Acta Botanica 2-2014.indd

ACTA BOT. CROAT. 73 (2), 2014 347

Acta Bot. Croat. 73 (2), 347–358, 2014 CODEN: ABCRA 25
ISSN 0365-0588
eISSN 1847-8476

Physiological and biochemical responses of
Fibigia triquetra (DC.) Boiss. to osmotic stress

VALERIJA VUJČIĆ*, SANDRA RADIĆ BRKANAC

Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6,
HR-10000 Zagreb, Croatia

Abstract – Water defi cit in the soil leads to osmotic stress in plants. The type of stress af-
fects plant water relations, osmolyte accumulation and oxidative stress balance. The pres-
ent study aimed to investigate the effects of osmotic stress on the Croatian perennial spe-
cies Fibigia triquetra (DC.) Boiss, adapted to a hot and dry habitat. Plants grown in culture
conditions were subjected to isoosmotic concentrations of mannitol and polyethylene gly-
col (PEG) and certain physiological and oxidative stress parameters were analyzed during
a period of 14 days. Dry weight and proline content in Fibigia triquetra shoots increased
in response to osmotic stress while the relative water content decreased. After an initial
rise, chlorophyll and carotenoid levels in treated plants dropped to untreated plant levels.
Oxidative damage to proteins and especially to lipids was evident upon PEG-induced os-
motic stress. Superoxide dismutase and ascorbate peroxidase appear to play an essential
protective role in stressed plants. Regardless of the osmotic agent, accumulation of heat-
shock proteins of 70 kDa was noticed under osmotic stress. The tolerance of the plant spe-
cies to osmotic stress seems to be associated with increased capacity of the antioxidative
system and effi cient photoprotective system.

Keywords: antioxidative enzymes, lipid peroxidation, mannitol, osmotic stress, oxidative
damage, polyethylene glycol, proline, stress proteins

Abbreviations: APOX – ascorbate peroxidase, CAT – catalase, DW – dry weight, FW –
fresh weight, MDA – malondialdehyde, PAGE – polyacrylamide gel electrophoresis, PEG –
polyethylene glycol, POX – pyrogallol peroxidase, RWC – relative water content, SOD –
superoxide dismutase

Introduction

Drought is one of the most common abiotic stressors limiting plant productivity. Under
such stress, water defi cit in plant tissue develops and the photosynthetic rate decreases due
to stomatal closure or metabolic impairment (REDDY et al. 2004). Plants have evolved differ-

VUJČIĆ V., RADIĆ BRKANAC S.

348 ACTA BOT. CROAT. 73 (2), 2014

ent mechanisms to limit water loss, including adaptations at morphological and physiologi-
cal levels – thick cuticle, sunken stomata, reduced leaf surface area, shoot growth inhibition,
enhanced root growth, changes in carbon metabolism and synthesis of compatible solutes
such as proline, glycine betaine, various sugars and polyols (XIONG and ZHU 2002, CHAVES
et al. 2009). Accumulation of proline is a common metabolic response of plants to various
abiotic stress conditions including water defi cit and salt stress (BARTELS and SUNKAR 2005,
HAYAT et al. 2012). Aside from acting as an osmoprotectant, proline has been reported to
play an important role in the removal of reactive oxygen species (ROS), maintaining protein
stability and the regulation of pH in the cytoplasm (HAYAT et al. 2012).

Inhibition of CO2 assimilation coupled with changes in photosystem activities and pho-
tosynthetic electron transport capacity results in increased generation of ROS which may
cause damage to membrane lipids and proteins and inactivate SH-containing enzymes (RED-
DY et al. 2004). This enhanced ROS production is however kept under tight control by a
versatile antioxidant system that includes enzymes such as superoxide dismutase (SOD),
catalase (CAT) and ascorbate peroxidase (APX) and low molecular weight antioxidants
such as ascorbic acid, tocopherol, carotenoids and other (CRUZ DE CARVALHO 2008). In addi-
tion to the activation of oxidative stress protection mechanisms, the combined effects of
multiple stress factors (drought, extreme temperatures, increased salinity etc.) often induce
synthesis of various stress proteins such as heat-shock proteins (HSPs) involved in the fold-
ing of nascent proteins and the refolding of denatured proteins. Among HSPs, the stress-
inducible heat shock protein 70 kDa (HSP70) has been proposed as a biomarker for moni-
toring environmental stressors (WANG et al. 2004).

Fibigia triquetra (DC.) Boiss. is a Croatian rare stenoendemic plant species growing on
sunny and dry habitats of calcareous rocks and thus often exposed to water defi cit (PEVALEK-
KOZLINA et al. 1997). Because of the natural habitat of F. triquetra, we assume that the plant
has developed tolerance to drought and high temperatures as well as to oxidative stress.
Osmotic stress in experimental conditions is often simulated by using polyethylene glycol
(PEG, non-ionic-impermeable osmoticum) and mannitol (non-ionic-permeable osmoti-
cum). Since mannitol partially enters the cell, the use of high molecular weight PEG (6000
or 8000) is recommended under laboratory conditions (VERSLUES et al. 2006). Several stud-
ies carried out with different osmotic agents correlated effi cient antioxidant defense with
tolerance mechanisms (RADIĆ et al. 2006, 2013). We hypothesized that F. trique tra tolerance
to osmotic stress could be associated with induction of the antioxidative system and HSP70
stress protein as well as effective osmoregulation mechanisms. Hence, this study reports on:
1) a comparative analysis of the changes in the activities of antioxidative enzymes, 2) analy-
sis of proline (compatible solute and antioxidant), and 3) a possible accumulation of HSP70
upon PEG- and mannitol-induced osmotic stress.

Material and methods
Plant material and osmotic treatments

F. triquetra seeds were collected from their natural habitat. The sterilized seeds were
inoculated in test tubes fi lled with 15 mL of MS ½ medium containing 0.1 g L–1 myoinositol
(Sigma–Aldrich), 0.1 mg L–1 thiamine × HCl (Sigma–Aldrich), 0.5 mg L–1 pyridoxine × HCl
(Sigma–Aldrich), 0.5 mg L–1 nicotinic acid (Sigma–Aldrich), 2.9 μM gibberellic acid (GA3;

RESPONSES OF FIBIGIA TRIQUETRA TO OSMOTIC STRESS

ACTA BOT. CROAT. 73 (2), 2014 349

Sigma–Aldrich), 0.5 μM 6-benzylaminopurine (BA; Sigma–Aldrich), 30 g L–1 sucrose (Ke-
mika) and 8 g L–1 agar (Sigma–Aldrich) (MURASHIGE and SKOOG 1962, PEVALEK-KOZLINA et
al. 1997). The pH value of nutrient medium was adjusted to 7 with 0.1 M KOH (Kemika)
and the medium was autoclaved at 118 kPa and 120 °C for 20 min. The cultures were grown
under a 16 h photoperiod of fl uorescent light (80 mE m–2 s–1) at 24±2 °C. Four-week old
plants were subcultured to liquid MS ½ medium and, following root initiation, were trans-
ferred to medium of the same composition supplemented with mannitol (Sigma–Aldrich) or
PEG (Sigma–Aldrich). Except for the control medium, osmotica were added to the medium
as 51 g L–1 mannitol or 168 g L–1 PEG 6000 corresponding to −1.0 MPa in both cases. The
water potentials of nutrient solutions were determined by a cryoscopic osmometer (Knauer).
Plants were exposed to mannitol- and PEG-mediated osmotic stress for a period of 14 days.
Shoot samples for all analyses were collected after 1, 7 and 14 days of experiment.

Growth parameters

Dry weight (DW) was measured after oven drying of the samples at 70 °C for 48 h.
Relative water content (RWC) was calculated as: RWC (%) = (FW − DW)/FW × 100 where
FW denotes fresh weight. Prior to determination of fresh weight, shoots were washed with
distilled water and dried with towels.

Proline content

Free proline content was measured by the method of BATES et al. (1973). Plant tissue was
homogenized in 3% (w/v) sulphosalycylic acid (Sigma–Aldrich) and centrifuged at 700 × g
for 3 min. After addition of ninhydrin reagent (Riedel-deHaën), mixtures were heated at 100
°C for 1 h and cooled in an ice-bath. The chromophore obtained was extracted from liquid
phase with toluene (Kemika) and the absorbance of the organic layer was read at 520 nm.
Proline concentration was determined from the calibration curve using L-proline as standard
and expressed as nmol g–1 FW.

Chlorophylls and carotenoids content

Chlorophyll a, b and total carotenoid contents were measured and calculated according
to LICHTENTHALER (1987). In brief, fresh leaves were homogenized with 80% (v/v) cold ac-
etone (Kemika), centrifuged at 5,000 × g for 10 min. The absorbances of the supernatant
were read at 663, 646 and 470 nm.

MDA content

Lipid peroxidation was determined by an estimation of the amount of malondialdehyde
(MDA) content with the use of the thiobarbituric acid method described by HEATH and
PACKER (1968). The crude extracts were mixed with 0.25% (w/v) thiobarbituric acid (Sig-
ma–Aldrich) solution containing 10% (w/v) trichloroacetic acid (Sigma–Aldrich), heated at
95 °C for 30 min and the reaction was stopped in an ice-bath. The cooled mixtures were
centrifuged at 10,000 × g for 10 min and the MDA content calculated from the absorbance
at 532 nm (correction was done by subtracting the absorbance at 600 nm for non-specifi c
turbidity) by using extinction coeffi cient of 155 mM–1 cm–1.

VUJČIĆ V., RADIĆ BRKANAC S.

350 ACTA BOT. CROAT. 73 (2), 2014

Carbonyl groups content
The amount of protein oxidation was estimated by the reaction of carbonyl groups

(C=O) with 2, 4-dinitrophenylhydrazine (Sigma–Aldrich), as described in LEVINE et al.
(1990). After the 2,4-dinitrophenylhydrazine reaction, the C=O content was calculated by
absorbance at 370 nm, using an extinction coeffi cient for aliphatic hydrazones of 22 mM–1
cm–1 and expressed as nmol mg–1 protein.

Enzyme determinations
Plant tissue was homogenized in 50 mM potassium phosphate (K2HPO4/KH2PO4) buf-

fer (pH 7.0) including 5 mM sodium ascorbate (Sigma-Aldrich), 1 mM ethylene diamine
tetraacetic acid (Sigma–Aldrich) and polyvinylpolypyrrolidone (PVPP, Sigma-Aldrich).
The homogenates were centrifuged (Sigma 3K18 centrifuge, Germany) at 22,000 × g for 20
min at 4 °C. Supernatant was used for enzyme activity and protein content assays. Total
soluble protein contents of the enzyme extracts were esti mated according to BRADFORD
(1976) using bovine albumin serum (BSA, Sigma) as standard. The activity of SOD was
assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazo-
lium (Sigma–Aldrich) following the method of GIANNOPOLITIS and RIES (1977). One unit of
SOD was taken as the volume of the enzyme extract causing 50% inhibition of nitroblue
tetrazolium reduction. CAT activity was determined by the decomposition of H2O2 and was
measured spectrophotometrically by following the decrease in absorbance at 240 nm (AEBI
1984). Activity was calculated using the extinction coeffi cient of 0.04 mM–1 cm–1 and mmol
H2O2 g

–1 FW min–1 was defi ned as unit of CAT. APX activity was done according to NA-
KANO and ASADA (1981). The ascorbate oxidation was followed at 290 nm and its concentra-
tion calculated using the molar extinction coeffi cient (e = 2.8 mM–1 cm–1). Corrections were
done for low, non-enzymatic oxidation of ascorbate by H2O2. One enzyme unit was defi ned
as mmol oxidized ascorbate g–1 FW min–1. The activity of POX was measured by monitor-
ing the formation of purpurogallin at 430 nm (e = 2.47 mM–1 cm–1) according to CHANCE
and MAEHLY (1955). The reaction mixture contained 50 mM potassium phosphate buffer
(pH 7), 1 mM H2O2, 20 mM pyrogallol (Sigma-Aldrich) and enzyme extract. The specifi c
enzyme activity for all enzymes was expressed as units per mg of protein.

Imunodetection of HSP70
To analyze HSP70, plant tissue were homogenized in Tris-HCl extraction buffer pH 8.0

containing 17.1% (w/v) sucrose (Kemika), 0.1% (w/v) ascorbic acid (Sigma-Aldrich), and
0.1% (w/v) cysteine- hydrochloride (Sigma-Aldrich) with addition of PVPP and then cen-
trifuged at 29,700 × g for 50 min. Total protein concentration in the supernatant was deter-
mined according to BRADFORD (1976). Aliquots of each homogenate were mixed with cor-
responding volumes of denaturing 0.065 M Tris-HCl buffer containing 6% (w/v) sodium
dodecyl sulfate (SDS, Sigma-Aldrich), 6% (v/v) b-mercaptoethanol (Sigma-Aldrich), 30%
(v/v) glycerol, and 0.01% (w/v) of bromphenol blue (Sigma-Aldrich). The extracts were
boiled for 2 min. Constant protein weights 9 or 12 mg of total protein per lane were ana-
lyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (Bio-Rad, Hercules, CA, USA)
and subsequent Western blotting at 60 V (Bio-Rad). The resolving gel was made at 10% of
polyacrylamide (w/v). Standard proteins of known molecular weights (Fermentas, Glen
Burnie, MD, USA) were run in the same gel. The membranes were blocked with 10% (w/v)
nonfat powdered milk solution made in phosphate-buffered saline (58 mM Na2HPO4, 17

RESPONSES OF FIBIGIA TRIQUETRA TO OSMOTIC STRESS

ACTA BOT. CROAT. 73 (2), 2014 351

mM NaH2PO4, 68 mM NaCl) pH 7.4 containing 1% (v/v) of Tween 20 (Sigma-Aldrich)
and incubated with a rabbit monoclonal antibody raised against the pea HSP70 (diluted
1:1000) overnight at 4 °C. The secondary antibody was an alkaline phosphatase–anti-rabbit
IgG (Sigma- Aldrich) diluted 1:2000. The membranes were developed with nitroblue tetra-
zolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich).

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) using Statistica 7.1.
(StatSoft, Inc.) software package, and differences between corresponding controls and ex-
posure treatment were considered as statistically signifi cant at p < 0.05. Each data point is
the average of six replicates (n = 6).

Results
Exposure of F. triquetra to PEG-induced osmotic stress caused a signifi cant decrease in

plant water status after only one day of exposure, while after 7- and 14-day periods a sig-
nifi cant drop in water content (between 7 and 9% compared to control) was evident in re-
sponse to both osmotica (Fig. 1 A). Mannitol induced a marked rise (a 32% rise compared

Fig. 1. Parameters: (A) RWC, (B) DW, (C) proline, (D) chlorophyll a, (E) chlorophyll b and (F) ca-
rotenoid contents evaluated in F. triquetra plants under control conditions (C) and upon
stresses caused by mannitol (M) and PEG (P) after 14- day growth period. Values are mean ±
SD based on six replicates. Bars with different letters are signifi cantly different at p < 0.05.

VUJČIĆ V., RADIĆ BRKANAC S.

352 ACTA BOT. CROAT. 73 (2), 2014

to control) in shoot DW after 7- and 14-day period while PEG caused more conspicuous
increase of DW (over 40% rise compared to control) which was noted even after the fi rst
day of stress (Fig. 1 B). Osmotic stress had a signifi cant effect on proline contents follow-
ing 7- and 14-day periods (Fig. 1 C) though PEG produced a more conspicuous rise in the
amino acid content (a 2.5-fold increase compared to control) than mannitol (35 and 73%
increase compared to control following the 7- and the 14-day period, respectively).

Content of chlorophyll a, chlorophyll b and carotenoids signifi cantly increased after 24 h
of plant exposure to mannitol and PEG as compared to control plants (Figs. 1 D–F). After
seven days of experiment, chlorophyll and carotenoid contents under mannitol treatment
were not signifi cantly different from respective values in control plants while in plants ex-
posed to PEG the content of these pigments was still signifi cantly elevated. However, at the
end of the experiment the contents of the studied photosynthetic pigments in osmotic-
stressed shoots were similar to those in control plants.

The level of lipid peroxidation, expressed as MDA content, in F. triquetra shoots sam-
pled after 24 h period increased by 43% in response to mannitol (Fig. 2 A). Following lon-
ger exposure (a 7- and a 14-day growth period), the MDA content of mannitol-treated
plants leveled with that of control. Unlike mannitol, PEG caused a signifi cant rise in the

Fig. 2. Parameters: (A) MDA, (B) C=O – carbonyl groups, (C) SOD, (D) CAT, (E) APOX and (F)
POX measured in F. triquetra plants under control conditions (C) and upon stresses caused
by mannitol (M) and PEG (P) after 14-day growth period. Values are mean ± SD based on
six replicates. Bars with different letters are signifi cantly different at p < 0.05.

RESPONSES OF FIBIGIA TRIQUETRA TO OSMOTIC STRESS

ACTA BOT. CROAT. 73 (2), 2014 353

Fig. 3. Patterns of heat-shock protein of 70 kDa (HSP70) in F. triquetra plants under control condi-
tions (C) and upon stresses caused by mannitol (M) and PEG (P) after 1-, 7- and 14-day
growth period.

extent of lipid peroxidation during the entire experimental period (increase by 40–60%,
compared to control). Regarding the level of carbonyl groups, PEG induced a much stron-
ger effect than the lower molecular weight osmoticum. Namely, the carbonyl group content
of PEG-treated F. triquetra was markedly elevated compared to control plants after a 7-day
period of exposure while mannitol did not show any signifi cant effect on the parameter
over the entire experimental period (Fig. 2 B).

The activity of SOD in the shoots of F. triquetra exposed to either mannitol or PEG re-
mained unchanged after 1- and 7-day growth periods and signifi cantly increased only at the
end of the experiment (Fig. 2 C). CAT activity of F. triquetra shoots was not affected by
either osmotica during the entire experimental period (Fig. 2 D). Regardless of the substrate
used, the activity of the peroxidases (APOX and POX) of F. triquetra shoots was markedly
induced by both mannitol and PEG over the entire experimental period (Figs. 2 E–F). How-
ever, after 1- and 14-day growth periods, the APOX activity of PEG-treated plants was over
30% higher than the enzyme activity of mannitol-treated plants. Under osmotic treatment,
the increase of POX activity in F. triquetra shoots exceeded 50% in comparison to control
after 1- and 7-day growth period. The rise in the activity of POX caused by osmotic stress
was even more expressed after a 14-day growth period – both mannitol and PEG caused a
2.5-fold increase in the enzyme activity compared to control.

Proteins of plant species F. triquetra separated by PAGE in the denatured conditions
were transferred to nitrocellulose membrane followed by immunodetection with HSP70
antibodies. After a 1-day period of exposure, the expression of the HSP70 stress protein
was already stronger in treated than in control plants (Fig. 3). The protein accumulated even
more in treated F. triquetra shoots following a longer period of exposure to mannitol and
PEG.

VUJČIĆ V., RADIĆ BRKANAC S.

354 ACTA BOT. CROAT. 73 (2), 2014

Discussion

The results presented here indicate that the use of PEG as an osmotic agent led to more
severe osmotic effects in F. triquetra shoots than when mannitol was used. Although both
agents produced similar effects on RWC following a longer growth period, PEG caused
signifi cant change in the parameter after only a 24 h period. The initially stronger effects of
high-molecular-weight PEG compared to low-molecular-weight mannitol on RWC could
be related to the inhibitory action of PEG on the capacity of roots to supply water to the
leaves (CHAZEN et al. 1995). Stronger effects of PEG on RWC, compared to mannitol, were
observed in the halophytic species Sesuvium portulacastrum exposed to osmotic stress for a
12-day period (SLAMA et al. 2007). Such effects of PEG were previously linked to greater
viscosity of PEG solutions while mannitol was assumed to attenuate osmotic gradient be-
tween the medium and the cell due to uptake by cells (HOHL and SCHOPFER 1991, SLAMA et
al. 2007).

Many studies have shown that osmotic stress caused by either use of osmotica or by the
withholding of water leads to a reduction i n plant RWC which is often accompanied with a
decrease of leaf FW or DW (FU and HUANG 2001, EGERT and TEVINI 2002, SLAMA et al.
2007). However, in our study, DW of F. triquetra shoots signifi cantly increased under both
mannitol and PEG treatment. Similar results with respect to DW and RWC were obtained
with drought-tolerant bean cultivar Phaseolus acutifolius grown on the medium with PEG
for a 14-day period (TÜRKAN et al. 2005). The increase in DW of stressed plants noted in our
study could, at least partly, be ascribed to increased accumulation of proline. However, re-
garding the level of proline accumulation in osmotically stressed F. triquetra shoots, espe-
cially under mannitol, the observed increase in DW might be the result of increased synthe-
sis of other organic compounds which are used in osmotic adjustment (HARE et al. 1998,
SLAMA et al. 2007, FAROOQ et al. 2009) or different proteins such as HSP proteins and dehy-
drins engaged in the adaptation to abiotic stress (WANG et al. 2004). A rise in proline noted
in our study was too low to suggest that the amino acid acts as an osmolyte (SLAMA et al.
2007). Thus, it is likely that proline accumulation in F. triquetra shoots is related to some
other proline roles such as ROS detoxifi cation, protection of cellular macromolecules or
maintenance of cellular pH. The greater accumulation of proline in response to PEG than to
mannitol, described here in response to osmotic stress, has also been observed in osmoti-
cally stressed rice (PANDEY et al. 2004).

The tolerance to drought may be closely related with effi cient photoprotective system
(FAROOQ et al. 2009). In the present study, dynamics of changes in chlorophyll and carot-
enoid contents of stressed F. triquetra over time was similar – the pigments showed an ini-
tial rise, chlorophyll a and carotenoids were still elevated under PEG-stress after 7 day-pe-
riod while after 14 day-period the levels of those pigments also leveled with control ones.
Increased or unaffected levels of chlorophylls and carotenoids under osmotic stress imply a
better photoprotection and capacity for light harvesting and are considered to be a defen-
sive response which restricts the harmful effects of drought (FAROOQ et al. 2009). Similar
results with respect to chlorophyll and carotenoid levels had been reported in drought-toler-
ant plant species exposed to drought stress conditions (FU and HUANG 2001, EGERT and
TEVINI 2002, KALEFETOGLU MACER and EKMEKCI 2009).

The results presented here indicate that osmotic stress increased antioxidative defense
in F. triquetra shoots, especially in the case of PEG. However, regarding parameters of oxi-
dative injury to vital biomolecules, F. triquetra proved to be less susceptible to mannitol-

RESPONSES OF FIBIGIA TRIQUETRA TO OSMOTIC STRESS

ACTA BOT. CROAT. 73 (2), 2014 355

than to PEG-induced osmotic stress. The indicator of lipid peroxidation, MDA, after initial
increase under both mannitol and PEG treatment, remained at high levels only in PEG-
stressed plants. The high-molecular PEG also caused transient increase in carbonyl groups
content indicating oxidative damage to proteins. The results suggest far greater ROS pro-
duction under PEG- than mannitol-stress despite the relatively higher induction of antioxi-
dative defense in response to PEG. Concerning chlorophyll and carotenoid contents under
PEG-stress, the targets for harmful action of ROS might not necessarily be thylakoid mem-
branes but possibly other ROS-generating sites like mitochondria, plasma membranes, cell
wall etc. (MORAN et al. 1994). Several plant species exposed to PEG-induced osmotic stress
showed the same pattern of change in MDA, C=O groups and the activities of antioxidative
enzymes described here i.e. the increase in the extent of lipid peroxidation or protein car-
bon ylation with simultaneous induction of antioxidative enzymes upon PEG action (SIVR-
ITEPE et al. 2008, PYNGROPE et al. 2013).

SOD, an enzyme that breaks down superoxide radicals to hydrogen peroxide and oxy-
gen, is often activated in response to osmotic stress especially in plants tolerant to the stress
conditions (BOR et al. 2003, TÜRKAN et al. 2005, HUANG et al. 2013, RADIĆ et al. 2013). SOD
activity in shoots of treated plants F. triquetra increased after 14 days of the experiment
which indicates that this species is well adapted to the conditions of osmotic stress and can
activate the mechanism of elimination of superoxide radicals. Hydrogen peroxide requires
further degradation enabled by the plant APOX, non-specifi c peroxidase (POX) and CAT.
Two enzymes engaged in the decomposition of hydrogen peroxide were induced in the F.
triquetra shoots, though in some plant species, depending on the type and severity of stress,
an increased activity of only one enzyme can be apparent. The main enzyme for degrading
H2O2 in chives and Centaurea ragusina leaves exposed to osmotic stress was APOX (EGERT
and TEVINI 2002, RADIĆ et al. 2013) while in pea plants exposed to drought it was nonspe-
cifi c peroxidase (MORAN et al. 1994). In our study, the activities of peroxidases, rather than
activity of CAT, seem to have a major role in the degradation of hydrogen peroxide pro-
duced upon osmotic stress. On the other hand, osmotic stress in cherry rootstock caused
activation of all three H2O2-degrading enzymes (SIVRITEPE et al. 2008).

A number of different proteins, including HSP proteins, are synthesized or accumulated
in response to osmotic stress (RIZHSKY et al. 2002). Studies on Arabidopsis and spinach
demonstrated that over-expression of HSP70 chaperones results in enhanced tolerance to
salt, water and high-temperature stress in plants (WANG et al. 2004). In our study, accumu-
lation of HSP70 in F. triquetra exposed to mannitol and PEG was evident after a period of
only 24 h. Similar results with respect to expression of HSP70 were obtained in Fucus ser-
ratus and Lemna minor upon osmotic stress conditions (IRELAND et al. 2004).

In conclusion, both osmotica, especially PEG, caused oxidative stress in F. triquetra,
but also a rapid activation of antioxidant and protective defense mechanisms (enzymatic
and non-enzymatic antioxidants, accumulation of HSP70). Based on the results and previ-
ous fi ndings on the plant’s morphology, it can be concluded that F. triquetra is relatively
tolerant to osmotic stress due to inducible defense systems.

Acknowledgements

This study was supported by Croatian Ministry of Science, Education and Sport, as part
of Project no. 119-1191196-1202.

VUJČIĆ V., RADIĆ BRKANAC S.

356 ACTA BOT. CROAT. 73 (2), 2014

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