Physiological and antioxidant responses of three leguminous species to saline environment during seed germination stage

The study investigated the physiological behaviors and antioxidant responses of Medicago sativa, Melilotus officinalis and Astragalus adsurgens to saline environment during seed germination stage. At 300 mM NaCl treatments, the final germination percentage of M. officinalis was much higher than that of M. sativa and of A. adsurgens and the initiation of germination process of A. adsurgens was 5 days later than the others. Moreover, after being treated in 300 mM NaCl, much weaker catalase (CAT) activity in seed of M. officinalis, peroxidase (POD) activity in seed of A. adsurgens and glutathione reductase (GR) activity in seed of M. sativa were found in the controls and salt treatments. Thus, oxidative stress could be an influential component of salt stresses on plant seeds during seed germination stage.


INTRODUCTION
High salinity is the most widespread abiotic stress and constitutes the most stringent factor that limits plant growth and development (Vernon and Bohnert, 1992).Germination is a key stage in the life cycle of plants in saline environments as it determines whether or not the plants can establish successfully in certain areas (Ungar, 1991).Studies on salt stress in seed germination have shown that during this stage the seeds are particularly sensitive to saline environments (Bewley and Black, 1982).Therefore, it is very important to understand the adaptive mechanisms of plants to saline environment during seed germination stage.
Plants vary greatly in their tolerance to salt.Some halophytes can complete their life cycle under hypersaline conditions (McKell, 1994), while glycophytes, though generally more sensitive to saline stress, range widely between species and even among varieties in their tolerances (Tobe et al., 2001).In general, non-halophytes and halophytes respond to salinity in a similar way during the germination stage; in some species, the initial *Corresponding author.E -mail: wanggrass@163.com.Tel/Fax: 86-514-87979037.germination process is often delayed under salt stress (Keiffer and Ungar, 1997;Khan and Ungar, 1997).The effect of salinity on seed germination can be attributed to an osmotic effect and/or specific ion toxicity, depending on the plant species (Petruzzelli et al., 1992;Poljakoff-Mayber et al., 1994).
Salt stress can stimulate formation of active oxygen species (AOS), such as superoxide, hydrogen peroxide and hydroxyl radicals.These activated oxygens injure the cellular components of proteins, membrane lipids and nucleic acids (Foyer et al., 1994).Malondialdehyde (MDA) is the decomposition product of polyunsaturated fatty acids of membranes and shows greater accumulation under salt stress (Gossett et al., 1994;Dionisio-Sese and Tobita 1998;Sudhakar et al., 2001).In order to avoid these oxidative injuries, plants have developed enzymatic systems for scavenging these highly active forms of AOS, superoxide is converted by SOD enzyme into H 2 O 2 , which is further scavenged by CAT and various peroxidases.Ascorbate peroxidase (APX) and GR also play a key role by reducing H 2 O 2 to water through the Halliwell-Asada pathway (Noctor and Foyer, 1998).However, under salt stress, the mechanisms developed by seeds to scavenge AOS are still poorly understood.
Medicago sativa, Melilotus officinalis and Astragalus adsurgens are all important leguminous forage species in China.During seed germination and seedling stage, M. sativa is more sensitive to salinity, while the mature plant bears with better tolerance to salt stress (Allen et al., 1986); M. officinali is a salt tolerant plant (Chen and Jia, 2001) and A. adsurgens is drought-resistant and exhibits high growth rates in the summer months of North China.We hypothesize that the adaptabilities of these three species seeds to saline environment are related to their ion toxicity and activities of antioxidative enzymes.Therefore, our work is to evaluate the germination percentage under salt stress of the three species and the effects of 300 mM NaCl on the activity of antioxidative enzymes and the lipid membrane peroxidation, in order to better understand their adaptive mechanisms in saline environment during seed germination stage.

Germination experiment
Seeds of M. sativa, M. officinalis and A. adsurgens were obtained from Forage Seed Laboratory (ISTA member Laboratory), China Agriculture University.Treatment solutions of NaCl were adjusted to 50, 100, 200 and 300 mM and a control (distilled water).Seeds of three species were germinated in 150 mm covered Petri dishes on two layers of filter paper moistened with 30 ml of treatment solution.
Four replicate dishes, each with 100 seeds, were used for each treatment and were placed in an incubator in constant darkness at 20°C.Every 2 days the solution in each Petri dish was renewed.Each day for 12 days, the germinant seeds were recorded and removed from the Petri dishes.Seeds were considered to have germinated when the emerging radicles were at least 2 mm in length.Final germination percentage was calculated as the total cumulative germination of a treatment group over the experimental period.Daily germination percentage was calculated as the one day germination percentage in a treatment group over the experiment period.

Determination of enzyme activity and MDA
Seeds of three species were moistened in 150 mm Petri dishes with a 300 mM NaCl solution and were placed in an incubator in constant darkness at 20°C.After one-, two-, or four-day treatment with 300 mM NaCl solution, the seeds of each treatment and of the control (moistened in distilled water for 1 day) were surface-dried with filter paper and 0.5 g of seed samples were homogenized with 50 mM sodium phosphate buffer (pH 7.0) containing 1mM ethylenediaminetetraacetic acid (EDTA) 1 mM ascorbate and 2% (w/v) polyvinylpyrrolido (PVP) at 4°C.Following centrifugation at 10,000 g for 15 min at 4°C, the supernatants were collected and used for assaying enzyme activity.Four replicate dishes were used for each treatment.Protein concentrations in the enzyme extract were determined by the method of Bradford (1976) using bovine serum albumin as a standard.Catalase (CAT, EC 1.11.1.6)activity was assayed through according to the method developed by Bergmeyer (1970).The final reaction mixture contained 50 mM sodium phosphate buffer (PH 7.0) and 2% H2O2.The activity was expressed as units (μmol H2O2 consumed per minute) per mg of protein.
Peroxidase (POD, EC 1.11.1.17)activity was determined according to Tatiana et al. (1999) with small modification.The reaction mixture contained 0.05 M sodium phosphate buffer (PH 5.5), 2% H2O2, 0.05 M guaiacol and 0.1ml enzyme extract.The formed tetraguaiacol was measured with a spectrometer at 470 nm.One unit of enzyme was defined as the amount of enzyme to decompose 1 μmol of H2O2 per min at 25°C.
Malondialdehyde content was measured by a thiobarbituric acid reaction according to Madhava Rao and Sresty (2000).

Statistical analysis
Data were determined by analysis of variance using SPSS software (10.0).Differences between treatment means were separated by the least significant difference (L.S.D.) at a 0.05 probability level.

Effects of NaCl on final germination and daily germination percentage
The final germination percentages of seeds of the three legume species were over 89% in distilled water, but the percentages decreased with the increasing NaCl concentration (Figure 1).In the 50 mM NaCl concentration treatment, final germination percentage for M. sativa seeds was significantly decreased by 8.7%, while for M. officinalis and A. adsurgens, the decrease was 7.3 and 0.5%, respectively.At 300 mM NaCl concentration, final germination percentage of M. officinalis was much higher than that of M. sativa and A. adsurgens and the final germination percentage values of M. sativa, M. officinalis and A. adsurgens were 41, 49 and 36%, respectively.
Seeds of the three species germinated rapidly in distilled water during the initial 2 days and the maximal values of daily germination percentage appeared on the first day for M. sativa and M. officinalis and the second day for A. adsurgens (Figure 2).With the increasing of NaCl concentration, the appearing of maximal values of daily germination percentage was postponed in all species.At 300 mM NaCl concentration, the delaying period of appearing of maximal values of daily germination percentage were 1 day for M. sativa, 2 days for M. officinalis and 5 days for A. adsurgens compared with the control.

Effects of NaCl on SOD, POD, CAT, APX, GR activities and MDA
NaCl treatment led to an increase of SOD activity in the seeds of M. sativa, but a decrease in the seeds of M. officinalis and A. adsurgens, compared with the control (Table 1).However, the SOD activity of the three species treated with a 300 mM NaCl solution decreased with time increasing.The average values of SOD activities were 2.8, 2.7 and 3.4 units / mg protein in M. sativa, M. officinalis and A. adsurgens, respectively, for 1, 2 or 4 day salt treatments.
A. adsurgens seeds displayed the highest CAT activity among the three species at control treatment, whereas the CAT activities of all three species seeds were inhibitted by salt solution (Table 1).The CAT activities of M. sativa seed were found to significantly decrease, not significantly change for M. officinalis seed and increase for A. adsurgens seed as the treating time lengthened.At 2 or 4 day treatment in 300 mM NaCl solution, the CAT activities of A. adsurgens seed were 0.23 or 0.22 units / mg protein, which was 2.0 or 2.2 times that in the seeds of M. sativa seed and 5.4 or 6.6 times that of the M. officinalis seed.
One-or two-day NaCl treatments led to an increase in the activity of POD in the seeds of M. sativa and M. officinalis.However salt did not change the POD activity in the seeds of A. adsurgens (Table 1).The average POD activities of M. sativa seed under all treatments were 3.6 times higher than those of M. officinalis seed and 30.2 times higher than those of A. adsurgens seed.
Salinity stress resulted in an increase in the APX activity of seeds of the three species (Table 1).However, with the lengthened stress time, the APX activity of the M. sativa and M. officinalis seeds decreased, while the APX activity in the A. adsurgens seed remained the same, though the treating time increased.After NaCl stress, the average seed APX activities of M. sativa were 2.5 and 3.2 times that in M. officinalis and A. adsurgens, respectively.
GR activity decreased after NaCl treatment with in M. sativa seed, while the GR activities in M. officinalis seed and A. adsurgens seed increased.The average seed GR activity of M. sativa under NaCl stress was only 29% of the GR activity of M. officinalis seed and 24% of the GR activity of A. adsurgens seed (Table 1).
NaCl stress led to a significant increase in the levels of MDA content in all species.At the 4th day treatment of 300 mM NaCl concentration, the seed MDA content increased by 36%, 35% and 16% of that of the control seed of M. sativa, M. officinalis and A. adsurgens, respectively (Table 1).

DISCUSSION
The effect of salinity on seed germination is due to an osmotic effect and/or ion toxicity.However, variation of adaptive mechanisms exists in different species (Rehman et al., 1996).Salt stress can induce both a reduction of seed germination and a delay of initiation of germination process in glycophytes and to a lesser extent in halophytes (El-Keblawy, 2004).In the present study, 50 mM NaCl treatment resulted in a significant decrease in the Afr.J. Biotechnol.final germination percentage for M. sativa seed.In a 300 mM NaCl concentration, final germination percentage of M. officinalis seed was much higher than that of M. sativa seed and A. adsurgens seed and a significant delay of initiation of germination process of A. adsurgens seed was found (Figure 1).These results indicated that in the seed germination stage, M. sativa was most sensitive to even low salt stress; M. officinalis could adapt to stronger salt stress; while the A. adsurgens seed possessed the important mechanism of delaying initiation of germination process in order to adapt to the saline environment.
There are increasing evidences that membrane injury under salt stress is related to a higher production of highly toxic AOS.Determining the MDA concentration and hence, the extent of membrane lipid peroxidation, has often been used as a tool to assess the severity of the oxidative stress (Amor et al., 2005).Our data show that, after 1 day treatment of the three species seeds with 300 mM NaCl, the membrane lipid peroxidation was induced.After 2 or 4 days salt stress for M. sativa and 1, 2 or 4 days salt stress for M. officinalis, the MDA contents in the seeds were much higher than in those of the control, while no significant increase was found in A. adsurgens seeds after salt stress (Table 1).Salt stress can cause plant membrane damage and stimulate formation of AOS such as superoxide, hydrogen peroxide and hydroxyl radicals.Among the AOS, superoxide is converted by SOD enzyme into H 2 O 2 , which is further scavenged by CAT and various peroxidases.APOX and GR also play a key role by reducing H 2 O 2 to water through the Halliwell-Asada pathway (Noctor and Foyer, 1998).In the present study, the diverse responses of SOD, CAT, POD, APX and GR enzyme activities to NaCl stress on 1, 2 or 4 days salt stressed seeds of M. sativa, M. officinalis and A. adsurgens suggest that oxidative stress could be an influential component of environmental stresses on plant seeds.
Salinity led to an increase of SOD activity of M. sativa seeds but it decreased that in the M. officinalis and A. adsurgens seeds after being treated with salt for 1 or 2 days (Table 1), which suggested that salinity induced more production of O 2 -in species of M. sativa which was counterbalanced by increasing activity of SOD.With the lengthening of the salt stress time, the SOD activities decreased in the three species seeds.Since SOD was inactivated by singlet oxygen and peroxyl radicals (Escobar et al., 1996), it could have been deactivated in the three species seeds by the increased levels of AOS.Under salt stress treatments, no great differences were found among the three species in the average values of SOD, which indicates that SOD is indispensable to scavenging AOS in seeds of the three species.
CAT, POD, APX and GR, as antioxidative enzymes, play a key role in scavenging H 2 O 2 in the cells (Noctor and Foyer, 1998).In our study, according to the active diversity of these enzymes in seeds of M. sativa, M. officinalis and A. adsurgens, we could conclude that the mechanisms in scavenging H 2 O 2 were different in the three species under stress conditions.In M. officinalis, the CAT activities of seeds were much weaker than in the M. sativa and A. adsurgens seeds both in the control and with salt treatments and were significantly inhibited in salt treatments.The POD and APX activities in seeds of M. officinalis were also feebler than in seeds of M. sativa at control and with salt treatments; however, they were significantly induced by salt.These results suggested that POD and APX could be more important than CAT in scavenging H 2 O 2 in seeds of M. officinalis under salt stress conditions.In M. sativa seed, POD and APX are also more important in scavenging H 2 O 2 under salt stress conditions due to the intenser activities at control and with salt treatments and induced the POD and APX activities to increase after short period salt stress.After 300 mM NaCl treatment for 4 days, the activities of CAT, POD and APX became weaker than those of the controls of the three species, resulting in the accumulation of H 2 O 2 and a higher lipid peroxidation level in the seeds.POD activity in seeds of A. adsurgens was much weaker than that in seeds of M. officinalis and M. sativa in control and under salt treatments, witnessing that POD might not play the key role of reducing H 2 O 2 at stress conditions in seeds of A. adsurgens.
In the antioxidant defense system pathway, monodehydroascorbate that is spontaneously dismutated to dehydroascorbate can react with glutathione to produce ascorbate and oxidized glutathione (GSSG) in a reaction catalyzed by dehydroascorbate reductase.GSSG is reduced by GR, requiring the consumption of NADPH.Singlet oxygen and hydroxyl ions are eliminated in the glutathione pathway (Bray et al., 2000).Bor et al., (2003) reported that Increased GR activity in leaves of sugar beet plants was closely related with salt tolerant capacity of these plants.In our study, the GR activity decreased in seeds of M. sativa after salt stress and increased in seeds of M. officinalis and A. adsurgens.This indicated that seeds of M. sativa could suffer from much severer toxicity of singlet oxygen and hydroxyl ions under salt stress condition.
In conclusion, the effects of NaCl on seed germination of the three species are due to osmotic stress, ion toxicity and oxidative stress.However, the adaptive mechanisms of the three species are variant.The seeds of M. sativa are susceptible to low level NaCl concentrations and suffer from much severer ion toxicity at high NaCl concentrations.As a salt tolerant plant, seeds of M. officinalis sustain salt stress of a higher concentration.Delaying initiation of germination process is the important mechanism of A. adsurgens to adapt to the saline environment.The membrane lipid peroxidation is induced by NaCl in seeds of the three species.The diverse responses of antioxidant enzyme activities to NaCl stress in seeds of M. sativa, M. officinalis and A. adsurgens suggest that oxidative stress could be an influential component of environmental stresses on plant seeds.

Figure 1 .
Figure 1.Final germination percentage for seeds germinated in contact with different NaCl in continuous darkness at 20 C for 12 d.Seeds were considered to have germinated when the emerging radicle was at least 2 mm.

Figure 2 .
Figure 2. Daily germination (%) for the 12 d when seeds were placed in contact with distilled water (controls) or solutions of NaCl in continuous darkness at 20 C in (A) M. sativa, (B) M. officinalis and (C) A. adsurgens.Seeds were considered to have germinated when the emerging radicle was at least 2 mm.

Table 1 .
Activities of enzymes and MDA contents in seeds moistened with 300 mmoml/L NaCl solution in constant darkness at 20°C for 1, 2 and 4 days and seeds moistened with distilled water in constant darkness at 20°C for 1 day were control.
Data represent mean values ± S.D. of analyses of 4 replicates.Means followed by the same letter are not significantly different at P = 0.05 within the same species.