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  (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy Research article Simultaneous determination of different endogenetic plant growth regulators incommon green seaweeds using dispersive liquid e liquid microextraction method Vishal Gupta a , Manoj Kumar a , Harshad Brahmbhatt b , C.R.K. Reddy a , * , Abhiram Seth c , Bhavanath Jha a a Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scienti  󿬁 c and Industrial Research (CSIR), Bhavnagar 364021, India b  Analytical Sciences, Central Salt and Marine Chemicals Research Institute, Council of Scienti  󿬁 c and Industrial Research (CSIR), Bhavnagar 364021, India c  Aquagri Processing Pvt Ltd, 18, Anand Lok, New Delhi 110049, India a r t i c l e i n f o  Article history: Received 18 July 2011Accepted 11 August 2011Available online 22 August 2011 Keywords: Plant growth regulatorGreen seaweedHigh performance liquid chromatographyDispersive liquid e liquid micro-extraction a b s t r a c t A simple and rapid HPLC-based method was developed for simultaneous determination of major classesof plant growth regulators (PGRs) in  Monostroma  and different species of   Ulva . The plant growth regu-lators determined included gibberellic acid (GA 3 ), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75,3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobilephase (60:40 methanol and 0.6% acetic acid in water), column temperature (35   C) and  󿬂 ow rate (1 ml/min). This method presented an excellent linearity (0.2 e 100  m g/ml) with limit of detection (LOD) as0.2  m g/ml for ABA, 0.5  m g/ml for KR and salicylic acid, and 1  m g/ml for IAA, IBA and GA 3 . The precision andaccuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plantmatrix was compensated after spiking and the resultant recoveries estimated were in the range of 80 e 120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimizedin this study determined IBA along with IAA for the  󿬁 rst time in the seaweed species investigated except Ulva linza  where the former was not detected. In all the species studied, ABA level was detected to be thehighest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, samplepreparation and analysis time were substantially reduced while allowing determination of more classesof PGRs simultaneously.   2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Plant growth regulators (PGRs) are structurally diverse group of naturally occurring substances that interacts with each other ina complex manner to regulate almost every aspect of plant life [1].They are highly functional and even at trace quantities triggera variety of basic physiological processes including cell division,enlargement and differentiation, organogenesis, seed dormancyand germination, leaf senescence and abscission as well as defenceagainst abiotic and biotic stresses [2]. Endogenic cytokininstogether with their riboside conjugate and auxins have beenreported from seaweed species such as  Porphyra perforata , Sargassum muticum  [3 e 5],  Laminaria japonica  [6],  Dictyota humi- fusa ,  Ulva fasciata  [7],  Undaria pinnati  󿬁 da  [8] and  Caulerpa paspa-loides  [9]. Recently, Yokoya et al. [10] quanti 󿬁 ed cytokinin, auxinand abscisic acid in common red algae from Brazilian coast. Theinherent growth regulating substances including both PGRs andminerals in seaweed were attributed to their application as fertil-izer in agriculture [11]. The foliar spray of seaweed extract or itsdirectapplicationtoroothasshownwiderangeofbene 󿬁 cialeffectson terrestrial crops ranging from early seed germination, improvedcrop performance, increased resistance toward abiotic and bioticstress and enhanced post harvest shelf-life of perishable products[12,13]. As a result, the seaweed industry has emerged with a newsectorof   ‘ phycosupplement ’  with an estimated global market valueof US$53 million [14]. Nowadays, the seaweed-based fertilizers aregaining importance over petrochemical based fertilizers because of thedetrimentaleffectscausedbypetrochemicalbasedfertilizersonsoil productivity upon prolonged use. The commonly usedseaweed-based fertilizers in the agriculture and horticulture aremainly prepared out of brown seaweeds namely  Ascophyllumnodosum ,  Ecklonia maxima ,  Macrocystis pyrifera  [12] owing to theirroundtheyearavailability.However,thefoliarsprayofredseaweed Kappaphycus alvarezii  sap on soyabean has resulted in an increaseof crop yields as high as 46% over control [15]. The analysis of thissap for PGRs has revealed the presence of IAA, GA 3 , kinetin andzeatin [16] in addition to several micro and macronutrients [17]. *  Corresponding author. Tel.: þ 91278 256 5801/256 3805x614; fax: þ 91278 2567562/256 6970. E-mail address:  crk@csmcri.org (C.R.K. Reddy). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$  e  see front matter    2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.plaphy.2011.08.004 Plant Physiology and Biochemistry 49 (2011) 1259 e 1263  Author's personal copy Further  󿬁 eld trials of this sap on other agricultural crops has alsoshown encouraging results over control and showed increasedyields of 30 e 40% in sugarcane, 26% in potato, 16% in barley andgram,and13 e 15%incorn(AbhiramSethpersonalcommunication).The genus  Ulva  with its worldwide distribution, wider adapt-ability to diverse environmental conditions, higher growth ratesand amenability for depolymerization makes it an attractive feed-stock for developing fertilizer and bio-re 󿬁 nery products. Theremoval of moisture from the biomass indeed is the bottlenecktoward the economy of energy industry therefore an integratedprocess involving extraction of liquid sap having agricultureapplications could make overall process much competitive.Development of such innovative processes will also be advanta-geous in utilizing the massive biomass of   Ulva  produced duringsudden outbreak as green tides or blooms recently reported inChina [18], the Baltic Sea [19] and Chile [20]. The analytical techniques employed for PGR analysis includehigh pressure liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography e mass spec-trometry(LC-MS),capillaryelectrophoresisetc.Ofthese,HPLCwithits inherent advantages such as wide application, short analysistimeandhighseparationef  󿬁 ciencymeritsitschoiceasabettertoolover others. However, intracellular traces of PGRs and coexistenceof many interfering substances make the analysis dif  󿬁 cult andnecessitate for sample preparation and puri 󿬁 cation prior to anal-ysis. The most commonly used pre-treatment techniques areliquid-liquid extraction (LLE), solid phase extraction (SPE), columnchromatography and solid phase microextraction (SPME). Of thesetechniques, dispersive liquid e liquid microextraction (DLLME)method developed by Razaee et al. [21] is relatively new and hasseveral advantageous over others such as 1) sample enrichment, 2)dispenses the need of puri 󿬁 cation through column, 3) low organicsolvent consumption and 4) overall short operation time. TheDLLME method together with HPLC coupled to ultraviolet detector(UV) has been employed for the analysis of polycyclic aromatichydrocarbons [22] and psycotropic drugs [23]. Lu et al. [1] for the 󿬁 rst time successfully demonstrated DLLME-HPLC-FLD as a power-ful pre-concentration method sensitive for detecting auxins.However, the requirement of rapid methodologies for samplederivatization has shifted the bottleneck for sample throughputfrom analysis to sample preparation. Therefore the objective of thisinvestigation was to optimize a method for simultaneous deter-mination of broad classes of plant growth regulators such as ABA,IAA, IBA, GA 3 , KR along with salicylic acid in common macrophyticmarine green algae  Monostroma  and  Ulva . 2. Results and discussion The interaction of different endogenetic hormone responsepathways plays a crucial role in regulating various developmentaland physiological processes. Therefore a method determiningsimultaneously different classes of endogenetic plant growthregulators (PGRs) will facilitate the understanding of variouscellular, physiological and biochemical responses. In this study,a simple and rapid HPLC based method was optimized for simul-taneous determination of majorclasses of PGRs such as auxins (IAAand IBA), ABA, GA 3  in common green seaweeds. The resolution of hydrophobic PGRs in HPLC analysis was found to signi 󿬁 cantly beaffected by mobile phase composition and concentration. Thedistinct separation of these PGRs was attained in mobile phaseconstituted with methanol: 0.6% acetic acid (60:40). The modi 󿬁 edmobilephasecompositionwasdistinctandsimplecomparedtothebinary mixtures used in majority of the studies in seaweed PGR analysis [7,10,24,25]. Also the modi 󿬁 ed composition was differentfrom those reported for PGR analysis in terrestrial plants [26,27]. Inaddition to mobile phase, the column temperature and  󿬂 ow ratewere the other determinants that offer better resolution of theanalytes. The separation column temperature and pump  󿬂 ow ratewas optimized as 35  C and 1.0 ml/min respectively. The standardswere detected at all the three wavelengths selected underthe optimized conditions and quanti 󿬁 cation was performed at208 nmforGA 3 andat265 nmforotherPGRs.Theretentiontimeof the standard analytes such as ABA, IAA, IBA, GA 3 , salicylic acid andKR was 3.91, 3.3, 4.95, 2.75, 5.39 and 6.59 min respectively(Supplementary Fig.1).The linear range along with the limit of detection (LOD) for thetargetcompoundsaresummarizedinTable1.TheLODwasfoundtobe lowest for ABA (0.2  m g/ml) followed by KR, salicylic acid (0.5  m g/ml), and IAA, IBA and GA 3  (1.0  m g/ml) (Table 1). The linear  󿬁 tting of the repeated data was found signi 󿬁 cant at  p  0.01 and the evalu-ation results of the linear model by regression variance analysis aresummarized in Table 2. The results revealed a good linear  󿬁 t. Theprecisionin theanalysiswasvalidated withinter/intra-dayanalysisin replicate and the relative deviation observed was in the range of 0.21 e 0.92%. The samples were found stable when stored at  20   Ceven for a month and the observed relative reductionwas 1.72% forall the targeted compounds except KR which was not recoveredafter storage (data not shown). The overall assay procedure hasshown a short separation time of 7 min only. Therefore this opti-mized method reduced the sample preparation and requiredanalysis time in addition to simultaneous pro 󿬁 ling of more classesof PGRs. These merits make this method a choice for highthroughput analysis over the other extraction and analysis strate-gies employed for seaweed PGR analysis [7,10].The methodprecisionandaccuracywasfoundtobecomparablewith those of the methods previously developed for simultaneousanalysis of PGRs from various plant matrices [1,2,28]. Recently, Luet al. [29] developed a method employing SPE strategy followed bythe analysis on pressurized capillary electrochromatography thatshowed LOD as lower as 0.2  m g/ml for endogenic (IAA) and ecto-genic hormones (BA, IAA, IPA, NAA and KT). The other extractionmethod based on SPME also showed LODs in the range of 0.121 e 0.442  m g/ml for IAA, ABA and IBA [2]. The comparison of themethod investigated in this study with other reported methods forPGR detection is illustrated in Table 3. The LODs for the sampleanalyzed herein were in the similar range as reported in some of the earlier reported methods thus demonstrating another advan-tage of dispensing the need of expensive cartridges. The earlierdeveloped methods were evaluated at a few real samples howeverthe method optimized in this study was evaluated on differentgreen seaweed species thereby giving precision to the analysis.Matrix effects were also compensated in this method to attainaccuracy in quanti 󿬁 cation. Signal recoveries were in the range of 80-92%forPGRsexceptGA 3 forwhichthesignalrecoverywas120%in  Ulva reticulata . The LOD and signal recovery was inferior in thisstudy compared to the report of Lu et al. [1] in which the samesample enrichment method was employed for analysis of auxins in Chlorella vulgaris  with FLD detection system. The method exploited  Table 1 Analytical performance data for major endogenetic plant growth regulators.Analyte Linear range( m g/ml)Regression equation  R  LOD( m g/ml)ABA 0.2 e 100  y ¼ 26,547  x  10,400 0.995 0.5GA 3  0.2 e 100  y ¼ 53,554  x þ 11,074 0.999 1IAA 0.2 e 100  y ¼ 96,168  x  17,311 0.995 1IBA 0.2 e 100  y ¼ 74,274  x  72,997 0.990 1KR 0.2 e 100  y ¼ 15,770  x  46,378 0.976 0.5Salicylic acid 0.2 e 100  y ¼ 69,670  x þ 40,625 0.971 0.5 V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259 e 1263 1260  Author's personal copy the natural  󿬂 uorescence properties of indole and naphthalenederivatives of IAA, IBA and NAA therefore selectively detectedauxins only. However, the same method with slight modi 󿬁 cationsin this study has shown its versatility for simultaneous analysis of major hormones in their natural state, dispensing the need of derivatization of extract prior to analysis or puri 󿬁 cation throughexpensive columns.The PGRs thereby detected were further identi 󿬁 ed using elec-tron spray ionization mass spectroscopy (ESI-MS). The precursorand product ions transition speci 󿬁 c for each PGR were identi 󿬁 edusing standard compounds. The main product ions obtained afterthe molecular fragmentation of standard compounds were used asdiagnostic product ions. The precursor ion ( m /  z  ) peak at 265, 175,203, 346 and 138matched well with the standard compounds ABA,IAA, IBA,GA 3  and SA respectivelywhen analyzed onESI- mode. The m /  z  peak forKR was recordedat348 with reference tothe standardwhen analyzed on ESI þ  mode. The product ion scan spectrum forIAA gavethe ion [M-COOH]  andits diagnostic transition ( m /  z  )was175.18 / 130.12. Similarly, IBA and SA showed ionized structureas [M-COOH]  and their precursor ion transitions ( m /  z  ) were203.01 / 158.03 and 138.14 / 93.08 respectively. The stableproduct ion for GA 3  was [M-C 14 H 22 O]  and its mass fragmentationwas 346.36 / 253.0 and the corresponding ionized state for ABAwas [M-C 5 H 8 O 2 ]  with mass fragmentation 265.23 / 164.98. ThediagnosticionstateforKRanalyzedonpositivemodecorrespondedto the [M-C 5 H 9 ONH] þ with precursor-to-product ion transition( m /  z  ) as 348.16 / 216.12.The auxins quanti 󿬁 ed in the present study were IAA and IBA(Table 4). IAA is the most prevalent auxin in plants but very little isknown about their metabolism and their conjugate intermediatesinseaweeds.Ofthefewreportsonauxinsfromseaweeds,IAAalongwith indole-3-acetamide (IAM), an intermediate of IAA synthesisthrough tryptophan mediated pathway was quanti 󿬁 ed [7,10].Interestingly, IAA along with another auxin IBA was detected in allspecies investigated in this study except  Ulva linza . Terrestrialplants investigated for auxins, other than IAA, showed  in-vivo synthesisofphenylaceticacid,halogenatedIAAas4-Cl-IAAandIBA[30]. The plant growth regulator IBA, considered as natural planthormone, has shown growth inducing properties more effectivethan IAA even at lower concentrations. But its metabolism throughIAA dependent or independent pathway is being debated. One of the possible synthesis routes for IBA may be through acetylation of IAA by a microsomal membrane fraction in the presence of acetylCoA and ATP [31]. The concentration of IAA in green seaweed U. fasciata was reported to be 800 pmol/g DW [7], 8.47  m g/kg Fwt in Undaria pinnati  󿬁 da [8]and1  m g/gFwtin Caulerpa sp. [1].Inanotherstudy, IAA content of 90 e 95  m g/kg Fwt was determined in kelpextract using HPLC as analytical technique [32]. For unicellulargreen alga  Chlorella vulgaris , IAA content was reported as 37 ng/gFwt [1]. The estimation of IAA together with IBA in this study(Table 4) apparently ascribed to the sensitivity of the DLLMEmethod toward auxins as also summarized by Lu et al. [1]. Also, thehormonal content estimated for  U. fasciata  (Table 4) in the presentstudy was signi 󿬁 cantly higher over the same studied by Stirk et al.[7]. Although we are tempted to attribute such high values to theef  󿬁 ciency of method employed, it could be even due to naturaloccurrence of higher amounts in the sample itself, which needs tobe investigated.The trend of endogenous ABA was similar to that of auxins andwashighestfor U.reticulata than U.linza andfollowedby U.taeniata , U. lactuca ,  U. fasciata  and  Monostroma oxyspermum  (Table 4).GA 3  was present in the range of 2.26  0.20 to 8.46   0.29nmole/g Fwt and was below limit of detection for  M. oxyspermum .Gibberellins are known for the regulation of plant growth andmainly the stem elongation in higher plants [33]. GA 3  was detectedand quanti 󿬁 ed byPrasad et al. [16] in the sap of   K. alvarezii  which isshown to have plant growth promoting capacity. The resultantquanti 󿬁 cation was 30.85 ppm by HPLC and 27.87 ppm by ESI-MStherefore signi 󿬁 ed the HPLC based analysis. In another study forplant growth regulator analysis, Giannarelli et al. [34] showedHPLC-MS/MS as a betterquanti 󿬁 cation tool than GC/MS analysis. Inseaweeds, most of the earlier studies on hormone analysis wererestricted tothe detection and quanti 󿬁 cation of auxins, cytokininesand abscisic acid [7,10,24]. The GA 3  in  Ulva  spp. detected in thepresentstudyalthoughintraceamounthighlightedthepotentialof the preconcentration method opted in this study.The cytokinin in the form of riboside conjugate of kinetin wasquanti 󿬁 ed in all the seaweed species but its relative ratio waslowest over the other PGRs studied. The endogenous KR contentranged from 0.76  0.02 to 3.35  0.53 nmole/g Fwt. Cytokinin andits conjugates have been shown to control numerous events in celldivision, enlargement and differentiation, chloroplast and vasculartissue development, shoot growth,  󿬂 owering and senescence [35].Recently, nineteen types of cytokinins including its isoprenoid andaromatic forms weredetected in a numberof seaweed species [24].The hormone kinetin or its conjugate which were earlier consid-ered as un-natural was detected in marine unicellular algae [36].The same was also identi 󿬁 ed in plant cell extract (coconut chunk)by modi 󿬁 ed method of HPLC/UV/EC [37] and its riboside conjugatein coconut water using LC-tandem mass spectrometry [38]. Prasad  Table 3 Comparison of the method optimized in this study with the previously reported methods for detection of plant growth promoting substances. ABA: Abscisic acid, BA: Benzyladenine, GA 3 : Gibberellic acid, IAA: Indole acetic acid, IBA: Indole butyric acid, IPA: Indole propionic acid, KR: Kinetin riboside, KT: Kinetin-6-furfurylaminopurine, NAA:Naphthaline acetic acid, SA: Salicylic acid, NR: Not required.Analytical method HPLC Pressurized CEC HPLC HPLC HPLCDetector UV UV Fluorescence UV UV Column  󿬁 ltration NR SPE NR SPE SPMEMobile phase Single Single Single Binary mixture Binary mixtureLOD ( m g/ml) 0.2 e 1.0 0.1 e 0.9 0.001 e 0.002 0.1 e 2.0 0.05 e 0.44PGRs detected IAA, IBA, ABA,GA 3 , KR, SAIAA, IPA, NAA,BA, KTIAA, IBA, IPA,NAAABA, IAA, GA 3  ABA, IAA, IBA, NAAReference This study 29 1 28 2  Table 2 Evaluationoflinearmodelfortheendogeneticplantgrowthregulatorsquanti 󿬁 edbyregression variance analysis at  p  0.01. SS E  is the residual sum of squares.PGRs SS E  Degree of freedomThe mean squares  F  -valueABA 3.127  10 12 7 668369.29 2032.14GA 3  4.772  10 10 5 97697.27 4148.68IAA 4.164  10 11 5 288587.13 1373.08IBA 3.872  10 11 5 278313.43 901.85KR 5.799  10 12 6 983118.76 307.57Salicylic acid 1.967  10 14 6 5.726 228.70 V. Gupta et al. / Plant Physiology and Biochemistry 49 (2011) 1259 e 1263  1261
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