Salinity can cause two distinct types of stress in plants, making it a major limiting factor for agricultural production. Salt stress in the soil generally involves osmotic stress, caused by the greater difficulty of water absorption, and ionic stress, associated with the effects of sodium ions on diverse cellular functions, decreasing nutrient absorption, enzyme activities, photosynthesis, and metabolism (Zhu 2001, Munns 2002). Thus, plants in saline soil must cope with physiological drought and ion toxicity, and also need to maintain intracellular ion balance and regulate pH outside the roots.

Salt marshes have often been considered to be wastelands. However, they are habitats for many halophytes with high economic and medicinal importance (Khan et al. 2000, Parida and Das 2005). It is necessary for plants growing well under saline conditions to have a greater salt tolerance, and this characteristic has influenced the ecological distribution of various plants (Flowers and Colmer 2008). By accumulating inorganic ions, halophytes absorb water by maintaining a high osmotic potential. Glycine betaine and proline are compatible solutes that accumulate in response to osmotic stress, and the accumulation of these osmolytes represents an important adaptive response to salt and drought stress (Di Martino et al. 2003, Moghaieb et al. 2004). It is essential to study plants with resistances to various saline environments in order to develop crops that can withstand increased salinity (Chinnusamy et al. 2005).

Increased salinization of arable land and decline of fresh water supplies have developed new interest in plant materials possessing salt tolerance, such as halophytes (Koyro et al. 2013). Halophytes are being used not only as food crops and fodder, but also as ground cover to protect against soil erosion. Through the root development, soil porosity increases, and the physical condition of the soil is subsequently improved over time (Rozema and Flowers 2008).

Suaeda japonica, S. malacosperma, Phargmites communis and Carex scabrifolia are the main species distributed on saline marsh habitats in Suncheon Bay in Korea. Most studies on salt marshes have focused on the environmental factors affecting vegetation characteristics and the distribution of halophytes (Kim et al. 1986, Lee 1988, Min and Kim 1999, Yang 1999, Kefu et al. 2002, Lee et al. 2003, Choi et al. 2004, Choi et al. 2012). However, their adaptive mechanisms under dry and saline conditions have not yet been clearly described.

The aim of this research was to study the organic and inorganic solute levels in salt marsh plants due to seasonal changes.

The study site is located in Suncheon Bay Ecological Park, Korea (N 34°52′17.1″, E 127°30′11.9″; Tokyo datum). The annual mean temperature and precipitation in the study area were 13.9°C and 1308 mm, respectively. This study site is a salt marsh where communities of Phragmites communis are predominate (Fig. 1).

Soil samples from 15–20 cm below the soil surface around the plant were collected to analyze the chemical characteristics of rhizospheric soil. The collected soil samples were air dried. Soil samples (5 g) were added to distilled water (25 mL) and were shaken for 1 h. The soil solution was then filtered through filter paper (Whatman No. 40, 110 mm; Whatman, Little Chalfont, England). Soil pH, total ionic contents (calculated as NaCl equivalents), and chloride contents were measured by using a pH meter (Orion US/710; Thermo Orion, Beverly, MA, USA), electronic conductivity meter (Mettler Check Mate 90; Mettler Toledo Inc., Columbus, OH, USA), and chloride titrator (Titrators DL 50; Mettler Toledo Inc.), respectively.

Exchangeable cations (Na⁺, K⁺, Ca²⁺, Mg²⁺) in soil solution were extracted by 1 N ammonium acetate (CH₃COONH₄) and quantified by using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Optima 7300 DV; Perkin Elmer, Waltham, MA, USA).

Carex scabrifolia Steud., P. communis Trin., S. japonica Makino, and S. malacosperma Hara were sampled on July 29, August 22, and September 28, 2011. After the direct determination of fresh weight in their habitats, leaf samples were dried in an oven at 70°C for 3 days to determine dry weight. Plant water (pw) content was measured as the difference between the fresh weight and dry weight.

The dried plant material was ground to a homogeneous powder and extracted with 95°C distilled water for 1 h, after which the sample was filtered with a GF/A filter (Whatman, 4.7 cm). Inorganic cations (Na⁺, K⁺, Ca²⁺ and Mg²⁺) were determined by ICP-AES (Optima 7300 DV; Perkin Elmer). The chloride content was measured using a chloride titrator (Titrators DL 50; Mettler Toledo Inc.).

Total soluble carbohydrates of the plants were assayed using the phenol-sulfuric acid method (Chaplin and Kennedy 1994). Plant extract (20 μL) was mixed with 580 μL of distilled water, 400 μL of 5% phenol, and 400 μL of sulfuric acid, and the solution was allowed to stand for 10 min before being shaken vigorously. Total carbohydrates were quantified by determining the absorbance at 490 nm by using a UV mini 1240 spectrophotometer (Shimadzu, Kyoto, Japan) after 30 more minutes. Glucose (2–40 μg in 200 μL) was used as a standard solution.

Osmolality was measured by cryoscopy using a Micro-Osmometer 3MO osmometer (Advanced Instruments, Needham Heights, MA, USA).

Free amino acids were quantified using an L-8900 amino acid analyzer (Hitachi, Tokyo, Japan).

The extract was diluted with 2 N H₂SO₄ (1:1 v/v) and 0.5 mL of the acidified extract was cooled in ice water for 1 h. Later, 0.2 mL of cold potassium tri-iodide solution was added and mixed by vortex, and the tubes were stored at 4°C for 15 min and centrifuged at 10,000 rpm for 15 min. The supernatant was aspirated with a fine-tipped glass tube. The periodide crystals were dissolved in 9 mL of 1,2-dichloroethane with vigorous shaking. After 2.5 h, the absorbance was determined at 365 nm in a spectrophotometer. Glycine betaine (200 μg in 1 N H₂SO₄ 1000 mL) was used as a standard solution.

Data were analyzed by analysis of variance (ANOVA) using SPSS ver. 19.0 (SPSS Inc., Chicago, IL, USA). Graphs show means with standard deviation (SD). A Duncan’s multiple range test was carried out to determine significant differences (P < 0.05, N = 3) among the groups.

The soil was sampled at three sites (Carex scabrifolia and Phragmites communis from site 1; Suaeda malacosperma from site 2; S. japonica from site 3) once a month from July to September in 2011. The habitats of the investigated plant species were weakly alkaline, with a pH between 6.9 and 8.1. Soil samples from site 1 (C. scabrifolia and P. communis) and site 3 (S. japonica) showed significant changes in pH, while site 2 (S. malacosperma) remained relatively constant during the study period. Soil pH was generally high in July and low in September (Fig. 2).

The total ion and Cl^- contents in soil of site 1 gradually increased, while they were low at site 2 and site 3 in August and high in September. The soil of site 2 (S. malacosperma) had a low total ion content of 133.7 μmol/g soil in August.

The exchangeable Ca²⁺, Mg²⁺ and K⁺ contents of the soil were relatively constant during the study period, but the exchangeable Na⁺ content was variable, showing a similar pattern to the Cl^- content (Fig. 3). Saline land of Korea’s west coast has an Na⁺ concentration of 160–200 μmol/g soil (Choo et al. 1999), which was relatively close to the ion content of site 1 (C. scabrifolia and P. communis) and site 3 (S. japonica) within the investigation area. The regions where S. malacosperma was found had lower Na⁺ contents than site 1 and site 3.

The ratio of water content to dry weight in leaves is described in Fig. 4. The ratio of water content to dry weight in C. scabrifolia and P. communis ranged 2.0–2.4 and 1.6–1.7, respectively. Both plants had constant water content in their leaves during the entire study period. However, the leaf water content of S. malacosperma (9.7–12.0) and S. japonica (11.3–14.3), belonging to Chenopodiaceae, decreased after August. Succulence is a typical morphological adaptation to osmotic stress and is contributed to salt regulation by increasing the vacuolar volume available for ion accumulation (Khan et al. 2000, Qi et al. 2009). Suaeda japonica and S. malacosperma maintained a high ratio of water and tissue succulence to avoid ion toxicity by increasing storage area and diluting accumulated Na⁺ and Cl^-.

All of the investigated species tended to accumulate soluble carbohydrates in their leaves, but the contents differed among the plant species (Fig. 5). Carex scabrifolia and P. communis had very high concentrations of soluble carbohydrates. The soluble carbohydrate contents of both plants were low in July and increased rapidly in September. Suaeda malacosperma and S. japonica had very low but constant soluble carbohydrate concentrations in their leaves; however, soluble carbohydrate concentration increased in September due to the decreased water contents in their leaves. Monocotyledonous families accumulate more soluble carbohydrates than dicotyledonous families in order to maintain a sufficiently low osmotic potential in saline habitats (Choo and Albert 1997, 1999).

The osmolality of C. scabrifolia and P. communis leaves slightly increased during the study period, but S. japonica and S. malacosperma showed a distinct increase in September. The osmolality was highest in September in all of the plant species (Fig. 6a). The total ion content of C. scabrifolia remained constant while P. communis slightly decreased, and that of S. japonica and S. malacosperma increased in September (Fig. 6a). According to the ecophysiological characteristics of each plant species, the cation patterns of the four investigated plant species remained constant throughout the study period. Carex scabrifolia accumulated similar concentrations of both Na⁺ and K⁺ ion in the leaves and showed efficient regulation of ionic uptake. The genus Carex and some other representatives of Cyperaceae, for example, could regulate their mineral metabolism especially well, being excluders in ion-rich and accumulators in ion-poor habitats. A balanced uptake of ions was accomplished by an efficient regulation metabolism that also prevented the uptake of potentially toxic amounts of ions (Choo and Albert 1997, 1999). Phragmites communis contained a high concentration of K⁺ ions despite the low exchangeable K+ in the soil. Suaeda japonica and S. malacosperma accumulated significantly higher amounts of Na⁺ and Cl^- than K⁺ in their leaves and had a small amount of soluble Ca²⁺ ions in their leaves (Fig. 6b). Deleterious effects of salinity are thought to result from low water potentials, ion toxicities, nutrient deficiencies, or a combination of these factors. Na⁺ in high concentrations in the soil reduces the amounts of available K⁺, Mg²⁺, and Ca²⁺; this is also the case when Na⁺ displaces membrane-bound Ca²⁺. In addition, Na⁺ may have a direct toxic effect, such as when it interferes with the function of potassium as a cofactor in various reactions (Cramer et al. 1985, Khan et al. 2000). Salt tolerance is often seen as the interaction of absorption, transport, and excretion processes, which allows for effective osmotic adjustment by means of salt accumulation. For members of the Chenopodiaceae (such as Suaeda japonica and S. malacosperma), the ability to withstand high external salt concentrations appears to depend on osmotic adjustment, most commonly with Na⁺ and Cl⁻, but, because these elements are toxic, their concentrations in the cytoplasm are maintained at substantially lower levels than in the vacuoles.

The content of free amino acids in the leaves of S. malacosperma and S. japonica was found to be very low and contained a small amount of proline. Phargmites communis accumulated higher levels of free amino acids in its leaves under saline conditions than the chenopodiaceous plants, and contained more amino acids, such as arginine, lysine, histidine, glycine, serine, and proline (Table 1).

Plants stressed by low water potential are known to accumulate amino acids such as arginine, lysine, histidine, glycine, and serine, as well as amide compounds such as glutamine and asparagine (Flores and Galston 1984, Pulich 1986, Hartzendorf and Rolletschek 2001). The accumulation of proline, a compatible solute, is a common metabolic response of higher plants to water deficits, salinity stress, and cold stress, and proline accumulation may play a major role in osmotic adjustment. As proline was shown to minimize cellular damage by enhancing the stability of proteins and membranes, proline content was shown to increase when a plant was subjected to salt stress. Proline is slowly accumulated until a plant reaches a critical salinity level. Exceeding this salt concentration, a greater reaction occurs and proline is accumulated in very high amounts (Voetberg and Sharp 1991, Rhodes and Hanson 1993). Based on the low content of proline in the investigated Suaeda malacosperma and S. japonica, proline did not seem to contribute to the osmotic regulation of this plant. However, C. scabrifolia and P. communis accumulated higher levels of proline in their leaves under salt conditions than the chenopodiaceous plant species.

The content of glycine betaine in the leaves of P. communis was very low. Suaeda japonica accumulated higher levels of glycine betaine in their leaves under saline conditions than C. scabrifolia and P. communis (Table 2). The osmotic potential in leaves decreases under saline conditions due to the accumulation of salt and increasing levels of glycine betaine, which represents an important adaptive response to osmotic stress. Thus, these plants maintain a low osmotic potential, which is important for the absorption of water under low soil water potential. Generally, chenopodiaceous plant species are known to accumulate glycine betaine for osmoregulation (McCue and Hanson 1990). If salt is taken up by a plant and accumulates in the cytoplasm of cells, it can lead to toxicity. For plants to maintain their ability to take up water and prevent dehydration, as well as positive functions in relation to the maintenance of membrane integrity and the stability of other cellular structures, their osmotic potential needs to be lower than that in the soil. In principle, this can be achieved by storing most of the NaCl in the vacuole and by synthesizing compatible solutes such as glycine betaine in the cytoplasm, which contribute to maintaining osmotic potential between the cytoplasm and vacuole (Parks et al. 2002, Shen et al. 2002, Wang and Showalter 2004).

Under the saline conditions observed within the study area, C. scabrifolia accumulated similar concentrations of Na⁺ and K⁺ ions in its leaves. This species showed an efficient regulation of ionic uptake and contained a very high concentration of soluble carbohydrates in order to maintain a sufficiently low osmotic potential in saline habitats. Phragmites communis contained a large quantity of K⁺ ions in its leaves and accumulated proline and free amino acids in response to salinity stress. In contrast, S. japonica and S. malacosperma retained high concentrations of inorganic ions to maintain a low water potential and a high ratio of water and tissue succulence in order to avoid ion toxicity by increasing the storage area and diluting accumulated Na⁺ and Cl^-. However, they maintained low free amino acid and water soluble carbohydrate contents. These ecophysiological characteristics enable the studied species to grow and adapt to saline environments. Further studies on the interactions of proline, ion contents, and glycine betaine in halophytic plant species are required to elucidate more of these ecophysiological characteristics. Studying the ecophysiological characteristics of halophytes under various environments is essential to select and to improve salt-resistant genes for breeding salt-tolerant plants that are able to withstand salt stresses. In addition, we have to domesticate and introduce halophytes to saline areas where they can be used. At the same time, we should study the various potential economic uses of halophytes.