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Plant Physiology and Biochemistry 80 (2014) 176e183

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Plant Physiology and Biochemistry

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

Research article

Interactive effect of brassinosteroids and cytokinins on growth,chlorophyll, monosaccharide and protein content in the green algaChlorella vulgaris (Trebouxiophyceae)

Andrzej Bajguz*, Alicja Piotrowska-NiczyporukUniversity of Bialystok, Institute of Biology, Department of Plant Biochemistry and Toxicology, Swierkowa 20 B, 15-950 Bialystok, Poland

a r t i c l e i n f o

Article history:Received 8 March 2014Accepted 11 April 2014Available online 21 April 2014

Keywords:BrassinosteroidsChlorophyllsCytokininsGrowthMonosaccharidesProteins

Abbreviations: BL, Brassinolide; BR, BrassinosteCytokinin; 6-deoxoCS, 6-Deoxocastasterone;Dehydroteasterone; 6-deoxoTE, 6-DeoxoteasteDeoxotyphasterol; DPU, 1,3-diphenylurea; 24-epiBL,24-Epicastasterone; 28-homoBL, 28-HomobrassiHomocastasterone; Kin, kinetin; TE, Teasterone; TY, T* Corresponding author. Tel.: þ48 857457293.

E-mail address: abajguz@uwb.edu.pl (A. Bajguz).

http://dx.doi.org/10.1016/j.plaphy.2014.04.0090981-9428/� 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

Interaction between brassinosteroids (BRs) (brassinolide, BL; 24-epibrassinolide, 24-epiBL; 28-homobrassinolide, 28-homoBL; castasterone, CS; 24-epicastasterone, 24-epiCS; 28-homocastasterone,28-homoCS) and adenine- (trans-zeatin, tZ; kinetin, Kin) as well as phenylurea-type (1,3-diphenylurea,DPU) cytokinins (CKs) in the regulation of cell number, phytohormone level and the content of chlo-rophyll, monosaccharide and protein in unicellular green alga Chlorella vulgaris (Trebouxiophyceae) wereexamined. Chlorella vulgaris exhibited sensitivity to CKs in the following order of their stimulatingproperties: 10 nM tZ > 100 nM Kin >1 mM DPU. Exogenously applied BRs possessed the highest bio-logical activity in algal cells at concentration of 10 nM. Among the BRs, BL was characterized by thehighest activity, while 28-homoCS - by the lowest. The considerable increase in the level of all endog-enous BRs by 27e46% was observed in C. vulgaris culture treated with exogenous 10 nM tZ. It can bespeculated that CKs may stimulate BR activity in C. vulgaris by inducing the accumulation of endogenousBRs. CKs interacted synergistically with BRs increasing the number of cells and endogenous accumula-tion of proteins, chlorophylls and monosaccharides in C. vulgaris. The highest stimulation of algal growthand the contents of analyzed biochemical parameters were observed for BL applied in combination withtZ, whereas the lowest in the culture treated with both 28-homoCS and DPU. However, regardless of theapplied mixture of BRs with CKs, the considerable increase in cell number and the metabolite accu-mulation was found above the level obtained in cultures treated with any single phytohormone inunicellular green alga C. vulgaris.

� 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Plant hormones such as abscisic acid, auxins, brassinosteroids(BRs), gibberellins and cytokinins (CKs) are signal molecules pre-sent in trace quantities and are actively involved in manybiochemical processes (Ogweno et al., 2010). BRs are plant hor-mones that play an important role in the plant growth and devel-opment (Bajguz and Tretyn, 2003). BRs are hydroxylatedderivatives of cholestane and their structural variations comprise

roid; CS, Castasterone; CK,6-deoxo-3-DT, 6-Deoxo-3-rone; 6-deoxo-TY, 6-24-Epibrassinolide; 24-epiCS,nolide; 28-homoCS, 28-yphasterol; tZ, trans-Zeatin.

served.

substitution patterns on rings A and B as well as the C-17 side-chain. According to the B ring oxidation stage, BRs can be dividedinto 7-oxalactone, 6-ketone and non B-ring oxidized BRs. In gen-eral, 7-oxalactone BRs have higher biological activity than 6-ketonecongeners, non-oxidized BRs have almost no activity in variousbioassays. Since their discovery, over 70 BR compounds have beenisolated from the plant kingdom (Bajguz and Tretyn, 2003; Bajguz,2009a, 2011). BRs, e.g. BL and CS have been also identified in uni-cellular green algae Chlorella vulgaris (Bajguz and Asami, 2004;Bajguz, 2009a; Stirk et al., 2013). BRs are important plant growthregulators in multiple developmental processes at nanomolar tomicromolar concentration, including ethylene biosynthesis, celldivision and elongation, membrane hyperpolarisation, DNA, RNAand protein synthesis, enzyme activity, photosynthesis and thebalance of other endogenous phytohormones in green alga andvascular plants (Bajguz and Czerpak, 1996a,b; Bajguz, 2000;Choudhary et al., 2012).

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183 177

The relationships between BRs and the other well-known planthormones have been extensively explored (Arteca et al., 1988).Indeed, BRs are involved in a complex signaling network via amodulation of the levels and sensitivity of other phytohormones(Müssig et al., 2002). Some recent studies have identified the spe-cific mechanisms of the coordinated action of BRs and several otherphytohormones, including jasmonic acid, abscisic acid, gibberellicacid, auxin, and ethylene (Müssig et al., 2002). For example, BRsenhance the level of abscisic acid in green algae C. vulgaris (Tre-bouxiophyceae) cultures exposed to heat stress (Bajguz, 2009b).BRs elevated ethylene biosynthesis in etiolated seedlings of Arabi-dopsis thaliana by increasing the stability of ACC synthase, and theBR effects were additive with those of cytokinin (CK) (Hansen et al.,2009). Physiological activity of BRs is largely consistent withphysiological influences exerted by auxins (Bajguz and Piotrowska-Niczyporuk, 2013). The signal transduction pathways of both phy-tohormones are connected and the physiological effects of thesegroups’ compounds are synergistic. For example, the application ofIAA to C. vulgaris cultures stimulated the level of all detectedendogenous BRs in C. vulgaris cells. Auxins cooperated synergisti-cally with BRs stimulating algal cell proliferation and endogenousaccumulation of proteins, chlorophylls and monosaccharides inC. vulgaris. The highest stimulation of algal growth and the contentsof analyzed biochemical parameters were observed for the mixtureof BL with IAA, whereas the lowest in the culture treated with both28-homoCS and IBA (Bajguz and Piotrowska-Niczyporuk, 2013).

However, many questions remain unclear about the mecha-nisms by which BRs interact with other groups of phytohor-mones. Unlike the cross-talk between BRs and theaforementioned hormones, the mechanisms of interplay be-tween BRs and CKs are still obscure. Microarray data havedemonstrated that Arabidopsis BR-deficient mutants cpd/cbb3,and dwf1-6 displayed altered transcript levels of the CK-regulated genes ARR7 and ARR5; however, these genes showedinconsistent responses to BR treatment in wild-type plants(Müssig et al., 2002). Furthermore, a BR deficiency did not causealtered transcript levels of CK biosynthetic genes. The results ofthese studies imply that BR-CK interactions are possibly medi-ated through various metabolic pathways and can be integratedin a complex signaling network (Kudryakova et al., 2013). Resultsobtained by Ogweno et al. (2010) indicate that ABA, BR and CKstimulated photosynthesis and the activity of antioxidant en-zymes in detached tomato leaves.

CKs can be classified into two groups: adenine- andphenylurea-type, according to the chemical structure. Naturallyoccurring CKs are adenine derivatives with an isoprene (e.g.trans-zeatin, tZ) or a heterocyclic (kinetin, Kin) group at the N6-position in the molecule. In addition, there is the class of thestructurally unrelated synthetic phenylurea-type cytokinins.These phytohormones, such as 1,3-diphenylurea (DPU) are highlyactive in many CK bioassays, e.g. in C. vulgaris culture(Piotrowska and Czerpak, 2009).

Synchronous and homogenous cell population of C. vulgaris is anespecially promising experimental system for examining the re-lationships between phytohormones in green algae. C. vulgaris isdistributed widely in freshwater and seawater and has a shortgrowth cycle, which make it ideal for biochemical (protein, chlo-rophyll and monosaccharide content) studies and it can be used todirectly observe phytohormone response at the cellular level,because the perception of signaling molecule and biochemicalresponse takes place within the same cell under controlled condi-tions (Bajguz and Piotrowska-Niczyporuk, 2013). Soluble-proteincontent in green algae, an important indicator of reversible andirreversible changes in metabolism, is known to respond to a widevariety of plant growth regulators. Photosynthesis enhancement is

an often observed feature in lower plant cells in response to BRsand CKs. For that reason, the changes in the accumulation of pig-ments involved in photosynthesis and the products of this processin C. vulgaris were studied (Bajguz, 2000; Piotrowska and Czerpak,2009; Bajguz and Piotrowska-Niczyporuk, 2013).

Therefore, the goal of this study was to investigate the interac-tion of the BR and CK in regulation of phytohormone level inC. vulgaris cells and effect of these two groups of plant hormones onthe growth and metabolite content in unicellular green alga.

2. Materials and methods

2.1. Plant material and growth conditions

The wild-type C. vulgaris Beijerinck (SAG 211-12) (Treboux-iophyceae) used in this study was obtained from the collection ofthe Institute of Biology at the University of Bialystok. The axeniccultures were cultivated for 48 h under controlled sterile conditionsat 25� 0.5 �C. Illuminationwas supplied during a 16-h photoperiod(8-h dark period) by a bank of fluorescent lights yielding a photonflux 50 mmol m�2 s�1 at the surface of the tubes. Complete syn-chronization has been obtained by a regular change of light anddark periods according to the method of Pirson and Lorenzen(1966).

The homogenous population of young synchronous cells wascollected by centrifugation (1000� g, 10 min) and used for subse-quent experiments. Synchronization of the culture was controlledby studying cell division and the diagrams of cell size distributionevery day at the beginning of light period using a microscope.Growth of cultures was initiated by introduction of inoculumscontaining about 106 algal cells. The culture mineral medium usedwas modified mineral Knop’s medium. The pH of the medium wasadjusted to 6.8 with 1 M NaOH. C. vulgaris cells were cultured in anErlenmeyer flasks (500mL) containing 250mLmedium and shakenin a rotary shaker. The effects of BRs and CKs on C. vulgaris growthand metabolite content were examined. The following exogenousBRs: brassinolide (BL), 24-epibrassinolide (24-epiBL), 28-homobrassinolide (28-homoBL) from 7-oxalactonic type as wellas castasterone (CS), 24-epicastasterone (24-epiCS), 28-homocastasterone (28-homoCS) from 6-ketone type of BRs wereused at concentration 10 nM. 1,3-Diphenylurea (DPU) representingof phenylurea-type CKs and adenine-type CKs: kinetin (Kin) andtrans-zeatin (tZ) were applied at range of concentrations 100 pM e

100 mM. In co-application experiments BRs (BL, 24-epiBL, 28-homoBL, CS, 24-epiCS or 28-homoCS) at concentration 10 nMwere mixed with CKs (Kin, tZ or DPU) at range of concentration100 pM e 100 mM. The algal cultures were conducted in sixreplications.

2.2. Number of cells

For growth profile of the algal culture, 100 mL samples werecollected after 48 h of cultivation. The number of cells was deter-mined by direct counting of cells in the growth medium using aBürker chamber (Lee and Low, 1993).

2.3. Brassinosteroid determination

Determination of endogenous BR levels in the cells of C. vulgaristreated only with 10 nM tZ and in the control culture was per-formed on extracts with internal 2H standards, which is widelyaccepted as the most accurate method of BR determination (Bajguz,2009a). Deuterium-labeled substrates used in this study werechemically synthesized: [2H6]BL, [2H6]CS, [2H6]TY, [2H6]TE, [2H6]6-deoxoCS, [2H6]6-deoxoTY, and [2H6]6-deoxoTE. Lyophilized plant

Fig. 1. The comparative effect of brassinosteroids (BR) with the cytokinins (CK) on thegrowth expressed as cell number of Chlorella vulgaris after 48 h of cultivation. Data arethe means of six independent experiments � SD.

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183178

materials from C. vulgaris cultures were extracted with 300 mL ofMeOHeCHCl3 (4:1) twice, and [2H6]BL, [2H6]CS, [2H6]TY, [2H6]TE,[2H6]6-deoxoCS, [2H6]6-deoxoTY, [2H6]6-deoxoTE (100 ng each)were added to the extract as internal standards. After evaporationof the solvent in vacuo, the extract was partitioned between CHCl3andwater three times. The CHCl3 - soluble fractionwas subjected tosilica gel chromatography (Sep-Pak Vac Silica, 35 mL, Waters, Mil-ford, MA, USA). The columnwas subsequently eluted with100mL ofCHCl3, 2% MeOH in CHCl3, and 7% (v/v) MeOH in CHCl3. Each 2% (v/v) MeOH and 7% (v/v) MeOH fractionwas purified by Sephadex LH-20 column chromatography (column volume of 200 mL). The col-umn was eluted with MeOHeCHCl3 (4:1). The effluents of elutionvolume/total column volume: 0.6e0.8 were collected as the BRfraction. After purification on an ODS cartridge (Sep-Pak Plus C18,Waters) with 20 mL of MeOH, eluates were subjected to ODS-HPLC(Pak ODS 10 � 30 mm þ 20 � 250 mm, Senshu Scientific, Tokyo) ata flow rate of 8mLmin-1with the solvents 90% (v/v) acetonitrile forthe eluate derived from the 2% (v/v) MeOH fraction and 65%(v/v)acetonitrile for the eluate derived from the 7% (v/v) MeOH fraction.HPLC purification from the 7% (v/v) MeOH fraction yielded a BLfraction (Rt 10e15 min), CS fraction (Rt 15e20 min), TE fraction(Rt35e45 min), TY fraction (Rt 45e55 min), and 6-deoxoCS fraction(Rt 65e80 min), and HPLC purification from the 2% (v/v) MeOHfraction yielded a 6-deoxoTE fraction (Rt 55e65 min) and 6-deoxoTY fraction (Rt 65e90 min). Each fraction was analyzed byGCeMS after derivatization. The presence of endogenous non-labeled BRs was examined by GC-SIM-MS (JMS AX 505W Instru-ment JEOL, Tokyo) after suitable derivatization. The BRs contentwas quantified by the standard isotope-dilution method (Masslynx4.1 software, Waters, Milford, MA, USA).

2.4. Determination of total chlorophylls

The content of chlorophylls in C. vulgaris was determined ac-cording to Wellburn (1994) method in 48 h of culture. Algal freshweight was homogenized in darkness using 99.9% methanol in theproportion 1:10 (w/v). Chlorophylls were extracted by heating in awater bath at 70 �C for 30 min and centrifuged (1000� g, 10 min).The supernatant was used for the photosynthetic pigment deter-mination. The absorbance of the extract was measured with aspectrophotometer at 652.4 and 665.2 nm for total chlorophyllsand quantified.

2.5. Determination of water-soluble proteins

The measurement of the content of proteins soluble in waterwas done after 48 h of cultivation by the method of Bradford (1976)calibrated with bovine serum albumin obtained from SigmaChemical Co. (USA) as the standard.

2.6. Monosaccharide determination

For monosaccharide’s concentration, 10 mL of the algal culturesamples were first collected by centrifugation. Next, the mono-saccharide content was estimated in 48 h of culture after extractionin ethanol according to the Somogyi (1954) method.

2.7. Replication and statistical analysis

Each treatment consisted of 6 replicates and each experimentwas carried out at least twice at different times. The data wereanalyzed by one-way analysis of variance (ANOVA) and the meanswere separated using Duncan’s multiple-range test (Statistica 6,StatSoft, USA). The level of significance in all comparisons wasp < 0.05.

3. Results

3.1. Growth of C. vulgaris in response to brassinosteroids andcytokinins

To test which concentration of CKs was the most active, thenumber of algal cells after DPU, Kin and tZ treatments at range ofconcentrations 100 pM e 100 mM, were examined after 48 h oftreatment (Fig. 1). Isoprene CK e tZ was the most active atconcentration 10 nM stimulating cell number by 126%. Adenine-type CK with heterocyclic ring (Kin) was the most effective at100 nM stimulating cell number by 106%. The highest increase incell number in response DPU (71%) was observed at concentra-tion of 1 mM. C. vulgaris exhibited sensitivity on CKs in thefollowing order of their stimulating properties: tZ > Kin > DPU.Therefore, adenine-type CKs were characterized by higher bio-logical activity in C. vulgaris culture in comparison withphenylurea-type CK (DPU).

The preliminary experiments showed that exogenous BRs usedat range of concentrations 1 fM e 1 mM (data not shown) werecharacterized by the highest biological activity in C. vulgaris at10 nM inducing the increase in cell number by 53e178% in 48 h ofcultivation. The stimulatory effect of BRs in green algae was ar-ranged in the following order: BL > 24-epiBL > 28-homoBL > CS > 24-epiCS > 28-homoCS (Fig. 1). Fig. 2 showingthe interactions of BRs with CKs used at single concentrationindicate that the highest stimulating properties was obtained bythe co-application of 10 nM tZ with 10 nM BL which belongs to 7-oxalactone type of BRs, where cell number increased by 382% incomparison with the control in 48 h of cultivation. 100 nM Kinmixed with 10 nM BL stimulated cell number by 362%, whereas themixture of 1 mMDPU with 10 nM BL e by 326% in comparison withthe control (Fig. 2). Among 6-ketone type of BRs, CS was shown tohave the highest stimulating influence on algal cell proliferationinducing 310% increase in the number of cells. Kin and DPU used atrange of concentrations 100 pM e 100 mM in combinations withdifferent BRs such as BL, 24-epiBL, 28-homoBL, CS, 24-epiCS and28-homoCS used at concentration of 10 nM showed weaker stim-ulating influence on algal growth expressed as the number of cells(Figs. 1 and 2).

Fig. 2. The effect of tZ, Kin and DPU in combination with BL on the growth expressedas cell number of Chlorella vulgaris after 48 h of cultivation. Treatment with at least oneletter the same are not significantly different according to Duncan’s test. Fig. 3. The comparative effect of brassinosteroids (BR) with the cytokinins (CK) on the

protein content in Chlorella vulgaris after 48 h of cultivation. Data are the means of sixindependent experiments � SD.

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183 179

3.2. Brassinosteroid content in C. vulgaris in response to tZ

The application of exogenous 10 nM tZ representing adenine-type CK stimulated the accumulation of all identified endogenousBRs in C. vulgaris cells (Table 1) after 48 h of cultivation. The level of6-deoxoTE increased by 30%, 6-deoxoTY by 34%, 6-deoxo-3-DT by41%, and 6-deoxoCS by 32%, respectively in relation to the controlculture. Therefore, the content of the total compounds of late C-6oxidation pathwaywas stimulated by 32% in response to exogenousapplication of 10 nM tZ. Similarly, the concentration of the totalcompounds of early C-6 oxidation pathwaywas enhanced by 33% intZ-treated cells. Among them, the level of TEwas stimulated by 28%,TY by 27%, and CS by 36%. The increase by 46% in BL content wasobtained in algal cells exposed to 10 nM tZ after 48 h of cultivation.

3.3. Protein content in response to brassinosteroids and cytokinins

The application of BRs alone to culture medium enhanced pro-tein level in C. vulgaris cells by 33e133% in relation to the control(Fig. 3) after 48 h of cultivation. Among CKs, 10 nM tZ was observedto have the most stimulating effect (36%) on protein content inC. vulgaris. In response to Kin and DPU, the 29% and 22% increase inprotein level was obtained. The stimulating effect of CKs on proteincontent was enhanced in the presence of BRs in the culture me-dium. For example, co-application of 10 nM tZ with 10 nM BLinduced 2.5-fold increase in protein content in algal cells (Fig. 4). 6-

Table 1The effect of tZ on the brassinosteroid level in C. vulgaris cells after the 48 h ofculture. Treatment with at least one letter the same are not significantly differentaccording to Duncan’s test.

Brassinosteroid content (fg cell�1)

Brassinosteroid Cytokinin treatment

10 nM tZ Control

6-Deoxoteasterone (6-deoxoTE) 0.196 � 0.07a 0.151 � 0.01g

6-Deoxo-3-Dehydroteasterone (6-deoxo-3-DT) 0.134 � 0.09b 0.095 � 0.04f

6-Deoxotyphasterol (6-deoxoTY) 0.173 � 0.07b 0.129 � 0.08b

6-Deoxocastasterone (6-deoxoCS) 0.294 � 0.03c 0.223 � 0.05c

Teasterone (TE) 0.245 � 0.05c 0.191 � 0.01h

Typhasterol (TY) 0.319 � 0.08d 0.251 � 0.06i

Castasterone (CS) 0.447 � 0.02e 0.329 � 0.08j

Brassinolide (BL) 0.124 � 0.05f 0.085 � 0.07k

ketone type of BRs was characterized by lower stimulating prop-erties in cells treated with exogenous CKs. Among them, CS at10 nM combinedwith 10 nM tZ induced the highest (215%) increasein protein production in comparison with control (Fig. 3). In thecontrast, the application of the mixtures of Kin or DPU with BRs toalgal cells resulted in lower accumulation of soluble-proteins after48 h of algal cultures.

3.4. Chlorophyll content in response to brassinosteroids andcytokinins

The increase in the total content of chlorophylls by 51%, 35% and28% in algal cells exposed to 10 nM tZ, 100 nM Kin and 1 mM DPU,respectively was obtained after 48 h of algal cultivation (Figs. 5 and6). Higher biological activity was observed in case of BRs whichinduced 63e198% increase in photosynthetic pigment accumula-tion in comparison with the control. C. vulgaris possessed much

Fig. 4. The effect of tZ, Kin and DPU in combination with BL on the protein content inChlorella vulgaris after 48 h of cultivation. Treatment with at least one letter the sameare not significantly different according to Duncan’s test.

Fig. 5. The comparative effect of brassinosteroids (BR) with the cytokinins (CK) on thechlorophyll content in Chlorella vulgaris after 48 h of cultivation. Data are the means ofsix independent experiments � SD.

Fig. 7. The comparative effect of brassinosteroids (BR) with the cytokinins (CK) on themonosaccharide content in Chlorella vulgaris after 48 h of cultivation. Data are themeans of six independent experiments � SD.

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183180

higher content of total chlorophylls in response to the BRs com-bined with CKs after 48 h of cultivation. Among 7-oxalactone typeof BRs, 10 nM BL applied together with 10 nM tZ induced 339%,whereas 10 nM CS belonging to 6-ketone type of BRs, combinedwith 10 nM tZ induced 232% increase in chlorophyll level in algalcells after 48 h of cultivation. Therefore, the strong synergistic re-lationships between BRs and CKs on photosynthetic apparatus inC. vulgaris were observed.

3.5. Monosaccharide content in response to brassinosteroids andcytokinins

BRs interact with CKs by affecting monosaccharide accumula-tion in C. vulgaris culture (Figs. 7 and 8) after 48 h of cultivation. Thesignificant increase in monosaccharide content by 50%, 33%, and29% in C. vulgaris cells treated with 10 nM tZ, 100 nM Kin and 1 mMDPU, respectively was observed in experiments after 48 h ofcultivation. However, exogenous CKs increased biological activity of

Fig. 6. The effect of tZ, Kin and DPU in combination with BL on the chlorophyll contentin Chlorella vulgaris after 48 h of cultivation. Treatment with at least one letter thesame are not significantly different according to Duncan’s test.

BRs because algal cells treated with 10 nM BL and 10 nM tZ con-tained about 3 times as many sugars as the cells of the controlculture (Fig. 8). Among BRs belonging to 6-ketone type, CS co-applied with tZ was characterized by the highest biological activ-ity inducing the increase in sugar content by 169% in comparisonwith the control. In contrast, adenine-type CK with heterocyclicring Kin and phenylurea-type CK (DPU) were characterized bylower stimulating properties in interaction with BRs in C. vulgaris.The treatment algal cultures with BRs without CKs possessedweaker stimulating effect on monosaccharides. For example, underthe influence of BL their content was stimulated by 189% and inresponse to CS by 98% in relation to control after 48 h of cultivation.

4. Discussion

4.1. Brassinosteroid content in C. vulgaris

Plant growth is known to be under the control of endogenoushormonal system and, in connection with this, it may be suggested

Fig. 8. The effect of tZ, Kin and DPU in combination with BL on the monosaccharidecontent in Chlorella vulgaris after 48 h of cultivation. Treatment with at least one letterthe same are not significantly different according to Duncan’s test.

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183 181

that CKs actively influence the content of different hormones inplants. There are numerous examples with different plant speciesproviding evidences for their ability to affect hormonal balance inplants (Yuldashev et al., 2012). It is widely accepted that the hor-mone level at any given site might be affected by the relative ratesof its synthesis, degradation, inactivation, and transport within theplant. Each of these factors can be considered in terms of their inputto, or output from, the level of free hormone (Piotrowska andBajguz, 2011). The previous study demonstrated that the level ofseven BRs, including TE, TY, 6-deoxoTE, 6-deoxoTY, 6-deoxoCS, CSand BL is stimulated by auxin (IAA) in wild-type C. vulgaris cells(Bajguz and Piotrowska-Niczyporuk, 2013). All compounds belongto the BR biosynthetic pathway. Similarly, our present resultsindicate that exogenous 10 nM tZ representing adenine-type CKstimulates the accumulation of all identified endogenous BRs by27e46% in C. vulgaris cells such as: 6-deoxoTE, 6-deoxoTY, 6-deoxoCS from early C-6 oxidation pathways, TE, TY and CS fromlate C-6 oxidation pathways and BL. The results indicate that tZ ischaracterized by higher properties of activation BR biosynthesis inthe cells of green alga in comparison with auxin (IAA) (Bajguz andPiotrowska-Niczyporuk, 2013). This is the first evidence on thestimulating effect of CKs on the contents of phytohormonesinvolved in BR biosynthetic pathway in unicellular green algaC. vulgaris. The findings suggest a possibility that CKs regulatedirectly the biosynthesis and accumulation of BRs in lower plants.

A straightforward route of hormone pathway interaction wouldbe modulation of the biosynthesis of one hormone by the other,which has been repeatedly observed in vascular plants (Hardtkeet al., 2007; Hansen et al., 2009; Yuldashev et al., 2012;Kudryakova et al., 2013). For example, fast and stable 2-fold accu-mulation of CKs was detected initially in roots and then in shoots of4-day-old wheat (Triticum aestivum) seedlings in the course of theirtreatment with 0.4 mM 24-epiBL (Yuldashev et al., 2012). Anotherstudy suggests that the exogenous application of BRs may elevatethe CK content in A. thaliana plant (Kudryakova et al., 2013).Moreover, evidence suggest that both BRs and CKs additively pro-mote ethylene biosynthesis by increasing the stability of a subset ofACC synthases (Hansen et al., 2009).

Our data support the idea that unicellular green algae possess acomplicated network of BR-CK interactions. It can be speculatedthat CKs may stimulate BR activity in C. vulgaris by inducing theaccumulation of endogenous BRs. However, it should bementionedthat more extensive investigations are required to confirm theworking scenario.

4.2. Growth of C. vulgaris in response to brassinosteroids andcytokinins

The most characteristic responses elicited by BRs and CKscomprise the stimulation of cell division in higher plants and algae(Sasse, 1985; Clouse and Sasse, 1998; Riou-Khamlichi et al., 1999;Hu et al., 2000; Nakaya et al., 2002). The analysis of dose-effectrelationships showed that the most optimal concentration for tZwas 10 nM, for Kin e 100 nM, whereas phenylurea-type cytokinin(DPU) was the most biologically active at 1 mM in C. vulgaris cultureafter 48 h of treatment. Green alga exhibited sensitivity on CKs inthe following order of their stimulating properties: Z > Kin > DPU.

The stimulatory effect of 10 nM BRs on the cell number in greenalgae was arranged in the following order: BL > 24-epiBL > 28-homoBL > CS > 24-epiCS > 28-homoCS. Our results confirm thatBRs are growth-promoting steroidal hormones (Sasse, 1985; Bajguzand Czerpak, 1998; Clouse and Sasse, 1998; Nakaya et al., 2002).However, they induce biological responses not only by itself, butalso by interaction with CKs in the promoting of cell division inC. vulgaris culture. Moreover, we suggest that CKs interact

synergistically with BRs in stimulating algal growth, i.e. when thetwo hormones were included in the same incubation medium, thenumber of algal cells wasmuch higher than the sum of that inducedby each hormone. Previous results confirm that auxins may alsoenhance stimulating properties of BRs on algal growth expressed asthe number of cells (Bajguz and Piotrowska-Niczyporuk, 2013).

Control of cell division in vascular plants, along with cellexpansion, by BRs in the presence of other phytohormones is wellknown. Two studies (Bach et al., 1991; Gaudinová et al., 1995) haveindicated that BR treatment alters the endogenous auxin andcytokinin levels in tobacco callus cultures, thus influencing thecontrol of cell division. For example, in the sequential response toplant growth regulators in young elongating tissue from peas andwheat the peak of sensitivity to 24-epiBL (1 mM) occurs after CKapplication in isolated wheat (Triticum vulgare) coleoptiles agedfrom 21 to 96 h (Sasse,1985). BRs have been shown to stimulate celldivision in the presence of auxin in cultured parenchyma cells ofHelianthus tuberosus (Clouse and Sasse, 1998). Furthermore, BRsstimulated the cell increase ratio for fresh weight the in culturesOnsoma paniculatum only in the presence of auxin (IAA) and aro-matic CK (benzyladenine) belonging to adenine-type CKs by 248e262% (Yang et al., 1999) Thus, it seems possible that the presence ofCKs widens the activity spectrum of BRs stimulating the growth ofC. vulgaris culture.

4.3. Protein content in response to brassinosteroids and cytokinins

Soluble-protein content in plants, an important indicator ofreversible and irreversible changes in metabolism, is known torespond to a wide variety of phytohormones (Bajguz andPiotrowska-Niczyporuk, 2013). The application of BRs and CKsalone to culture medium enhanced the protein level in C. vulgariscells. The stimulating effect of BRs on protein contentwas enhancedin the presence of CKs in the culture medium. Our results are inagreement with Yang et al. (1999) indicating the increase in thelevel of soluble proteins in cultured O. paniculatum cells in responseto BL and benzyladenine. High protein content, increased nitratereductase and carbonic anhydrase activities was also observed inleaves of Vigna radiata sprayed with Kin and 28-homoBL(Fariduddin et al., 2004). Similarly, exogenously applied auxinsenhanced the stimulating properties of BRs on the protein accu-mulation in green alga C. vulgaris cells (Bajguz and Piotrowska-Niczyporuk, 2013). In contrast, the addition of BR or CK aloneslightly increased the content of proteins. Probably, the increase inprotein level may be induced by elevated and potentially prolongedexpression of target genes in response to the addition of bothhormones. Previous studies conducted on the alga C. vulgarisconfirmed the stimulating role of BRs and CKs (applied alone) in theenhancement of the content of nucleic acids and proteins (Bajguzand Czerpak, 1996a; Bajguz, 2000; Piotrowska and Czerpak,2009). The shortening of the developmental cycle and the in-crease by two or three times in DNA, RNA and protein level during36 h cultivation of the algae suggest an unusual increase in the rateof the processes of transcription and translation (Bajguz andCzerpak, 1996a; Bajguz, 2000; Piotrowska and Czerpak, 2009).The results of study regarding the interaction of the BR and CKsignaling pathways indicate that BRs mixed with CKs may activatethe synthesis of many proteins and involved in metabolism ofvascular plants (Zubo et al., 2008, 2011; Kudryakova et al., 2013).

4.4. Chlorophyll content in response to brassinosteroids andcytokinins

Photosynthesis enhancement is an often observed feature inplant cells in response to BRs and CKs (Fariduddin et al., 2004; Xia

A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183182

et al., 2009; Jiang et al., 2012). Therefore, the changes in the level ofpigments involved in photosynthesis were studied in C. vulgariscells in response to these two groups of phytohormones. The in-crease in the total content of chlorophylls in algal cells exposed toCKs without BRs and BRs without CKs was observed in 48 h of algalcultivation. On the other hand, C. vulgaris possessed much highercontent of total chlorophylls in response to the BRs combined withCKs. Similarly, previous studies indicate that BRs cooperated syn-ergistically with exogenous auxins in stimulation of chlorophyllcontent in C. vulgaris cultures (Bajguz and Piotrowska-Niczyporuk,2013). High accumulation of chlorophyll level and increase in netphotosynthesis rate was also observed in leaves of V. radiatasprayed with Kin and 28-homoBL (Fariduddin et al., 2004). Theobtained results confirm the stimulating effect of BRs on photo-synthesis and photosynthetic pigment contents in vascular plants(Xia et al., 2009) and unicellular algae (Bajguz and Czerpak, 1996b).Moreover, BRs may interact with CO2 enrichment and have positiveeffect of photosynthetic apparatus in leaves of young plants ofcucumber (Cucumis sativus) (Jiang et al., 2012). Stimulating effect ofphenylurea- and adenine-type CKs on photosynthetic pigments inalgal cells is well known (Czerpak and Bajguz,1997; Piotrowska andCzerpak, 2009). CK treatment abolishes the lag phase in chlorophyllsynthesis and accelerates its rate, inhibits chlorophyll breakdownby chlorophyllase, Mg-dechelatase and peroxidase-linked chloro-phyll bleaching in higher plants (Costa et al., 2005).

4.5. Monosaccharide content in response to brassinosteroids andcytokinins

An increase in chlorophyll content in C. vulgaris under the in-fluence of BRs in combination with CKs correlates well with higheraccumulation of monosaccharides. All adenine- (tZ, Kin) andphenylurea-type (DPU) CKs interacted with BRs (BL, 24-epiBL, 28-homoBL, CS, 24-epiCS, 28-homoCS) in stimulation of sugar con-tent in algal cells. This response is similar to the synergistic re-sponses previously reported (Bajguz and Piotrowska-Niczyporuk,2013) between BRs and auxins in the culture of C. vulgaris. Obtainedresults are comparable with that reported for a number of higherplant tissues and green algae, indicating that BRs and CKs influenceaccumulation of carbohydrates (Bajguz and Czerpak, 1996a,b;Piotrowska and Czerpak, 2009). Exogenous CKs may increase bio-logical activity of BRs because algal cells treated with BL and tZ at10 nM contained the highest level of monosaccharides in com-parison with the control culture. The stimulating effect of BRs andCKs on sugar content is well known. Microarray and photosynthesisanalysis of transgenic plants T. aestivum and Oryza sativa withenhanced BR biosynthetic pathway revealed evidence of enhancedCO2 assimilation, enlarged glucose pools in the flag leaves, andincreased assimilation of glucose to starch in the seed (Wu et al.,2008). A broad stimulating effect of CKs on monosaccharide levelhas been also showed in Beta vulgaris, when cytokinins induced 9-fold increase in glucose, fructose, galactose and ribose content (Ivicet al., 2001).

5. Conclusion

The data presented in this paper suggest that exogenouslyapplied CKs may change the phytohormone balance in C. vulgariscells increasing the level of all identified endogenous BRs. Co-application experiments suggest a novel relationships betweenBRs and CKs in unicellular green alga. This interaction is synergisticbecause the effect of two hormones applied simultaneously ex-ceeds the sum of each effect on algal growth and metabolite con-tent. We have demonstrated that BR induced response of algal cellsis enhanced by exogenous CKs several fold. This is the first evidence

demonstrating a synergistic relationship of BRs and CKs in lowerplants. Nonetheless, our work provides an important starting pointfor future dissection of what appears to be complex mechanisms ofBR-CK cross-talks in plants.

Contribution

In this paper we have the same contribution.

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