Cultured halophytic and endemic plant species Centaurea ragusina

Дата канвертавання15.04.2016
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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




Department of Botany

Faculty of Science

University of Zagreb

Rooseveltov trg 6

HR-10000 Zagreb

Key words: Centaurea ragusina L., peroxidase, drought


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.


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 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.


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, 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.

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.

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.


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.

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.


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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


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.







































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