Response of in vitro cultured halophytic and endemic plant species
Centaurea ragusina L. to drought
Response of in vitro cultured Centaurea ragusina L. to drought
SANDRA RADIĆ
MARIJA PROLIĆ
BRANKA PEVALEK-KOZLINA
Department of Botany
Faculty of Science
University of Zagreb
Rooseveltov trg 6
HR-10000 Zagreb
Croatia
Key words: Centaurea ragusina L., peroxidase, drought
Abstract
Background and Purpose: Centaurea ragusina L. is an endemic Croatian species morphologically
well adapted to dry and sunny habitats. The effects of drought induced by 300 mM mannitol on
relative plant growth rate, soluble peroxidase (POD) activity and isoperoxidase pattern in C.
ragusina plants cultured in vitro were evaluated.
Material and Methods: Rooted plantlets originated from shoots grown in vitro on Murashige and
Skoog half-strength (MS ½) medium supplemented with 2.5 M indole-3-butyric acid. The plants
were transferred to the same composition basal medium without or with 300 mM mannitol and
harvested after 5, 10 or 15 days. Fresh weight, protein content, peroxidase activity and isoenzyme
pattern in C. ragusina roots and shoots were analyzed.
Results and Conclusion: After 5, 10 and 15 days in stress conditions the relative plant growth was
suppressed for 107, 79 and 52 %, respectively, in comparison to control plants. The content of
soluble proteins in shoots and roots of treated and non-treated plants was similar, except after 10
days when it was elevated under water-stress conditions. Whether it was assayed with guaiacol or
pyrogallol, drought significantly increased the peroxidase activity in both roots and shoots,
especially after 10 days of stress conditions.
Six different peroxidase isozymes were distinguished in shoots after 10 days of experiment. Two of
these acidic (anioinic) isozymes were slow migrating and four fast migrating with much stronger
intensity of the bands in drought-stressed plants. Also, drought induced presence of additional
isozyme of slower migration towards anode thus confirming the culmination of stress after 10 days
of experiment. These results suggest peroxidases to be important part of C. ragusina defense
mechanism and their involvement in adaptation to stress conditions.
Radić et al.
INTRODUCTION
Centaurea ragusina L. is a Croatian endemic plant species growing in the gapes of the vertical
limestone cliffs above the Adriatic Sea, in a geographically limited area of southern Dalmatian coast
and some islands. It is a halophytic species with xeromorphic leaves. Thick cuticle, dense hair cover,
glandular trichomes and stomata on both sides have evidently arisen as the consequence of aridity
and salinity in natural habitat (1, 2).
Water deficit stress causes cellular dehydration and imposes an osmotic stress on plants, whereas
salinity imposes both an osmotic and ionic stress (3). Besides morphological adaptive mechanisms,
plants can adapt on water stress by tolerance mechanisms (osmoregulation and accumulation of
compatible solutes) while halophytes can exclude salt and/or compartmentalize it in vacuoles (4, 5).
It is well-known that drought also induces oxidative stress through the production of active oxygen
species (AOS) during stress (6). Plants cope with higher level of AOS by synthesizing antioxidants
and enhancing antioxidative enzymes – peroxidases, superoxide dismutases and catalases. In
contrast to ascorbate peroxidases, guaiacol (nonspecific) peroxidases are characterized by their
broad specifity with respect to an electron donor (guaiacol, pyrogallol, benzidine). Peroxidases
oxidize different cellular components in the presence of hydrogen-peroxide and are thought to
participate in a great number of physiological processes, such as the biosynthesis of lignin and
ethylene, plant development and organogenesis, response to wounding and pathogens (7, 8). But,
they are also responsive to a variety of environmental stresses, including drought (9).
We analyzed peroxidase (POD) activity in shoots and roots of C. ragusina grown on MS ½ medium
(10) in the presence of 300 mM mannitol, assuming that it is tolerant to drought and oxidative stress,
due to life in its natural habitat. To evaluate possible differences in enzymatic stress response of
5
Radić et al.
POD, we measured POD activity with two substrates, guaiacol and pyrogallol. Since plant
peroxidases exist in different isoforms (11), their electrophoretic pattern was analyzed also.
6
Radić et al.
MATERIAL AND METHODS
C. ragusina seeds were collected from natural stations on island of Palagruža. The sterilized seeds
were inoculated in test tubes filled with 15 mL of MS ½ medium containing 0.1 g L-1 myo-inositol,
0.1 mg L-1 thiamineHCl, 0.5 mg L-1 pyridoxineHCl, 0.5 mg L-1 nicotinic acid, 2.9 M gibberellic
acid (GA3), 0.5 M 6-benzylaminopurine (BA), 30 g L-1 sucrose and 8 g L-1 agar. The shoots
isolated from the seedlings were first subcultured on the same media. After 4 weeks in culture,
shoots were transferred to liquid MS ½ medium containing 2.5 M indole-3-butyric acid (IBA).
Rooted plantlets were transferred to media supplemented with IBA and with or without 300 mM
mannitol. Fresh weight of the plants was measured at the beginning (first day) and after 5, 10 or 15
days of experiment. Relative growth rates (RGR) were estimated as the ratio (mn  mi)/mi, where mn
was the fresh weight of the plant after n days (n=5, 10, 15) and mi the initial fresh weight.
Extraction of soluble proteins and peroxidase activities
Shoot or root (100 mg) samples were homogenized in 1 mL of ice cold 100 mM Tris-HCl buffer,
pH 8.0 (12) with addition of insoluble polyvinylpyrrolidone (PVP). The centrifugation was carried
out for 50 min at 29 700 g at 4C (Sigma 3K18 Centrifuge). The supernatant was used for
determination of soluble protein content which was estimated by the method of Bradford (13).
Shoot (250 mg) or root (100 mg) samples were homogenized in 1 mL of ice cold phosphate buffer
pH 7.0 (14). The homogenates were centrifuged at 29 700 g (4C, 50 min). POD activity was
determined in the supernatant using either guaiacol or pyrogallol as electron donors (15). Guaiacol
POD (GPOD) activity was calculated following the increase in absorbance at 470 nm, due to the
formation of tetraguaiacol (26.6 mM-1 cm-1). The reaction mixture contained 50 mM potassium
phosphate (pH 7.0), 18 mM guaiacol, 5 mM H2O2 and extract (50 L) in a total volume of 1 mL.
The pyrogallol oxidation (PPOD) was estimated by monitoring the increase in absorbance at 430 nm,
7
Radić et al.
using an extincion coefficient of 2.47 mM-1 cm-1, in the reaction mixture (1 mL) that consisted of 50
mM potassium phosphate (pH 7.0), 20 mM pyrogallol, 1 mM H2O2 and 50 L extract. Peroxidase
activity was expressed on a fresh matter basis and on a protein basis (specific activity).
Activity gel analysis
For the separation of POD isozymes, non-denaturating PAGE was performed on 10% acrylamide
gels at 4C for 6 hours (16). The gels were then equilibrated with 50 mM sodium phosphate buffer
(pH 7.0) for 30 min and the bands visualized in the same buffer containing 4 mM H2O2 and 20 mM
pyrogallol (17). The gels were scanned and their pictures drawn schematically.
Statistical analysis
Mean values and standard errors (SE) of at least 5 replicates for each measurement were calculated..
Drought-stressed and control plants were compared with Student's t-test at the 0.05 level of
probability. Data presented here are pooled from three independent experiments.
8
Radić et al.
RESULTS
Mannitol induced drought suppressed the relative plant growth significantly (Figure 1). After 5 days
of stress, C. ragusina plantlets exhibited even negative RGR value indicating the loss of water from
cells. However, RGR was less affected in response to stress after 10 and 15 days (71 and 52%,
respectively, in comparison to control plants).
After 10 days of stress, content of soluble proteins was elevated, especially in shoots of treated
plants (Figure 2 A, B). Otherwise, protein content of shoots and roots showed almost no difference
beetwen control and stress conditions.
Peroxidase activity, assayed with either guaiacol or pyrogallol, was markedly increased both in
shoots and roots of stressed plants after 10 and 15 days (Figure 3, 4). We noticed slight difference
between GPOD and PPOD activities after 5 days when activity of GPOD was higher in control plants
(Figure 4A). Also, PPOD exhibited 10 times higher values than GPOD activity. During all
experiment peroxidase activity was two times (PPOD) or three times (GPOD) higher in roots than in
shoots. Peroxidase activity, expressed on a fresh matter basis, correlated very good with specific
peroxidase activity, except after 10 days when specific POD activity was almost equalized in shoots
of control and stressed plants (data not shown). We considered the decrease of corresponding
specific activity was mainly due to the increase of soluble protein content after 10 days of stress.
As electrophoretic patterns stained with either guaiacol or pyrogallol were identical, native gels
stained only with pyrogallol were presented. Change in the peroxidase isoenzyme pattern in shoots of
treated plants was in accordance with the activity change in time (Figure 5A). Native PAGE
revealed the presence of seven peroxidase isoforms (PI) in shoots after 10 days. Six of them, were
present both in control and treated plants but accumulation of PI 4,5,6 and 7 was prominent in the
latter. Also, a new isoform (PI 3) was observed after 10 days of treatment with mannitol.
9
Radić et al.
In general, the isoenzyme pattern of roots was not so consistent with the change in peroxidase
activity of roots during treatment (Figure 5B). After 10 days of experiment, PI 7 accumulated more
in stressed roots than in control. PI 3 appeared as a new isoform and PI 5 was present in control, but
not in stressed roots. However, after 15 days native PAGE showed the presence of seven isoforms in
control versus five in stressed roots, while activity was higher in stressed roots.
10
Radić et al.
DISCUSSION
Cell wall and osmotic adjustments are involved in the early inhibition of growth in plant tissues
exposed to osmotic stress (18). It is not yet resolved whether, over the timescale in days, water status
or hormonal signals control the plant growth in dry soil (5). The RGR of C. ragusina plantlets
decreased in response to osmotic stress which was consistent with several studies that reported
negative correlation between an osmotic stress and plant growth (19, 20, 21). After 10 and 15 days
of stress, RGR values were less affected suggesting plants’ adaptive and/or defense mechanisms had
been recruited.
Soluble protein content was reported to decrease (22, 9), remain unchanged (23) or even increase
(24) in plants subjected to drought. In this study, protein content was elevated after 10 days of stress
excluding serious oxidative damage of proteins due to osmotic stress (9). On the other hand, many
different proteins are even synthesized and/or accumulated in response to water deficit including
dehydrins, heat shock proteins and detoxyfying enzymes (25, 26).
Besides their many physiological roles, peroxidases also protect many cellular components against
detrimental effect of active oxygen species. Some species that can adapt to moderate drought stress
exhibit increase in activities of superoxide dismutase, catalase (27) and peroxidase (24). Other
studies reported decrease (28) and no changes (23) in POD activity in response to drought stress.
Markedly enhanced peroxidase activity in shoots and roots of C. ragusina after 10 days of stress
correlates with elevated soluble proteins sugessting de novo synthesis of peroxidase isoenzymes.
That hypothesis was confirmed by native anionic PAGE showing accumulation of several isoforms
and appearance of new ones after 10 days of stress. The inconsistency between root electrophoretic
pattern and activity after15 days suggests cathionic (basic) peroxidases could be involved.
11
Radić et al.
Higher root versus shoot peroxidase activities are not unusual as found by Verma and Dubey (29).
Roots are often the first part of the plant sensing a water deficit, due to their role in absorbing water
and minerals (3). Knorzer at al. (30), working with soybean cells, observed some differences in
guaiacol and pyrogallol isoenzyme induction, which was not the case here.
Regarding these results, we can confirm the importance of POD antioxidant role in C. ragusina
resistance to drought. For better understanding of complex response to drought, we should continue
investigations with respect to other detoxifying enzymes and antioxidants.
12
Radić et al.
REFERENCES
1. BAČIĆ T, VLADOVIĆ D, POPOVIĆ Ž 1997 Contribution to knowledge about the leaf
anatomy of the Croatian endemic taxon Centaurea ragusina L. subsp. ragusina.
Nat Croat 4: 367-380
2. PEVALEK-KOZLINA B 1998 In vitro propagation of Centaurea ragusina L., a Croatian
endemic species. Acta Biol Cracov 40: 21-24
3. JIN S, CHEN C C S, PLANT A L 2000 Regulation by ABA of osmotic-stress-induced
changes in protein synthesis in tomato roots. Plant Cell Environ 23: 51-60
4. WANG H L, LEE P D, LIU L F, SU J C 1999 Effect of sorbitol induced osmotic stress on the
changes of carbohydrate and free amino acid pools in sweet potato cell suspension cultures.
Bot Bull Acad Sin 40: 219-225
5. MUNNS R 2002 Comparative physiology of salt and water stress. Plant Cell Environ
25: 239-250
6. SMIRNOFF N 1993 The role of active oxygen in the response of plants to water deficit and
desiccation. New Phytol 125: 27-58
7. AMAKO K, CHEN G X, ASADA K 1994 Separate assays specific for ascorbate peroxidase
and guaiacol peroxidase and for the chlorolplastic and cytosolic isozymes of ascorbate
peroxidase in plants. Plant Cell Physiol 35: 497-504
8. VIANELLO A, ZANCANI M, NAGY G, MACRÌ F 1997 Guaiacol peroxidase associated to
soybean root plasma membranes oxidizes ascorbate. J Plant Physiol 150: 573-577
9. MORAN J F, BECANA M, ITURBE-ORMAETXE I, FRECHILLA S, KLUCAS R V,
APARICIO-TEJO P 1994 Drought induces oxidative stress in pea plants. Planta 194: 346-352
13
Radić et al.
10. MURASHIGE T, SKOOG F 1962 A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol Plant 15: 473-497
11. GASPAR T, PENEL C, HAGEGE D, GREPPIN H 1991 Peroxidases in plant growth,
differentiation and developmental processes. In: Biochemical, Molecular and Physiological
Aspects of Plant Peroxidases, J. Lobarzewsky, H. Greppin, C. Penel and T. Gaspar (ed).
University M. Curie-Sklodowska, Poland and University of Geneva, Switzerland, p 249-280
12. STAPLES R C, STAHMANN M A 1964 Changes in proteins and several enzymes in susceptible
bean leaves after infection by the bean rust fungus. Phytopatol 54: 760-764
13. BRADFORD M M 1976 A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254
14. PETERS J L, CASTILLO F J, HEATH R L 1988 Alternation of extracellular enzymes in pinto
bean leaves upon exposure to air pollutants, ozone and sulfur dioxide. Plant Physiol 89: 159-164
15. CHANCE B, MAEHLY A C 1955 Assay of catalases and peroxidases. In: Methods in
Enzymology, S. P. Colowick and N. O. Kaplan (ed). Academic press, New York, p 764-775
16. LAEMMLI U K 1970 Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680-685
17. MITTLER R, ZILINSKAS B 1993 Detection of Ascorbate Peroxidase Activity in Native Gels by
Inhibition of the Ascorbate-Dependent Reduction of Nitroblue Tetrazolium. Anal Biochem 212:
540-546
18. NEUMANN P 1997 Salinity resistance and plant growth revisited. Plant Cell Environ 20: 11931198
19. LE THIEC D, MANNINEN S 2003 Ozone and water deficit reduced growth of Aleppo pine
seedlings. Plant Physiol Biochem 41: 55-63
14
Radić et al.
20. COSTA FRANCA M G, PHAM THI A T, PIMENTEL C, ROSSIELLO R P, ZUILY-PODIL Y,
LAFFRAY D 2000 Differences in growth and water relations among Phaseolus vulgaris
cultivars in response to induced drought stress. Environ Exper Bot 43: 227-237
21. PÉREZ-ALFOCEA F, ESTAÑ M T, CARO M, GUERRIER G 1993 Osmotic adjustment in
Lycopersicon esculentum and L. pennellii under NaCl and polyethylene glycol 6000 iso-osmotic
stress. Physiol Plant 87: 493-498
22. SEEL W E, HENDRY G A F, LEE J A 1992 The combined effect of dessication and irradiance
on mosses from xeric and hydric habitats. J Exp Bot 43: 1023-1030
23. EGERT M, TEVINI M 2002 Influence of drought on some physiological parameters
symptomatic for oxidative stress in leaves of chives (Allium schoenoprasum). Environ Exper Bot
48: 43-49
24. TKALEC M, MLINAREC J, VIDAKOVIĆ-CIFREK Ž, JELENČIĆ B, REGULA I 2001 The
effect of salinity and osmotic stress on duckweed Lemna minor L. Acta Bot Croat 60: 237-241
25. CAMPALANS A, MESSEGUER R, GODAY A, PAGÈS M 1999 Plant responses to drought,
from ABA signal transduction events to the action of the induced proteins. Plant Physiol
Biochem 37: 327-340
26. SMIRNOFF N 1998 Plant resistance to environmental stress. Curr Opin Biotech 9: 214-219
27. JAGTAP V, BHARGAVA S 1995 Variation in the antioxidant metabolism of drought tolerant
and drought susceptible varieties of Sorghum bicolor (L.) Moench. exposed to high light, low
water and high temperature stress. J Plant Physiol 145: 195–197
28. ZHANG J, KIRKHAM M B 1996 Enzymatic responses of the ascorbate-glutathione cycle to
drought in sorghum and sunflower plants. Plant Sci 113: 139-147
29. VERMA S, DUBEY R S 2003 Lead toxicity induces lipid peroxidation and alters the activities of
antioxidant enzymes in growing rice plants. Plant Sci 00 (article in press)
15
Radić et al.
30. KNÖRZER O C, DURNER J, BÖGER P 1996 Alternations in the oxidative system of
suspension-cultured soybean cells (Glycine max) induced by oxidative stess. Physiol Plant 97:
388-396
16
Radić et al.
FIGURE 1. C. ragusina growth expressed as relative growth rate during growth period
of 15 days on MS ½ containing 300 mM mannitol. Each value is the mean of 6 replicates±
standard error. Bars indicate standard errors. * indicates significant difference (p<0.05).
FIGURE 2. Soluble protein content of C. ragusina shoots (A) and roots (B) during growth
period of 15 days on MS ½ without (control) or with 300 mM mannitol. Each value is the mean
of six replicates±standard error. Bars indicate standard errors. * indicates significant difference
(p<0.05).
FIGURE 3. Pyrogallol peroxidase activity, expressed on a fresh matter basis, in shoots (A) and
roots (B) of C. ragusina during growth period of 15 days on MS ½ containing 300 mM mannitol.
Each value is the mean of six replicates±standard error. Bars indicate standard errors. * indicates
significant difference (p<0.05).
FIGURE 4. Guaiacol peroxidase activity, expressed on a fresh matter basis, in shoots (A) and
roots (B) of C .ragusina during growth period of 15 days on MS ½ containing 300 mM mannitol.
Each value is the mean of 5-6 replicates±standard error. Bars indicate standard errors. * indicates
significant difference (p<0.05).
FIGURE 5. Schematic representation of the banding pattern of peroxidase isoforms in shoots (A) and
roots (B) of C. ragusina during growth period of 15 days on MS ½ without (1) or with (2) 300 mM
mannitol.
17
Radić et al.
Relative growth rate
2,0
1,5
1,0
*
0,5
*
*
0,0
-0,5
control
5
10
15
Days
mannitol
FIGURE 1.
18
Soluble proteins (mg/g FW)
Radić et al.
A
7
6
5
4
3
2
1
0
*
5
control
10
15
Days
Soluble proteins (mg/g FW)
mannitol
B
7
6
5
4
3
2
1
0
control
5
10
15
Days
mannitol
FIGURE 2.
19
Radić et al.
A
Peroxidase activity
(mol/min g FW)
250
200
150
*
100
50
0
5
control
10
15
Days
mannitol
B
Peroxidase activity
(mol/min g FW)
250
*
200
*
150
100
50
0
5
control
10
15
Days
mannitol
FIGURE 3.
20
Radić et al.
A
Peroxidase activity
(mol/min g FW)
20
15
10
*
5
*
0
5
10
control
15
Days
mannitol
Peroxidase activity
(mol/min g FW)
20
*
15
B
*
*
10
5
0
5
control
10
15
Days
mannitol
FIGURE 4.
21
Radić et al.
A A
PI
5.day
10.day
15.day
B B
1
1
1
B
2
1
2
3
4
5
6
7
2
2
PI
5.day
10.day
15.day
1
1
1
2
2
2
1
1
2
2
33
44
5
6
7
FIGURE 5.
22