidopsis thaliana), the plastidial isoform of phosphoglucose isomerase (PGI1) mediates photosynthesis, metabolism, and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Microbial volatile compounds (VCs) with molecular masses of <45 Da promote photosynthesis, growth, and starch overaccumulation in leaves through PGI1-independent mechanisms. Exposure to these compounds in leaves enhances the levels of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (GPT2) transcripts. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized the responses of wild-type (WT), GPT2-null gpt2-1, PGI1-null pgi1-2, and pgi1-2gpt2-1 plants to small fungal VCs. In addition, we characterized the responses of pgi1-2gpt2-1 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. Fungal VCs promoted increases in growth, starch content, and photosynthesis in WT and gpt2-1 plants. These changes were substantially weaker in VC-exposed pgi1-2gpt2-1 plants but reverted to WT levels with vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves and showed that knocking out GPT2 reduced the expression of photosynthesis-related proteins in pgi1-2 plants. Histochemical analyses of GUS activity in plants expressing GPT2-GUS under the control of the GPT2 promoter showed that GPT2 is mainly expressed in root tips and vascular tissues around hydathodes. Overall, the data indicated that the PGI1-independent response to microbial VCs involves resetting of the photosynthesis-related proteome in leaves through long-distance GPT2 action.

New Water-Soluble Cytokinin Derivatives and Their Beneficial Impact on Barley Yield and Photosynthesis.

Solubility of growth regulators is essential for their use in agriculture. Four new cytokinin salts─6-benzylaminopurine mesylate (1), 6-(2-hydroxybenzylamino)purine mesylate (2), 6-(3-hydroxybenzylamino)purine mesylate (3), and 6-(3-methoxybenzylamino)purine mesylate (4)─were synthesized, and their crystal structures were determined to clarify structural influence on water solubility. The mesylates were several orders of magnitude more water-soluble than the parent CKs. The new salts significantly reduced chlorophyll degradation and impairment of photosystem II functionality in barley leaf segments undergoing artificial senescence and had pronounced effects on the leaves' endogenous CK pools, maintaining high concentrations of functional metabolites for several days, unlike canonical CKs. A foliar treatment with 1 and 3 increased the harvest yield of spring barley by up to 8% when compared to treatment with the parent CKs while also increasing the number of productive tillers. This effect was attributed to the higher bioavailability of the mesylate salts and the avoidance of dimethyl sulfoxide exposure.

Carbon dioxide expanded liquid: an effective solvent for the extraction of quercetin from South African medicinal plants.

BACKGROUND: Quercetin is one of the most important bioflavonoids having positive effects on the biological processes and human health. Typically, it is extracted from plant matrices using conventional methods such as maceration, sonication, infusion, and Soxhlet extraction with high solvent consumption. Our study aimed to optimize the environmentally friendly carbon dioxide-based method for the extraction of quercetin from quince fruit with an emphasis on extraction yield, repeatability, and short extraction time. RESULTS: A two-step design of experiments was used for the optimization of the key parameters affecting physicochemical properties, including CO2/co-solvent ratio, co-solvent type, temperature, and pressure. Finally, gas expanded liquid combining CO2/ethanol/H2O in a ratio of 10/81/9 (v/v/v) provided the best extraction yield. Extraction temperature 66 °C and pressure 22.3 MPa were the most suitable conditions after careful optimization, although both parameters did not significantly affect the process. It was confirmed by experiments in various pressure and temperature conditions and statistical comparison of obtained data. The optimized extraction procedure at a flow rate of 3 mL/min took 30 min. The repeatability of the extraction method exhibited an RSD of 20.8%. CONCLUSIONS: The optimized procedure enabled very fast extraction in 30 min using environmentally friendly solvents and it was successfully applied to 16 different plant samples, including 14 bulbs and 2 fruits from South Africa. The quercetin content in extracts was quantified using ultra-high performance liquid chromatography (UHPLC) with tandem mass spectrometry. UHPLC hyphenated with high-resolution mass spectrometry was used to confirm chemical identity of quercetin in the analyzed samples. We quantified quercetin in 11 samples of all 16 tested plants. The quercetin was found in Agapanthus praecox from the Amaryllidaceae family and its presence in this specie was reported for the first time.

Topolin cytokinins enhanced shoot proliferation, reduced hyperhydricity and altered cytokinin metabolism in Pistacia vera L. seedling explants.

The effect of 10 µM meta-topolin (mT) and meta-topolin riboside (mTR) on in vitro proliferation and anomalies of Pistacia vera L. were evaluated and compared to that of 6-benzylaminopurine (BA). The highest proliferation rate (15.6) was recorded in the mT-medium, with a value 6 times higher than in BA-medium. Moreover, the lowest percentage of hyperhydric usable shoots (58,9%) and callus weight (46,9%) were found in mTR-treated shoots. Shoot tip as well as leaf necrosis were not influenced by cytokinin (CK) type. Image analysis was used to evaluate photosynthetic efficiency as well as anthocyanin index. Photosynthesis was more efficient with BA and mTR but the higher anthocyanin accumulation in BA-treated shoots suggests more stress. Endogenous CKs and their metabolites were determined in seedlings and, for the first time, the metabolism of exogenous BA, mT and mTR was studied in pistachio. The stimulating effect on cis-zeatin and its riboside and the appearance of BA and traces of ortho-topolin and para-topolin as natural CKs are discussed. The quantitative and qualitative CK metabolite analyses provides some initial clues as to why topolin would be superior to BA in terms of proliferation rate and avoiding hyperhydricity and allowed a better understanding of the effect of exogenous administration of CK.

Enhanced Yield of Pepper Plants Promoted by Soil Application of Volatiles From Cell-Free Fungal Culture Filtrates Is Associated With Activation of the Beneficial Soil Microbiota.

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants' responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota.

Caged Phytohormones: From Chemical Inactivation to Controlled Physiological Response.

Plant hormones, also called phytohormones, are small signaling molecules regulating a wide range of growth and developmental processes. These unique compounds respond to both external (light, temperature, water, nutrition, or pathogen attack) and internal factors (e.g., age) and mediate signal transduction leading to gene expression with the aim of allowing plants to adapt to constantly changing environmental conditions. Within the regulation of biological processes, individual groups of phytohormones act mostly through a web of interconnected responses rather than linear pathways, making elucidation of their mode of action in living organisms quite challenging. To further progress with our knowledge, the development of novel tools for phytohormone research is required. Although plenty of small molecules targeting phytohormone metabolic or signaling pathways (agonists, antagonists, and inhibitors) and labeled or tagged (fluorescently, isotopically, or biotinylated) compounds have been produced, the control over them in vivo is lost at the time of their administration. Caged compounds, on the other hand, represent a new approach to the development of small organic substances for phytohormone research. The term "caged compounds" refers to light-sensitive probes with latent biological activity, where the active molecule can be freed using a light beam in a highly spatio/temporal-, amplitude-, or frequency-defined manner. This review summarizes the up-to-date development in the field of caged plant hormones. Research progress is arranged in chronological order for each phytohormone regardless of the cage compound formulation and bacterial/plant/animal cell applications. Several known drawbacks and possible directions for future research are highlighted.

Soil nutrient status of KwaZulu-Natal savanna and grassland biomes causes variation in cytokinin functional groups and their levels in above-ground and underground parts of three legumes.

Cytokinins (CKs) are involved in several developmental stages in the life-cycle of plants. The CK content in plants and their respective organs are susceptible to changes under different environmental conditions. In the current study, we profiled the CK content in the above and underground organs of three legumes (Lessertia frutescens, Mucuna pruriens and Pisum sativum) grown in soils collected from four locations (Ashburton, Bergville, Hluhluwe and Izingolweni) in KwaZulu-Natal province, South Africa. The quantified CK contents in the three legumes were categorized on the basis of their side chains (isoprenoid, aromatic and furfural) and modifications (e.g. free bases and glucosides). Legume and soil types as well as their interaction significantly influenced the concentrations of CKs. Lessertia frutescens, Mucuna pruriens and Pisum sativum had CK content that ranged from 124-653, 170-670 and 69-595 pmol/g DW, respectively. Substantial quantity (> 600 pmol/g DW) of CK were observed in plants grown in Bergville (above-ground part of Lessertia frutescens) and Izingolweni (underground part of Mucuna pruriens) soils. A total of 28 CK derivatives observed in the legumes comprised of isoprenoid (22), aromatic (5) and furfural (1) side-chain CKs. However, the 16 CK derivatives in Mucuna pruriens were isoprenoid-type based on the side-chain. Generally, a higher ratio of cis-zeatin (cZ) relative to the trans-zeatin (tZ) was evident in the above-ground part of Lessertia frutescens and Pisum sativum for the four soil treatments. In terms of functional and physiological importance of the CKs, the free bases (active form) and ribosides (translocation form) were the most abundant CK in Lessertia frutescens and Pisum sativum. However, N-glucoside, a deactivation/detoxicification product was the most dominant CK in Mucuna pruriens from Hluhluwe and Izingolweni soils. The total CKs in the underground parts of the legumes had a positive significant correlation with the total phosphorus and nitrogen content in the plant as well as the soil nitrogen. Overall, the CK profiles of the legumes were strongly influenced by the soil types. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01021-2.

Interactive effects of plant growth-promoting rhizobacteria and a seaweed extract on the growth and physiology of Allium cepa L. (onion).

Detrimental effects caused by the overuse of synthetic agrochemicals have led to the development of natural biostimulants such as seaweed extracts and plant growth-promoting rhizobacteria (PGPR) being used as an alternative, environmentally-friendly technology to improve crop growth and increase agricultural yields. The present study aimed to investigate the interactions between PGPR and a commercial seaweed extract on the growth and biochemical composition of onion (Allium cepa). A pot trial was conducted under greenhouse conditions where onion plants were treated individually with the two PGPR, namely Bacillus licheniformis (BL) and Pseudomonas fluorescens (PF) and a seaweed extract Kelpak® (KEL) and combinations of KEL + BL and KEL + PF. Growth and yield parameters were measured after 12 weeks. KEL-treated plants showed the best growth response and overcame the inhibitory effects of BL treatment. KEL-treated plants also had the highest chlorophyll content. PGPR application improved the mineral nutrition of onion with these plants having the highest mineral content in the leaves and bulb. All biostimulant treatments increased the endogenous cytokinin and auxin content with the highest concentrations generally detected in the PF-treated plants. These results suggest that co-application of different biostimulant classes with different modes of action could further increase crop productivity with an improvement in both growth and nutrition content being achieved in onion with the co-application of a seaweed extract and PGPR.

A stable isotope dilution method for a highly accurate analysis of karrikins.

BACKGROUND: Karrikins (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. RESULTS: We 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). 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.