Interactive effect of brassinosteroids and cytokinins

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 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).
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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
177
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
178
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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.
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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.
179
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.
180
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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.
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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
181
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
182
A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 80 (2014) 176e183
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
demonstrating a synergistic relationship of BRs and CKs in lower
plants. Nonetheless, our work provides an important starting point
for future dissection of what appears to be complex mechanisms of
BR-CK cross-talks in plants.
Contribution
In this paper we have the same contribution.
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