Abscisic acid, gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima Wendy A. Stirk, Danuše Tarkowská, Veronika Turečová, Miroslav Strnad & J van Staden Journal of Applied Phycology ISSN 0921-8971 Volume 26 Number 1 J Appl Phycol (2014) 26:561-567 DOI 10.1007/s10811-013-0062-z
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Author's personal copy J Appl Phycol (2014) 26:561–567 DOI 10.1007/s10811-013-0062-z
Abscisic acid, gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima Wendy A. Stirk & Danuše Tarkowská & Veronika Turečová & Miroslav Strnad & J van Staden
Received: 25 February 2013 / Revised and accepted: 30 May 2013 / Published online: 15 June 2013 # Springer Science+Business Media Dordrecht 2013
Abstract The seaweed extract Kelpak® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak®. Kelpak® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak®, ranging from 0.31 to 20.70 pg mL−1 Kelpak®. Eighteen GAs were found in E. maxima and Kelpak® with concentrations from 187.54 to 565.96 pg mL−1 Kelpak®. The biologically active GAs (GA1, GA3, GA4, GA5, GA6 and GA7) comprised less than 3 % in Kelpak®. Although GA13 (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and W. A. Stirk (*) : J. van Staden Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg Campus, P/Bag X01, Scottsville, Pietermaritzburg 3209, South Africa e-mail:
[email protected] D. Tarkowská : V. Turečová : M. Strnad Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany AS CR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic M. Strnad Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic
Kelpak®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL−1 Kelpak® and 62.84 to 567.51 pg CS mL−1 Kelpak®. It is likely that this cocktail of natural PGRs present in Kelpak® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak® application to plants. Keywords Abscisic acid . Agricultural biostimulant . Brassinosteroids . Gibberellins . Plant growth regulators Abbreviations ABA BL CS GAs MRM PGRs UPLC-MS/MS
Abscisic acid Brassinolide Castasterone Gibberellins Multiple-reaction monitoring Plant growth regulators Ultra performance liquid chromatography tandem mass spectrometry
Introduction The first liquid seaweed extract for agricultural use was developed in the late 1940s and sold as Maxicrop (Craigie 2011). There are now numerous other liquid and powdered seaweed extracts made mainly from brown seaweeds that are sold as plant biostimulants (Khan et al. 2009). Seaweed extracts elicit a wide range of responses in plants such as increased root and shoot growth, improved nutrient uptake, increased flower and fruit set leading to higher yields, delayed senescence and longer shelf life of fruits (Metting et al. 1990; Crouch and van Staden 1994; Khan et al. 2009). Treated plants also have better resistance to insect and
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pathogen attack and to abiotic stress such as drought and frost (Metting et al. 1990; Crouch and van Staden 1994; Khan et al. 2009; Craigie 2011). As seaweed extracts are applied at relatively low application rates either as a soil drench or foliar spray, the numerous beneficial effects cannot be accounted for by an increase in the supply of macro- and micronutrients present in the extract (Crouch and van Staden 1994; Craigie 2011). Instead, plant growth regulators (PGRs) and other elicitor molecules such as oligomers and polysaccharides are considered as the active ingredients as they are able to elicit physiological responses at low concentrations (Crouch and van Staden 1994; Khan et al. 2009; Craigie 2011). Due to the wide range of physiological responses obtained with seaweed extract application, it is probable that a number of active compounds are present in the seaweed extract (Crouch and van Staden 1994). Since 1979, Kelp Products (Pty) Ltd. (Simon’s Town, South Africa) have been harvesting the kelp Ecklonia maxima (Osbeck) Papenfuss from the nutrient-rich cool temperate waters along the west coast of South Africa. They harvest approximately 100 t dry weight (DW) annually to make the seaweed biostimulant Kelpak® (Anderson et al. 2003). While most other seaweed extracts are made using either alkaline or acidic solutions at elevated temperatures (Craigie 2011), Kelpak® is made using a “cell burst method” which relies on rapid changes in pressure to rupture the cells, thus releasing the cellular contents. No high temperatures or chemicals which may be detrimental to the cellular compounds are used during the extraction process (Stirk and van Staden 1997). Kelpak® contains numerous cytokinins (free bases, O-glucoside derivatives and aromatic cytokinins), auxins (indole-3acetic acid, four amino acid conjugates and three other conjugates; Stirk et al. 2004) and polyamines (putrescine and spermine; Papenfus et al. 2012). The ethylene precursor 1-aminocyclopropane-1-carboxylic acid has also been detected in Kelpak® (Nelson and van Staden 1985). The aim of the present study was to quantify levels of other PGRs, i.e. abscisic acid (ABA), gibberellins (GAs) and brassinosteroids that may be present in Kelpak®.
Materials and methods Samples Kelpak® was obtained from Kelp Products (Pty) Ltd. Samples were selected to include different harvest dates during early spring (August 2008, 2009 and 2010) and late summer (February 2009 and 2010). These samples had been stored for either 2, 8, 14, 20 or 26 months at room temperature prior to analysis. There were two replicates per season selected from different harvests and processing batches (Table 1). In addition, the hormone content of E. maxima was analysed where the seaweeds were washed and separated into stipe and fronds in the Kelp Products (Pty) Ltd.
J Appl Phycol (2014) 26:561–567
factory. Two samples were prepared from multiple plants coming from two processing batches of material collected in August 2010 (3 Aug 2010 and 4 Aug 2010). These samples were received at the Research Centre for Plant Growth and Development, University of KwaZulu-Natal, within 48 h of harvesting and were immediately lyophilized, ground to a fine powder and stored at −70 °C until analysis. ABA analysis Samples of E. maxima (100 mg DW) and 5 mL Kelpak® were analysed for ABA using a modified method described by Yokoya et al. (2010). To check the recovery during purification and to validate the determination, 20 pmol of deuterium-labelled ABA ([(+)-3′,5′,5′,7′,7′,7′-2H6-ABA] synthesized using a previously described method; Prinsen et al. 1995) was added to all samples. Kelpak® samples were directly purified by solid-phase extraction on Oasis® HLB cartridges (60 mg, 3 mL, Waters, USA) while E. maxima samples were extracted for 1 h in 1 mL cold methanol/water/acetic acid (80/19/1 v/v/v), centrifuged (20,000× rpm, 10 min, 4 °C), and the supernatants were collected. The pellets were re-extracted for another hour. The combined supernatants were purified on Oasis® HLB cartridges. All samples were evaporated to dryness and derivatised by methylation with diazomethane. The samples were further subjected to ABA-specific immunoaffinity chromatography as described by Hradecká et al. (2007). The eluents were evaporated to dryness and analysed by ultra performance tandem mass spectrometry (UPLC-MS/MS; Micromass, UK; Turečková et al. 2009). GA analysis Quantification of GAs was performed according to the method of Urbanová et al. (2013) with some modifications. Aliquots of either E. maxima (15 mg DW) or 500 μL Kelpak® were extracted overnight in 1 mL 80 % acetonitrile containing 5 % formic acid with the addition of 19 internal GA standards ([2H2]GA1, [2H2]GA3, [2H2]GA4, [2H2]GA5, [2H2]GA7, [2H2]GA8, [2H2]GA9, [2H2]GA12, [2H2]GA12ald, [2H2]GA15, [2H2]GA19, [2H2]GA20, [2H2]GA24, [2H2]GA29, [2H2]GA34, [2H2]GA44, [2H2]GA51 and [2H2]GA53; OlChemIm, Olomouc, Czech Republic). After centrifugation at 19,000 rpm Table 1 ABA content in the seaweed extract Kelpak® harvested over a 2-year period and stored for up to 26 months. The values of the two samples from each harvest are given (replicate 1; replicate 2) Harvest dates
Storage time
Replicate 1
Replicate 2
3 Aug 2010 3 Feb 1010 8 Aug 2009 8 Feb 2009 14 Aug 2008
4 Aug 2010 10 Feb 2010 28 Aug 2009 13 Feb 2009 25 Aug 2008
