Plant Physiology and Biochemistry 71 (2013) 290e297

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Plant Physiology and Biochemistry

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Research article

Synergistic effect of auxins and brassinosteroids on the growth and

regulation of metabolite 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: The relationships between brassinosteroids (BRs) (brassinolide, BL; 24-epiBL; 28-homoBL; castasterone,
Received 5 July 2013 CS; 24-epiCS; 28-homoCS) and auxins (indole-3-acetic acid, IAA; indole-3-butyric acid, IBA; indole-3-
Accepted 8 August 2013 propionic acid, IPA) in the regulation of cell number, phytohormone level and metabolism in green
Available online 20 August 2013
alga Chlorella vulgaris were investigated. Exogenously applied auxins had the highest biological activity
in algal cells at 50 mM. Among the auxins, IAA was characterized by the highest activity, while IBA e by
Keywords:
the lowest. BRs at 0.01 mM were characterized by the highest biological activity in relation to auxin-
Auxins
treated and untreated cultures of C. vulgaris. The application of 50 mM IAA stimulated the level of all
Brassinosteroids
Chlorophylls
detected endogenous BRs in C. vulgaris cells. The stimulatory effect of BRs in green algae was arranged in
Growth the following order: BL > 24-epiBL > 28-homoBL > CS > 24-epiCS > 28-homoCS. Auxins cooperated
Monosaccharides synergistically with BRs stimulating algal cell proliferation and endogenous accumulation of proteins,
Proteins chlorophylls and monosaccharides in C. vulgaris. The highest stimulation of algal growth and the con-
tents 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. However, regardless of the applied mixture
of BRs with auxins, the considerable increase in cell number and the metabolite accumulation was found
above the level obtained in cultures treated with any single phytohormone. Obtained results confirm that
both groups of plant hormones cooperate synergistically in the control of growth and metabolism of
unicellular green alga C. vulgaris.
Ó 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction
Brassinosteroids (BRs) are hydroxylated derivatives of choles-
tane and their structural variations comprise substitution patterns
on rings A and B as well as the C-17 side-chain. According to 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, nonoxidized
BRs reveal almost no activity in various bioassays. Since their
discovery, over 70 BR compounds have been isolated from the
Abbreviations: AX, auxin; BL, brassinolide; BR, brassinosteroid; CS, castasterone;
4-Cl-IAA, 4-chloroindole-3-acetic acid; 6-deoxoTE, 6-deoxoteasterone; 6-deoxo-TY,
6-deoxotyphasterol; 24-epiBL, 24-epibrassinolide; 24-epiCS, 24-epicastasterone;
28-homoBL, 28-homobrassinolide; 28-homoCS, 28-homocastasterone; IAA, indole-
3-acetic acid; IBA, indole-3-butyric acid; IPA, indole-3-propionic acid; TE, teaster-
one; TY, typhasterol.
* Corresponding author.
E-mail address: abajguz@uwb.edu.pl (A. Bajguz).
plant kingdom [1e3]. BRs have been also identified in microalgae
[2,4]. BRs are important plant growth regulators in multiple
developmental processes at nanomolar to micromolar concentra-
tion, including cell division, cell elongation, vascular differentia-
tion, reproductive development and modulation of gene
expression. They also influence various other developmental pro-
cesses like germination of seeds, rhizogenesis, flowering, senes-
cence, abscission and maturation. BR application includes ethylene
biosynthesis, membrane hyperpolarisation, enhanced DNA, RNA
and protein synthesis, increased invertase activity, stimulation of
photosynthetic activity and changes in the balance of other
endogenous phytohormones [5e8]. BRs stimulate plant growth
under unfavorable conditions, such as salinity, low and high
temperature, drought, heavy metals action or nutrient deficiency.
BRs are not only implicated in plant response to abiotic stresses
but also have medicinal applications. At present, our knowledge of
the effects of BRs in animals or human is still rather fragmentary.
However, it is known that BRs have an anabolic action, anticancer,
antiproliferative properties [9,10].

0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2013.08.003
 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297 291

The relationships between BRs and the other well-known plant Table 1
hormones have been extensively explored. BR can induce ethylene The effect of 50 mM IAA on the brassinosteroid level in C. vulgaris cells after 48 h of
cultivation. Treatment with at least one letter the same are not significantly different
production [11,12] and enhance the level of abscisic acid in according to Duncan’s test. Data are the means of six independent
Chlorella vulgaris cultures exposed to heat stress [13]. Physiological experiments
SD.
activity of BRs is largely consistent with physiological influences
Brassinosteroid content (fg cell1)
exerted by auxins [14]. The signal transduction pathways of both
phytohormones are connected and the physiological effects of Brassinosteroid Auxin treatment

these groups’ compounds are synergistic. It has been shown that 50 mM IAA Control
both growth substances promote segment elongation, increase 6-Deoxoteasterone 0.175
0.03a 0.151
0.01g
fresh weight and electrogenic proton extrusion in Vigna angularis 6-Deoxotyphasterol 0.135
0.07b 0.129
0.08b
epicotyls [15] and ethylene production [11]. BRs synergistically 6-Deoxocastasterone 0.241
0.09c 0.223
0.05c
increase seedling sensitivity to auxin and show that combined Teasterone 0.213
0.06c 0.191
0.01h
Typhasterol 0.267
0.01d 0.251
0.06i
treatment with both hormones can increase the magnitude and Castasterone 0.339
0.02e 0.329
0.08j
duration of gene expression [16]. Probably, BR and auxin signaling Brassinolide 0.098
0.05f 0.085
0.07k
pathways converge at the level of the transcriptional regulation of
the common target genes. For example, BRs have been shown to
regulate the expression of PIN genes, which encode essential
2.2. Growth of C. vulgaris in response to auxins and
components in the polar transport of auxin. Similarly, auxins may
brassinosteroids
have a role in the control of gene expression involved in BR
signaling [17].
To test which concentration of auxin was the most active, the
In Arabidopsis thaliana, auxin and BRs interact synergistically
number of algal cells after IAA, IBA and IPA treatments at range of
in lateral root development and hypocotyl elongation [17,18]. In
concentrations 1e100 mM, were examined (Fig. 1). All auxins tested
addition, it has been reported that subnanomolar concentrations
were the most effective in induction of culture growth after 48 h of
of brassinolide (BL) promote primary root elongation in
treatment at concentration of 50 mM with an increase in the
conjunction with auxin [19]. BRs operate by stimulating auxin
number of cells by 52e83%. C. vulgaris exhibited sensitivity on
transport from the junction between the root and the hypocotyl,
auxins in the following order of their stimulating properties:
towards the root tip, which explains why lateral roots are
IAA > IPA > IBA. The preliminary experiments showed that exog-
induced upon treatment with BR [18]. However, many questions
enous BRs used at range of concentrations 1 fMe1 mM (data not
remain unclear about the mechanisms by which BRs interact
shown) were characterized by the highest biological activity in
with auxins in lower plants, such as unicellular green alga C.
C. vulgaris at 1 nM inducing the increase in cell number by 53e178%
vulgaris which contain BRs [2,4] and auxins [20]. Synchronous
in 48 h of cultivation. The stimulatory effect of BRs in green algae
and homogenous cell population of C. vulgaris is an especially
was arranged in the following order: BL > 24-epiBL > 28-
promising experimental system for examining the relationships
homoBL > CS > 24-epiCS > 28-homoCS. The highest stimulating
between phytohormones in green algae. C. vulgaris is distributed
properties was obtained by the co-application of 50 mM IAA with
widely in freshwater and seawater and has a short growth cycle,
1 nM BL which belongs to 7-oxalactone type of BRs, where cell
which make it ideal for biochemical studies and it can be used to
number increased by 321% in comparison with the control in 48 h
directly observe phytohormone response at the cellular level,
of cultivation. Among 6-ketone type of BRs, CS was shown to have
because the perception of signaling molecule and biochemical
the highest stimulating influence on algal cell proliferation
response takes place within the same cell under controlled
inducing 202% increase in the number of cells (Fig. 2A, B, C). IBA and
conditions [7,9,13].
IPA used at range of concentrations 1e100 mM in combinations with
The aim of our study was to determine the level of endogenous
BRs in unicellular green alga C. vulgaris treated with exogenous
auxins. The part of the aim of study was to test how the length of
the auxin side chain affects on biological activity of C. vulgaris.
The nature of interaction of selected BRs with auxins in regulation
of algal growth expressed as cell number and the contents of
chlorophylls, proteins as well as monosaccharides were also
examined.

2. Results

2.1. Brassinosteroid content in C. vulgaris

Exogenous IAA stimulated the accumulation of endogenous

BRs in C. vulgaris cells (Table 1). The level of 6-deoxoTE increased
by 16%, 6-deoxoTY by 5% and 6-deoxoCS by 8%, respectively in
relation to the control culture. Therefore, the content of the total
compounds of early C-6 oxidation pathway was stimulated by
11% in response to exogenous application of IAA. On the other
hand, the concentration of the total compounds of late C-6
oxidation pathway was enhanced by 7% in IAA-treated cells.
Among them, the level of TE was stimulated by 11.5%, TY by 6%,
and CS only by 3% (not statistically significant). The increase Fig. 1. The comparative effect of auxins (AX) with the brassinosteroids (BR) on the
by 15% in BL content was obtained in algal cells exposed to growth expressed as cell number of Chlorella vulgaris. Data are the means of six in-
50 mM IAA. dependent experiments
SD.
292 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297
Fig. 2. The effect of IAA (A), IPA (B) and IBA (C) in combination with BL and CS on the growth expressed as cell number of Chlorella vulgaris. Treatment with at least one letter the
same are not significantly different according to Duncan’s test.
different BRs such as BL, 24-epiBL, 28-homoBL, CS > 24-epiCS and
28-homoCS used at concentration of 1 nM showed weaker stimu-
lating influence on algal growth (Fig. 2).
2.3. Protein content in response to auxins and brassinosteroids
The application of BRs alone to culture medium enhanced
protein level in C. vulgaris cells by 33e133% in relation to the
control (Fig. 3). Among auxins, 50 mM IAA was observed to have
the most stimulating effect (57%) on protein content in C. vulgaris.
In response to IPA and IBA, the 38% and 13% increase in protein
level was obtained. The stimulating effect of auxins on protein was
enhanced in the presence of BRs in the culture medium. For
example, co-application of 50 mM IAA with 1 nM BL induced 3-fold
increase in protein content in algal cells. 6-Ketone type of BRs was
characterized by lower stimulating properties in cells treated with
exogenous auxins. Among them, CS at 1 nM combined with 50 mM
IAA induced the highest (211%) increase in protein production in
comparison with control (Fig. 4A, B, C). In the contrast, the
application of the mixtures of IPA or IBA with BRs to algal cells
resulted in lower accumulation of soluble-proteins in 48 h of algal
cultures.
Fig. 3. The comparative effect of auxins (AX) with the brassinosteroids (BR) on the
protein content in Chlorella vulgaris. Data are the means of six independent
experiments
SD.
2.4. Chlorophyll content in response to auxins and brassinosteroids
The increase in the total content of chlorophylls by 80%, 57% and
30% in algal cells exposed to 50 mM IAA, IPA and IBA, respectively
was obtained in 48 h of algal cultivation. Higher biological activity
was observed in case of BRs which induced 63e198% increase in
photosynthetic pigment accumulation in comparison with the
control (Fig. 5). C. vulgaris possessed much higher content of total
chlorophylls in response to the BRs combined with auxins. Among
7-oxalactone type of BRs, BL applied together with IAA induced
378%, whereas CS belonging to 6-ketone type of BRs, combined
with IAA induced 283% increase in chlorophyll level in algal cells in
48 h of cultivation (Fig. 6A, B, C). Therefore, the strong synergistic
relationships between BRs and auxins on photosynthetic apparatus
in C. vulgaris were observed.
2.5. Monosaccharide content in response to auxins and
brassinosteroids
BRs interact with auxin by affecting sugar accumulation in
C. vulgaris culture (Fig. 7). The significant increase in mono-
saccharide content by 72%, 51%, and 24% in C. vulgaris cells treated
with IAA, IPA and IBA, respectively was observed in experiments.
However, exogenous auxins increase biological activity of BRs
because algal cells treated with BL and IAA at 50 mM contained
about 3.5 times as many monosaccharides as the cells of the control
culture. Among BRs belonging to 6-ketone type, CS co-applied with
IAA was characterized by the highest biological activity inducing
the increase in sugar content by 273% in comparison with the
control (Fig. 8A, B, C). In contrast, IPA and IBA were characterized by
lower stimulating properties in interaction with BRs in C. vulgaris.
The treatment algal cultures with BRs without auxins 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.
3. Discussion
3.1. Brassinosteroid content in C. vulgaris
BRs are unique steroidal phytohormones widely distributed
throughout the plant kingdom. They exhibit a broad spectrum of
physiological functions of plant growth and development in
vascular plants as well as unicellular green algae [7,9,10,13]. The
importance of BRs in such a diverse range of developmental pro-
cesses implies the existence of mechanisms that strictly control the
 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297
Fig. 4. The effect of IAA (A), IPA (B) and IBA (C) in combination with BL and CS on the protein content in Chlorella vulgaris. Treatment with at least one letter the same are not
significantly different according to Duncan’s test.
endogenous BR levels and their distribution in the target cells or
tissues. It is widely accepted that the hormone level at any given
site might be affected by the relative rates of its synthesis,
destruction, 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 [21].
The previous study demonstrated that seven BRs, including TE,
TY, 6-deoxoTE, 6-deoxoTY, 6-deoxoCS, CS and BL occur in wild-type
C. vulgaris cells [2]. All compounds belong to the BR biosynthetic
pathway. Despite the importance of BRs in algal biology, the signal
that initiates BR biosynthesis remains unknown. However, present
results indicate that exogenous IAA stimulates the accumulation of
endogenous BRs by 5e16% in C. vulgaris cells such as: 6-deoxoTE, 6-
deoxoTY, 6-deoxoCS from early C-6 oxidation pathways, TE, TY and
CS from late C-6 oxidation pathways and BL. This is the first evi-
dence on the stimulating effect of auxins on the contents of phy-
tohormones involved in BR biosynthetic pathway in unicellular
green alga C. vulgaris. The findings suggest a possibility that auxins
regulate directly the biosynthesis and accumulation of BRs in
C. vulgaris.
A straightforward route of hormone pathway interaction would
be modulation of the biosynthesis of one hormone by the other,
Fig. 5. The comparative effect of auxins (AX) with the brassinosteroids (BR) on the
chlorophyll content in Chlorella vulgaris. Data are the means of six independent
experiments
SD.
293
which has been repeatedly observed in vascular plants. For
example, both auxin and BRs promote ethylene biosynthesis [22].
In many instances, such as microtubule rearrangement or tropisms,
ethylene can even largely replace auxin or BR [23]. With respect to
auxin biosynthesis, the consensus suggests that auxin levels do not
change dramatically in response to BR [18]. Moreover, consistent
with the idea that BR does not enhance auxin responses through
promoting auxin biosynthesis, which could have explained the
slow kinetics of the BR response, BR still enhances physiological
responses in auxin-saturated backgrounds [17]. However, BR
biosynthesis genes are under negative feedback control [24,25],
which is thought to aid in maintaining BR levels at a homeostatic
level [26e28]. Furthermore, it remains possible that BR-auxin
cross-talk involves auxin action through its regulation of BR accu-
mulation or sensitivity. Our data support the idea that plants
possess a complicated network of auxin-BR interactions, which
modulates the balance between the BR and auxin level through
feedback control mechanisms. However, it should be mentioned
that more extensive investigations are required to confirm the
working scenario.
3.2. Growth of C. vulgaris in response to auxins and
brassinosteroids
Auxins and brassinosteroids are essential phytohormones that
synergistically regulate physiological and developmental processes
in plants such as cell division [29,30]. All auxin tested were the
most effective in induction of culture growth after 48 h of treat-
ment at concentration of 50 mM. C. vulgaris exhibited sensitivity on
auxins in the following order of their stimulating properties:
IAA > IPA > IBA. The stimulatory effect of 0.01 mM BRs on the cell
number in green algae was arranged in the following order:
BL > 24-epiBL > 28-homoBL > CS > 24-epiCS > 28-homoCS. Our
results confirm that BRs are growth-promoting steroidal hormones.
However, they induce biological responses not only by itself, but
also by interaction with auxins in promoting of cell division in
C. vulgaris culture. Moreover, we suggest that auxins cooperate
synergistically with BRs 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.
Control of cell division in vascular plants, along with cell
expansion, by BRs in the presence of auxin is well known [30]. BRs
have been shown to stimulate cell division in the presence of auxin
in cultured parenchyma cells of Helianthus tuberosus [22].
Furthermore, BRs stimulated the growth only in the presence of
294 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297
Fig. 6. The effect of IAA (A), IPA (B) and IBA (C) in combination with BL and CS on the chlorophyll content in Chlorella vulgaris. Treatment with at least one letter the same are not
significantly different according to Duncan’s test.
auxin in some plant species, stimulating a debate on auxin-
dependency of brassinosteroid activities in the stimulation of cell
elongation and proliferation [22,29,31]. The most convincing evi-
dence for a role of BR in cell division is provided by a study that
identified CYCLIN D3 (CYCD3) as a target of BR-induced transcrip-
tion [32,33]. Thus, the presence of BR widens the activity spectrum
of auxin. Moreover, auxin and BRs share a number of early target
genes, many of which are involved in growth related processes [16].
Therefore, the auxin treatments have dramatic BR effects on growth
response in C. vulgaris culture.
3.3. Protein content in response to auxins and brassinosteroids
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. The application of
auxins and BRs alone to culture medium enhanced the protein level
in C. vulgaris cells. The stimulating effect of auxins on protein
content was enhanced in the presence of BRs in the culture me-
dium. Our results are in agreement with [34] indicating the in-
crease in the level of soluble proteins in cultured Onsoma
paniculatum cells in response to BL and IAA. In contrast, the addi-
tion of BR or auxin alone slightly increased the content of proteins.
Fig. 7. The comparative effect of auxins (AX) with the brassinosteroids (BR) on the
monosaccharide content in Chlorella vulgaris. Data are the means of six independent
experiments
SD.
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 in the enhancement of the content of
nucleic acids and proteins. The shortening of the developmental
cycle and the increase by two or three times of its efficiency
during 36 h cultivation of the algae suggest an unusual increase in
the rate of the processes of transcription and translation. At the
same time a diversified impact of BRs, depending on their chem-
ical structure, upon the content of DNA, RNA and proteins, was
demonstrated [7]. Moreover, other studies indicated that auxins
may stimulate brassinosteroid perception by regulating the level
of brassinosteroid receptor in rice. Auxin treatment increased
expression of the brassinosteroid receptor gene BRI1 in A. thaliana.
The promoter of BRI1 has an auxin-response element that is tar-
geted by auxin-response factor transcription factors. Auxin pre-
treatment increased the sensitivity to BRs of brassinosteroid-
responsive genes. For example, auxins control the degree of
brassinosteroid perception by regulating the expression of gene
for BR receptor [35,36].
3.4. Chlorophyll content in response to auxins and brassinosteroids
Photosynthesis enhancement is an often observed feature in
plant cells in response to BRs and auxins. For that reason,
changes in the production of pigments involved in photosyn-
thesis were studied under the influence of auxins and BRs. The
increase in the total content of chlorophylls in algal cells
exposed to auxins without BRs and BRs without auxins 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 auxins. The obtained
results confirm the stimulating effect of BRs on photosynthesis
and photosynthetic pigment contents in vascular plants and
unicellular algae [6,37]. BRs could prevent loss of photosynthetic
pigments by an activation of enzymes participating in chloro-
phyll biosynthesis or an induction of their synthesis. For
example, BRs promote photosynthesis and growth by positively
regulating synthesis and activation of a variety of photosyn-
thetic enzymes including Rubisco in Cucumis sativus [37] and
C. vulgaris [6]. Another study indicated that BRs may cooperate
with other phytohormones in regulation of photosynthetic
apparatus. Genome-wide analysis suggest that BRs and gibber-
ellins are responsible for stimulation of the expression of light-
regulated genes and genes involved in cell wall synthesis and
photosynthesis/chloroplast function [38].
 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297
Fig. 8. The effect of IAA (A), IPA (B) and IBA (C) in combination with BL and CS on the monosaccharide content in Chlorella vulgaris. Treatment with at least one letter the same are
not significantly different according to Duncan’s test.
3.5. Monosaccharide content in response to auxins and
brassinosteroids
The stimulation of the chlorophyll content in response to BRs
with or without auxins may probably enhance the photosynthesis
process contributing significantly to the production of mono-
saccharides which are building substances for plant and play a key
role as source of energy [6]. In this study, we demonstrated that
BRs interact with auxin by affecting sugar accumulation in
C. vulgaris culture. This finding is comparable with that reported
for a number of higher plant tissues and green algae, indicating
that BRs and auxins influence accumulation of carbohydrates [5,6].
Exogenous auxins increase biological activity of BRs because algal
cells treated with BL and IAA at 50 mM contained the highest level
of monosaccharides in comparison with the control culture. The
stimulating effect of BRs and auxins on sugar content is well
known. Microarray and photosynthesis analysis of transgenic
plants Triticum aestivum and Oryza sativa with enhanced BR
biosynthetic pathway revealed evidence of enhanced CO2 assimi-
lation, enlarged glucose pools in the flag leaves, and increased
assimilation of glucose to starch in the seed [39]. The stimulating
effect of BRs on the level of monosaccharides in C. vulgaris cells
may be enhanced by exogenous auxin application.
4. Conclusion
The data presented in this paper suggest that all BRs work in a
synergistic manner with the active auxins tested (IAA, IBA, and
IPA) in promoting algal growth and metabolite accumulation.
Auxins increase the activity of BRs in stimulation of algal growth
and metabolite accumulation. The stimulatory effect of auxins on
biochemical parameters was arranged in the following order
IAA > IPA > IBA. Our results confirm previous studies indicating
that among auxins IAA represents the highest biological activity in
algal cultures [40]. In the case of auxins, a decisive influence on
their activity is exerted upon carboxyl group in the aliphatic chain
with a strong negative charge, which is located at a distance of
54e55 nm from the aromatic ring with a positive charge in the
center. This electrical and structural combination of molecules in
phytohormone compound is necessary to demonstrate the rela-
tion to specific cell receptors of plants and induction characteristic
auxin response [41]. The lengthening of the aliphatic chain at the
indole ring of auxin causes decreasing the metabolic activity.
Therefore IAA possesses higher activity in microalgae in com-
parison with IBA, whose aliphatic chain has two more atoms of
carbon in length.
295
This interpretation is in agreement with others [16,17,36] who
have demonstrated the effectiveness of increased BR stimulation in
the presence of auxins in other plants. Our results suggest that
auxins may stimulate the synthesis of BRs in C. vulgaris culture and
increase algal sensitivity to exogenous BRs. On the other hand, one
of the major functions of BR appears to be its involvement with
auxin, generally stimulating its activity. Results obtained from
other experiments demonstrated that auxin-induced ethylene
production is enhanced by several-fold [11]. Moreover, BRs prob-
ably may prevent the conjugation of endogenous auxins, such as
IAA into inactive forms [42].
Summarizing, exogenously applied auxins may change the
phytohormone balance in C. vulgaris cells increasing the level of
endogenous BRs. Co-application experiments suggest a novel
relationships between BRs and auxins 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 auxins several fold.
This is the first evidence demonstrating a synergistic relationship
of BRs and auxins in green algae. Nonetheless, our work provides
an important starting point for future dissection of what appears
to be complex mechanisms of BR-auxin cross-talks in lower
plants.
5. Materials and methods
5.1. 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 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 syn-
chronization has been obtained by a regular change of light and
dark periods according to the method of [43].
The homogenous population of young synchronous cells was
collected by centrifugation (1,000 g, 10 min) and used for sub-
sequent 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
microscope (Nikon Eclipse 50i). Growth of cultures was initiated
by introduction of inoculums containing about 106 algal cells.
The culture mineral medium used was modified mineral Knop’s
296 A. Bajguz, A. Piotrowska-Niczyporuk / Plant Physiology and Biochemistry 71 (2013) 290e297
medium in the following nutritive solution: 0.5 g KNO3, 0.5 g
Ca(NO3)2$4H2O, 0.2 g KH2PO4, 0.15 g MgSO4$7H2O, 0.01 g FeS-
O4$7H2O, 0.003 g H3BO3, 0.002 g MnCl2$H2O, 0.0003 g NH4VO3,
0.0002 g ZnSO4$7H2O, 0.0001 g (NH4)6Mo7O24$7H2O, 1 L distilled
water [44]. 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. Using air pumps cell
suspension was bubbled by atmospheric air at 1 L min1 to
provide necessary CO2.
The effects of BRs and auxins on C. vulgaris growth and metabolite
content were examined. The following exogenous BRs: brassinolide
(BL), 24-epibrassinolide (24-epiBL), 28-homobrassinolide (28-
homoBL) from 7-oxalactonic type as well as castasterone (CS), 24-
epicastasterone (24-epiCS), 28-homocastasterone (28-homoCS)
from 6-ketone type of BRs were used at concentration 0.01 mM.
However, auxins such as: indole-3-acetic acid (IAA), indole-3-
propionic acid (IPA) as well as indole-3-butyric acid (IBA) were
applied at range of concentrations 1e100 mM.
5.2. Brassinosteroid determination
Determination of endogenous BR levels in the cells of
C. vulgaris treated only with 50 mM IAA and in the control culture
was performed on extracts with internal 2H standards, which is
widely accepted as the most accurate method of BR determina-
tion [2]. Deuterium-labeled substrates used in this study were
chemically synthesized: [2H6]BL, [2H6]CS, [2H6]TY, [2H6]TE, [2H6]
6-deoxoCS, [2H6]6-deoxoTY, and [2H6]6-deoxoTE. Lyophilized
plant 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 parti-
tioned betweenCHCl3 and water three times. The CHCl3 e soluble
fraction was subjected to silica gel chromatography (Sep-Pak Vac
Silica, 35 mL, Waters, Milford, MA, USA). The column was sub-
sequently 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 LH-20 column chromatog-
raphy (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 purifi-
cation 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 min1 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 frac-
tion. HPLC purification from the 7% (v/v) MeOH fraction yielded a
BL fraction (Rt 10e15 min), CS fraction (Rt 15e20 min), TE frac-
tion (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
6-deoxoTY fraction (Rt 65e90 min). Each fraction was analyzed
by GCeMS after derivatization. The presence of endogenous non-
labeled BRs was examined by GC-SIM-MS (JMS AX 505W In-
strument JEOL, Tokyo) after suitable derivatization.
5.3. 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 deter-
mined by direct counting of cells in the growth medium using a
Bürker chamber [45].
5.4. Determination of total chlorophylls
The content of chlorophylls in C. vulgaris was determined ac-
cording to Wellburn [46] method in cultures after 48 h cultivation.
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
(1,000 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. The chlorophyll (Chl) content was
determined as follows: Chl ¼ (1.44$A665.2 þ 24.93$A652.4).
5.5. 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 [47]
calibrated with bovine serum albumin obtained from Sigma
Chemical Co. (USA) as the standard.
5.6. Monosaccharide determination
10 mL algal sample was collected after 48 h cultivation and
centrifuged. The monosaccharide content was estimated after
extraction in ethanol according to the Somogyi [48] method.
5.7. 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.
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