Journal of Experimental Botany, Vol. 52, No. 355, pp. 351-360,
February 2001
© 2001 Oxford University Press
Original Papers |
In situ and in vitro senescence induced by KCl stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism
Biology Department, Cell Biology Centre, University of Aveiro, 3800 Aveiro, Portugal
Received 21 August 2000; Accepted 5 September 2000
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Abstract |
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Sunflower (Helianthus annuus L. cv. SH222) plants and calli were exposed to KCl stress for three weeks. Calli were more tolerant to KCl than plants. KCl stress decreased NO-3, Mn, Fe and B levels in whole plants and P, Ca and Mg in shoots. NO-3, P, Ca, Mg, Mn, and B levels decreased in 100 mM-stressed calli. Chlorophyll content, Fm and (Fm-F0)/Fm ratio decreased in stressed leaves, while F0 increased only in leaves exposed to severe stress (100 and 150 mM). Membrane permeability and lipid peroxidation increased in plants under all stress conditions and in 100 and 150 mM stressed calli, but remained unchanged in 25 mM stressed calli. Salt stress also induced changes relating to antioxidant enzymes: plants under all stress conditions showed a decrease in catalase, peroxidase and SOD activities. Calli under moderate stress (25 mM KCl) showed an increase of catalase, peroxidase and SOD activities, but the activities of peroxidase and SOD decreased when calli were exposed to higher KCl concentrations. The decrease of antioxidant enzyme activities is in tune with lipid peroxidation and membrane permeability increases. On the other hand, calli adapted for 6 months to 100 mM KCl showed an increase of these enzyme activities compared to unstressed calli, while MDA production and membrane permeability were not significantly affected.
Key words: Helianthus annuus, KCl stress, mineral deficiencies, oxidative stress, tissue culture, senescence.
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Introduction |
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Induced premature senescence is often quantified by decreases of soluble protein contents, changes of chl a/ chl b ratio (Lin and Kao, 1998
; Lutts et al., 1996) and by membrane permeability and/or lipid peroxidation increases (Dhindsa and Matowe, 1981; Dhindsa et al., 1981). Lipid peroxidation induced by active AOS is considered to be an important mechanism of membrane deterioration (Irigoyen et al., 1992; Shalata and Tal, 1998). Plants have evolved non-enzymatic and enzymatic protection mechanisms that efficiently scavenge AOS and prevent damaging effects of free radicals (Irigoyen et al., 1992; Shalata and Tal, 1998). Enzymatic protection is partly performed by SOD that eliminates O2·- radicals and by catalase and peroxidases that degrade H2O2 influencing the level of lipid peroxidation.
Senescence induced by salt stress is also related to the accumulation of toxic ions or to nutrient depletion. For example, some nutrients are involved in photosynthesis and in protein synthesis regulation and their depletion may lead to serious inhibition of these processes. In particular, Mg deficiency may lead to decreases of chlorophyll synthesis and variable fluorescence. Another example is calcium, a second messenger and an important element in cell wall and in membrane structure/stability (White, 1998), which deficiency may lead to serious cell damage. Also, Zn, Cu and Mn deficiencies were reported to affect SOD isoenzyme activities (Yu and Rengel, 1999). Maintenance of membrane integrity and the selective uptake of essential minerals as well as ion compartmentation are some of the parameters that were previously related with salt tolerance acquisition (Salama et al., 1994).
In the present report, the effects of KCl stress on sunflower plant and calli growth, nutritional imbalance and senescence parameters were studied. It is also proposed a correlation between changes of antioxidant enzyme activities (SOD, catalase and peroxidase) and membrane permeability and lipid peroxidation in in vitro and in situ KCl-stressed cells.
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Materials and methods |
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Plant material and growth conditions
Sunflower (Helianthus annuus L. cv. SH222) seeds were supplied by SCLEPAL, Portugal. Six groups of 5-d-old seedlings (n=60 for each group) were grown on aerated Long Ashton medium (according to Santos and Cadeira, 1999), supplemented, respectively, with 0 mM KCl (-0.06 MPa), 10 mM (-0.09 MPa), 25 mM (-0.14 MPa), 50 mM (-0.29 MPa), 100 mM (-0.52 MPa), and 150 mM (-0.63 MPa). Plants were grown at 22±2 °C, with light supplied by Osram 36 W lamps giving an intensity of 98±2 µmol m-2 s-1 and a photoperiod of 16 h. Plant fresh and dry weights were measured at 0, 3, 8, 15, and 21 d of culture (n=15).
One-month-old calli, obtained on modified MS (Murashige and Skoog, 1962) medium (MSmod) supplemented with NAA 0.5 mg dm-3, BA 0.5 mg dm-3 and GA3 0.1 mg dm-3 (according to Santos and Caldeira, 1999), were used for KCl stress studies. Calli were grown in a growth chamber at 22±1 °C, with light supplied by Osram 18 W lamps giving an intensity of 93±1 µmol m-2 s-1 and a photoperiod of 16 h. Six groups of calli (±0.1 g fresh weight) were grown on MSmod medium containing, respectively, 0 mM (-0.19 MPa), 10 mM (-0.21 MPa), 25 mM (-0.27 MPa), 50 mM (-0.38 MPa), 100 mM (-0.57 MPa), and 150 mM (-0.76 MPa) KCl. Calli samples (n=15) from all stress conditions were harvested for measurement of fresh and dry weights at 0, 3, 8, 15, and 21 d after stress imposition.
Calli that survived to 100 mM KCl, after treatment, were maintained in this salinity for 6 months.
Determination of salt resistance
Resistance to KCl was estimated as ID50, which is defined as the KCl concentration required in the growth medium to inhibit dry weight gain of plants or calli by 50%, 2 weeks after salt exposure.
Mineral analysis
Ion content was determined in roots, shoots and in calli. Dried tissues were mineralized and NO-3, SO42- and Cl- quantification was performed by a capillary electrophoresis system (Waters Quanta 4000) (Santos and Caldeira, 1999). For the analysis of all other elements (B, Ca, Cu, Fe, K, Mg, Mn, P, and Zn) dried tissues were treated according to Evans and Bucking (Evans and Bucking, 1976) and elements were determined by Induced Coupled Plasma Spectroscopy (Jobin Ivon JY70 Plus) according to Santos and Caldeira (Santos and Caldeira, 1999). Results were averaged from six individuals in two independent assays with three replicates each.
Determination of MDA concentration and membrane permeability
Lipid peroxidation was determined by malondialdehyde (MDA) content (according to Dhindsa and Matowe, 1981): 0.25 g of tissue samples were homogenized in 5 cm3 trichloroacetic acid 0.1% and centrifuged at 10 000 g for 10 min. The supernatant was collected and 1 cm3 was mixed with 4 cm3 20% trichloroacetic acid and 0.5% thiobarbituric acid. The mixture was heated at 95 °C (30 min), quickly cooled and centrifuged at 10 000 g for 10 min. The supernatant was used to determine MDA concentration at 532 nm.
Membrane permeability was determined by conductivity (Lt/L0) and by UV absorbance (RLR) as described previously (Lutts et al., 1996): samples of plants and calli were washed in de-ionized water to remove surface-adhered electrolytes. Tissue segments were placed in tubes with 10 cm3 de-ionized water and incubated overnight at 25 °C (85 rpm). Electrolyte leakage analysis was performed by conductivity of the bath solution before and after autoclaving. Solute leakage was determined by measuring the absorbance (280 nm) of the bathing solutions before and after freezing the tissues (Redmann et al., 1986).
Chlorophyll concentration and fluorescence parameters in plants
Chlorophyll content was determined from expanded and young leaves according to Arnon (Arnon, 1949).
Plants were dark adapted for 20 min in a growth chamber at 22±2 °C. Fluorescence was monitored in expanded and young leaves using a Plant Efficiency Analyser (Hansatech Instruments Ltd., UK) by illuminating leaves with a peak wavelenght 650 nm and a saturating light intensity of 3000 µmol m-2 s-1. The basal non-variable chlorophyll fluorescence (F0) and the maximal fluorescence induction (Fm) were determined and then the maximum quantum yield of PSII ((Fm-F0)/Fm) (Maxwell and Johnson, 2000) was estimated.
Antioxidant enzymes and soluble protein content
Tissue samples (0.5 g) were homogenized at 4 °C in 1 cm3 of K-phosphate buffer 0.05 M (pH 7.8) containing 0.1 mM ethylenediamine tetraacetic acid, 5 mM cysteine, 1% polyvinyl pyrrolidone, and 0.2% Triton X-100 (Olmos et al., 1994). Homogenates were filtered and centrifuged at 8000 g for 15 min at 4 °C. The supernatant was dialysed at 4 °C for 24 h against 1 dm3 10 mM K-phosphate buffer (pH 7.8) containing 10 µM leupeptin and 100 µM phenylmethylsulphonyl fluoride. The dialysed samples were centrifuged at 2500 g for 10 min at 4 °C and used for enzymatic determination (Olmos et al., 1994). Catalase (EC 1.11.1.6) was determined according to Aebi (Aebi, 1974). Guaiacol peroxidase was determined (Putter, 1974). For superoxide dismutase (EC 1.15.1.1) samples were treated, and SOD activity was determined, by the method described earlier (Osswald et al., 1992). Soluble proteins were determined according to Bradford (Bradford, 1976).
Fisher and t-tests
Results presented are average means for at least three independent assays with three replicates each. Variance analysis was performed by Fisher test and the means were statistically tested using a 2-sided t-test. Statistical significance is assumed at P<0.05.
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Results |
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Plant and calli growth
KCl stress decreased sunflower plant and calli growth rates (Fig. 1a
–c). This decrease was evident not only in shoot and root fresh and dry weights, but also in leaf areas (data not shown). For concentrations higher than 25 mM, shoots were more affected than roots (Fig. 1a, b). Stressed leaves were small and chlorotic and soon became necrotic in plants exposed to 100 and 150 mM KCl. Plants exposed to 150 mM died 3 weeks after stress imposition. After 15 d, negative correlation were established between shoot (y=-0.00321x0.4306, R=0.69), root (y=-0.00153x0.2372, R=0.78) and calli growth (y=-0.00098x0.163; R=0.93) and KCl concentration in the media. These negative correlations gave an ID50 of 40.1 mM KCl for shoots, of 57 mM KCl for roots and of 72 mM KCl for calli suggesting that calli are more tolerant to KCl stress than plants.
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Nutrient content in plants and calli
Severe KCl stress decreased NO-3 levels in the whole plant (Table 1a). Phosphorus, Ca and Mg decreased only in stressed shoots. K and Cl-, as expected, increased in the whole plant. With respect to micronutrients, Mn, Fe and B decreased in stressed roots and leaves. Phosphorus, NO-3, Ca, and Mg decreased in KCl-stressed calli, while K and Cl- levels increased (Table 1b). Only B and Mn decreased in 100 mM-stressed calli while other micronutrients were not affected (Table 1b).
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Chlorophyll content and fluorescence parameters
Table 2 shows that basal fluorescence (F0) was not significantly affected in moderate (25 mM) stressed leaves, but increased during treatments in 100 and 150 mM-stressed leaves. Maximal values were obtained in 150 mM-stressed expanded leaves at the end of treatment. Contrarily, Fm decreased with KCl stress, in particular in 100 and 150 mM-stressed leaves leading, together with the increase of F0, to a decrease of (Fm-F0)/Fm ratio. In expanded leaves this reduction was significant for all KCl concentrations at the end of treatment while in young leaves this effect was only significant for 100 and 150 mM KCl.
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Chlorosis was observed in plants exposed to KCl stress, and this was confirmed by a decrease of chlorophyll content (Fig 2a
, b). At the end of treatment chl a/ chl b ratio was lower in 25 mM-stressed leaves than in unstressed, but at the same time this ratio increased in both 100 and 150 mM stressed leaves (Fig. 2c, d).
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Determination of lipid peroxidation and membrane permeability
Malondialdehyde (MDA) production increased significantly with leaf ageing and this effect was enhanced by KCl stress. For example, at day 21 unstressed leaves had a MDA production 1.4 times higher than at day 8, while leaves exposed to 150 mM produced five times more MDA than unstressed leaves (Fig. 3b). Also, MDA production in unstressed plants was always higher in shoots than in roots (Fig. 3a, b).
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MDA production in calli was not significantly affected by tissue ageing (Fig. 3c
). Also, there were no significant differences of MDA production between unstressed and 25 mM-stressed calli during the whole treatment in contrast to what happened between unstressed and 25 mM-stressed roots and leaves. MDA production increased however in 100 and 150 mM-stressed calli which is similar to what happened in plants. At the end of treatment, 150 mM-stressed calli had a MDA production 2.4 times higher than unstressed calli.
Similar to MDA formation, the increase of solute and electrolyte leakage (quantified by Lt/Lo and RLR) also increased with plant ageing (Fig. 3d, e, g, h). It can be seen, by the increases of Lt/Lo and RLR, that KCl stress increased membrane damage and this effect was more evident in leaves than in roots (Fig. 3d, e, g, h). In 150 mM-stressed plants the increments of solute and electrolyte leakage were more evident during the first weeks, being higher in leaves. In 100 mM-stressed plants, solute and electrolyte leakage increases were more significant during the last week of treatment, being also more evident in leaves. Calli exposed to 100 and 150 mM KCl also suffered increases of solute and electrolyte leakage, but to a lesser extent than stressed plants (Fig. 3f, i). Solute and electrolyte leakage increases were evident at the end of the first week in 150 mM-stressed calli while they were only evident at the second or third week in 100 mM-stressed calli. Calli exposed to 25 mM never showed any changes in MDA production or in membrane permeability (Fig. 3c, f, i).
KCl adapted-calli for 6 months produced only 1.2 times more MDA than unstressed calli, and solute and electrolyte leakage was not significantly different from unstressed calli (Table 3).
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Antioxidant enzymes in stressed plants and calli and in KCl-adapted calli
Catalase activity was higher in unstressed calli than in shoots while SOD and peroxidase activities were lower (Fig. 4a, d). All stress conditions decreased catalase, peroxidase and SOD activities in shoots (Fig. 4a–f). Minimum values were obtained in 150 mM-stressed shoots at day 21 for catalase, peroxidase and SOD. Significant decreases of SOD and peroxidase activities were already observed at day 3 in stressed shoots. By contrast, moderate stress increased catalase, peroxidase and SOD activities in calli, while higher KCl concentrations decreased peroxidase and SOD activities not affecting significantly the activity of catalase (Fig. 4d, e, f).
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Unstressed shoots had, in average, 17.2±1.2 mg protein g-1 FW and this content was not significantly affected by moderate stress. Only 150 mM KCl decreased the content of protein to 10.3±1.5 mg protein g-1 FW in shoots. Calli exposed to 100 and 150 mM KCl also suffered a slight decrease of soluble protein content: unstressed calli had an average 13.2±0.6 mg protein g-1 FW, but 100 mM and 150 mM stress decreased protein contents to 11.3±1.1 and 9.5±0.8 mg protein g-1 FW, respectively. KCl adapted-calli showed an increase of peroxidase, catalase and SOD activities respectively to unstressed calli with the same age (Table 3
) while protein content was not significantly affected.
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Discussion |
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Plant and calli growth in the presence of KCl
In this paper it is demonstrated that KCl stress accelerates the development of senescence characteristics in sunflower cells and that these changes are accompanied by decreases of antioxidant enzyme activities. Shoots are more sensitive to KCl than roots having not only a higher reduced growth but also higher membrane degradation.
Sunflower calli are more tolerant to KCl stress than plants, which is shown by the higher ID50 exhibited by calli. Also, KCl stress induced higher increases of MDA production and solute and electrolyte leakage in plants than in calli. This fact, together with the higher activities of antioxidant enzymes observed in calli, may justify the higher tolerance to KCl exhibited by in vitro cells.
It is still a question why, in some genotypes, cells growing in vitro are more tolerant to salt than intact plants. When one compares salt tolerance between calli and plants, several factors must be taken in account: (1) plants have different stages of differentiation during their life cycle that may affect their salt tolerance while calli differentiation can usually be controlled; (2) due to transpiration mesophyll cells may be exposed to higher concentrations of K+ than the concentration present in the medium; (3) culture media used in vitro are usually richer in salts and sugars than those used for plant growth and, therefore, in vitro cells may not suffer so easily from macro and micronutrient deficiencies. On the other hand, in vitro cells may be indirectly selected for higher saline concentrations and reduced osmotic potentials. These aspects may contribute to the higher salt tolerance observed in calli.
Nutritional imbalance, lipid peroxidation and antioxidant metabolism
In several species, the increase of Cl- concentration in the medium leads to a reduction of nitrate levels in tissues (Grattan and Grieve, 1994). The same effect was found for KCl-stressed sunflower plants and calli. However, once the pool of free amino acid increased with increasing KCl stress (Santos, 1998), the decrease of nitrate levels may not only be due to a decrease of N uptake, but also to an increase of its reutilization by cells mainly in proline synthesis (Santos, 1998).
KCl stress decreased Ca levels in shoots. This decrease may, besides other effects, significantly affect membrane structure and permeability, cell wall composition and other physiological processes. Cytoplasmic calcium acts as a second messenger in plant cells, being involved in signalling pathways mediation and it was reported to be a primary event in response to ABA, a hormone that was postulated to play a central role in signalling stress responses (Ingram and Bartels, 1996; Netting, 2000).
KCl also induced the accumulation of high levels of K in sunflower cells. Potassium uptake seems to be similarly regulated in 100 mM-stressed and adapted calli because both systems accumulated similar amounts of K. It was previously proposed, for other species, that K+ influx regulation may be due to the allosteric effect of internal K+ in the cell (Benlloch et al., 1994). However, regulation of K+ uptake may follow a more complicated pathway than the allosteric response (Benlloch et al., 1994). Recently, experiments with split roots have shown that long-distance signals must also play a role (Maathuis and Amtmann, 1999), and it was postulated that hormones could be involved in this regulation. For example, Montero et al. suggested that ABA inhibited Cl- and Na+ uptake, and stimulated the uptake of K+ in NaCl-tolerant bush bean plants (Montero et al., 1997). Also, stimulation of K+ uptake was also reported to be promoted by GA3, a hormone that was also associated with antisenescence in salt-stressed cells (Prakash and Prathapasenan, 1990). It is therefore important to study the effect of KCl stress on plant hormones and its influence on K+ uptake regulation.
On the other hand, there is no definitive study on the effects of KCl on micronutrient accumulation in plants. Micronutrient accumulation depends on the tissue nature, the micronutrient concentration in the medium and also on the type of salt stress (Grattan and Grieve, 1994). In sunflower, micronutrient accumulation with salinity also depends on the culture system used. The behaviour of plants and calli was different with respect to the accumulation of micronutrients with increasing KCl concentration in the medium: in fact, except for B and Mn, KCl-stressed calli were not affected in their micronutrient contents. This fact may be due not only to different cellular organization, but also to the fact that the MS medium is richer in salts than the culture medium used for plant growth.
Similarly to what has been reported for this (Santos and Caldeira, 1999) and other species exposed to NaCl (Chen et al., 1991; Irigoyen et al., 1992; Lutts et al., 1996), KCl stress induced changes in sunflower cells that represent a hastening of the natural senescence. On the other hand, KCl tolerance seemed to correlate with the stimulation of antioxidant enzymes and the enhanced ability to remove AOS. In fact, 25 mM-tolerant and 100 mM-adapted calli showed increases of antioxidant enzyme activities, while MDA production and membrane permeability did not differ from unstressed calli. On the contrary, in all stressed plants the activities of these enzymes decreased, together with increases of MDA production and membrane permeability.
Shalata and Tal suggested that, as AOS induce peroxidation of membrane lipids, resistance to environmental stress may depend on the inhibition of AOS production or the enhancement of antioxidant levels (Shalata and Tal, 1998). One of the plant responses to AOS production is the increase of antioxidant enzyme activities providing protection from oxidative damage induced by several environmental stresses. The higher tolerance of some genotypes to environmental stresses has been associated with higher activities of antioxidant enzymes. For example, the wild NaCl-tolerant species Lycopersicon pennellii had lower lipid peroxidation than the cultivated species L. esculentum, and higher activities of SOD, peroxidase and catalase (Shalata and Tal, 1998), suggesting that the wild species is better protected against oxidative damage. Lopez et al. reported an increase of peroxidase activity in salt-stressed radish cells (Lopez et al., 1996). Also, NaCl-sensitive pea cultivars suffered a decrease of Mn-SOD activity, while in tolerant cultivars the activity of this isoenzyme increased (Olmos et al., 1994). The deficiency of Cu, Zn and Mn also affected Cu/ZnSOD and MnSOD activities in lupin leaves (Yu and Range, 1999) and in bush beans (Mehlhorn and Wenzel, 1996). KCl stress induced deficiency of Mn in both 100 mM-stressed plants and calli, but Cu decreased only in plants. On the other hand, SOD and other antioxidant enzyme activities were more affected in stressed plants. It must be further clarified if there are different mechanisms of removing AOS in stressed calli and plants that may justify the influence of the cell system to salt stress response.
Fluorescence parameters and chlorophyll content
Some antioxidant enzymes (e.g. MnSOD) are located in plastids and, as postulated by Osswald et al., compartmentation of the singlet oxygen (formed within thylakoid membranes) and the detoxifying enzymes, localized in the stroma, must occur (Osswald et al., 1992). This compartmentation implies the maintenance of membrane integrity, and if one of these conditions is destroyed, the plastid loses integrity affecting photosynthesis.
Photosynthetic parameters such as chlorophyll content and photosynthetic rate have previously been used as senescence parameters (Buchanan-Wollaston, 1997). KCl stress severely affected chlorophyll concentrations. In leaves exposed to severe stress, not only was the total chlorophyll content drastically reduced, but the chl a/chl b ratio increased showing that chl b was being degraded at a higher rate than chl a. This can be explained by the fact that the first step in chl b degradation involves its conversion to chl a (Fang et al., 1998).
The stability of F0, found for moderate KCl stress, indicates that this low concentration of salt has no significant changes in the reaction centres. However, the variations found for higher KCl concentrations indicate losses of energy transference from pigments to the reaction centre (probably due to damage of the LHC centre associated to the PSII). Changes in (Fm-F0)/Fm indicate variations in the photochemical efficiency of the PSII (Maxwell and Johnson, 2000). A reduction of this ratio in KCl-stressed plants is also due to a reduction of Fm that may reflect an increased energy dissipation due to a destruction of photosynthetic apparatus. Previous studies showed that salt and osmotic stress induced changes in the photosynthetic apparatus (Bhorer and Dorffling, 1993; Lutts et al., 1996), and the membrane permeability properties of chloroplasts. Dissociation of the light-harvesting antennae from the PSII core and the denaturation of the PSII reaction centre have been postulated for rice NaCl-stressed leaves (Lutts et al., 1996) and for drought-stressed cotton (Genty et al., 1987) and sunflower (Conroy et al., 1988) plants. This fact may be the result of chlorophyll degradation and/or synthesis deficiency together with a decrease of thylakoid membrane integrity. Several data sets point to this conclusion: (1) a reduction in Mg and Mn contents together with a reduction of chlorophyll contents observed in KCl stressed leaves; (2) an increase of membrane permeability and of lipid peroxidation, probably due to changes in membrane composition/structure. It must be emphasized that KCl stress decreased Ca levels while increased K, affecting the sites of transport and Ca2+ allocation in membranes and most probably, affecting plastid membrane structure.
Acquisition of salt tolerance
Calli that survived to 100 mM KCl were subcultured on the same medium for 6 months. At the end of this time it was possible to select some calli adapted to this salinity that had, nevertheless, a retarded growth. Tolerance to KCl has been reported previously (Rush and Epstein, 1981) for some tomato genotypes and by Sumaryati et al. in protoplast-derived colonies of haploid Nicotiana plumbaginifolia L., that regenerated into plants (Sumaryati et al., 1992). In all cases, selected cells accumulated high levels of K+, similar to what happens with KCl-tolerant sunflower calli. This fact suggests that KCl-adapted calli developed alternative mechanisms of tolerating high K+ level that were not present in KCl-stressed cells and that may contribute to KCl adaptation. In adapted calli, MDA production and membrane permeability reached values that were similar to those of unstressed calli while antioxidant enzymes were much more active.
These data support the idea that salt tolerance/adaptation may be correlated with a stimulation of antioxidant enzymes and that these may control lipid peroxidation and reduce membrane degradation. In fact, in these adapted cells, catalase, peroxidase, and SOD activities were higher than those found for unstressed calli. On the contrary in 100 mM-stressed calli the activities of these enzymes decreased during the whole treatment. Further studies must be done in order to clarify if these changes in antioxidant enzymes are a direct effect of K+ and/or Cl- in cell, or a response to a primary effect induced by KCl, such as nutrient imbalance.
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Notes |
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1 To whom correspondence should be addressed. Fax: +351 2344 26408. E-mail: csantos{at}bio.ua.pt
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Abbreviations |
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AOS, active oxygen species; BA, benzyladenine; GA3, gibberelic acid; ID, inhibition dose; MDA, malondialdehyde; MS, Murashige and Skoog medium; NAA, naphthalene acetic acid; RLR, relative leakage ratio; SOD, superoxide dismutase.
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