Introduction

An Azolla–Anabaena symbiosis is considered as an important biofertilizer due to its ability to fix atmospheric nitrogen. The fern Azolla has a symbiotic relationship with the cyanobacterium Anabaena azollae which grows in the dorsal leaf lobe cavity of host Azolla (Peters 1978). This association is agronomically important and A. azollae is able to fix atmospheric nitrogen at a rate of 1.1 Kg N ha-1 d-1 (Watanabe et al. 1977). This is sufficient to meet the entire nitrogen requirement of rice crop within a few weeks (Lumpkin 1987). Azolla has a global distribution and it has a variety of other uses such as animal feed, human food, hydrogen fuel, production of biogas, medicine, water purifier, and weed control and is thus known as “green gold mine”(Wagner 1997).

However, salinity is a major problem for the growth and development of plants. Approximately 100 million hectares of the worldwide land has been adversely affected by salinity (Ghassemi et al. 1995). It is estimated that in the next 25 years, there will be a loss of 30% in the farmable land, and by the year 2050, this will increase to 50% (Zhu 2001). Salinity in general leads to an imbalance of the cellular ions resulting in ion toxicity, osmotic stress, and production of reactive oxygen species (Cheeseman 1988; Cramer et al. 1994). Salt stress is generally associated with osmotic stress, and high exogenous salt concentrations also lead to the production of reactive oxygen species (ROS) which are highly reactive and can react with proteins and lead to denaturation of proteins and cause lipid peroxidation (Navari-Izzo et al. 1996). However, plants have evolved effective scavenging mechanisms involving the antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and catalase to counter the stress factor (Dionisio-Sese and Tobita 1998; Matysik et al. 2002). Non-enzymatic antioxidants such as proline also play a significant role against singlet oxygen and free radical damage (Alia et al. 2000).

However, to ensure future productivity of the agricultural regions, we need to select and characterize salt-tolerant plants. It is in this context that improvement in salinity tolerance of Azolla plants becomes important. Although inhibitory effect of salinity on Azolla has been studied in considerable detail in the past (Rai and Rai 1999; Masood et al. 2006), very few attempts have been made to improve the salinity tolerance in Azolla. Induction of increased salt tolerance in Azolla pinnata has been attempted by Rai and Rai (1999). Although they have been able to adapt A. pinnata to higher salt concentrations by pre-exposing them to sub-lethal concentrations of NaCl, the complex sequence of physiological events that follow the adaptation process has not been elucidated in detail. A proper understanding of the adaptation mechanism of Azolla will help in improving the salt tolerance of Azolla plants and their possible exploration as successful bio-inoculants in saline paddy fields. It is not certain as yet if adaptation process in Azolla is a result of efficient detoxification of free radicals. Salt-tolerant plants, besides being able to regulate the ion and water movements, should also have a better antioxidant system for efficient removal of ROS (Rout and Shaw 2001). The antioxidant enzymes play a vital role in tolerance to different environmental stresses and an efficient antioxidant system leads to better tolerance or resistance to environmental stresses including salinity. Several physiological studies have correlated salt tolerance with the accumulation of ions also (Pantalone et al. 1998). Thus, the complex sequence of events involved in salt adaptation is important for the survival due to salinity and provide vital clues for improving salt tolerance. Therefore, the present study was conducted to study the adaptation to salinity by Azolla microphylla plants pre-exposed to salinity and mechanism(s) leading to salt adaptation with an emphasis on the ion metabolism and antioxidant enzymes.

Materials and methods

Plant material and growth conditions

A. microphylla plants maintained at the Centre for Conservation and Utilization of Blue Green Algae, Indian Agricultural Research Institute, New Delhi, were used in the present study. Plants were washed thoroughly and cleaned of contaminating organisms. Surface sterilization was done using a solution of mercuric chloride (0.1% for 30 s) followed by dipping of the plants into a large volume of sterile distilled water. Plants were then transferred into beakers (500 ml) containing combined N-free Espinase and Watanabe medium (1976). The cultures were grown at 26°C, under a 16:8 (light/dark) photoperiod with light from a combination of incandescent and cool white light fluorescent lamps at a photon fluence rate of 95 μmol m-2 s-1.

Salt treatment and biomass estimation

Plants were raised in 500-ml beakers containing 200 ml combined nitrogen-free Espinase and Watanabe medium, and each beaker was inoculated with 0.75 g of fresh Azolla fronds. A. microphylla fronds pre-exposed to NaCl concentration of 2 dsm-1 for 7 days and subsequently incubated in medium containing NaCl at a final concentration of 13 dsm-1 have been designated as pre-exposed plants. Directly exposed plants have been raised without a pre-incubation of 21 days but exposed to NaCl concentration of 4 and 9 dsm-1, respectively. Azolla plants that were not exposed to any NaCl treatment were taken as control. For determining biomass, fresh Azolla plants were harvested and blotted using a blotting paper, and their fresh weight was recorded. Dry weight was determined by placing the samples in a hot air oven at 90°C for 24 h to a constant weight.

Estimation of Na+ and K+ contents

To determine the Na+ and K+ ions, plants grown in saline medium were washed and blotted on filter paper. Na+ and K+ were extracted by digesting the fronds with HNO3:HClO4 mixture (1:1, v/v) in a boiling water bath for 30 min, and Na+ and K+ were determined using flame photometer (Systronics, India). The Na+ and K+ contents were expressed on the basis of dry weight.

Estimation of proline

Proline content was estimated by the method of Bates et al. (1973). Proline was extracted from the 100 mg fronds with 2 ml 40% methanol. One ml extract was mixed with 1 ml of a mixture of glacial acetic acid and orthophosphoric acid (6 M) (3:2, v/v) and ninhydrin (25 mg). After an hour of incubation at 100°C, the tubes were cooled, and 5 ml toluene was added. Absorbance of the upper phase was determined spectrophotometrically at 528 nm, and the proline content was determined using standard curve.

Estimation of lipid peroxidation

The level of lipid peroxidation was determined as malondialdehyde (MDA) content by the method of Heath and Packer (1968). Fresh leaves (0.1 g) were ground in 0.1% TCA using a mortar and pestle and centrifuged at 10,000 rpm for 5 min. The supernatant was collected in a separate test tube to which 4 ml of 0.5% Thiobarbituric acid (TBA) was added. The mixture was heated at 95°C for 30 min. It was then cooled quickly in an ice bath and re-centrifuged at 5000 rpm for 5 min to suspend the turbidity. The absorbance of the supernatant was read at 532 and 600 nm, respectively and corrected for unspecific turbidity by substracting the value at 600 nm. The blank was 0.5% TBA reagent. Concentration of MDA was calculated using an extinction coefficient (ε) of 155 mM-1.

Estimation of superoxide dismutase activity

The fronds (0.1 g fresh weight) were homogenized in a chilled mortar and pestle in ice-cold homogenization buffer (Phosphate buffer, 100 mM, pH 7.0) containing ascorbate (1 mM), ethylene diamine tetraacetic acid (1 mM), and DL-dithiothreitol (1 mM). The homogenate was centrifuged at 10,000 rpm for 20 min. Activity of superoxide dismutase (SOD) was assayed by measuring its ability to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT) according to Stewart and Bewley (1980). One unit of SOD was defined as that being present in the volume of extract that caused the inhibition of the photo-reduction of NBT by 50% and is expressed as units per milligram protein.

Estimation of ascorbate peroxidase activity

The ascorbate peroxidase activity was estimated according to the method of Nakano and Asada (1984). Azolla fronds (0.1 g fresh weight) were homogenized in a chilled mortar and pestle in ice-cold homogenization buffer as mentioned above. Ascorbate peroxidase (APX) activity was determined by following the rate of hydrogen peroxide-dependent oxidation of ascorbic acid. Reaction mixture containing 1.5 ml, 50 mM phosphate buffer (pH 7.0), 0.1 ml 0.5 mM ascorbate, 0.1 ml of 0.5% H2O2, 0.1 ml of 3 mM EDTA, and 100 µl of the enzyme extract was allowed to run for 3 min at 25°C. One enzyme unit determines the amount of enzyme necessary to decompose 1 µmol ascorbate per milligram of protein per minute at 25°C and expressed as units per milligram protein.

Estimation of catalase activity

Catalase (CAT) activity was determined according to the method of Aebi (1984). Azolla fronds (0.1 g fresh weight) were homogenized in a chilled mortar and pestle in ice-cold homogenization buffer as mentioned above. Catalase activity was determined by monitoring the disappearance of hydrogen peroxide measuring a decrease in the absorbance at 240 nm. The reaction was carried out in a reaction mixture containing 1.0 ml of the reaction buffer, 0.1 ml of EDTA, 0.1 ml of the enzyme extract and 0.1 ml of H2O2. One unit of enzyme determines the amount of enzyme necessary to decompose 1 µ mol of H2O2 per milligram protein.

Estimation of protein

A. microphylla (100 mg fresh weight) plants were homogenized in 5 ml phosphate buffer (0.2 M, pH 7.0), and the homogenate was centrifuged at 5000 rpm. The pellet obtained was mixed with 5 ml of 10% trichloroacetic acid (w/v), and recentrifuged. Subsequently, the pellet was repeatedly washed with the buffer to remove the trichloroacetic acid and allowed to dry at room temperature. The dried pellet was dissolved in 1 ml of 0.1 N NaOH and vortexed. Protein was estimated according to the method described by Bradford (1976).

Statistical analysis

All experiments were conducted thrice with triplicate samples. Data were analyzed using one-way analysis of variance and Duncan's multiple range test.

Results and discussion

Increasing soil salinity is a serious problem affecting the agricultural productivity worldwide. Azolla is commonly found in the paddy fields and has biofertilizer potential due to its symbiotic relationship with the nitrogen-fixing cyanobacterium A. azollae. Therefore, any adverse effect due to salinity may lead to loss of productivity and disturbed nitrogen balance in the paddy field ecosystems. Strategies are therefore needed to enhance the salinity tolerance potential of these organisms to better exploit them as bio-inoculants. The present study deals with the possible mechanisms of salinity adaptation of A. microphylla pre-exposed to lower concentrations of NaCl. Table 1 shows the growth in terms of biomass production by A. microphylla exposed to various levels of salinity. Plants exposed directly to salinity treatment (13 dsm-1) did not survive beyond a day and perished. However, plants exposed to 4 and 9 dsm-1 NaCl were able to survive and produce biomass. Interestingly, plants pre-exposed to 2 dsm-1 NaCl for 7 days and exposed to a salinity concentration of 13 dsm-1 were able to grow and survive. The rate of biomass production was, however, significantly lower as compared with salinity exposure at 4 dsm-1 NaCl (p > 0.05). Reduction in growth and biomass production of NaCl pre-exposed Azolla plants is in agreement with the findings of Rai and Rai (1999) who observed very little tolerance with A. pinnata which could not survive beyond an NaCl concentration of 30 mM and appears to be highly sensitive. Masood et al. (2006) observed adverse effect of NaCl on the growth of Azolla plants and found A. pinnata to be a tolerant strain as compared with Azolla filiculoides. Plants including Azolla show differences in their response to salinity. A. microphylla has the potential to tolerate relatively higher levels of salinity, and varietal differences in relation to salinity tolerance in Azolla is known (Haller et al. 1974).

Table 1 Effect of salinity treatment on biomass accumulation and lipid peroxidation of A. microphylla
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We further investigated the possible mechanisms for the induction of salt tolerance in A. microphylla due to pre-exposure to low concentrations of NaCl. In this context, the MDA production was studied which is often used as an indicator of oxidative damage (Table 1). In directly exposed plants, lipid peroxidation levels increased significantly, but the content of MDA was lower in pre-exposed plants (p > 0.05). It therefore appears that the extent of membrane damage was not so severe in pre-exposed A. microphylla plants as compared with plants exposed directly to NaCl. Sreenivasulu et al. (2000) observed an increase in MDA content in sensitive seedlings of foxtail millet exposed to salinity. Lower levels of lipid peroxidation due to enhanced activities of antioxidant enzymes under salt stress have been reported in salt-tolerant wild tomato species (Shalata et al. 2001). Excessive accumulation of superoxide radical and hydrogen peroxide in directly exposed plants could be one of the reasons for enhanced lipid peroxidation and consequent sensitivity to salinity whereas the pre-exposure to NaCl may have resulted in the modulation of the scavenging mechanisms leading to lower levels of accumulation of lipid peroxidation products.

Significant differences in the endogenous K+ and Na+ content (p > 0.05) of directly exposed and pre-exposed plants were noticed (Table 2). In directly exposed A. microphylla plants, the internal Na+ content increased by 95.7%, whereas in NaCl pre-exposed A. microphylla plants, the endogenous Na+ content increased by 38.8%. Azolla plants directly exposed to 60 mM NaCl showed higher Na+ content as compared with unadapted plants (Rai and Rai 1999). Sreenivasulu et al. (2000) observed lower amount of Na+ ions in salt-tolerant strains of Foxtail millet. Significantly lower levels of Na+ content in the leaves of soybean plants pretreated with salinity for a specific period of time has been observed by Umezava et al. (2000). Similarly, the concentration of K+ in pre-exposed plants increased over the value of control as well as directly exposed plants. The pre-exposed plants showed 36.5% higher K+ than directly exposed plants. However, in directly exposed plants, K+ content increased marginally by 19.8%. Failure to maintain a favorable K+ to Na+ ratio can inhibit enzyme functions (Greenway and Munns 1980). The observed positive effects of pretreatment on the regulation of ion accumulation may involve a Na+/K+ selection system at the cellular level. Munns et al. (2000) found that in Triticum turgidum lower Na+ uptake and high K+/ Na+ selectivity is probably associated with salt tolerance. Similarly, in guava seedlings, relatively high salt tolerance is associated with their ability to maintain a sufficient level of K+/Na+ (Ebert et al. 2002). Many transport systems in plants are reported to be modulated by salt stress (Tester and Davenport 2003; Zhu 2003). Rai et al. (2006) observed that varietal difference in salinity tolerance could be due to the genetic difference in the rate of salt accumulation. Differences in the internal Na+ content of pre-exposed and directly exposed plants and production of proline therefore may have adaptive response of the plants to salinity and probably accounts for salt tolerance in Azolla plants.

Table 2 Effect of salinity treatment on proline accumulation and Na+/K+ ratio of A. microphylla
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Proline accumulates in larger amounts than any other amino acids due to salinity stress (Ashraf and Harris 2004). Significant differences regarding the levels of proline was also found in pre-exposed and directly exposed plants (Table 2). Concentration of proline in pre-exposed plants increased significantly as compared with plants exposed directly to 9 dsm-1 NaCl (p > 0.05). Khelil et al. (2007) observed that, in salt-sensitive tomato cultivar salt adaptation was possible due to regulation of ion transport and synthesis of compatible solutes such as proline. Proline is also involved in osmoregulation and protection of proteins from dehydration and can also act as an enzymatic regulator during stress conditions (Rontain et al. 2002). Increase in proline content in the seedlings of Chenopodium quinoa lead to salinity tolerance (Ruffino et al. 2010). Colmer et al. (1996) observed that in Sorghum bicolor root tips, maintenance of net K+ to Na+ selectivity led to enhancement of proline accumulation in the root tip.

Antioxidant enzymes are crucial to tolerance and lead to better tolerance or resistance to environmental stresses including salinity. Therefore, we investigated the response of antioxidant enzymes of plants undergoing adaptation. The SOD activity increased significantly due to NaCl treatment (Table 3). However, in pre-exposed plants, the increase in the SOD activity was even more as compared with directly exposed plants (p > 0.05). Higher activity of SOD is an indication of efficient detoxification of superoxide radical. Acar et al. (2001) observed higher levels of constitutive and induced levels of SOD in barley cultivars tolerant to salinity. Increase in SOD activity in Chrysanthememum morifolium were found to be correlated with the tolerance of wheat to salinity stress (Hossain et al. 2004). Salt-tolerant plants appear to have protection mechanism against salt-induced free radical production through the induction of SOD activity (Hernandez et al. 2010). Therefore, pre-exposure may have resulted in better dismutating capacity in A. microphylla plants as compared with directly exposed plants. Similarly, significant increase in the APX activity was also noticed due to NaCl treatment (p > 0.05). It is evident from the results that pre-exposure had resulted in better capacity to decompose H2O2 than directly exposed plants. Stimulation in APX activity has been shown to be associated with salt tolerance in many plants (Hernandez et al. 1999; Shalata and Tal 1998; Sudhakar et al. 2001; Yazici et al. 2007). CAT activity increased in the directly exposed plants and considerable increase in CAT activity in pre-exposed A. microphylla plants was also noticed (p > 0.05). CAT activity has been found to be one of the most effective antioxidant enzymes next to SOD in preventing cellular damage (Scandalios 1993). Bor et al. (2003) observed a high level of CAT activity in salt-tolerant beet [Beta maritima] leading to better protection against oxidative damage. Increase in CAT activity has been reported by Daneshmond et al. (2010) in the wild species of potato exposed to salinity. Therefore, the activation of antioxidant enzymes in pre-exposed A. microphylla plants could be the mechanism conferring adaptation to salinity. Pre-exposure to salinity has been reported to ensure better survival of plants upon subsequent stress conditions (Atreya et al. 2009). Furthermore, Amzallag et al. (1990) described that Sorghum responded to salinity by using preexisting resistance or adaptation. Modulation of gene expression by ROS particularly O*- and H2O2 due to salinity pre-treatment and induction of various genes and enzymes related to oxidative damage could be the reasons behind the adaptation to salinity.

Table 3 Effect of salinity treatment on antioxidative enzymes of A. microphylla
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Conclusion

A. microphylla plants exposed directly to 4 and 9 dsm-1 NaCl were able to survive and produce biomass as compared with plants exposed to 13 dsm-1 NaCl which perished and did not survive beyond a day. However, plants pre-exposed to 2 dsm-1 for 7 days and subsequently exposed to 13 dsm-1 were able to grow and produce biomass, although at a slow rate. The pattern of biomass production, lipid peroxidation, proline accumulation, and the response of antioxidant enzymes such as SOD, POD, and APOX as well as Na+/K+ ratio have been distinctly different due to direct exposure to salinity or pre-exposure to salinity. Salt tolerance could be induced in highly sensitive Azolla plants by pre-incubating them in non-lethal NaCl concentrations. The studies indicate that complex mechanism(s) involved in salt adaptation in A. microphylla may involve low rate of lipid peroxidation coupled with efficient regulation of ion transport and modulation of antioxidant enzymes.