JXB Advance Access originally published online on June 18, 2004
Journal of Experimental Botany 2004 55(403):1743-1750; doi:10.1093/jxb/erh188
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RESEARCH PAPER |
Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress
Departamento de Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín (CSIC). Profesor Albareda no 1, 18008 Granada, Spain
* To whom correspondence should be addressed. Fax: +34 958 129600. E-mail: juanmanuel.ruiz{at}eez.csic.es
Received 23 March 2004; Accepted 29 April 2004
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Abstract |
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This study investigated several aspects related to drought tolerance in arbuscular mycorrhizal (AM) soybean plants. The investigation included both shoot and root tissues in order to reveal the preferred target tissue for AM effects against drought stress. Non-AM and AM soybean plants were grown under well-watered or drought-stressed conditions, and leaf water status, solute accumulation, oxidative damage to lipids, and other parameters were determined. Results showed that AM plants were protected against drought, as shown by their significantly higher shoot-biomass production. The leaf water potential was also higher in stressed AM plants (–1.9 MPa) than in non-AM plants (–2.5 MPa). The AM roots had accumulated more proline than non-AM roots, while the opposite was observed in shoots. Lipid peroxides were 55% lower in shoots of droughted AM plants than in droughted non-AM plants. Since there was no correlation between the lower oxidative damage to lipids in AM plants and the activity of antioxidant enzymes, it seems that first the AM symbiosis enhanced osmotic adjustment in roots, which could contribute to maintaining a water potential gradient favourable to the water entrance from soil into the roots. This enabled higher leaf water potential in AM plants during drought and kept the plants protected against oxidative stress, and these cumulative effects increased the plant tolerance to drought.
Key words: Arbuscular mycorrhizal symbiosis, drought, osmotic adjustment, oxidative damage
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Drought stress is considered to be one of the most important abiotic factors limiting plant growth and yield in many areas (Kramer and Boyer, 1997
) and arbuscular mycorrhizal (AM) symbiosis can protect host plants against its detrimental effects (for reviews see Augé, 2001; Ruiz-Lozano, 2003). Although mycorrhizal effects on plant water relations are not as dramatic and consistent as those on P acquisition and host growth, it is accepted that modest changes, if sustained, can have meaningful effects on plant fitness (Augé, 2001). Several studies on the topic have demonstrated that the contribution of the AM symbiosis to plant drought tolerance results from a combination of physical, nutritional, physiological, and cellular effects (Ruiz-Lozano, 2003). This appears to be due in many instances to differences in tissue hydration between AM and non-AM plants: one treatment group manages either to absorb more water or lose less water as the soil dries (Augé et al., 2001a). However, this seems not to be the only mechanism by which AM symbiosis enhances drought tolerance of plants. Additional mechanisms have been proposed, such as enhanced osmotic adjustment and leaf hydration or reduced oxidative damage caused by the reactive oxygen species (ROS) generated during drought (Ruiz-Lozano, 2003). In fact, it has been shown that mycorrhizal colonization and drought interact in modifying free amino acid and sugar pools in roots (Augé et al., 1992). A greater osmotic adjustment has also been reported in leaves of mycorrhizal basil plants than in non-mycorrhizal ones during a lethal drought period (Kubikova et al., 2001). In the same way, AM plants had postponed declines in leaf water potential (
) during drought stress (Davies et al., 1992; Subramanian et al., 1997; El-Tohamy et al., 1999) and leaf returned to non-AM level more quickly in AM than non-AM maize plants after the relief of drought (Subramanian et al., 1997). In contrast, leaf was similar in AM and non-AM plants when water was not limiting (Ebel et al., 1996; Bryla and Duniway, 1997). Finally, mycorrhizal lettuce plants showed increased superoxide dismutase (SOD) activity under drought stress and this correlated to plant protection against drought (Ruiz-Lozano et al., 1996, 2001a). In the same way, AM soybean plants subjected to drought had lower oxidative damage to lipids and proteins in nodules than non-AM plants, and this was linked to protection against nodule senescence (Ruiz-Lozano et al., 2001b; Porcel et al., 2003).
While several studies have focused on the physiological, biochemical, and molecular responses of shoot plant tissues to drought, few studies have considered the root systems at the same time, although this organ constitutes the primary site of drought perception and plays an important role in drought stress resistance and recovery. Root size and architecture are important factors for determining yield performance, particularly under conditions of limited water availability (Price et al., 2000). Roots are also actively involved in the perception and transduction of the drought stress signal and the information seems to be transferred to the whole plant via growth regulators [ABA (abscisic acid) or ethylene] that induce stomatal closure (Dubos and Plomion, 2003).
This study extends to the root system previous investigations on soybean shoot and nodule tissues in order to elucidate the preferred target tissue for AM effects against drought stress. For that, the effects of AM symbiosis on solute accumulation and on the oxidative damage to lipids and antioxidant activities in root and shoots of soybean plants were evaluated.
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Materials and methods |
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Experimental design and statistical analysis
The experiment consisted of a randomized complete block design with two inoculation treatments: (i) control non-mycorrhizal plants; and (ii) plants inoculated with the mycorrhizal fungus Glomus intraradices Schenck and Smith. Ten replicates of each treatment were done totalling 20 pots (one plant per pot), so that half of them were cultivated under well-watered conditions throughout the entire experiment while the other half were drought-stressed for 10 d before harvest.
Data were subjected to analysis of variance (ANOVA) with mycorrhizal treatment, water supply, and mycorrhizal treatment – water supply interaction as sources of variation, and followed by Duncan's multiple-range test (Duncan, 1955). Percentage values were arcsin transformed before statistical analysis.
Soil and biological materials
Loamy soil was collected from the Zaidin Experimental Station (Granada, Spain), sieved (2 mm), diluted with quartz-sand (<1 mm) (1:1, soil:sand, v/v) and sterilized by steaming (100 °C for 1 h d–1 for three consecutive days). The soil had a pH of 8.1 (water, 10 g soil in 25 ml water); 1.81% organic matter, available nutrient concentrations (mg kg–1): N, 2.5; P, 6.2 (NaHCO3-extractable P); K, 132.0. The soil texture was made up of 35.8% sand, 43.6% silt, and 20.5% clay.
Soybean (Glycine max L. cv. Williams) seeds were sterilized in a 15% H2O2 solution for 8 min, then washed several times with sterile water to remove any trace of chemical that could interfere with seed germination, and placed on sterile vermiculite at 25 °C to germinate. Three-day-old seedlings were transferred to plastic pots containing 600 g of the sterilized soil/sand mixture. A suspension (1 ml seed–1) of the diazotrophic bacterium Bradyrhizobium japonicum, strain USDA 110 (109 cell ml–1), was sprinkled over all seedlings at the time of planting.
Mycorrhizal inoculum was bulked in an open-pot culture of Zea mays L. and consisted of soil, spores, mycelia, and colonized root fragments. The AM species was Glomus intraradices Schenck and Smith, isolate EEZ 6, BEG 121. Ten grams of inoculum were added to the appropriate pots at sowing time just below the soybean seedlings.
Growth conditions
Plants were grown in a controlled environmental chamber with 70–80% relative humidity, day/night temperatures of 25/15 °C, and a photoperiod of 16 h at a photosynthetic photon flux density of 460 µmol m–2 s–1 (Li-Cor, Lincoln, NE, USA, model LI-188B).
Soil moisture was measured with a ML2 ThetaProbe (AT Delta-T Devices Ltd, Cambridge, UK), which measures volumetric soil moisture content by responding to changes in the apparent dielectric constant of moist soil. Water was supplied daily to maintain constant soil water content close to field capacity (17% volumetric soil moisture, as determined experimentally using a pressure plate apparatus and applying a pressure of one-third atmosphere for 48 h, and then determining the volumetric soil moisture) during the first 5 weeks of plant growth. At this time half of the plants were allowed to dry until soil water content reached 70% field capacity (3 d needed), which corresponded to 10% volumetric soil moisture (also determined experimentally in a previous assay). Plants were maintained under such conditions for an additional 10 d. To control the level of drought stress, the soil water content was measured daily with the ThetaProbe ML2 (at the end of the afternoon) and the amount of water lost was added to each pot to return soil water content to the desired 10% of volumetric soil moisture (70% of field capacity). However, during the 24 h period between each rewatering the soil water content progressively decreased, reaching a minimum value of 55% of field capacity.
Each week throughout the experiment plants received 10 ml of Hewitt's nutrient solution lacking N and P (Hewitt, 1952). Three weeks after planting, non-AM plants received nutrient solution amended with 0.35 mM K2HPO4 (Goicoechea et al., 1997). That P concentration was chosen to obtain well-watered plants of similar size and P content in both plant treatments.
Parameters measured
Biomass production: At harvest (48 d after planting), the shoot and root systems were separated and the shoot dry weight (DW) measured after drying in a forced hot-air oven at 70 °C for 2 d.
Symbiotic development: The percentage of mycorrhizal root infection was estimated by visual observation of fungal colonization after clearing washed roots in 10% KOH and staining with 0.05% trypan blue in lactophenol (v/v), according to Phillips and Hayman (1970). The extent of mycorrhizal colonization was calculated according to the gridline intersect method (Giovannetti and Mosse, 1980).
Leaf : The mid-day leaf was determined with a C-52 thermocouple psychrometer chamber and a HR-33T dew point microvoltmeter (Wescor Inc., Logan, UT, USA). Leaf discs corresponding to the third youngest leave were cut, placed inside the psychrometer chamber and allowed to reach temperature and water vapour equilibrium for 30 min before measurements were made by the dew point method.
Proline and total soluble sugars: Free proline and total soluble sugars were extracted from 1 g of fresh roots and leaves (Bligh and Dyer, 1959). The methanolic phase was used for the quantification of both substances. Proline was estimated by spectrophotometric analysis at 515 nm of the ninhydrin reaction, according to Bates et al. (1973). Soluble sugars were analysed by 0.1 ml of the alcoholic extract reacting with 3 ml freshly prepared anthrone (200 mg anthrone + 100 ml 72% (w:w) H2SO4) and placed in a boiling water bath for 10 min according to Irigoyen et al. (1992). After cooling, the absorbance at 620 nm was determined in a Shimadzu UV-1603 spectrophotometer. The calibration curve was made using glucose in the range of 20–400 µg ml–1.
Hydrogen peroxide concentration and oxidative damage to lipids: For the determination of the hydrogen peroxide concentration, aliquots (0.5 g) of roots and leaves were homogenized in an ice-cold mortar in HCl 25 mM and filtered through four layers of nylon cloth. The supernatants were adjusted to pH 7.0 for subsequent H2O2 quantification, which was performed by the 4-aminoantipyrine method (Frew et al., 1983).
Lipid peroxides were extracted by grinding 0.5 g of roots or leaves in an ice-cold mortar and 6 ml of 100 mM potassium phosphate buffer (pH 7). Homogenates were filtered through one Miracloth layer and centrifuged at 15 000 g for 20 min. The chromogen was formed by mixing 200 ml of supernatants with 1 ml of a reaction mixture containing 15% (w/v) trichloroacetic acid (TCA), 0.375% (w/v) 2-thiobarbituric acid (TBA), 0.1% (w/v) butyl hydroxytoluene, and 0.25 N HCl, and by incubating the mixture at 100 °C for 30 min (Minotti and Aust, 1987). After cooling to room temperature, tubes were centrifuged at 800 g for 5 min and the supernatant was used for spectrophotometric reading at 532 nm. Lipid peroxidation was estimated as the content of 2-thiobarbituric acid-reactive substances (TBARS) and expressed as equivalents of malondialdehyde (MDA) according to Halliwell and Gutteridge (1989). The calibration curve was made using MDA in the range of 0.1–10 nmol. A blank for all samples was prepared by replacing the sample with extraction medium, and controls for each sample were prepared by replacing TBA with 0.25 N HCl. In all cases, 0.1% (w/v) butyl hydroxytoluene was included in the reaction mixtures to prevent artieactual formation of TBARS during the acid-heating step of the assay.
Preparation of extracts from roots and shoots: Enzymes were extracted at 0–4 °C from 1 g (fresh weight (FW)) of root or shoot tissues using a mortar and pestle with 50 mg polyvinylpolypyrrolidone (PVPP) and 10 ml of the following optimized medium: 50 mM K-phosphate buffer pH 7.8 containing 0.1 mM EDTA for SOD, catalase (CAT), and ascorbate peroxidase (APX) (Gogorcena et al., 1995). The same medium supplied with 10 mM ß-mercaptoethanol was used for glutathione reductase (GR) (Moran et al., 1994). Extracts were filtered through four layers of nylon cloth and centrifuged at 20 000 g, 20 min, 0–4 °C. The supernatants were kept at –70 °C for subsequent enzymatic assays.
Soluble protein was determined by the dye binding microassay (Bio-Rad, Madrid, Spain) using BSA as the standard (Bradford, 1976).
Enzyme assays: Total SOD activity (EC 1.15.1.1
[EC]
) was measured according to Beyer and Fridovich (1987) based on the ability of SOD to inhibit the reduction of nitroblue tetrazolium (NBT) by superoxide radicals generated photochemically. One unit of SOD was defined as the amount of enzyme required to inhibit the reduction rate of NBT by 50% at 25 °C. CAT activity (EC 1.16.1.6
[EC]
) was measured by the disappearance of H2O2 (Aebi, 1984). The reaction mixture (3 ml) contained 10.6 mM H2O2. The reaction was initiated by adding 25 µl of the extract and monitoring the change in absorbance at 240 nm and 25 °C for 3 min. APX activity (EC 1.11.1.11
[EC]
) was measured in a 1 ml reaction volume containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM hydrogen peroxide, and 0.5 mM ascorbate. Adding the H2O2 started the reaction and the decrease in absorbance at 290 nm was recorded for 1 min to determine the oxidation rate of ascorbate (Amako et al., 1994). Finally, GR activity (EC 1.6.4.2
[EC]
) was determined by the procedure of Carlberg and Mannervik (1985). The reaction mixture (1 ml) contained 0.1 M HEPES pH 7.8, 1 mM EDTA, 3 mM MgCl2, 0.5 mM oxidized glutathione, 0.2 mM NADPH, and 150 µl of the enzyme extract. The rate of NADPH oxidation was monitored by the decrease in absorbance at 340 nm for 2 min. Two blanks, one without the enzyme extract and the other without oxidized glutathione, were used as controls.
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Results |
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Plant biomass and mycorrhizal colonization
Under well-watered conditions, shoot DWs of AM and non-AM soybean plants were similar (Table 1). Drought stress decreased plant growth in both treatments (a decrease of 57% in non-AM plants and 42% in AM plants). In any case, drought-stressed AM plants showed enhanced shoot DW (27%) as compared to non-AM plants. Root DW was similar in AM and non-AM soybean plants at whatever water regime.
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No mycorrhizal colonization was observed in plants not provided with AM inoculum. Mycorrhizal plants showed about 50% of mycorrhizal root length under both well-watered and drought-stressed conditions.
Leaf
The leaf determined at the end of the drought period was similar in AM and non-AM plants cultivated under well-watered conditions (Table 1). Drought stress decreased , but the decrease was larger in non-AM plants (–2.5 MPa) than in AM plants (–1.9 MPa). The time-course of leaf during the entire drought period showed a similar pattern for AM and non-AM plants, both under well-watered and under drought-stress conditions, with droughted non-AM plants always exhibiting lower leaf than the corresponding AM plants (Fig. 1).
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Proline and total soluble sugar accumulation
Accumulation of proline increased considerably in roots as a consequence of drought stress and AM plants accumulated 14% more proline in roots than non-AM plants (Fig. 2; Table 2). In shoots, drought stress also induced the accumulation of proline. However, in such plant tissue, AM plants accumulated 39% less proline than non-AM plants.
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Under well-watered conditions, total soluble sugars in roots were higher in AM plants than in non-AM plants (Fig. 3; Table 2). Drought stress increased sugar accumulation in both treatments, but no significant differences between treatments were found under such conditions according to ANOVA. In shoots, total soluble sugar was similar in both treatments under well-watered conditions. Drought increased the sugar content in non-AM plants by (116%), while AM plants showed a sugar content similar to that for well-watered conditions.
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Hydrogen peroxide accumulation and oxidative damage to lipids
No significant differences among the different treatments were found in hydrogen peroxide concentration in roots or shoots (data not shown). Roots of all treatments accumulated about 150 nmol H2O2 g–1 FW, while shoots accumulated about 10 times more.
In roots, the oxidative damage to lipids increased as a consequence of drought only in non-AM plants (Fig. 4; Table 2). AM plants showed similar levels of lipid peroxidation under both water conditions. However, under drought conditions roots of AM plants exhibited 13% less lipid peroxides than roots of non-AM plants. In shoots, the different behaviours of AM and non-AM plants were more evident. Drought enhanced lipid peroxidation in non-AM plants by 78%, while lipid peroxidation in shoots of AM plants remained unaffected. In any case, under drought conditions shoots of AM plants had 55% less lipid peroxides than shoots of non-AM plants.
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Antioxidant activities
In roots, SOD activity was similar in the different treatments, except for drought-stressed AM roots, which had significantly lower SOD (Table 3). In shoots, AM plants had lower SOD activity than non-AM plants when cultivated under well-watered conditions and higher activity when cultivated under drought-stress conditions.
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CAT activity showed an opposite behaviour in roots and in shoots (Table 3). In roots, CAT activity only increased in AM plants as a consequence of drought. In shoots, the CAT activity of AM plants was higher than in non-AM plants under well-watered conditions, but under drought stress conditions the CAT activity of AM plants decreased, reaching a value similar to that in non-AM plants.
APX was always higher in non-AM plants than in AM plants (Table 3). Drought stress increased the APX activity in shoots of both AM and non-AM plants compared with well-watered conditions. Mycorrhizal roots had significantly lower APX regardless of whether they had been grown under well-watered or drought-stressed conditions.
GR activity was notably enhanced by drought stress in roots of non-AM plants and decreased in roots of AM plants (Table 3). Drought-stressed AM and non-AM shoots had similar GR activities, while under well-watered conditions the GR activity increased by 40-fold in non-AM compared with AM plants.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Water deficit has profound effects on crop production. Even plants with an optimum water supply experience transient water-shortage periods, where water absorption cannot compensate for water loss by transpiration (Kramer and Boyer, 1997
). Arbuscular mycorrhizal symbiosis has been shown to increase plant tolerance to water deficit, although the exact mechanisms involved are still a matter of debate (Augé, 2001; Ruiz-Lozano, 2003).
This study investigated physiological and biochemical aspects related to water relations and drought tolerance in AM and non-AM plants subjected to drought stress. AM plants showed a higher tolerance to the drought stress imposed (only for 10 d) than non-AM plants, as shown by their enhanced shoot biomass production (27%), higher (less negative) leaf under such conditions, or lower lipid peroxidation.
Drought-stressed plants have been shown to accumulate organic osmolytes such as sugars and amino acids (proline) that are known to contribute to the host-plant tolerance under water-deficit conditions (Schellembaum et al., 1998; Trotel-Aziz et al., 2000). The enhanced sugar content in AM roots under well-watered conditions may be due to the sink effect of the mycorrhizal fungus demanding sugars from shoot tissues. Under drought the sugar content in roots was similar in both treatments, suggesting that osmotic adjustment occurred. In contrast, in shoots the sugar content of droughted AM plants was considerably lower than in non-AM plants. Schellembaum et al. (1998) suggested that the AM fungus can be a strong competitor for root-allocated carbon under conditions limiting photosynthesis. These authors proposed that the lower hexose accumulation in leaves of mycorrhizal plants in drought could be due to a lower availability of photosynthates for storage in these tissues. However, another explanation is also possible, that AM shoots were less strained by drought than non-AM ones. The lower accumulation of compatible solutes may indicate that the plants more successfully avoided drought stress (Augé, 2001). In fact, proline, the other osmoregulator measured in this study, also accumulated less in shoots of AM plants than in non-AM plants. The higher leaf in stressed AM plants (–1.9 MPa) than in non-AM plants (–2.5 MPa) also supports this last hypothesis (Subramanian et al., 1995). In contrast, in roots proline accumulated more in AM plants than in non-AM ones. The accumulation of proline and total soluble sugar in roots could have provided the root with an osmotic mechanism to maintain a favourable gradient for water entrance into the roots (Irigoyen et al., 1992) leading, therefore, to a lower stress injury in the plant.
In addition to acting as an osmoprotectant, proline also serves as a sink for energy to regulate redox potentials, as a hydroxyl radical scavenger, as a solute that protects macromolecules against denaturation, and as a means of reducing acidity in the cell (Smirnoff, 1993; Kishor et al., 1995). This can be important for AM plants since drought also induces an oxidative stress, which, it has been pointed out, is responsible for many of the degenerative reactions caused by drought. The oxidation of membrane lipids is a reliable indication of uncontrolled free-radical production and hence of oxidative stress (Noctor and Foyer, 1998). Accordingly, the amount of lipid peroxides was quantified in roots and shoots. In roots, the lipid peroxidation in AM plants subjected to drought was 13% lower than in droughted non-AM plants. In shoots, lipid peroxidation was 55% lower in droughted AM plants than in droughted non-AM plants. Curiously, the hydrogen peroxide concentration measured in this study was similar in all treatments. However, it should be remembered that H2O2 is involved in virtually all major areas of aerobic biochemistry (e.g. respiratory and photosynthetic electron transport; oxidation of glycolate, xanthine, and glucose) and is produced in copious quantities by several enzyme systems, even under optimal conditions (Noctor and Foyer, 1998). Moreover, in some circumstances, the destructive power and signalling potential of ROSs such as H2O2 are utilized as an effective means of defence (Levine et al., 1994; Foyer et al., 1997).
The activities of four antioxidant enzymes were measured for correlation with the oxidative damage to lipids. Results showed that there was no relationship between the antioxidant activities and the decrease in lipid peroxidation in roots and shoots of droughted AM plants. In addition, only shoot SOD and shoot APX activities showed a significant interaction between mycorrhization and water regime, while no significant interaction was observed for the other activities. In general, the results obtained for the four antioxidant activities agree with previous results obtained in roots of soybean plants inoculated with G. mosseae (Porcel et al., 2003). The only exception was found in relation to the GR activity that, in the present study involving G. intraradices, was lower in roots of droughted AM plants than in the corresponding non-AM plants, while in the previous study, involving G. mosseae, the GR activity increased in AM plants (Porcel et al., 2003). However, it must be borne in mind that the AM fungi utilized in both studies were different and that dissimilar behaviour of AM fungi in relation to several plant enzymatic activities has been often reported (Azcón et al., 1996; Azcón and Tobar, 1998; Calvente, 2003). In contrast to the GR activity, the lower oxidative damage to lipids in the AM plants seems to be a consistent effect of AM symbiosis, regardless of the fungal species involved in the association (Ruiz-Lozano et al., 2001b; Porcel et al., 2003; the present results).
In addition to the above-discussed drought-tolerance mechanisms, the AM contribution to plant drought tolerance might also have occurred through drought avoidance mechanisms such as hyphal water uptake (Hardie, 1985; Ruiz-Lozano and Azcón, 1995; Marulanda et al., 2003) or increased water uptake related to mycorrhizal changes in root morphology (Kothari et al., 1990) or soil structure (Augé et al., 2001a). Such mycorrhizal effects could allow plants to remain more hydrated than non-AM plants as soil dries (Augé et al., 2001b). Data from the present study, such as the higher mid-day leaf in AM than in non-AM plants, the lower accumulation of soluble sugar and proline in shoots of AM than in non-AM plants, or the lower lipid peroxidation in AM than in non-AM plants without a correlation with enhanced antioxidant activities in such plants, suggest that possibility.
The overall data show that both root and shoot tissues are influenced by AM symbiosis by means of drought-avoidance and drought-tolerance mechanisms. It seems that first the AM symbiosis enhances osmotic adjustment in roots which could contribute to maintaining a gradient favourable to the water passing from soil into the roots. This enables higher leaf in AM plants during drought and keeps the plants protected against oxidative stress, and these cumulative effects increase the plant drought tolerance.
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Acknowledgements |
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This work and R. Porcel were financed by CICYT-FEDER (Project AGL2002–03952).
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