Hormone profiles in microalgae: Gibberellins and brassinosteroids

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Plant Physiology and Biochemistry 70 (2013) 348e353

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Hormone profiles in microalgae: Gibberellins and brassinosteroids

W.A. Stirk a,*, P. Bálint b, D. Tarkowská c, O. Novák c, M. Strnad c,d, V. Ördög a,b,J. van Staden a

aResearch Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, P/Bag X01, Scottsville 3209,South Africab Institute of Plant Biology, Faculty of Agricultural and Food Sciences, University of West Hungary, Mosonmagyaróvár H-9200, Hungaryc Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany AS CR, �Slechtitel�u 11, CZ-783 71 Olomouc, Czech RepublicdCentre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, �Slechtitel�u 11, CZ-783 71 Olomouc, CzechRepublic

a r t i c l e i n f o

Article history:Received 15 April 2013Accepted 28 May 2013Available online 13 June 2013

Keywords:BrassinolideBrassinosteroidsCastasteroneEndogenous hormonesGibberellinsGA6

Microalgae

Abbreviations: ABA, abscisic acid; BL, brassinolidcastasterone; cZ, cis-zeatin; DHZ, dihydrozeatin; GAsacetic acid; IAM, indole-3-acetamide; iP, isopentenyltion; MACC, Mosonmagyaróvár Algal Culture CollectioMS, ultraperformance tandem mass spectrometry.* Corresponding author. Tel.: þ27 (0) 33 260 5135;

E-mail address: [email protected] (W.A. Stirk).

0981-9428/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2013.05.037

a b s t r a c t

Endogenous gibberellins and brassinosteroids were quantified in 24 axenic microalgae strains from theChlorophyceae, Trebouxiophyceae, Ulvophyceae and Charophyceae microalgae strains after 4 days inculture. This is the first report of endogenous gibberellins being successfully detected in microalgae. Be-tween18 and 20 gibberellinswere quantified in all strainswith concentrations ranging from342.7 pgmg�1

DW in Raphidocelis subcapitata MACC 317e4746.1 pg mg�1 DW in Scotiellopsis terrestris MACC 44. Slowergrowing strains (S. terrestrisMACC 44, Gyoerffyana humicolaMACC 334, Nautococcus mamillatusMACC 716and Chlorococcum ellipsoideumMACC 712) exhibited the highest gibberellin contentswhile lowest levels ofgibberellinswere found in faster growing strains (R. subcapitataMACC317 and Coelastrum excentricaMACC504). In all strains, the active gibberellin detected in the highest concentration was GA6, the predominantintermediateswereGA15 andGA53 and themain biosynthetic end productswereGA13 andGA51. Gibberellinprofiles were similar in all strains except for the presence/absence of GA12 and GA12ald. To date this is thesecond report of endogenous brassinosteroids in microalgae. Brassinosteroids were detected in all 24strainswith concentrations ranging from117.3 pgmg�1 DW in R. subcapitataMACC317e977.8 pgmg�1 DWin Klebsormidium flaccidum MACC 692. Two brassinosteroids, brassinolide and castasterone were deter-mined in all the strains. Generally, brassinolide occurred in higher concentrations than castasterone.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Plant hormones are ubiquitous and essential in regulatinggrowth and development in multicellular plants as well as con-trolling their responses to environmental cues and stresses [1].Although research on the occurrence and function of plant hor-mones in microalgae is lagging far behind that of the more complexmulticellular plants, there is increasing evidence that plant hor-mones are present in microalgae [2]. We recently quantifiedendogenous auxins and cytokinins in 24 axenic microalgae strains[3]. These strains included the more closely related Chlorophyceae(17 strains), Trebouxiophyceae (5 strains) and Ulvophyceae (1

e; BRs, brassinosteroids; CS,, gibberellins; IAA, indole-3-adenine; LOD, limit of detec-n; tZ, trans-zeatin; UPLC-MS/

fax: þ27 (0) 33 260 5897.

son SAS. All rights reserved.

strain) within the Chlorophyta clade and one strain from thedivergent Streptophyta clade from which land plants evolved [4].Two auxins, i.e. indole-3-acetic acid (IAA) and indole-3-acetamide(IAM), were present in all 24 strains. Nineteen cytokinins werealso detected in these 24 strainswith cis-zeatin (cZ) forms being themost abundant. Moderate amounts of isopentenyladenine (iP)forms and lowconcentrations of trans-zeatin (tZ) and dihydrozeatin(DHZ) forms were measured [3]. Considering the other classes ofplant hormones, abscisic acid (ABA) has been detected and quan-tified in microalgae [5,6]. To date, gibberellins (GAs) have not beenfound in microalgae [2] and there is only one report of brassinos-teroids (BRs) identified in the microalga Chlorella vulgaris [7].

Gibberellins control various aspects of plant growth such as seedgermination, stem elongation, leaf expansion and flower and seeddevelopment [8]. Over 130 GAs have been identified in plants andother organisms. The first gibberellin was isolated from Gibberellafujikuroi, a fungal rice pathogen and more GAs have since beenisolated from other fungi and bacteria. However, the biosyntheticpathway differs a bit from that in plants [8]. Gibberellins have alsobeen identified in some tree ferns while functional homologs that

W.A. Stirk et al. / Plant Physiology and Biochemistry 70 (2013) 348e353 349

form complexes with GAs have been identified in Lycophytes, e.g.Selaginella moellendorffii and Selaginella kraussiana [8]. Recently 18GAs were detected in the stipe and frond of the kelp Eckloniamaxima (Phaeophyceae) [9].

Brassinosteroids are now recognized as a relatively new class ofplant hormones. They have a duel function, acting as growth-promoting hormones and playing a role in protection againstenvironmental stresses [10]. Brassinosteroids arewidely distributedin the plant kingdom [10] with over 70 BRs isolated from plants [11]including angiosperms (both dicotyledons and monocotyledons),gymnosperms as well as from a few lower order plants such asEquisetum arvense (Pteridophyte) [12], Marchantia polymorpha(Bryophyte) [13], the freshwater alga Hydrodictyon reticulatum(Chlorophyceae) [14], the seaweed E. maxima (Phaeophyceae) [9]and the microalgae C. vulgaris (Trebouxiophyceae) [7].

There is evidence for cross-talk between plant hormones [1].Gibberellins and auxins act synergistically with GA levels positivelyregulated by auxin. There are antagonistic effects between GAs andABA as well as GAs and cytokinins [8,15]. Brassinosteroids have asynergistic effectwith auxins and interactwith ABA, have an additiveeffect with GAs and increase ethylene production [11]. Thus, it isimportant to have an overview of the total hormone complement inmicroalgae in order to improve their growth and responses toexternal factors when grown in culture. We previously reported onthe endogenous auxin and cytokinin profiles in 24microalgae strains[3]. The aimof thepresent studywas to quantify endogenousGAs andBRs in the same 24 strains to confirm their presence in microalgae.

2. Results

2.1. Growth in culture

As the starting density was 10 mg L�1 DW for all the cultures,biomass accumulation measured on day 4 was used as a measure ofgrowth rates. The fastest growing strain with the highest biomassaccumulation was Raphidocelis subcapitata MACC 317 and the

Table 1Taxonomic details, culture origin and biomass accumulation in the 24 microalgal strains

Class Genus and species

Chlorophyceae Stigeoclonium nanum (Dillwyn) Kütz.Chlorococcum ellipsoideum Deason et BoldGyoerffyana humicola Kol et ChodatMonoraphidium contortum (Thur.) Komárková-LegneroNautococcus mamillatus KorschikovPoloidion didymos PascherProtosiphon botryoides G.A. KlebsAcutodesmus acuminatusa (Lagerh.) TsarenkoAcutodesmus incrassatulusb (Bohlin) TsarenkoDesmodesmus armatusc (R. Chodat) E. HegewaldScotiellopsis terrestris (Reisigl) Pun�coch. et KalinaRaphidocelis subcapitata (Korshikov) G. Nygaard,J. Komárek, Kristiansen et SkulbergChlamydomonas reinhardtii P.A. Dang.Protococcus viridis C. AgardhCoelastrum microporum NägeliSpongiochloris excentrica StarrCoccomyxa sp.

Trebouxiophyceae Chlorella pyrenoidosa ChickChlorella vulgaris BeyerinckChlorella minutissima Fott et NovákováMyrmecia bisecta ReisiglStichococcus bacillaris Nägeli

Ulvophyceae Ulothrix sp.Charophyceae Klebsormidium flaccidum (Kütz.) P.C. Silva,

K.R. Mattox et W.H. Blackw.

a Formerly Scenedesmus acuminatus.b Formerly Scenedesmus incrassatulus.c Formerly Scenedesmus armatus.

slowest growing strain with the lowest biomass accumulation wasGyoerffyana humicola MACC 334 (Table 1).

2.2. Gibberellins

Between18and20GAsweredetected in the24microalgal strainsanalyzed after 4 days growth in culture. Total GA concentrationsranged from 342.7 pg mg�1 DW in R. subcapitata MACC 317 to4746.1 pgmg�1 DW in Scotiellopsis terrestrisMACC44.Whenplottedagainst biomass which was used as a measure of growth, slowergrowing strains exhibited the highest GA contents, e.g. S. terrestrisMACC 44, G. humicolaMACC 334 andNautococcus mamillatusMACC716, while faster growing strains, e.g. R. subcapitata MACC 317 andCoelastrum excentrica MACC 504, showed the lowest GA levels(Fig. 1).

Gibberellin profiles were similar in all strains except for thepresence/absence of GA12 and GA12ald. While these two GAs wereboth present in high concentrations in 16 strains, five strains con-tained high concentrations of GA12 but with GA12ald being belowthe limit of detection (LOD), 1 strain had only GA12ald that occurredin a high concentrationwith GA12 below the LOD and both GA12 andGA12ald were below the LOD in two strains (Table 2). Biologicallyactive GAs (GA1, GA3, GA4, GA5, GA6 and GA7) contributed between5 and 33% of the total GA content, the metabolic end products (GA13and GA51) contributed between 7 and 40% and the intermediatesmade up the biggest proportion (between 36 and 91%) of the totalGA content (Table 2; Fig. 1). In all 24 strains, the active GA detectedin the highest concentration was GA6, the predominant in-termediates were GA15 and GA53 and the biosynthetic end products(GA13 and GA51) occurred in relatively high amounts (Table 2).

2.3. Brassinosteroids

Using the recently developed sensitive mass spectrometrybased method for simultaneous profiling of many of the knownbrassinosteroid precursors and metabolites in small amounts of

analyzed after 4 days in culture.

MACC Origin DW mg L�1

790 Czech Republic, soil 635712 Czech Republic, soil 320334 Brazil, soil 239

vá 700 Czech Republic, soil 470716 Czech Republic, soil 277545 Brazil, soil 42732 Czech Republic, soil 46141 Czech Republic, soil 630

730 Brazil, soil 43059 Czech Republic, soil 65744 Czech Republic, soil 385

317 Czech Republic, soil 1020

772 Brazil, soil 559219 Brazil, soil 76551 Czech Republic, soil 525

504 Czech Republic, soil 884535 Brazil, soil 429

3 Russia, soil 559755 Czech Republic, soil 681361 Brazil, soil 594594 Czech Republic, soil 284505 Czech Republic, soil 376777 Brazil, soil 413692 Ukraine, soil 443

STRAIN (MACC)

GIB

BER

ELLI

NS

(pg

mg-1

DW

)

0

500

1000

1500

2000

2500

317

504

219

755 59 790 41 361 3

772 51 700 32 692

730

535

545 44777

505

712

594

716

334

faster growth rate slower growth rate

CatabolitesActive GAs 4303.7 pg mg DW

Intermediates

Fig. 1. Gibberellin content in 24 microalgal strains harvested after 4 days in culture.The stains with the larger biomass accumulation are nearest the Y axis and those withthe least biomass accumulation furthest from the Y axis.

W.A. Stirk et al. / Plant Physiology and Biochemistry 70 (2013) 348e353350

plant tissue, two of the most common BRs i.e. brassinolide (BL) andcastasterone (CS) were detected in all 24 microalgal strainsanalyzed. Generally BL occurred in higher concentrations than CSand in a few exceptions, in similar amounts e.g. Poloidion didymosMACC 545 and R. subcapitata MACC 317 (Table 3). The total bras-sinosteroid concentration was variable, with the lowest concen-tration measured in R. subcapitata MACC 317 (117.3 pg mg�1 DW)while the brassinosteroid content was approximately 8-fold higherin Klebsormidium flaccidumMACC 692 (977.8 pgmg�1 DW; Table 3).

3. Discussion

This is the first report of endogenous GAs inmicroalgae. Apart forthepresenceor absence ofGA12ald andGA12, similarGAprofileswerepresent in all 24microalgae strains analyzed. These GAswere similarto those detected in the seaweed E. maxima [9] but occurred inmuchhigher concentrations in the microalgae. There was an apparenttrend between growth (as measured by biomass accumulation) andGA content with slower growing strains having higher endogenousGA contents and the faster growing strains having lowerGA contents.This trendwas especially apparentwhen considering the biologicallyactive GAs (Fig. 1). It will be necessary to carry out growth experi-ments with exogenously supplied GAs to confirm this observationand to determine which GAs elicit the strongest responses.

The concentrations of the active GAs depend on the rates ofbiosynthesis and deactivation [8]. Between 18 and 20 GAs weredetected in the 24 microalgae analyzed in the present study. This issimilar to higher order plants with 23 GAs detected in Arabidopsisthaliana, 12 in Oryza sativa (rice) and 14 in Brassica napus [16]. Onlya fewGAs are biologically active i.e. GA1, GA3, GA4, GA5, GA6 and GA7with GA1 and GA4 being themost widespread active forms in plants[8]. This was different in microalgae analyzed in the present studywhere GA6 was the active GA that was consistently detected in thehighest concentration (Table 2). Other GA forms found includedprecursors of the various biosynthetic pathways and deactivationforms.

In plants, GAs are formed from geranylgeranyl diphosphate via anumber of intermediates such as ent-kaurene, which are oxidized toform GA12ald that is then converted to GA12. GA12 is modified byvarious oxidation steps to produce biologically active forms withthese final steps in the GA biosynthetic pathways being speciesspecific [15]. Themain pathways in plants are either GA12/ GA15/

GA24 / GA9 / GA4 / GA34 or GA12 / GA53 / GA44 / GA19 /

GA20/ GA1. In addition,minor pathways have been observedwhereGA20 is converted to either GA6 or GA3 via GA5 [8,16,17]. As analternative regulatory pathway, GA19 may be hydroxylated to pro-duce the inactive GA13 [16]. Based on the concentration of the GAspresent in the 24 strains analyzed in the present study, it wouldappear that the main biosynthetic pathway in microalgae isGA12 / GA53 / GA44 / GA19 / GA20 / GA5 / GA6. This wouldinclude the possibility of forming the deactivation metabolite GA13from GA19 and a subsidiary pathway for the conversion ofGA20 / GA1. The GA15 /./GA4 route seems to be a minorbiosynthetic pathway in microalgae although GA15 generallyoccurred in high concentrations in all the strains analyzed. In addi-tion, GA9 may be metabolized to produce GA51 as a catabolite inparallel to the formation of GA4 from GA9 (Table 2).

Two BRswere detected in all 24microalgal strains analyzed after4 days in culture. These strains come from diverse groups withinthe Chlorophyta clade (23 strains) and one strain from the Strep-tophyta clade and thus highlight the ubiquitous distribution of BRsin microalgae. Brassinosteroid concentrations were higher in thesemicroalgae than in the brown kelp E. maxima (as were the GAs) [9]but within a similar range as in the green freshwater algaH. reticulatum [14]. The concentration of BL in C. vulgaris wascalculated as approximately 0.7 fg cell�1 [18]. Among all the BRS,castasterone is the most widely distributed, followed by BL [10]. Upto 13 different BRs have been identified in a single angiospermspecies with few BRs identified in the three lower order plantsinvestigated [10]. Seven BRs (BL, CS, 6-deoxocastasterone, typhas-terol, 6-deoxtyphasterol, teasterone and 6-deoxoteasterone) werepresent in C. vulgaris [7] while only two BRs (BL and CS) weredetected in the 24 strains analyzed. The difference in the BR profilesmay be explained by the age/growth phase of the microalgal cul-tures e the samples analyzed in the present study where harvestedon day 4, i.e. in the exponential growth phase while no mention ismade of the age of the C. vulgaris culture [7].

Brassinosteroids are found in all plant organs with reproductivetissues such as pollen and immature seeds generally having higherlevels of BRs than more mature vegetative tissues. Brassinosteroidlevels in pollen and immature seeds range from 1 to 100 ng g�1 FWwhile in shoots and leaves, concentrations range from 0.01 to0.1 ng g�1 FW [10,19]. Exogenous application of various BRs (10�12

to 10�8 M) stimulated growth of C. vulgaris over a 36 h period,significantly increasing cell number as well as increasing the pro-tein, DNA and RNA content of the cells [20]. Organic and inorganicphosphorus, chlorophyll a and b and monosaccharide content werealso increased which in turn influenced the rate of photosynthesisand sugar and glycolate secretion. Brassinolide was more activewith a weaker response from CS [21]. However, there was noapparent trend in the endogenous BR content and biomass pro-duction in the 24 actively growing microalgae strains analyzed inthe present study. Further investigations on the changing contentof the endogenous BRs as the cultures age may shed some light ontheir possible role that they play in microalgae.

Grafting experiments with growth-promoting BR-deficient peaand tomato mutants and various tissue excision experimentsshowed that BRs do not undergo long-distance transport like otherplant hormones. This implies that BRs are produced in situ withevidence that their biosynthesis and deactivation is controlled byfeedback regulation in response to endogenous BRs [22]. However,there is limited transport of BRs between different tissues within anorgan with BR receptors on both the exterior and interior cellmembranes. This implies that BRs are transported, either passivelyor more likely by an active carrier from their intracellular sites ofbiosynthesis to exterior sites of perception in adjacent cells [22].When exogenously applied BL was applied to C. vulgaris cultures,

Table 2Gibberellins quantified in 24 axenic microalgal strains analyzed after 4 days in culture. Results are shown asmean� SD (n¼ 3). LOD indicates that the concentrationwas belowthe limit of detection.

Species Stigeocloniumnanum

Chlorococcumellipsoideum

Gyoerffyanahumicola

Monoraphidiumcontortum

Nautococcusmamillatus

Poloidondidymos

Protosiphonbotryoides

Acutodesmusacuminatus

MACCgibberellin

790 712 334 700 716 545 32 41

pg mg�1 DW

GA1a 2.4 ± 0.2 4.6 ± 0.8 4.5 ± 0.2 2.8 ± 0.2 4.7 ± 0.2 3.0 ± 0.1 4.8 ± 0.4 5.2 ± 0.1

GA3a 0.3 ± 0.0 1.9 ± 0.1 1.9 ± 0.0 1.4 ± 0.2 0.7 ± 0.0 1.7 ± 0.2 3.9 ± 0.2 0.2 ± 0.0

GA4a 5.9 ± 0.0 9.6 ± 0.1 15.2 ± 0.5 7.6 ± 0.2 11.4 ± 0.5 7.4 ± 0.3 10.2 ± 0.2 6.6 ± 0.2

GA5a 7.6 ± 0.3 23.1 ± 0.8 34.4 ± 1.9 14.5 ± 0.5 29.7 ± 0.8 15.5 ± 0.5 13.2 ± 0.5 10.3 ± 0.1

GA6a 116.1 ± 4.0 271.2 ± 18.1 383.3 ± 5.5 162.0 ± 7.0 295.7 ± 11.8 150.5 ± 3.4 180.2 ± 2.6 154.6 ± 3.8

GA7a 1.1 ± 0.1 1.3 ± 0.0 3.4 ± 0.1 1.1 ± 0.0 1.8 ± 0.1 2.3 ± 0.5 0.9 ± 0.0 0.9 ± 0.0

GA8 11.5 � 1.5 25.5 � 2.3 5.7 � 0.2 11.7 � 0.3 20.8 � 1.2 4.0 � 0.6 3.0 � 0.1 5.3 � 0.2GA9 5.6 � 0.2 21.4 � 1.5 86.3 � 2.0 32.7 � 0.8 65.7 � 5.0 41.3 � 1.7 5.8 � 0.7 4.2 � 0.1GA12 163.4 � 19.5 <LOD <LOD 194.2 � 26.2 229.0 � 12.2 159.9 � 7.4 77.2 � 4.1 89.2 � 5.3GA12ald <LOD <LOD 494.4 � 16.5 <LOD 403.3 � 19.4 106.7 � 2.5 86.8 � 7.6 129.9 � 14.8GA13

b 67.3 � 0.5 348.7 � 5.9 62.5 � 2.8 156.4 � 1.8 263.1 � 12.3 53.7 � 2.5 20.3 � 0.9 14.9 � 0.8GA15 97.8 � 1.2 218.1 � 7.9 745.3 � 13.6 91.7 � 3.5 146.8 � 4.6 98.4 � 2.4 54.2 � 3.4 62.8 � 3.5GA19 0.7 � 0.0 1.3 � 0.1 1.4 � 0.1 0.8 � 0.1 1.7 � 0.1 1.0 � 0.0 2.8 � 0.1 1.4 � 0.2GA20 1.1 � 0.0 2.0 � 0.1 7.4 � 0.3 1.3 � 0.1 4.7 � 0.2 1.7 � 0.3 1.1 � 0.0 0.9 � 0.2GA24 3.6 � 0.3 6.8 � 0.7 17.9 � 1.8 4.7 � 0.3 7.3 � 0.8 4.5 � 0.6 4.0 � 0.4 5.4 � 0.6GA29 5.2 � 0.2 5.9 � 0.3 17.9 � 0.7 11.6 � 1.0 2.9 � 0.2 3.7 � 0.4 3.0 � 0.1 4.9 � 0.8GA34 1.3 � 0.1 1.2 � 0.1 2.7 � 0.2 1.4 � 0.2 3.1 � 0.5 0.9 � 0.1 0.8 � 0.1 0.9 � 0.1GA44 14.3 � 0.9 53.0 � 2.7 77.7 � 8.3 43.0 � 0.5 81.9 � 3.9 45.2 � 1.1 44.4 � 2.8 16.8 � 0.3GA51

b 60.2 � 5.5 91.7 � 3.6 313.7 � 8.1 46.0 � 2.5 609.8 � 5.6 157.2 � 4.3 244.7 � 14.5 23.1 � 0.5GA53 36.9 � 1.7 138.5 � 1.8 16.6 � 1.5 95.9 � 2.0 107.4 � 1.2 91.5 � 1.3 9.0 � 1.0 4.6 � 0.7

Species Acutodesmusincrassatulus

Desmodesmusarmatus

Scotiellopsisterrestris

Chlamydomonasreinhardtii

Raphidocelissubcapitata

Protococcusviridis

Coelastrummicroporum

Spongiochlorisexcentrica

MACCGibberellin

730 59 44 772 317 219 51 504pg mg�1 DW

GA1a 12.6 ± 0.7 2.0 ± 0.2 14.4 ± 0.5 7.6 ± 0.2 6.5 ± 0.3 2.8 ± 0.1 4.4 ± 0.3 2.5 ± 0.2

GA3a 2.0 ± 0.1 1.6 ± 0.0 1.8 ± 0.0 1.1 ± 0.0 0.6 ± 0.0 0.8 ± 0.0 2.7 ± 0.2 0.3 ± 0.0

GA4a 13.8 ± 0.1 8.9 ± 0.2 10.6 ± 0.2 3.4 ± 0.0 5.8 ± 0.1 5.7 ± 0.2 9.1 ± 0.2 5.8 ± 0.1

GA5a 24.1 ± 1.7 5.7 ± 0.1 10.8 ± 0.4 9.2 ± 0.2 6.6 ± 0.1 4.7 ± 0.3 8.2 ± 0.1 4.3 ± 0.2

GA6a 189.9 ± 5.2 135.3 ± 8.3 205.6 ± 14.8 145.9 ± 7.2 87.1 ± 2.5 124.2 ± 4.5 169.9 ± 11.2 94.3 ± 3.3

GA7a 1.7 ± 0.1 1.8 ± 0.0 2.7 ± 0.1 1.9 ± 0.0 0.9 ± 0.0 1.6 ± 0.0 1.2 ± 0.1 1.1 ± 0.1

GA8 12.1 � 1.0 1.6 � 0.4 10.7 � 0.1 15.4 � 1.1 3.4 � 0.5 6.0 � 0.7 2.6 � 0.7 3.3 � 0.5GA9 14.1 � 2.2 4.3 � 0.3 47.2 � 1.8 13.0 � 2.2 33.8 � 1.2 4.4 � 0.2 42.8 � 0.4 14.6 � 0.7GA12 117.0 � 10.5 80.1 � 5.3 312.6 � 28.3 118.9 � 13.1 88.8 � 4.8 177.0 � 15.1 170.3 � 14.8 93.8 � 2.7GA12ald 148.1 � 16.2 93.0 � 4.4 407.0 � 50.0 <LOD <LOD 82.3 � 5.4 119.2 � 3.8 86.3 � 1.2GA13

b 210.1 � 8.7 6.3 � 0.2 31.9 � 2.1 66.5 � 4.1 12.4 � 0.6 18.0 � 0.5 44.6 � 3.0 12.1 � 0.2GA15 129.6 � 3.3 158.5 � 2.1 3452.9 � 178.1 115.8 � 5.8 32.6 � 1.6 32.5 � 1.4 473.8 � 24.6 14.7 � 0.2GA19 2.3 � 0.1 0.5 � 0.0 1.1 � 0.3 0.9 � 0.1 0.3 � 0.0 1.0 � 0.1 3.4 � 0.2 0.9 � 0.1GA20 1.4 � 0.0 1.8 � 0.1 1.6 � 0.3 1.1 � 0.0 4.5 � 0.2 1.5 � 0.1 4.8 � 0.1 0.6 � 0.0GA24 4.9 � 0.4 4.3 � 0.2 5.2 � 0.4 3.8 � 0.3 2.5 � 0.3 8.4 � 0.6 14.2 � 1.5 4.4 � 0.2GA29 2.0 � 0.1 2.1 � 0.0 12.8 � 1.2 1.7 � 0.1 1.4 � 0.0 2.1 � 0.4 6.2 � 1.4 1.6 � 0.2GA34 1.2 � 0.0 1.2 � 0.2 2.1 � 0.1 1.2 � 0.2 0.5 � 0.0 0.7 � 0.1 0.7 � 0.0 1.8 � 0.1GA44 60.6 � 1.0 23.8 � 0.8 24.3 � 3.9 24.3 � 0.9 21.0 � 0.2 18.7 � 0.9 19.5 � 2.2 41.9 � 0.8GA51

b 91.7 � 2.7 207.2 � 11.1 164.8 � 9.4 142.3 � 5.6 30.3 � 1.3 110.1 � 3.8 169.1 � 5.6 29.2 � 1.4GA53 63.4 � 2.2 4.4 � 0.6 26.4 � 1.7 46.6 � 1.6 2.8 � 0.3 3.6 � 0.3 5.2 � 0.4 1.5 � 0.1

Species Coccomyxasp.

Chlorellapyrenoidosa

Chlorellavulgaris

Chlorellaminutissima

Myrmeciabisecta

Stichococcusbacillaris

Ulothrix sp. Klebsormidiumflaccidum

MACCGibberellin

535 3 755 361 594 505 777 692pg mg�1 DW

GA1a 2.9 ± 0.2 7.4 ± 0.3 2.1 ± 0.0 7.3 ± 0.5 21.2 ± 1.3 3.1 ± 0.1 6.9 ± 0.3 16.2 ± 0.5

GA3a 1.2 ± 0.1 2.4 ± 0.1 0.6 ± 0.0 0.6 ± 0.0 4.1 ± 0.1 1.7 ± 0.0 0.5 ± 0.0 2.5 ± 0.0

GA4a 11.3 ± 0.3 9.2 ± 0.3 6.9 ± 0.2 8.5 ± 0.1 20.2 ± 0.2 11.4 ± 0.6 12.6 ± 0.4 6.2 ± 0.0

GA5a 10.0 ± 0.2 5.2 ± 0.1 4.7 ± 0.2 10.5 ± 0.1 18.3 ± 0.3 32.0 ± 0.9 26.2 ± 0.9 10.5 ± 0.5

GA6a 198.1 ± 1.9 141.7 ± 9.6 114.9 ± 2.3 114.5 ± 3.4 321.6 ± 8.1 218.8 ± 10.0 166.6 ± 17.6 163.4 ± 4.4

GA7a 2.4 ± 0.1 1.3 ± 0.0 0.6 ± 0.0 1.0 ± 0.0 2.1 ± 0.1 2.3 ± 0.2 1.1 ± 0.2 1.6 ± 0.0

GA8 5.8 � 0.4 8.2 � 0.7 9.4 � 0.9 5.2 � 0.9 5.3 � 0.3 7.7 � 0.2 20.5 � 1.6 9.4 � 0.7GA9 12.4 � 0.8 31.5 � 0.8 10.0 � 0.2 11.1 � 0.5 34.7 � 1.5 10.7 � 3.2 58.0 � 7.9 7.5 � 0.3GA12 242.5 � 25.1 70.3 � 4.9 128.9 � 7.4 275.7 � 13.5 443.5 � 27.4 260.3 � 29.6 <LOD 79.9 � 4.1GA12ald 221.2 � 17.1 79.1 � 10.2 238.6 � 33.8 <LOD 479.5 � 9.5 415.5 � 16.9 <LOD 263.0 � 8.6GA13

b 15.8 � 0.6 12.3 � 0.2 39.1 � 1.1 12.6 � 0.3 28.8 � 0.2 11.4 � 0.8 58.9 � 3.2 35.2 � 2.2GA15 226.9 � 14.8 114.4 � 9.4 42.8 � 2.2 117.3 � 1.5 246.8 � 6.6 132.6 � 8.2 119.4 � 3.8 61.2 � 3.7GA19 0.8 � 0.0 1.5 � 0.2 0.6 � 0.3 0.6 � 0.0 5.8 � 0.5 0.9 � 0.0 1.1 � 0.1 1.00 � 0.1GA20 1.2 � 0.1 3.0 � 0.0 1.9 � 0.1 4.4 � 0.2 7.4 � 0.6 15.6 � 1.2 1.6 � 0.0 3.0 � 0.1GA24 5.4 � 0.3 13.7 � 0.7 3.1 � 1.7 9.7 � 0.5 22.3 � 2.7 11.7 � 0.4 6.2 � 0.4 5.1 � 0.5GA29 1.5 � 0.1 3.7 � 0.2 6.4 � 0.4 3.7 � 0.2 6.6 � 0.3 3.8 � 0.2 4.9 � 0.4 7.8 � 0.3GA34 0.9 � 0.1 1.5 � 0.1 1.9 � 0.3 0.5 � 0.1 3.2 � 0.1 1.2 � 0.0 1.0 � 0.1 1.2 � 0.1GA44 28.1 � 1.0 33.4 � 3.4 3.6 � 0.1 33.8 � 1.2 49.0 � 0.6 41.3 � 0.7 14.5 � 1.9 38.5 � 1.7GA51

b 168.4 � 6.3 57.1 � 1.6 129.6 � 5.8 399.0 � 2.3 244.0 � 6.5 182.3 � 6.2 234.1 � 9.1 26.3 � 4.7GA53 5.8 � 0.1 10.4 � 0.8 69.6 � 1.7 19.8 � 0.7 85.4 � 2.7 15.2 � 1.0 66.3 � 1.0 46.2 � 3.1

a Bioactive Gas are in bold font.b Final metabolic products (catabolites) are in italics.

W.A. Stirk et al. / Plant Physiology and Biochemistry 70 (2013) 348e353 351

Table 3Brassinosteroids (BRs) quantified in 24 microalgal strains analyzed after 4 days inculture. Results are shown as mean � SD (n ¼ 3).

Species Strain Brassinolide Castasterone Total BRcontent

MACC pg mg�1 DW

Stigeoclonium nanum 790 168.7 � 0.8 144.9 � 12.1 313.6Chlorococcum ellipsoideum 712 168.7 � 14.5 105.7 � 5.4 274.4Gyoerffyana humicola 334 270.9 � 32.8 201.1 � 19.4 472.0Monoraphidium contortum 700 284.9 � 17.2 195.0 � 4.1 479.9Nautococcus mamillatus 716 115.8 � 14.5 99.9 � 12.2 215.7Poloidion didymos 545 167.3 � 15.6 172.8 � 19.3 340.1Protosiphon botryoides 32 100.6 � 3.2 74.0 � 15.8 174.6Acutodesmus acuminatus 41 125.1 � 7.8 105.5 � 4.2 230.6Acutodesmus incrassatulus 730 124.8 � 12.6 92.6 � 6.1 217.4Desmodesmus armatus 59 125.1 � 5.7 109.3 � 0.2 234.4Scotiellopsis terrestris 44 336.9 � 40.0 235.9 � 4.9 572.8Raphidocelis subcapitata 317 58.6 � 6.8 58.7 � 7.6 117.3Chlamydomonas reinhardtii 772 162.9 � 4.2 153.8 � 15.3 316.7Protococcus viridis 219 211.6 � 11.8 134.8 � 8.3 346.4Coelastrum microporum 51 199.2 � 19.7 158.3 � 18.6 357.5Spongiochloris excentrica 504 131.2 � 10.6 108.5 � 1.8 239.7Coccomyxa sp. 535 205.8 � 3.9 177.1 � 0.5 382.9Chlorella pyrenoidosa 3 253.0 � 14.7 158.0 � 4.8 411.0Chlorella vulgaris 755 193.3 � 14.3 151.7 � 4.9 345.0Chlorella minutissima 361 306.5 � 15.5 215.3 � 19.4 521.8Myrmecia bisecta 594 202.4 � 44.5 164.3 � 32.7 366.7Stichococcus bacillaris 505 291.8 � 7.0 242.7 � 9.3 534.5Ulothrix sp. 777 84.9 � 5.3 74.2 � 3.5 159.1Klebsormidium flaccidum 692 548.7 � 2.7 429.1 � 30.8 977.8

W.A. Stirk et al. / Plant Physiology and Biochemistry 70 (2013) 348e353352

endogenous BL, auxin (IAA), cytokinin (zeatin) and ABA contentincreased in C. vulgaris [17]. Thus an intriguing question is whetherendogenous BRs are secreted by microalgae into the media and ifsimilar exterior BR receptor sites occur in microalgae. If so, thedensity of the culture could potentially affect the concentration ofother plant hormones in the cells. Theoretically, this could providea means of communication between microalgal cells.

In conclusion, this is the first report of positive detection ofendogenous GAs in microalgae with slower growing strains havinghigher GA contents and faster growing strains having lower GA con-tents. Based on the GAs detected in the microalgae samples, it wouldappear that GA biosynthesis favors the GA12/ GA53/ GA44/ GA19/ GA20 / GA5 / GA6 route with GA6 being the predominant activeGA detected. Brassinosteroids were also present in all the strainsanalyzed with BL and CS, being detected. Endogenous auxin andcytokinin contents were previously quantified in these microalgalstrains [3]. The role(s) that these plant hormones play in microalgaegrowth and physiology processes needs to be further perused in orderto successfully use plant hormones to enhancemedium- to large-scalemicroalgae cultivation.

4. Materials and methods

4.1. Cultures and growth conditions

Twenty-four axenic microalgal strains from the Mosonmagyar-óvár Algal Culture Collection (MACC) were inoculated into BristolLiquid Media [23] from agareagar stock cultures. Cultures weremaintained for seven days at 25 � 2 �C in a 14:10 h light: dark cycleilluminated from below with 130 mmol m�2 s-�1 light intensity andaerated during the light period with 1.5% CO2-enriched sterile, hu-midified air [24]. In addition, dilute algal suspensions were sprayedonto enriched, solidified nutrient media and incubated at 25 � 2 �Cfor 7 days to check that microalgal cultures were axenic [3].

Axenic cultures were inoculated into four flasks containing250 mL Bristol medium to give a starting density of 10 mg L�1 DW.The cultures were combined and harvested after four days growth

in the conditions described above. A small sample (10e50 mL) wasremoved from the combined sample in order to determine thebiomass of the suspension (mg L�1 DW) as described by Stirk et al.[3]. The remaining algal suspension was frozen at �70 �C untilanalysis for plant hormones.

4.2. Gibberellin analysis

Samples were analyzed for GA content using the method pre-viously described [16] with some modifications. Briefly, samples of500 mL algae culture were sonificated for 5 min and extractedovernight with 1 mL 80% acetonitrile containing 5% formic acid and19 internal GA standards ([2H2]GA1, [2H2]GA3, [2H2]GA4, [2H2]GA5,[2H2]GA6, [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, CzechRepublic). The extracts were centrifuged at 19 000 rpm for 10 minat 4 �C. The supernatants were further purified using ion exchangecartridges (Waters, Milford, MA, USA) and analyzed by ultra-performance chromatography-tandem mass spectrometry (UPLC-MS/MS; Micromass, Manchester, U.K). Gibberellins were detectedusing multiple-reaction monitoring mode of the transition of theprecursor ion [M�H]- to the appropriate product ion. The datawasacquired by Masslynx 4.1 software (Waters, Milford, MA, USA) andthe standard isotope-dilution method was used to quantify the GAlevels in the microalgae samples.

4.3. Brassinosteroid analysis

Sampleswere analyzed for BR content as previously described [25]with a few modifications. Briefly, samples of 1 mL algae culture weresonificated for 5 min and extracted overnight with stirring in ice-cold80% methanol and the addition of 50 pmol of [2H6]brassinolide, [2H6]castasterone, [2H3]24-epibrassinolide and [2H3]24-epicastasterone asinternal standards (OlChemIm, Olomouc, Czech Republic). Followingcentrifugation, samples were further purified on polyamide SPE col-umns (Supelco, Bellefonte, PA, USA) and polymer-based reverse phasecolumns (Phenomenex, Torrance, CA, USA) and then analyzed byUPLC-MS/MS (Micromass, Manchester, UK). The data was analyzedusing Masslynx 4.1 software (Waters, Milford, MA, USA) and BRcontent quantified by the standard isotope-dilution method.

Acknowledgments

The University of KwaZulu-Natal, the National Research Foun-dation (South Africa) and the EU funding Operational ProgramResearch and Development for Innovations (ED0007/01/01) aregratefully acknowledged for financial support.

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