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Response of Olea europaea ssp. maderensis in vitro shoots exposed to osmotic stress
Scientia Horticulturae 97 (2003) 411±417
Short communication
Response of Olea europaea ssp. maderensis in vitro shoots exposed to osmotic stress Gina Brito, Armando Costa, Henrique M.A.C. Fonseca, ConceicËaÄo V. Santos* Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal Accepted 17 October 2002
Abstract Olea europaea ssp. maderensis shoots (2.5 cm) were grown for 2 months on Driver and Kuniyuki medium (DKW) supplemented with 4.4 mM benzyladenine (BA) and 0.4 mM indole-3-butyric acid (IBA). Osmotic stress was induced for 1 month in shoots by the addition of sorbitol (0.1 and 0.2 M) to DKW. Moderate stress (0.1 M) did not affect shoot growth, and induced osmotic adjustment by increasing K content without water lost. Also no decrease in chlorophyll ¯uorescence was detected although chlorophyll content decreased. However, at 0.2 M sorbitol (a more severe stress) growth was reduced. This sorbitol concentration increased tissue osmolality and lipid peroxidation, but decreased water and soluble protein contents. It also decreased chlorophyll content and chlorophyll ¯uorescence, by increasing basal ¯uorescence (Fo) and decreasing maximal ¯uorescence (Fm) values. Both sorbitol concentrations decreased Mg and Zn contents while 0.2 M decreased K and Cu contents in shoots. Rooting was reduced by moderate stress (0.1 M) and completely inhibited by 0.2 M sorbitol. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Olea europaea ssp. maderensis; Olive; Osmotic stress; Mineral composition; Senescence
1. Introduction Olive (Olea europaea L.) is an important crop plant with large distribution in regions that frequently suffer from low water availability. The subspecies O. europaea ssp. maderensis (wild olive) is a native plant of Porto Santo island in the Madeira Archipelago. This island Abbreviations: BA: benzyladenine; chl: chlorophyll; DKW: Driver and Kuniyuki medium; IBA: indole-3butyric acid; MDA: malondialdehyde * Corresponding author. Tel.: 351-23-4370780; fax: 351-23-4426408. E-mail address: [email protected] (C.V. Santos). 0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 2 1 6 - 9
412
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
faces a dramatic problem of deserti®cation with either the disappearance of most of its native plants, or their reduction to a very restricted number as it is the case of wild olive. Reforestation programs involving native plants in these threatened environments must consider the selection of plants resistant to desiccation. The validation of reforestation programs using micropropagation (Santos et al., 2002) implies the study of the survival of those plants to osmotic stress. Some parameters are frequently used to assay the resistance of plants to salt and water stress. Growth rates, chlorophyll content and ¯uorescence are invaluable parameters used to determine effects on biomass production and photosynthetic ef®ciency (Epron, 1997; Santos et al., 2001). Also, one of the ®rst effects of stress in cells is the decrease in membrane integrity that in¯uences membrane permeability, and therefore solute content. This membrane integrity is usually calculated by measuring the production of malondialdehyde (MDA) in stressed cells (e.g. Shalata and Tal, 1998). Finally, changes of soluble protein contents are important to understand the impact of stress on cell proteolysis and protein synthesis (Santos and Caldeira, 1999). Some of these parameters were used by several authors (e.g. Santos and Caldeira, 1999; Santos et al., 2001) to characterise the degree of senescence induced by water or salt stress in sun¯ower. In the present study we use the physiological parameters described previously to characterise the response of wild olive in vitro shoots to osmotic stress and analyse the in¯uence of stress on rooting capacity. 2. Materials and methods 2.1. Plant material and culture conditions Cuttings of O. europaea ssp. maderensis were obtained from adult plants growing in Porto Santo, and sterilised explants (with axillary buds) were then grown on Driver and Kuniyuki medium (DKW) (Driver and Kuniyuki, 1984) supplemented with 4.4 mM benzyladenine (BA) and 0.4 mM indole-3-butyric acid (IBA) according to the protocol described by Santos et al. (2002). Shoots with an approximate length of 2.5 cm were transferred to DKW with 4.4 mM BA, 0.4 mM IBA, containing 0, 0.1 and 0.2 M sorbitol, respectively. Each treatment consisted of ®ve ¯asks containing four shoots each, giving a total of 20 replicates. After 1 month the shoots were collected for determination of the growth rate, photosynthetic ef®ciency, water content, osmolality, protein content and mineral composition as described below: Growth rate. Calculated as the dry weight ratio relative to the unstressed treatments. Photosynthetic ef®ciency. Chlorophyll concentration was determined according to Arnon (1949) by homogenising tissue in acetone (80%). Chlorophyll ¯uorescence was monitored using a Plant Ef®ciency Analyser (Hansatech, UK) according to the conditions described by Santos et al. (2001). The variable component of chlorophyll (Fv) was obtained by subtracting the basal non-variable chlorophyll ¯uorescence (Fo) from the maximal chlorophyll ¯uorescence (Fm) (Maxwell and Johnson, 2000). Water content and osmolality. Water content was determined by the difference between fresh and dry weights, after drying samples at 60 8C. For osmolality analysis, samples were
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
413
submitted to freeze/unfreeze cycles to assure membrane rupture (Santos et al., 2001). Osmolality was determined using an osmometer (Knauer) at 20 8C. Protein content. Soluble proteins were extracted using a 50 mM Tris±buffer pH 8.0, containing 2 mM EDTA, 10 mM mercaptoethanol and 0.1 mM phenilmethylsulphonyl ¯uoride. Soluble protein content was determined by the Bradford method (Bradford, 1976). Mineral composition. Nutrient composition was determined in stressed and unstressed leaves. For K, P, Ca, Mg, Mn, Fe, B, Cu and Zn analysis, dried tissue was treated according to Evans and Bucking (1976) and elements were determined by inductively coupled plasma spectroscopy (ICPS). Membrane integrity. The degree of lipid peroxidation was used to calculate membrane integrity. Lipid peroxidation was determined by quantifying the production of MDA according to Dhindsa and Matowe (1981): 0.25 g of tissue samples were homogenised in 5 ml trichloroacetic acid 0.1% (w/v) and centrifuged at 10,000 g for 10 min. The supernatant was collected and 1 ml was mixed with 4 ml 20% trichloroacetic acid containing 0.5% (v/v) thiobarbituric acid. The mixture was heated at 95 8C for 30 min and then cooled and centrifuged at 10,000 g for 10 min. The supernatant was used to determine MDA concentration at 532 nm. Rooting ef®ciency. After 1 month of stress, three groups (containing 15 shoots each) were transferred to rooting medium consisting of DKW supplemented with 20.7 mM IBA (Santos et al., 2002) and having 0, 0.1 and 0.2 M sorbitol. Number of shoots that developed roots and root length was determined 45 days after transfer. Statistical analysis. Data were averaged from two independent analyses and were analysed using the uni-factorial test (ANOVA) to assay signi®cant differences among averages (P < 0:05), using the program Statistica1 (Statsoft, Norman, OK). 3. Results and discussion Osmotic stress reduced growth (Fig. 1) and induced some senescence characteristics in O. europaea ssp. maderensis shoots. Stressed shoots had a reduced number of nodes and leaves (data not shown). Survival rates were 100% in all stress conditions although it was possible to observe leaf necroses and decay in some shoots exposed to 0.2 M sorbitol. Shoots turned light green when growing in the presence of sorbitol, suggesting that chlorophyll content was affected. There was an increase of tissue osmolality in both sorbitol treatments, but a reduction of water content was only observed in 0.2 M stressed shoots (Table 1). The increased osmolality observed in 0.1 M-stressed shoots indicates that leaves under stress accumulated osmotically active substances to reduce cell water potential with no water lost. The increase of K content (Fig. 2) may justify the higher increase of osmolality in these stressed shoots. Potassium accumulates in several plants in response to salt and osmotic stress (e.g. Santos and Caldeira, 1999), and is reported to play an osmotic role and to contribute to the higher tolerance to stress exhibited by some genotypes/cells, respectively, to others that do not accumulate this ion (e.g. Torrecillas et al., 1994; Santos and Caldeira, 2000).
414
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
Fig. 1. Effect of sorbitol (0.1 and 0.2 M) on O. europaea ssp. maderensis growth. Values are expressed in percentage to unstressed shoots. Mean values with the same letter do not differ significantly at P < 0:05. Bars represent the mean S:D:
Osmotic stress also decreased chla and chlb contents and it increased slightly chla/chlb ratio (Table 2). Severe stress (0.2 M) also reduced photosynthetic ef®ciency, an increase of Fo and decrease of Fv, Fm and of Fv/Fm ratio in stressed shoots (Table 2) were observed. This reduction of photosynthetic ef®ciency in stressed plants was accompanied by decreases of membrane integrity, shown by the increase of MDA production (Table 1). The decrease of chlorophyll content may be the result of chlorophyll degradation, by the activation of chlorophyllases that convert chlb into chla or be due to chlorophyll synthesis de®ciency together with changes of thylakoid membrane structure. Bussis et al. (1998) indicated that expanded leaves exposed to water de®cit started to degrade their photosynthetic apparatus, possibly to mobilise resources for the production of new acclimated leaves. The observed decrease of chlorophyll content and photosynthetic rate may also be correlated with decrease (observed in the same leaves) of Mg (Fig. 2), an element that plays an important role in the photosynthetic apparatus and process. The stability of Fo and Fm found in 0.1 M stressed leaves indicate that this concentration of sorbitol does not induce changes in the photosynthetic reaction centres. The lack of drought-induced damage to Photosystem II (PSII) photochemistry, as indicated by the maintenance of Fv and Fm values, has already been reported for some species (e.g. Santos et al., 2001). However, PSII seems to be sensitive to more severe drought stress induced by Table 1 Effect of sorbitol (0.1 and 0.2 M) on water content (%) and osmolality in O. europaea ssp. maderensis shootsa Sorbitol 0.0 M Water content (%) Osmolality (mOsm kg 1) MDA (mmol g FW 1) a
75.5 0.8 b 162 5 a 0.8 0.1 a
0.1 M 74.8 0.6 b 229 9 b 0.7 0.2 a
Values (mean S:D:) within rows with the same letter do not differ at P < 0:05.
0.2 M 73.3 0.2 a 280 12 c 2.8 0.2 b
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
415
Fig. 2. (a)±(c) Contents of K, P, Ca, Mg, Zn, Mn, Cu, Fe and B in O. europaea ssp. maderensis shoots exposed to 0, 0.1 and 0.2 M sorbitol. (d) Soluble protein contents of O. europaea ssp. maderensis shoots exposed to 0, 0.1 and 0.2 M sorbitol. For each parameter, mean values with the same letter do not differ significantly at P < 0:05. Bars represent the mean S:D:
0.2 M sorbitol. In fact Fv/Fm ratio decreased in severely stressed shoots indicating a signi®cant increase of energy dissipation in the photosynthetic apparatus, together with a reduction of Fm and Fv. Previous studies showed that salt and osmotic stress induced changes in the photosynthetic apparatus (e.g. Epron, 1997; Santos et al., 2002), and membrane permeability properties of chloroplasts. In O. europaea ssp. maderensis shoots, osmotic stress increased MDA production (Table 1), an indication of membrane degradation that correlates with the decrease of photosynthetic activity. It is widely reported that active oxygen species bring Table 2 Effect of sorbitol (0.1 and 0.2 M) on fluorescence parameters and on chlorophyll content in O. europaea ssp. maderensis leavesa Sorbitol 0.0 M Fo Fm Fv Fv/Fm chla (mg cm 2) chlb (mg cm 2) chla/chlb a
1016.3 2590.0 1573.8 0.6076 15.787 6.530 2.4174
0.1 M
57.83 a 375.80 b 363.47 b 0.17 b 0.048 c 0.528 a 0.061 a
1120.5 2440.0 1019.5 0.4178 5.737 2.211 2.5945
0.2 M
171.42 ab 309.76 b 265.41 b 0.12 b 0.126 b 0.127 b 0.204 a
1352.8 1684.8 332.0 0.1971 3.899 1.403 2.7789
Values (mean S:D:) within rows with the same letter do not differ significantly at P < 0:05.
184.33 b 161.92 a 27.32 a 0.05 a 0.084 a 0.063 a 0.028 b
416
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
Table 3 Effect of sorbitol (0.1 and 0.2 M) on rooting capacity of O. europaea ssp. maderensis shootsa Half strength DKW with 20.7 mM IBA Unstressed shoots Percentage rooting Number of roots per shoot Total root length (cm) a
75 15.6 c 2.2 0.6 b 2.5 0.5 b
Stressed shoots (0.1 M) 25 10.0 b 1.0 0.0 a 1.2 0.6 a
Stressed shoots (0.2 M) 0 0.0 a ± ±
Values (mean S:D:) within rows with the same letter do not differ significantly at P < 0:05.
about peroxidation of membrane lipids leading to membrane damage (Shalata and Tal, 1998). Protection from oxidative damage is, partly, due to the activity of antioxidant enzymes such as superoxide desmutase, catalase and peroxidase. The connection between some of the responses observed under water stress conditions, and some antioxidant enzymes, namely with peroxidase and superoxide desmutase isoenzymes is presently being studied. Shoots of wild olive were maintained in the presence of sorbitol for adaptation and rooting, in order to, in the end, transfer acclimated tolerant plants to the ®eld. Osmotic stress reduced the capacity of shoots to develop roots (Table 3). Only 25 10:0 of the shoots adapted to 0.1 M developed roots in contrast to the 75 15 of rooted shoots under control conditions and these were smaller (1:2 0:6 cm) than those developed under unstressed conditions (2:5 0:5 cm). This reduction may be due to the inhibitory effect of low osmotic potential in rooting capacity that was already reported for other species such as sun¯ower (Santos and Caldeira, 2000). Acknowledgements The authors thank FCT (Project Ref. PNAT/1999/AGR/15011).
References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1±10. Bradford, M.M., 1976. Rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. 72, 248±254. Bussis, D., Kauder, F., Heineke, D., 1998. Acclimation of potato plants to polyethylene glycol-induced water deficit. I. Photosynthesis and metabolism. J. Exp. Bot. 49, 1349±1360. Dhindsa, R.S., Matowe, W., 1981. Drought tolerance in two mosses: correlated with enzymatic defence against lipid peroxidation. J. Exp. Bot. 32, 79±91. Driver, D., Kuniyuki, A., 1984. In vitro propagation of Paradox Walnut rootstock. HortScience 19, 507±509. Epron, D., 1997. Effects of drought on photosynthesis and on the termotolerance on photosystem II in seedlings of cedar (Cedrus atlantica and C. libani). J. Exp. Bot. 48, 1835±1841. Evans, F., Bucking, W., 1976. Mineral analysis. In: Nitsche, J.P. (Ed.), Modern Methods in Forest Genetics, Proceedings in Life Science. Springer, Berlin, pp. 165±188. Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescenceÐa practical guide. J. Exp. Bot. 51, 659±668.
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Santos, C.V., Caldeira, G., 1999. Comparative responses of Helianthus annuus plants and calli exposed to NaCl. I. Growth rate and osmotic regulation in intact plants and calli. J. Plant Physiol. 155, 769±777. Santos, C.V., Caldeira, G., 2000. Comparative responses of Helianthus annuus plants and calli exposed to NaCl. II. Selection of stable tolerant calli cell lines and evaluation of osmotic adjustment and morphogenic capacity. J. Plant Physiol. 156, 68±74. Santos, C.V., Campos, A., Azevedo, H., Caldeira, G., 2001. Nutritional imbalance and senescence induced in plants and calli exposed to KCl. J. Exp. Bot. 52 (335), 351±360. Santos, C.V., Brito, G., Fonseca, H.M.A.C., Costa, A., 2002. In vitro plantlet regeneration of Olea europaea ssp. maderensis. Sientia Horticulturae. Shalata, A., Tal, M., 1998. The effects of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiol. Plant. 104, 169±174. Torrecillas, S., Alarcon, Blanco, S., Bolarin, M. 1994. Osmotic adjustment in leaves of Lycopersicon esculentum and Lycopersicum pennellii in response to saline water irrigation. Biol. Plant. 36 (2), (1994) 247±254.
Short communication
Response of Olea europaea ssp. maderensis in vitro shoots exposed to osmotic stress Gina Brito, Armando Costa, Henrique M.A.C. Fonseca, ConceicËaÄo V. Santos* Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal Accepted 17 October 2002
Abstract Olea europaea ssp. maderensis shoots (2.5 cm) were grown for 2 months on Driver and Kuniyuki medium (DKW) supplemented with 4.4 mM benzyladenine (BA) and 0.4 mM indole-3-butyric acid (IBA). Osmotic stress was induced for 1 month in shoots by the addition of sorbitol (0.1 and 0.2 M) to DKW. Moderate stress (0.1 M) did not affect shoot growth, and induced osmotic adjustment by increasing K content without water lost. Also no decrease in chlorophyll ¯uorescence was detected although chlorophyll content decreased. However, at 0.2 M sorbitol (a more severe stress) growth was reduced. This sorbitol concentration increased tissue osmolality and lipid peroxidation, but decreased water and soluble protein contents. It also decreased chlorophyll content and chlorophyll ¯uorescence, by increasing basal ¯uorescence (Fo) and decreasing maximal ¯uorescence (Fm) values. Both sorbitol concentrations decreased Mg and Zn contents while 0.2 M decreased K and Cu contents in shoots. Rooting was reduced by moderate stress (0.1 M) and completely inhibited by 0.2 M sorbitol. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Olea europaea ssp. maderensis; Olive; Osmotic stress; Mineral composition; Senescence
1. Introduction Olive (Olea europaea L.) is an important crop plant with large distribution in regions that frequently suffer from low water availability. The subspecies O. europaea ssp. maderensis (wild olive) is a native plant of Porto Santo island in the Madeira Archipelago. This island Abbreviations: BA: benzyladenine; chl: chlorophyll; DKW: Driver and Kuniyuki medium; IBA: indole-3butyric acid; MDA: malondialdehyde * Corresponding author. Tel.: 351-23-4370780; fax: 351-23-4426408. E-mail address: [email protected] (C.V. Santos). 0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 2 1 6 - 9
412
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
faces a dramatic problem of deserti®cation with either the disappearance of most of its native plants, or their reduction to a very restricted number as it is the case of wild olive. Reforestation programs involving native plants in these threatened environments must consider the selection of plants resistant to desiccation. The validation of reforestation programs using micropropagation (Santos et al., 2002) implies the study of the survival of those plants to osmotic stress. Some parameters are frequently used to assay the resistance of plants to salt and water stress. Growth rates, chlorophyll content and ¯uorescence are invaluable parameters used to determine effects on biomass production and photosynthetic ef®ciency (Epron, 1997; Santos et al., 2001). Also, one of the ®rst effects of stress in cells is the decrease in membrane integrity that in¯uences membrane permeability, and therefore solute content. This membrane integrity is usually calculated by measuring the production of malondialdehyde (MDA) in stressed cells (e.g. Shalata and Tal, 1998). Finally, changes of soluble protein contents are important to understand the impact of stress on cell proteolysis and protein synthesis (Santos and Caldeira, 1999). Some of these parameters were used by several authors (e.g. Santos and Caldeira, 1999; Santos et al., 2001) to characterise the degree of senescence induced by water or salt stress in sun¯ower. In the present study we use the physiological parameters described previously to characterise the response of wild olive in vitro shoots to osmotic stress and analyse the in¯uence of stress on rooting capacity. 2. Materials and methods 2.1. Plant material and culture conditions Cuttings of O. europaea ssp. maderensis were obtained from adult plants growing in Porto Santo, and sterilised explants (with axillary buds) were then grown on Driver and Kuniyuki medium (DKW) (Driver and Kuniyuki, 1984) supplemented with 4.4 mM benzyladenine (BA) and 0.4 mM indole-3-butyric acid (IBA) according to the protocol described by Santos et al. (2002). Shoots with an approximate length of 2.5 cm were transferred to DKW with 4.4 mM BA, 0.4 mM IBA, containing 0, 0.1 and 0.2 M sorbitol, respectively. Each treatment consisted of ®ve ¯asks containing four shoots each, giving a total of 20 replicates. After 1 month the shoots were collected for determination of the growth rate, photosynthetic ef®ciency, water content, osmolality, protein content and mineral composition as described below: Growth rate. Calculated as the dry weight ratio relative to the unstressed treatments. Photosynthetic ef®ciency. Chlorophyll concentration was determined according to Arnon (1949) by homogenising tissue in acetone (80%). Chlorophyll ¯uorescence was monitored using a Plant Ef®ciency Analyser (Hansatech, UK) according to the conditions described by Santos et al. (2001). The variable component of chlorophyll (Fv) was obtained by subtracting the basal non-variable chlorophyll ¯uorescence (Fo) from the maximal chlorophyll ¯uorescence (Fm) (Maxwell and Johnson, 2000). Water content and osmolality. Water content was determined by the difference between fresh and dry weights, after drying samples at 60 8C. For osmolality analysis, samples were
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
413
submitted to freeze/unfreeze cycles to assure membrane rupture (Santos et al., 2001). Osmolality was determined using an osmometer (Knauer) at 20 8C. Protein content. Soluble proteins were extracted using a 50 mM Tris±buffer pH 8.0, containing 2 mM EDTA, 10 mM mercaptoethanol and 0.1 mM phenilmethylsulphonyl ¯uoride. Soluble protein content was determined by the Bradford method (Bradford, 1976). Mineral composition. Nutrient composition was determined in stressed and unstressed leaves. For K, P, Ca, Mg, Mn, Fe, B, Cu and Zn analysis, dried tissue was treated according to Evans and Bucking (1976) and elements were determined by inductively coupled plasma spectroscopy (ICPS). Membrane integrity. The degree of lipid peroxidation was used to calculate membrane integrity. Lipid peroxidation was determined by quantifying the production of MDA according to Dhindsa and Matowe (1981): 0.25 g of tissue samples were homogenised in 5 ml trichloroacetic acid 0.1% (w/v) and centrifuged at 10,000 g for 10 min. The supernatant was collected and 1 ml was mixed with 4 ml 20% trichloroacetic acid containing 0.5% (v/v) thiobarbituric acid. The mixture was heated at 95 8C for 30 min and then cooled and centrifuged at 10,000 g for 10 min. The supernatant was used to determine MDA concentration at 532 nm. Rooting ef®ciency. After 1 month of stress, three groups (containing 15 shoots each) were transferred to rooting medium consisting of DKW supplemented with 20.7 mM IBA (Santos et al., 2002) and having 0, 0.1 and 0.2 M sorbitol. Number of shoots that developed roots and root length was determined 45 days after transfer. Statistical analysis. Data were averaged from two independent analyses and were analysed using the uni-factorial test (ANOVA) to assay signi®cant differences among averages (P < 0:05), using the program Statistica1 (Statsoft, Norman, OK). 3. Results and discussion Osmotic stress reduced growth (Fig. 1) and induced some senescence characteristics in O. europaea ssp. maderensis shoots. Stressed shoots had a reduced number of nodes and leaves (data not shown). Survival rates were 100% in all stress conditions although it was possible to observe leaf necroses and decay in some shoots exposed to 0.2 M sorbitol. Shoots turned light green when growing in the presence of sorbitol, suggesting that chlorophyll content was affected. There was an increase of tissue osmolality in both sorbitol treatments, but a reduction of water content was only observed in 0.2 M stressed shoots (Table 1). The increased osmolality observed in 0.1 M-stressed shoots indicates that leaves under stress accumulated osmotically active substances to reduce cell water potential with no water lost. The increase of K content (Fig. 2) may justify the higher increase of osmolality in these stressed shoots. Potassium accumulates in several plants in response to salt and osmotic stress (e.g. Santos and Caldeira, 1999), and is reported to play an osmotic role and to contribute to the higher tolerance to stress exhibited by some genotypes/cells, respectively, to others that do not accumulate this ion (e.g. Torrecillas et al., 1994; Santos and Caldeira, 2000).
414
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
Fig. 1. Effect of sorbitol (0.1 and 0.2 M) on O. europaea ssp. maderensis growth. Values are expressed in percentage to unstressed shoots. Mean values with the same letter do not differ significantly at P < 0:05. Bars represent the mean S:D:
Osmotic stress also decreased chla and chlb contents and it increased slightly chla/chlb ratio (Table 2). Severe stress (0.2 M) also reduced photosynthetic ef®ciency, an increase of Fo and decrease of Fv, Fm and of Fv/Fm ratio in stressed shoots (Table 2) were observed. This reduction of photosynthetic ef®ciency in stressed plants was accompanied by decreases of membrane integrity, shown by the increase of MDA production (Table 1). The decrease of chlorophyll content may be the result of chlorophyll degradation, by the activation of chlorophyllases that convert chlb into chla or be due to chlorophyll synthesis de®ciency together with changes of thylakoid membrane structure. Bussis et al. (1998) indicated that expanded leaves exposed to water de®cit started to degrade their photosynthetic apparatus, possibly to mobilise resources for the production of new acclimated leaves. The observed decrease of chlorophyll content and photosynthetic rate may also be correlated with decrease (observed in the same leaves) of Mg (Fig. 2), an element that plays an important role in the photosynthetic apparatus and process. The stability of Fo and Fm found in 0.1 M stressed leaves indicate that this concentration of sorbitol does not induce changes in the photosynthetic reaction centres. The lack of drought-induced damage to Photosystem II (PSII) photochemistry, as indicated by the maintenance of Fv and Fm values, has already been reported for some species (e.g. Santos et al., 2001). However, PSII seems to be sensitive to more severe drought stress induced by Table 1 Effect of sorbitol (0.1 and 0.2 M) on water content (%) and osmolality in O. europaea ssp. maderensis shootsa Sorbitol 0.0 M Water content (%) Osmolality (mOsm kg 1) MDA (mmol g FW 1) a
75.5 0.8 b 162 5 a 0.8 0.1 a
0.1 M 74.8 0.6 b 229 9 b 0.7 0.2 a
Values (mean S:D:) within rows with the same letter do not differ at P < 0:05.
0.2 M 73.3 0.2 a 280 12 c 2.8 0.2 b
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
415
Fig. 2. (a)±(c) Contents of K, P, Ca, Mg, Zn, Mn, Cu, Fe and B in O. europaea ssp. maderensis shoots exposed to 0, 0.1 and 0.2 M sorbitol. (d) Soluble protein contents of O. europaea ssp. maderensis shoots exposed to 0, 0.1 and 0.2 M sorbitol. For each parameter, mean values with the same letter do not differ significantly at P < 0:05. Bars represent the mean S:D:
0.2 M sorbitol. In fact Fv/Fm ratio decreased in severely stressed shoots indicating a signi®cant increase of energy dissipation in the photosynthetic apparatus, together with a reduction of Fm and Fv. Previous studies showed that salt and osmotic stress induced changes in the photosynthetic apparatus (e.g. Epron, 1997; Santos et al., 2002), and membrane permeability properties of chloroplasts. In O. europaea ssp. maderensis shoots, osmotic stress increased MDA production (Table 1), an indication of membrane degradation that correlates with the decrease of photosynthetic activity. It is widely reported that active oxygen species bring Table 2 Effect of sorbitol (0.1 and 0.2 M) on fluorescence parameters and on chlorophyll content in O. europaea ssp. maderensis leavesa Sorbitol 0.0 M Fo Fm Fv Fv/Fm chla (mg cm 2) chlb (mg cm 2) chla/chlb a
1016.3 2590.0 1573.8 0.6076 15.787 6.530 2.4174
0.1 M
57.83 a 375.80 b 363.47 b 0.17 b 0.048 c 0.528 a 0.061 a
1120.5 2440.0 1019.5 0.4178 5.737 2.211 2.5945
0.2 M
171.42 ab 309.76 b 265.41 b 0.12 b 0.126 b 0.127 b 0.204 a
1352.8 1684.8 332.0 0.1971 3.899 1.403 2.7789
Values (mean S:D:) within rows with the same letter do not differ significantly at P < 0:05.
184.33 b 161.92 a 27.32 a 0.05 a 0.084 a 0.063 a 0.028 b
416
G. Brito et al. / Scientia Horticulturae 97 (2003) 411±417
Table 3 Effect of sorbitol (0.1 and 0.2 M) on rooting capacity of O. europaea ssp. maderensis shootsa Half strength DKW with 20.7 mM IBA Unstressed shoots Percentage rooting Number of roots per shoot Total root length (cm) a
75 15.6 c 2.2 0.6 b 2.5 0.5 b
Stressed shoots (0.1 M) 25 10.0 b 1.0 0.0 a 1.2 0.6 a
Stressed shoots (0.2 M) 0 0.0 a ± ±
Values (mean S:D:) within rows with the same letter do not differ significantly at P < 0:05.
about peroxidation of membrane lipids leading to membrane damage (Shalata and Tal, 1998). Protection from oxidative damage is, partly, due to the activity of antioxidant enzymes such as superoxide desmutase, catalase and peroxidase. The connection between some of the responses observed under water stress conditions, and some antioxidant enzymes, namely with peroxidase and superoxide desmutase isoenzymes is presently being studied. Shoots of wild olive were maintained in the presence of sorbitol for adaptation and rooting, in order to, in the end, transfer acclimated tolerant plants to the ®eld. Osmotic stress reduced the capacity of shoots to develop roots (Table 3). Only 25 10:0 of the shoots adapted to 0.1 M developed roots in contrast to the 75 15 of rooted shoots under control conditions and these were smaller (1:2 0:6 cm) than those developed under unstressed conditions (2:5 0:5 cm). This reduction may be due to the inhibitory effect of low osmotic potential in rooting capacity that was already reported for other species such as sun¯ower (Santos and Caldeira, 2000). Acknowledgements The authors thank FCT (Project Ref. PNAT/1999/AGR/15011).
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