Plant Physiology and Biochemistry 80 (2014) 176e183

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Research article

Interactive effect of brassinosteroids and cytokinins on growth, chlorophyll, monosaccharide and protein content in the green alga Chlorella vulgaris (Trebouxiophyceae) Andrzej Bajguz*, Alicja Piotrowska-Niczyporuk University 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

a b s t r a c t

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

Interaction between brassinosteroids (BRs) (brassinolide, BL; 24-epibrassinolide, 24-epiBL; 28homobrassinolide, 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 chlorophyll, monosaccharide and protein in unicellular green alga Chlorella vulgaris (Trebouxiophyceae) were examined. Chlorella vulgaris exhibited sensitivity to CKs in the following order of their stimulating properties: 10 nM tZ > 100 nM Kin >1 mM DPU. Exogenously applied BRs possessed the highest biological activity in algal cells at concentration of 10 nM. Among the BRs, BL was characterized by the highest activity, while 28-homoCS - by the lowest. The considerable increase in the level of all endogenous BRs by 27e46% was observed in C. vulgaris culture treated with exogenous 10 nM tZ. It can be speculated that CKs may stimulate BR activity in C. vulgaris by inducing the accumulation of endogenous BRs. CKs interacted synergistically with BRs increasing the number of cells and endogenous accumulation of proteins, chlorophylls and monosaccharides in C. vulgaris. The highest stimulation of algal growth and the contents of analyzed biochemical parameters were observed for BL applied in combination with tZ, whereas the lowest in the culture treated with both 28-homoCS and DPU. However, regardless of the applied mixture of BRs with CKs, the considerable increase in cell number and the metabolite accumulation was found above the level obtained in cultures treated with any single phytohormone in unicellular green alga C. vulgaris. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Brassinosteroids Chlorophylls Cytokinins Growth Monosaccharides Proteins

1. Introduction Plant hormones such as abscisic acid, auxins, brassinosteroids (BRs), gibberellins and cytokinins (CKs) are signal molecules present in trace quantities and are actively involved in many biochemical processes (Ogweno et al., 2010). BRs are plant hormones that play an important role in the plant growth and development (Bajguz and Tretyn, 2003). BRs are hydroxylated derivatives of cholestane and their structural variations comprise

Abbreviations: BL, Brassinolide; BR, Brassinosteroid; CS, Castasterone; CK, Cytokinin; 6-deoxoCS, 6-Deoxocastasterone; 6-deoxo-3-DT, 6-Deoxo-3Dehydroteasterone; 6-deoxoTE, 6-Deoxoteasterone; 6-deoxo-TY, 6Deoxotyphasterol; DPU, 1,3-diphenylurea; 24-epiBL, 24-Epibrassinolide; 24-epiCS, 24-Epicastasterone; 28-homoBL, 28-Homobrassinolide; 28-homoCS, 28Homocastasterone; Kin, kinetin; TE, Teasterone; TY, Typhasterol; tZ, trans-Zeatin. * Corresponding author. Tel.: þ48 857457293. E-mail address: [email protected] (A. Bajguz). http://dx.doi.org/10.1016/j.plaphy.2014.04.009 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

substitution patterns on rings A and B as well as the C-17 sidechain. According to the B ring oxidation stage, BRs can be divided into 7-oxalactone, 6-ketone and non B-ring oxidized BRs. In general, 7-oxalactone BRs have higher biological activity than 6-ketone congeners, non-oxidized BRs have almost no activity in various bioassays. Since their discovery, over 70 BR compounds have been isolated from the plant kingdom (Bajguz and Tretyn, 2003; Bajguz, 2009a, 2011). BRs, e.g. BL and CS have been also identified in unicellular green algae Chlorella vulgaris (Bajguz and Asami, 2004; Bajguz, 2009a; Stirk et al., 2013). BRs are important plant growth regulators in multiple developmental processes at nanomolar to micromolar concentration, including ethylene biosynthesis, cell division and elongation, membrane hyperpolarisation, DNA, RNA and protein synthesis, enzyme activity, photosynthesis and the balance of other endogenous phytohormones in green alga and vascular plants (Bajguz and Czerpak, 1996a,b; Bajguz, 2000; Choudhary et al., 2012).

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The relationships between BRs and the other well-known plant hormones have been extensively explored (Arteca et al., 1988). Indeed, BRs are involved in a complex signaling network via a modulation of the levels and sensitivity of other phytohormones (Müssig et al., 2002). Some recent studies have identified the specific mechanisms of the coordinated action of BRs and several other phytohormones, including jasmonic acid, abscisic acid, gibberellic acid, auxin, and ethylene (Müssig et al., 2002). For example, BRs enhance the level of abscisic acid in green algae C. vulgaris (Trebouxiophyceae) cultures exposed to heat stress (Bajguz, 2009b). BRs elevated ethylene biosynthesis in etiolated seedlings of Arabidopsis thaliana by increasing the stability of ACC synthase, and the BR effects were additive with those of cytokinin (CK) (Hansen et al., 2009). Physiological activity of BRs is largely consistent with physiological influences exerted by auxins (Bajguz and PiotrowskaNiczyporuk, 2013). The signal transduction pathways of both phytohormones are connected and the physiological effects of these groups’ compounds are synergistic. For example, the application of IAA to C. vulgaris cultures stimulated the level of all detected endogenous BRs in C. vulgaris cells. Auxins cooperated synergistically with BRs stimulating algal cell proliferation and endogenous accumulation of proteins, chlorophylls and monosaccharides in C. vulgaris. The highest stimulation of algal growth and the contents of analyzed biochemical parameters were observed for the mixture of BL with IAA, whereas the lowest in the culture treated with both 28-homoCS and IBA (Bajguz and Piotrowska-Niczyporuk, 2013). However, many questions remain unclear about the mechanisms by which BRs interact with other groups of phytohormones. Unlike the cross-talk between BRs and the aforementioned hormones, the mechanisms of interplay between BRs and CKs are still obscure. Microarray data have demonstrated that Arabidopsis BR-deficient mutants cpd/cbb3, and dwf1-6 displayed altered transcript levels of the CKregulated genes ARR7 and ARR5; however, these genes showed inconsistent responses to BR treatment in wild-type plants (Müssig et al., 2002). Furthermore, a BR deficiency did not cause altered transcript levels of CK biosynthetic genes. The results of these studies imply that BR-CK interactions are possibly mediated through various metabolic pathways and can be integrated in a complex signaling network (Kudryakova et al., 2013). Results obtained by Ogweno et al. (2010) indicate that ABA, BR and CK stimulated photosynthesis and the activity of antioxidant enzymes in detached tomato leaves. CKs can be classified into two groups: adenine- and phenylurea-type, according to the chemical structure. Naturally occurring CKs are adenine derivatives with an isoprene (e.g. trans-zeatin, tZ) or a heterocyclic (kinetin, Kin) group at the N6position in the molecule. In addition, there is the class of the structurally unrelated synthetic phenylurea-type cytokinins. These phytohormones, such as 1,3-diphenylurea (DPU) are highly active in many CK bioassays, e.g. in C. vulgaris culture (Piotrowska and Czerpak, 2009). Synchronous and homogenous cell population of C. vulgaris is an especially promising experimental system for examining the relationships between phytohormones in green algae. C. vulgaris is distributed widely in freshwater and seawater and has a short growth cycle, which make it ideal for biochemical (protein, chlorophyll and monosaccharide content) studies and it can be used to directly observe phytohormone response at the cellular level, because the perception of signaling molecule and biochemical response takes place within the same cell under controlled conditions (Bajguz and Piotrowska-Niczyporuk, 2013). Soluble-protein content in green algae, an important indicator of reversible and irreversible changes in metabolism, is known to respond to a wide variety of plant growth regulators. Photosynthesis enhancement is

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an often observed feature in lower plant cells in response to BRs and CKs. For that reason, the changes in the accumulation of pigments involved in photosynthesis and the products of this process in 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 interaction of the BR and CK in regulation of phytohormone level in C. vulgaris cells and effect of these two groups of plant hormones on the 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) (Trebouxiophyceae) used in this study was obtained from the collection of the Institute of Biology at the University of Bialystok. The axenic cultures were cultivated for 48 h under controlled sterile conditions at 25  0.5  C. Illumination was supplied during a 16-h photoperiod (8-h dark period) by a bank of fluorescent lights yielding a photon flux 50 mmol m2 s1 at the surface of the tubes. Complete synchronization has been obtained by a regular change of light and dark periods according to the method of Pirson and Lorenzen (1966). The homogenous population of young synchronous cells was collected by centrifugation (1000 g, 10 min) and used for subsequent experiments. Synchronization of the culture was controlled by studying cell division and the diagrams of cell size distribution every day at the beginning of light period using a microscope. Growth of cultures was initiated by introduction of inoculums containing about 106 algal cells. The culture mineral medium used was modified mineral Knop’s medium. The pH of the medium was adjusted to 6.8 with 1 M NaOH. C. vulgaris cells were cultured in an Erlenmeyer flasks (500 mL) containing 250 mL medium and shaken in a rotary shaker. The effects of BRs and CKs on C. vulgaris growth and metabolite content were examined. The following exogenous BRs: brassinolide (BL), 24-epibrassinolide (24-epiBL), 28homobrassinolide (28-homoBL) from 7-oxalactonic type as well as castasterone (CS), 24-epicastasterone (24-epiCS), 28homocastasterone (28-homoCS) from 6-ketone type of BRs were used at concentration 10 nM. 1,3-Diphenylurea (DPU) representing of phenylurea-type CKs and adenine-type CKs: kinetin (Kin) and trans-zeatin (tZ) were applied at range of concentrations 100 pM e 100 mM. In co-application experiments BRs (BL, 24-epiBL, 28homoBL, CS, 24-epiCS or 28-homoCS) at concentration 10 nM were mixed with CKs (Kin, tZ or DPU) at range of concentration 100 pM e 100 mM. The algal cultures were conducted in six replications. 2.2. Number of cells For growth profile of the algal culture, 100 mL samples were collected after 48 h of cultivation. The number of cells was determined by direct counting of cells in the growth medium using a Bürker chamber (Lee and Low, 1993). 2.3. Brassinosteroid determination Determination of endogenous BR levels in the cells of C. vulgaris treated only with 10 nM tZ and in the control culture was performed on extracts with internal 2H standards, which is widely accepted as the most accurate method of BR determination (Bajguz, 2009a). Deuterium-labeled substrates used in this study were chemically synthesized: [2H6]BL, [2H6]CS, [2H6]TY, [2H6]TE, [2H6]6deoxoCS, [2H6]6-deoxoTY, and [2H6]6-deoxoTE. Lyophilized plant

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materials from C. vulgaris cultures were extracted with 300 mL of MeOHeCHCl3 (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 evaporation of the solvent in vacuo, the extract was partitioned between CHCl3 and water three times. The CHCl3 - soluble fraction was subjected to silica gel chromatography (Sep-Pak Vac Silica, 35 mL, Waters, Milford, MA, USA). The column was subsequently eluted with100 mL of CHCl3, 2% MeOH in CHCl3, and 7% (v/v) MeOH in CHCl3. Each 2% (v/ v) MeOH and 7% (v/v) MeOH fraction was purified by Sephadex LH20 column chromatography (column volume of 200 mL). The column was eluted with MeOHeCHCl3 (4:1). The effluents of elution volume/total column volume: 0.6e0.8 were collected as the BR fraction. 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) at a flow rate of 8 mL min-1 with the solvents 90% (v/v) acetonitrile for the 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 BL fraction (Rt 10e15 min), CS fraction (Rt 15e20 min), TE fraction(Rt 35e45 min), TY fraction (Rt 45e55 min), and 6-deoxoCS fraction (Rt 65e80 min), and HPLC purification from the 2% (v/v) MeOH fraction yielded a 6-deoxoTE fraction (Rt 55e65 min) and 6deoxoTY fraction (Rt 65e90 min). Each fraction was analyzed by GCeMS after derivatization. The presence of endogenous nonlabeled BRs was examined by GC-SIM-MS (JMS AX 505W Instrument JEOL, Tokyo) after suitable derivatization. The BRs content was quantified by the standard isotope-dilution method (Masslynx 4.1 software, Waters, Milford, MA, USA). 2.4. Determination of total chlorophylls The content of chlorophylls in C. vulgaris was determined according to Wellburn (1994) method in 48 h of culture. Algal fresh weight was homogenized in darkness using 99.9% methanol in the proportion 1:10 (w/v). Chlorophylls were extracted by heating in a water bath at 70  C for 30 min and centrifuged (1000 g, 10 min). The supernatant was used for the photosynthetic pigment determination. The absorbance of the extract was measured with a spectrophotometer at 652.4 and 665.2 nm for total chlorophylls and quantified.

3. Results 3.1. Growth of C. vulgaris in response to brassinosteroids and cytokinins To test which concentration of CKs was the most active, the number of algal cells after DPU, Kin and tZ treatments at range of concentrations 100 pM e 100 mM, were examined after 48 h of treatment (Fig. 1). Isoprene CK e tZ was the most active at concentration 10 nM stimulating cell number by 126%. Adeninetype CK with heterocyclic ring (Kin) was the most effective at 100 nM stimulating cell number by 106%. The highest increase in cell number in response DPU (71%) was observed at concentration of 1 mM. C. vulgaris exhibited sensitivity on CKs in the following order of their stimulating properties: tZ > Kin > DPU. Therefore, adenine-type CKs were characterized by higher biological activity in C. vulgaris culture in comparison with phenylurea-type CK (DPU). The preliminary experiments showed that exogenous BRs used at range of concentrations 1 fM e 1 mM (data not shown) were characterized by the highest biological activity in C. vulgaris at 10 nM inducing the increase in cell number by 53e178% in 48 h of cultivation. The stimulatory effect of BRs in green algae was arranged in the following order: BL > 24-epiBL > 28homoBL > CS > 24-epiCS > 28-homoCS (Fig. 1). Fig. 2 showing the interactions of BRs with CKs used at single concentration indicate that the highest stimulating properties was obtained by the co-application of 10 nM tZ with 10 nM BL which belongs to 7oxalactone type of BRs, where cell number increased by 382% in comparison with the control in 48 h of cultivation. 100 nM Kin mixed with 10 nM BL stimulated cell number by 362%, whereas the mixture of 1 mM DPU with 10 nM BL e by 326% in comparison with the control (Fig. 2). Among 6-ketone type of BRs, CS was shown to have the highest stimulating influence on algal cell proliferation inducing 310% increase in the number of cells. Kin and DPU used at range of concentrations 100 pM e 100 mM in combinations with different BRs such as BL, 24-epiBL, 28-homoBL, CS, 24-epiCS and 28-homoCS used at concentration of 10 nM showed weaker stimulating influence on algal growth expressed as the number of cells (Figs. 1 and 2).

2.5. Determination of water-soluble proteins The measurement of the content of proteins soluble in water was done after 48 h of cultivation by the method of Bradford (1976) calibrated with bovine serum albumin obtained from Sigma Chemical Co. (USA) as the standard. 2.6. Monosaccharide determination For monosaccharide’s concentration, 10 mL of the algal culture samples were first collected by centrifugation. Next, the monosaccharide content was estimated in 48 h of culture after extraction in ethanol according to the Somogyi (1954) method. 2.7. Replication and statistical analysis Each treatment consisted of 6 replicates and each experiment was carried out at least twice at different times. The data were analyzed by one-way analysis of variance (ANOVA) and the means were separated using Duncan’s multiple-range test (Statistica 6, StatSoft, USA). The level of significance in all comparisons was p < 0.05.

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

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Fig. 2. The effect of tZ, Kin and DPU in combination with BL on the growth expressed as cell number of Chlorella vulgaris after 48 h of cultivation. Treatment with at least one letter the same are not significantly different according to Duncan’s test.

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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 six independent experiments  SD.

3.2. Brassinosteroid content in C. vulgaris in response to tZ The application of exogenous 10 nM tZ representing adeninetype CK stimulated the accumulation of all identified endogenous BRs in C. vulgaris cells (Table 1) after 48 h of cultivation. The level of 6-deoxoTE increased by 30%, 6-deoxoTY by 34%, 6-deoxo-3-DT by 41%, and 6-deoxoCS by 32%, respectively in relation to the control culture. Therefore, the content of the total compounds of late C-6 oxidation pathway was stimulated by 32% in response to exogenous application of 10 nM tZ. Similarly, the concentration of the total compounds of early C-6 oxidation pathway was enhanced by 33% in tZ-treated cells. Among them, the level of TE was stimulated by 28%, TY by 27%, and CS by 36%. The increase by 46% in BL content was obtained 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 protein 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 observed to have the most stimulating effect (36%) on protein content in C. vulgaris. In response to Kin and DPU, the 29% and 22% increase in protein level was obtained. The stimulating effect of CKs on protein content was enhanced in the presence of BRs in the culture medium. For example, co-application of 10 nM tZ with 10 nM BL induced 2.5-fold increase in protein content in algal cells (Fig. 4). 6-

ketone type of BRs was characterized by lower stimulating properties in cells treated with exogenous CKs. Among them, CS at 10 nM combined with 10 nM tZ induced the highest (215%) increase in protein production in comparison with control (Fig. 3). In the contrast, the application of the mixtures of Kin or DPU with BRs to algal cells resulted in lower accumulation of soluble-proteins after 48 h of algal cultures. 3.4. Chlorophyll content in response to brassinosteroids and cytokinins The increase in the total content of chlorophylls by 51%, 35% and 28% 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 and 6). Higher biological activity was observed in case of BRs which induced 63e198% increase in photosynthetic pigment accumulation in comparison with the control. C. vulgaris possessed much

Table 1 The effect of tZ on the brassinosteroid level in C. vulgaris cells after the 48 h of culture. Treatment with at least one letter the same are not significantly different according to Duncan’s test. Brassinosteroid content (fg cell1) Brassinosteroid

Cytokinin treatment

6-Deoxoteasterone (6-deoxoTE) 6-Deoxo-3-Dehydroteasterone (6-deoxo-3-DT) 6-Deoxotyphasterol (6-deoxoTY) 6-Deoxocastasterone (6-deoxoCS) Teasterone (TE) Typhasterol (TY) Castasterone (CS) Brassinolide (BL)

0.196 0.134 0.173 0.294 0.245 0.319 0.447 0.124

10 nM tZ        

0.07a 0.09b 0.07b 0.03c 0.05c 0.08d 0.02e 0.05f

Control 0.151 0.095 0.129 0.223 0.191 0.251 0.329 0.085

       

0.01g 0.04f 0.08b 0.05c 0.01h 0.06i 0.08j 0.07k

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

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

higher content of total chlorophylls in response to the BRs combined with CKs after 48 h of cultivation. Among 7-oxalactone type of BRs, 10 nM BL applied together with 10 nM tZ induced 339%, whereas 10 nM CS belonging to 6-ketone type of BRs, combined with 10 nM tZ induced 232% increase in chlorophyll level in algal cells after 48 h of cultivation. Therefore, the strong synergistic relationships between BRs and CKs on photosynthetic apparatus in C. vulgaris were observed. 3.5. Monosaccharide content in response to brassinosteroids and cytokinins BRs interact with CKs by affecting monosaccharide accumulation in C. vulgaris culture (Figs. 7 and 8) after 48 h of cultivation. The significant increase in monosaccharide content by 50%, 33%, and 29% in C. vulgaris cells treated with 10 nM tZ, 100 nM Kin and 1 mM DPU, respectively was observed in experiments after 48 h of cultivation. However, exogenous CKs increased biological activity of

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

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

BRs because algal cells treated with 10 nM BL and 10 nM tZ contained about 3 times as many sugars as the cells of the control culture (Fig. 8). Among BRs belonging to 6-ketone type, CS coapplied with tZ was characterized by the highest biological activity inducing the increase in sugar content by 169% in comparison with the control. In contrast, adenine-type CK with heterocyclic ring Kin and phenylurea-type CK (DPU) were characterized by lower stimulating properties in interaction with BRs in C. vulgaris. The treatment algal cultures with BRs without CKs possessed weaker stimulating effect on monosaccharides. For example, under the influence of BL their content was stimulated by 189% and in response 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 endogenous hormonal 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 monosaccharide content in Chlorella vulgaris after 48 h of cultivation. Treatment with at least one letter the same are not significantly different according to Duncan’s test.

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that CKs actively influence the content of different hormones in plants. There are numerous examples with different plant species providing evidences for their ability to affect hormonal balance in plants (Yuldashev et al., 2012). It is widely accepted that the hormone level at any given site might be affected by the relative rates of its synthesis, degradation, inactivation, and transport within the plant. Each of these factors can be considered in terms of their input to, or output from, the level of free hormone (Piotrowska and Bajguz, 2011). The previous study demonstrated that the level of seven BRs, including TE, TY, 6-deoxoTE, 6-deoxoTY, 6-deoxoCS, CS and BL is stimulated by auxin (IAA) in wild-type C. vulgaris cells (Bajguz and Piotrowska-Niczyporuk, 2013). All compounds belong to the BR biosynthetic pathway. Similarly, our present results indicate that exogenous 10 nM tZ representing adenine-type CK stimulates the accumulation of all identified endogenous BRs by 27e46% in C. vulgaris cells such as: 6-deoxoTE, 6-deoxoTY, 6deoxoCS from early C-6 oxidation pathways, TE, TY and CS from late C-6 oxidation pathways and BL. The results indicate that tZ is characterized by higher properties of activation BR biosynthesis in the cells of green alga in comparison with auxin (IAA) (Bajguz and Piotrowska-Niczyporuk, 2013). This is the first evidence on the stimulating effect of CKs on the contents of phytohormones involved in BR biosynthetic pathway in unicellular green alga C. vulgaris. The findings suggest a possibility that CKs regulate directly the biosynthesis and accumulation of BRs in lower plants. A straightforward route of hormone pathway interaction would be modulation of the biosynthesis of one hormone by the other, which has been repeatedly observed in vascular plants (Hardtke et al., 2007; Hansen et al., 2009; Yuldashev et al., 2012; Kudryakova et al., 2013). For example, fast and stable 2-fold accumulation of CKs was detected initially in roots and then in shoots of 4-day-old wheat (Triticum aestivum) seedlings in the course of their treatment with 0.4 mM 24-epiBL (Yuldashev et al., 2012). Another study suggests that the exogenous application of BRs may elevate the CK content in A. thaliana plant (Kudryakova et al., 2013). Moreover, evidence suggest that both BRs and CKs additively promote ethylene biosynthesis by increasing the stability of a subset of ACC synthases (Hansen et al., 2009). Our data support the idea that unicellular green algae possess a complicated network of BR-CK interactions. It can be speculated that CKs may stimulate BR activity in C. vulgaris by inducing the accumulation of endogenous BRs. However, it should be mentioned that more extensive investigations are required to confirm the working scenario. 4.2. Growth of C. vulgaris in response to brassinosteroids and cytokinins The most characteristic responses elicited by BRs and CKs comprise 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-effect relationships showed that the most optimal concentration for tZ was 10 nM, for Kin e 100 nM, whereas phenylurea-type cytokinin (DPU) was the most biologically active at 1 mM in C. vulgaris culture after 48 h of treatment. Green alga exhibited sensitivity on CKs in the following order of their stimulating properties: Z > Kin > DPU. The stimulatory effect of 10 nM BRs on the cell number in green algae was arranged in the following order: BL > 24-epiBL > 28homoBL > CS > 24-epiCS > 28-homoCS. Our results confirm that BRs are growth-promoting steroidal hormones (Sasse, 1985; Bajguz and Czerpak, 1998; Clouse and Sasse, 1998; Nakaya et al., 2002). However, they induce biological responses not only by itself, but also by interaction with CKs in the promoting of cell division in C. vulgaris culture. Moreover, we suggest that CKs interact

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synergistically with BRs in stimulating algal growth, i.e. when the two hormones were included in the same incubation medium, the number of algal cells was much higher than the sum of that induced by each hormone. Previous results confirm that auxins may also enhance stimulating properties of BRs on algal growth expressed as the number of cells (Bajguz and Piotrowska-Niczyporuk, 2013). Control of cell division in vascular plants, along with cell expansion, by BRs in the presence of other phytohormones is well known. Two studies (Bach et al., 1991; Gaudinová et al., 1995) have indicated that BR treatment alters the endogenous auxin and cytokinin levels in tobacco callus cultures, thus influencing the control of cell division. For example, in the sequential response to plant growth regulators in young elongating tissue from peas and wheat the peak of sensitivity to 24-epiBL (1 mM) occurs after CK application in isolated wheat (Triticum vulgare) coleoptiles aged from 21 to 96 h (Sasse, 1985). BRs have been shown to stimulate cell division in the presence of auxin in cultured parenchyma cells of Helianthus tuberosus (Clouse and Sasse, 1998). Furthermore, BRs stimulated the cell increase ratio for fresh weight the in cultures Onsoma paniculatum only in the presence of auxin (IAA) and aromatic CK (benzyladenine) belonging to adenine-type CKs by 248e 262% (Yang et al., 1999) Thus, it seems possible that the presence of CKs widens the activity spectrum of BRs stimulating the growth of C. vulgaris culture. 4.3. Protein content in response to brassinosteroids and cytokinins Soluble-protein content in plants, an important indicator of reversible and irreversible changes in metabolism, is known to respond to a wide variety of phytohormones (Bajguz and Piotrowska-Niczyporuk, 2013). The application of BRs and CKs alone to culture medium enhanced the protein level in C. vulgaris cells. The stimulating effect of BRs on protein content was enhanced in the presence of CKs in the culture medium. Our results are in agreement with Yang et al. (1999) indicating the increase in the level of soluble proteins in cultured O. paniculatum cells in response to BL and benzyladenine. High protein content, increased nitrate reductase and carbonic anhydrase activities was also observed in leaves of Vigna radiata sprayed with Kin and 28-homoBL (Fariduddin et al., 2004). Similarly, exogenously applied auxins enhanced the stimulating properties of BRs on the protein accumulation in green alga C. vulgaris cells (Bajguz and PiotrowskaNiczyporuk, 2013). In contrast, the addition of BR or CK alone slightly increased the content of proteins. Probably, the increase in protein level may be induced by elevated and potentially prolonged expression of target genes in response to the addition of both hormones. Previous studies conducted on the alga C. vulgaris confirmed the stimulating role of BRs and CKs (applied alone) in the enhancement of the content of nucleic acids and proteins (Bajguz and Czerpak, 1996a; Bajguz, 2000; Piotrowska and Czerpak, 2009). The shortening of the developmental cycle and the increase by two or three times in DNA, RNA and protein level during 36 h cultivation of the algae suggest an unusual increase in the rate of the processes of transcription and translation (Bajguz and Czerpak, 1996a; Bajguz, 2000; Piotrowska and Czerpak, 2009). The results of study regarding the interaction of the BR and CK signaling pathways indicate that BRs mixed with CKs may activate the synthesis of many proteins and involved in metabolism of vascular plants (Zubo et al., 2008, 2011; Kudryakova et al., 2013). 4.4. Chlorophyll content in response to brassinosteroids and cytokinins Photosynthesis enhancement is an often observed feature in plant cells in response to BRs and CKs (Fariduddin et al., 2004; Xia

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et al., 2009; Jiang et al., 2012). Therefore, the changes in the level of pigments involved in photosynthesis were studied in C. vulgaris cells in response to these two groups of phytohormones. The increase in the total content of chlorophylls in algal cells exposed to CKs without BRs and BRs without CKs was observed in 48 h of algal cultivation. On the other hand, C. vulgaris possessed much higher content of total chlorophylls in response to the BRs combined with CKs. Similarly, previous studies indicate that BRs cooperated synergistically with exogenous auxins in stimulation of chlorophyll content in C. vulgaris cultures (Bajguz and Piotrowska-Niczyporuk, 2013). High accumulation of chlorophyll level and increase in net photosynthesis rate was also observed in leaves of V. radiata sprayed with Kin and 28-homoBL (Fariduddin et al., 2004). The obtained results confirm the stimulating effect of BRs on photosynthesis 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 positive effect of photosynthetic apparatus in leaves of young plants of cucumber (Cucumis sativus) (Jiang et al., 2012). Stimulating effect of phenylurea- and adenine-type CKs on photosynthetic pigments in algal cells is well known (Czerpak and Bajguz, 1997; Piotrowska and Czerpak, 2009). CK treatment abolishes the lag phase in chlorophyll synthesis and accelerates its rate, inhibits chlorophyll breakdown by chlorophyllase, Mg-dechelatase and peroxidase-linked chlorophyll bleaching in higher plants (Costa et al., 2005). 4.5. Monosaccharide content in response to brassinosteroids and cytokinins An increase in chlorophyll content in C. vulgaris under the influence of BRs in combination with CKs correlates well with higher accumulation of monosaccharides. All adenine- (tZ, Kin) and phenylurea-type (DPU) CKs interacted with BRs (BL, 24-epiBL, 28homoBL, CS, 24-epiCS, 28-homoCS) in stimulation of sugar content in algal cells. This response is similar to the synergistic responses previously reported (Bajguz and Piotrowska-Niczyporuk, 2013) between BRs and auxins in the culture of C. vulgaris. Obtained results are comparable with that reported for a number of higher plant tissues and green algae, indicating that BRs and CKs influence accumulation of carbohydrates (Bajguz and Czerpak, 1996a,b; Piotrowska and Czerpak, 2009). Exogenous CKs may increase biological activity of BRs because algal cells treated with BL and tZ at 10 nM contained the highest level of monosaccharides in comparison with the control culture. The stimulating effect of BRs and CKs on sugar content is well known. Microarray and photosynthesis analysis of transgenic plants T. aestivum and Oryza sativa with enhanced BR biosynthetic pathway revealed evidence of enhanced CO2 assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed (Wu et al., 2008). A broad stimulating effect of CKs on monosaccharide level has been also showed in Beta vulgaris, when cytokinins induced 9fold increase in glucose, fructose, galactose and ribose content (Ivic et al., 2001). 5. Conclusion The data presented in this paper suggest that exogenously applied CKs may change the phytohormone balance in C. vulgaris cells increasing the level of all identified endogenous BRs. Coapplication experiments suggest a novel relationships between BRs and CKs in unicellular green alga. This interaction is synergistic because the effect of two hormones applied simultaneously exceeds the sum of each effect on algal growth and metabolite content. We have demonstrated that BR induced response of algal cells is enhanced by exogenous CKs several fold. This is the first evidence

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