EffECT Of salT sTREss ON sOmE swEET CORN ( Zea mays l . var . saccharata ) gENOTypEs

An experiment was carried out hydroponically under laboratory conditions to investigate the effect of salt stress on several physiological and biochemical parameters of three sweet corn (Zea mays L. var. saccharata) genotypes: lines 6-13, C-6 (pollen source) and their heterotic F1 hybrid “Zaharina”. The degree of salinity tolerance among these genotypes was evaluated at three different sodium chloride (NaCl) concentrations: 0 mM, 100 mM, 125 mM and 150 mM. Seed germination, plant growth and biochemical stress determining parameters such as malondialdehyde (MDA), proline content and hydrogen peroxide (H2O2) levels were compared between seedlings of lines and hybrid. The obtained results indicated that both lines and hybrid have similar responses at different salinity levels for all examined traits. All the seedlings’ growth parameters, such as germination percentage, root length, shoot length, root and shoot fresh and dry weight, decreased with increasing salinity level. MDA, proline and H2O2 increased at different saline conditions in comparison to the control. Based on the results, of the three genotypes examined, the hybrid Zaharina, followed by line C-6, was more salt-sensitive than line 6-13 in salt stress condition.


INTRODUCTION
Soil salinity, as one of main abiotic stresses in nature, limits agricultural productivity on nearly 20% of the cultivated area and half of the irrigated area worldwide (Zhu, 2001).Salt stress has been reported to cause an inhibition of growth and development, and reduction in photosynthesis, respiration and protein synthesis in sensitive species (Meloni et al., 2003;Ashraf and Foolad, 2007;Tuteja et al., 2012).Like other abiotic stresses, salt stress also leads to oxidative stress through an increase in reactive oxygen species (ROS), such as superoxide (O 2 -), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH ) (Alscher et al., 1997;Mittler, 2002;Neill et al., 2002).These ROS are highly reactive and can alter normal cellular metabolism through oxidative damage to lipids, proteins and nucleic acids (Alscher et al., 1997;Imlay, 2003).During salt stress, plants adapt to oxidative stress by accumulating certain protective solutes like proline, glycine betaine, polyols, trehalose, etc. (Sakamoto and Murata, 2002).Proline plays a predominant role in protecting plants from osmotic stress.Malondialdehyde (MDA) content, a product of lipid peroxidation, has been considered to be an indicator of oxidative damage and has been widely utilized to differentiate salt-tolerant and salt-sensitive cultivars (Luna et al., 2000;Hernandez and Almansa, 2002;Meloni et al., 2003;Filippou et al., 2014).
Maize is moderately sensitive to salinity and considered as the most salt-sensitive of the cereals (Mass and Hoffman, 1977).Sweet corn (Zea mays L. saccharata) is one of the most popular vegetable crops in countries such as the USA and Canada.In Bulgaria, it is being increasingly cultivated in many areas, expanding to areas with a high potential for the accumulation of salts in the soil profile.Sweet corn has a short breeding history, and a different genetic basis than field maize (Tracy, 1997;Letrat and Pulam, 2007).Direct selection of superior salinity-tolerant genotypes under field conditions has been hindered by the significant influence of environmental factors.Salinity is often accompanied by changes in other soil physical and chemical properties, and interaction between these stresses with salinity can occur, stimulating genotype by environment interactions and making breeding progress more difficult.Therefore, the development and evaluation of new sweet corn lines and hybrids with the potential to provide useful initial material for plant breeding, in particular tolerant to abiotic stresses, is one of the most important maize research programs at the Institute of Plant Physiology and Genetics (IPPG), Bulgarian Academy of Sciences.Some evidence suggests that resistance to oxidative stress may, at least in part, be involved in salt-stress tolerance (Badawi et al., 2004;Neto et al., 2006;Filippou et al., 2014).The objective of the present study was to identify the effect of salinity stress on three sweet corn genotypes in the seedling stage subjected to different concentrations of NaCl by examining physiological characteristics, such as seed germination and growth parameters, and the intracellular regulation of osmolytes (proline con-tent), lipid peroxidation (based on MDA content) and hydrogen peroxide (H 2 O 2 level).

maTERIals aND mETHODs plant material and experimental design
This study was a laboratory-conducted experiment and carried out in the IPPG, Bulgarian Academy of Sciences, Sofia, Bulgaria.Two inbred lines 6-13 and C-6 (pollen source) of sweet corn carrying the su gene and their heterotic F 1 hybrid Zaharina were used.Both parents are part of a set of selfed lines that were bred and screened following a program of production of highly homogeneous, homozygous inbreds in the IPPG.The F 1 hybrid Zaharina was patented in 2010 and nowadays is largely grown in the Sofia region.The lines and hybrid were characterized as mid-season, yellow-seeded plants (Kraptchev et al., 2010).All experiments were carried out using seeds produced in the same year and under the same climatic conditions.Each experiment was repeated at least three times.

germination experiment
Three sweet corn genotypes were evaluated against salt stress at germination and seedling growth stages for 14 days under laboratory conditions.Twenty-five seeds of each genotype were surface-sterilized with 70% ethanol for 5 min followed by immersion in 15% (v/v) sodium hypochlorite solution for 15 min and then germinated in rolled moistened paper towels in darkness (25±1°C) as previously described (Vassilevska et al., 2014).The number of seeds germinated was recorded gradually from 16 to 96 h (4 h interval).Germination was considered to have occurred when the seed had developed a radicle at least 5 mm long.The experiment was performed with four replicates for each experimental unit.
Total germination was expressed as a percent of that in the control treatment for each genotype and then data underwent statistical analysis.
Salt stress was realized by subjecting the seeds to 15-ml salt solutions of 100, 125 and 150 mM NaCl.In addition, 15 ml of distilled water without NaCl was used as control.
After germination, when cotyledons fully emerged, the healthy and uniform seedlings were transferred to 600 mL plastic beakers filled with half-strength Hoagland's solution and grown in a controlled growth chamber "Forma Scientific" model 3744 at 25 ± 2 o C with a 16-h light (250 μmol m -2 s -1) and 8-h dark regime.After 10 days of growth, the length of roots and shoots was measured and seedlings were treated with NaCl solution.Four levels of salinity (0 − distilled water as control, 100, 125 and 150 mM) were generated by dissolving NaCl to half-strength Hoagland's solution.The salinity stress period created by NaCl was 3 days.

growth parameters
At the end of the experiment (14 days), the plants were "smoothly" uprooted and the root system was washed under running tap water.Plant growth in terms of shoot and root length, shoot and root fresh weight (Fw) and dry weight (Dw) were recorded.water content of the roots and shoots was determined by weight change.For dry weight de-termination, samples were oven-dried at 70°C for 72 h and then weighed.Each set of experiments was performed three times.

Biochemical analysis proline content
Free proline content in the leaves was determined by the method by Bates et al. (1973), and the absorbance was read at 520 nm against toluene blank.Proline concentration was determined using a calibration curve and expressed as μmol proline g −1 Fw.

Hydrogen peroxide (H 2 O 2 ) content
Fresh leaf material was homogenized in 0.1% w/v cold trichloroacetic acid.The homogenate was centrifuged at 15000 g for 30 min.The supernatant obtained was used for the determination of H 2 O 2. Hydrogen peroxide was measured spectrophotometrically after reaction with KI (Alexieva et al., 2001).The reaction mixture consisted of 0.5 ml 0.1% trichloroacetic acid, leaf extract, and 1 ml reagent (1 M KI in fresh bidistilled water).The reaction was allowed to proceed for 1 h in the dark with shaking, and absorbance was measured at 390 nm.The amount of H 2 O 2 was calculated using a standard curve prepared with known concentration of H 2 O 2 .

assay of lipid peroxidation
The level of lipid peroxidation was determined by estimating the malondialdehyde (MDA) content in 500 mg in leaf and stem fresh weight according to Cakmak and Horst (1991).MDA is a product of lipid peroxidation by thiobarbituric acid reaction.The con-centration of MDA was calculated from the absorbance at 532 nm (correction was done by subtracting the absorbance at 600 nm for unspecific turbidity) by using an extinction coefficient of 155 mM −1 cm −1 .

statistical analysis
The data collected were analyzed by analysis of variance (ANOVA) technique.Duncan's New Multiple Range test at 5% level of probability was used to test the significance of means (Steel and Torrie, 1980).

REsUlTs aND DIsCUssION
Background information on the germination behavior of the seed material used in the following experiments is given in Fig. 1.Seed germination from line C-6 and hybrid Zaharina was almost 99-100% at 72h after sowing.The seeds from the 6-13 line germinated slowly but higher germination also occurred within 72 h (Fig. 1A).The results showed that the germination abilities of the three maize genotypes were significantly affected by salt concentrations (Fig. 1 B).The germination percentage declined significantly with salinity  stress treatment compared to the control.At different salt concentrations, C-6 and C-13 had the highest and lowest germination percentage of 87% and 34%, respectively.C-6 demonstrated better tolerance to salt stress than the other two genotypes in germination percentage; non-salt stressed, (control) plants of line C-6 showed germination ability of 99% (Fig. 1).The germination capacity of seeds as a qualitative product of the germination process is known as an alternative measure of plant status, reflecting the metabolic activity in tissues (Flower and Ludlow, 1986;Ranal and Santana, 2006;Munns and Tester, 2008;Farsiani and Ghobadi, 2009).According to Khayatnezhad and Gholamin (2011), salinity affects germination in two ways: i) enough salt in the medium may decrease the osmotic potential to a point that retards or prevents the uptake of water necessary for the mobilization of nutrient requirement for germination, and ii) the salt constituents or ions may be toxic to the embryo.The decrease in germination percentage indicated a disturbance in the normal germination process, which includes three partial processes, including imbibition, the activation process and intra-seminal growth that is completed with embryo protrusion (Labouriau 1983).
The current results are in accordance with the findings of Rahman et al. (2000), which showed that the maize cultivars were significantly more tolerant to salt stress at germination than at later stages of growth.
The length of both root and shoot was inhibited by salinity stress (Table 1).Inhibition in plant growth was more pronounced in both lines whose growth was reduced to 72.5% and 76.4% with 150 mM NaCl (root length) and to 67.4% and 68.7% (shoot length) compared to the control, while the reduction in growth of hybrid Zaharina was about 81%.Similar results were reported by Khayatnezhad and Gholamin (2011).NaCl inhibits growth by reducing both cell division and cell enlargement (Yasseen et al., 1987).
The increase of salt concentration caused a significant decrease in the growth of the three corn genotypes (Table 2).Both lines and hybrid had different responses in root and shoot fresh and dry weights at different levels of NaCl.Generally, increasing salinity levels decreased plant growth parameters in all of the genotypes, but C-6 had been less damaged and its root and shoot fresh and dry weights were greater than line C-13 and hybrid Zaharina at 150 mM NaCl (Table 2).It was established that high salinity affects plants in several ways: water stress, ion toxicity, nutritional disorders, oxidative stress, alteration in the metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Hasegava et al., 2000;Munns, 2002;Zhu, 2007).Together, these effects reduced plant growth development and survival (Carillo et al., 2011;Filippou et al., 2014).Our results are in agreement with the reports of other authors revealing negative relationships between vegetative growth parameters and increasing salinity (Ashraf and Rauf, 2001;Carpici et al., 2009;Hussein et al., 2012).The results of the comparisons between the three genotypes (Tables 1, 2) allow sweet corn to be added to the list of species for which intraspecific variation in salinity tolerance has been shown to exist.Salinity stress stimulated the accumulation of proline (Fig. 2).when subjected to 150 mM NaCl treatment, the proline content in both lines increased 75% (line 6-13) and 35% (line C-6) as compared to the hybrid Zaharina.It has been reported that proline concentration increases under conditions of salinity up to 100 times the normal level, which makes up to 80% of the total amino acid pool (Thomas et al., 1992;Neto et al., 2006).According to Claussen (2005), proline content in plant tissues is both a reflection and measure of stress-induced damage at the cellular level.Accumulation of proline under stress protects the cell by balancing the osmotic strength of cytosol with that of the vacuole and external environment (Aspinall and Paleg, 1981).In addition, it may interact with cellular macromolecules (enzymes), and stabilize the structure and function of such macromolecules (Jain et al., 2001).
In the present work, the effect of salinity stress on the proline content was shown to be more pronounced in line 6-13 than in C-6 and the hybrid.It could therefore be concluded that the homozygous inbred 6-13 is more tolerant to salt stress than the two other genotypes (Fig. 2).The marked difference between sweet corn genotypes in responding to salinity stress is indicative for their key role in determining the plant's adaptation reaction to stress.This result is consistent with the observations that at different levels of salt stress, each plant genotype behaves differently according to its genetic makeup (Neto et al., 2006;Oraki et al., 2012).
The data obtained show that in all three genotypes as the salt concentration increased, both H 2 O 2 and MDA concentrations increased significantly (Figs. 3, 4).The responses of both lines and F 1 hybrid to salt stress indicated differences.For all salt concentrations, C-6 had the highest H 2 O 2 content.At maximum level of salinization (150 mM), the MDA level in the C-6 line increased to about 81% in comparison to the control.Due to the increase in salinity, MDA accumulation in line 6-13 and the hybrid increased about 2 times, showing that the salinization is associated with lipid peroxidation mechanisms.The increase in the value of proline, H 2 O 2 and MDA was associated in all three sweet corn genotypes to inherent differences in salt tolerance in maize genotypes.Similar results have been reported by other authors about maize (Carpichi et al., 2009;Hussein et al., 2014), sunflower (Vassilevska-Ivanova et al., 2014), and canola (Rasheed et al., 2014).
Our results show that in sweet corn there is a correlation between genotypes and salt tolerance.with regard to different parameters, such as those related to antioxidant defense (accumulation of proline, H 2 O 2 and MDA), the hybrid Zaharina was more salt-sensitive than lines C-6 and 6-13 because it exhibited lower levels of proline and higher levels of malondialdehyde at higher salt levels (Figs. 2, 4).Therefore, it could be suggested that the inbred 6-13 seems to be a very promising parent to breed and select new salt-tolerant sweet corn hybrids.Such a suggestion needs to be further explored by involving a larger set of sweet corn lines and hybrids.Our research also faced the lack of information about the genetic diversity and heterotic models in sweet corn germplasm.The current results demonstrate that it is possible to screen sweet corn genotypes for salt tolerance under controlled  conditions using fairly small plants.Full evaluation of the potential usefulness of this approach in a breeding program directed at improving the tolerance of sweet corn to salinity stress must await the determination of the heritability of this trait.A study of the heritability of salt stress in sweet corn lines and hybrids is underway at present.
authors' contributions: Lydia Shtereva and Roumiana Vassilevska-Ivanova designed and organized the study, and conducted plant growth and plant development experiments under laboratory condition.Lydia Shtereva and Tanja Kartzeva performed biochemical experiments.Roumiana Vassilevska-Ivanova and Lydia Shtereva wrote the manuscript, and bear the basic responsibility for the final content.All authors have read and approved the final manuscript.

Conflict of interest disclosure:
There is no conflict of interests between all three co-authors.
fig. 1. Germination responses of sweet corn lines and hybrid under optimal (A) and saline (B) conditions.
Fig. 3. fig. 3. H 2 O 2 content in leaf extracts of sweet corn inbred lines and their F 1 hybrid affected by NaCl.Values are mean ± SE obtained from four replicates.

Table 1 . Root and shoot growth responses (mean length per seedling in cm) of sweet corn lines and hybrid grown under optimal and stress conditions; percentage control values are given in parenthesis. Treatment length (cm) line 6-13 line C-6 Hybrid "Zaharina"
Values are means ± SE obtained from four replicates.The means with the same letters do not differ statistically by Duncan's multiple range test (P ≤ 0.05).

Table 2 .
Plantlet's growth responses (mean fresh and dry weight g/5 plants) of sweet corn lines and hybrid grown under optimal and stress conditions