We have done a comparative study of ion status, growth and biochemical parameters in shoots and roots of seablite (Suaeda altissima (L.) Pall.) and spinach (Spinacia oleracea L.) grown with different salinity levels in the medium (0.5 - 750 mМ). A distinctive feature of the halophyte was a high Na+ content in tissues at its low concentration in the medium (0.5 mM). In these conditions, Na+ accumulation in seablite roots was four-fold higher than in spinach roots, and Na+ content in seablite leaves was almost 20-fold higher than in spinach. Together with an increase in sodium concentration in the medium, K+ content decreased six-fold in seablite leaves, while in spinach it did not decrease so drastically. We can suppose that in the halophyte, some processes occur only in the presence of sodium, and these functions of sodium cannot be fully fulfilled by potassium. Analysis of protein and total nitrogen content in tissues shows that at high salinity, the ability to synthesize non-protein nitrogen-containing compounds increases in the halophyte and decreases in the glycophyte. Data on proline content dynamics show that its increase in tissues of spinach (salinity levels 150 and 250 mМ) and seablite (salinity levels 0.5 and 750 mМ) is an indicator of plant injury. In seablite and spinach, proline is not a major osmoregulator. Its concentration both in roots and leaves was no more than 2.5 μmol/g fresh weight. The data presented in this work concern the accumulation and distribution of Na+, Cl?, K+ and ions, as well as growth and biochemical parameters. Our data show that the development of adaptation reactions in the whole plants in the conditions of high salinity is determined by morphofunctional systems and their interaction.
Overcoming the negative effects of high soil salinity on plants is a serious problem that is being tackled by professionals from various fields. We can assume that overall, the biochemical and physiological mechanisms of plant salt tolerance on the cellular level are deciphered. It is currently known that one of the main strategies for plant adaptation to high salt concentrations on the cellular level is to maintain low concentrations of Na+ and Cl− ions in the cytoplasm. This is reached through the selectivity of transport systems in the plasma membrane that transfer K+ and Na+ into the cell, and due to the export of sodium from the cytoplasm. The activity of these transport systems contributes significantly to determining the cytoplasmic K+/Na+ ratio, which in turn determines salt tolerance [1,2]. Also, due to osmolyte synthesis in the cytoplasm and compartmentation of Na+ and Cl− ions into the vacuole, cells maintain the level of osmotic potential needed to absorb water in high salinity conditions [
However, plant resistance to high salt concentrations is largely determined by the efficiency of mechanisms functioning on the whole plant level. High salinity of soils causes osmotic stress and ion imbalance, which leads to a decrease in growth and productivity of plants susceptible to high concentrations of NaCl [
We can claim that salt tolerance in plants depends on the distribution of Na+ and Cl− in organs and tissues, the activity of ion transport systems in different tissues, and on the transport pathways of Na+ and Cl− ions in the whole plant [
The tolerance strategies described above are, to some degree, characteristic of halophytes as well as glycophytes [
High salt tolerance in dicotyledonous halophytes, for example, from the Chenopodiaceae family, is due to their ability to accumulate up to 1 - 1.5 M of sodium ions in shoot vacuoles in high salinity conditions, as well as their ability to use Na+ to maintain turgor and act instead of K+ [9,10]. In high salinity conditions, the halophytes show increase in growth rate, caused by the effect of sodium on cell extension growth and plant water balance. Monocotyledonous halophytes absorb less sodium, as compared to dicotyledonous halophytes, and are able to maintain a high concentration of K+ in shoot tissues [
A major mechanism of salt tolerance in the glycophytes is the limitation of inward flow of sodium and chloride into the roots and their transport into shoots [
It is supposed that the main distinction between halophytes and glycophytes is that the halophytes are capable of withstanding salt shock. This allows them to reach a new metabolically stable state faster than glycophytes, and continue growth in salinity conditions [
Proline plays a major role in reactions to stress, including salt stress. It has a polyfunctional physiological effect: It plays a role as an osmoregulator, a protector compound, and performs other functions that help maintain cell homeostasis [
In spite of the high number of works on salt tolerance, further study is needed to evaluate the role of different protective systems and their interaction on the whole plant level in the formation of salt tolerance.
Presented here is a comparative study of the effects of salinity on the accumulation and distribution of ions, proline, protein, and total nitrogen in roots and leaves of seablite and spinach. Seablite and spinach differ in their salt tolerance: Seablite is a succulent euhalophyte [
Experiments were performed on 50 - 60-day-old seablite (Suaeda altissima (L.) Pall.) and spinach (Spinacia oleracea L., cultivar “Matador”) plants of the Chenopodiaceae family.
Plants were grown with solution culture, as described in [9,16], with the use of Robinson and Dounton mineral solution [
All the parameters were determined separately for roots and leaves.
100 - 150 mg of dry plant material were ground in a porcelain mortar and transferred into a glass tube. To determine cation content, 5 - 10 ml of 0.0025 N HCl were added to the tube, to determine anion content 5 - 10 ml of distilled water were added; the tubes were then placed into a thermostat. Extraction was performed for 4 - 5 hours at a temperature of 80˚C. Potassium, sodium and nitrate content in the extracts were determined potentiometrically (pH Meter, Model 3320, “Jenway”, England) with ion-selective electrodes (“NIKO”, Russia).
An aliquote of the water extract was placed into a titration flask. Then, 5 ml of isopropyl alcohol, 0.05 ml of 1 N HNO3, 0.05 ml of α-nitroso-β-naphthol, and 0.2 ml of diphenyl carbosone were added to the titration flask. The mixture was titrated with 0.01 N Hg(NO3)2 until the solution had become purple. The normality of the Hg(NO3)2 solution was determined with a 0.01 N NaCl solution.
Extraction of free proline and determining its content was performed according to the method of Bates et al. [
Preparation of ninhdrin reagent: 1.25 g of ninhdrin (Sigma, USA) was reacted with 20 ml of 6 M phosphoric acid and 30 ml of glacial acetic acid in a glass-stoppered flask for 0.5 hour at 100˚C.
Total nitrogen content was determined in dried (55˚C - 60˚C) and ground samples with a semiautomated CNHanalyzer (“Carlo-Erba”, Italy).
200 mg of dry plant material was ground in a porcelain mortar and transferred into a glass tube. After adding 5 - 10 ml of distilled water, the tube was placed into a water bath (80˚C). Extraction was performed for one hour. The water extract was quantitatively transferred into a 25 ml volumetric flask, washing it multiple times with water and filtering through a paper filter. 15 ml of the extract was used to determine protein content.
To each 5 ml of water extract, placed into centrifugation tubes, 2 ml of 50% TCA was added, and the mixture was then incubated for 30 - 40 minutes with constant stirring. The content of the tubes was then centrifuged for 15 minutes at 12,000 - 15,000 rpm. The supernatant was discarded, and the pellet was washed successively with 5% and 1% TCA solutions. In the leaf extracts, after treatment with 1% TCA, the protein pellet was washed with 80% acetone. After each stage of washing, centrifugation was repeated. The final protein pellet was slightly dried with air and dissolved in 1 ml of 1% NaOH solution. To completely dissolve the protein, the tubes were placed into a thermostat and kept at 40˚C for 12 hours. The protein solution was then used to determine protein content with the Lowry’s method [
After extracting water-soluble proteins, alkali-soluble proteins were extracted from the plant material [
After extraction with water and alkali, 70% ethanol was added to the plant material in portions of 3 - 5 ml and the mixture was incubated for 30 minutes with constant shaking [
Total protein content was calculated as a sum of its content in water, alkali, and ethanol extracts.
Statistical Analysis was performed in Excel 7.0. The tables and figures represent the average values of 3 - 10 individual experiments and their standard deviations.
Under stress, growth parameters reflect the physiological state of the plant. Data on biomass accumulation by seablite and spinach plants show that upon an increase in salt content up to 250 mM, fresh and dry mass of all organs in the halophyte increases more than two-fold. When the salt concentration in the medium is further increased to 750 mM, organ mass decreases 4 - 5-fold (Figures 1(a) and (c)). These results support the known data that NaCl concentration of 250 mM in the medium is optimal for seablite growth and development [
Measurement of K+ and Na+ accumulation in seablite and spinach organs (Figures 2 and 3) distinctly shows
different reactions in the halophyte and glycophyte. Upon an increase in NaCl concentration in the nutrition medium, sodium content in seablite and spinach tissues increases. However, in all experiments seablite tissues accumulated substantially more sodium ions than spinach. Upon an increase in NaCl content in the nutrition medium from 0.5 to 250 mM, Na+ content in seablite roots increased from 80 to 300 µmol/g fresh weight, while in spinach roots it increased from 20 to 120 µmol/g fresh
weight. In leaves, the difference in sodium content between the halophyte and the glycophyte is even greater, and reach 500 µmol/g fresh weight for seablite. Seablite is a salt-accumulating halophyte. It adapts to high salinity by accumulating high quantities of ions in vacuoles of shoot cells in order to create a gradient of water potential along the axis of the plant [
Upon an increase in sodium concentration in the medium from 0.5 to 250 mM, K+ content in seablite roots did not change, while in spinach roots it decreased twofold (Figures 2(a) and 3(a)). At the same time, in seablite leaves K+ content drastically decreased (from 350 to 60 µmol/g fresh weight, six-fold), and did not decrease so strongly in spinach (Figures 2(b) and 3(b)). A possible reason for this is that, contrary to the glycophyte, in the halophyte Na+ can replace a large amount of K+ as an osmotic in leaves. It is known that high NaCl concentrations in the medium cause potassium deficit in salt-sensitive plants [
A distinct feature of the halophyte is a high Na+ content in its tissues at low concentrations of this ion in the medium (СNa+ = 0.5 mM; CK+/CNa+ ~ 14, where CK+ and CNa+-K+ and Na+ concentration in the medium). In these conditions, Na+ content in seablite roots is four-fold higher than in spinach roots, and Na+ content in leaves is more than 20-fold higher than in spinach leaves (Figures 2 and 3).
A qualitative parameter characterizing the accumulation of an ion may be its concentration coefficient (ki), which is equal to Cin/Cout, where Cin is the ion concentration in the tissue, mM, and Cout is the ion concentration in the medium, mM (
Sodium is an important element for halophytes of the Chenopodiaceae family. An important question is, what processes benefit from the significant concentration of this ion in seablite organs, when its concentration in the environment is low? Works concerned with studying salt
Note. Concentration coefficient ki = Cin/Cout, where Cin-ion concentration in the tissues, mM per 1 kg fresh weight; Cout-ion concentration in the medium, mM. Presented are average ki values and their relative errors.
tolerance mechanisms in halophytes are mainly aimed at finding the possible functions for sodium. For example, it has been found that in C4-plants, high concentrations of NaCl stimulated oxygen evolution by PSII, while the same concentrations of kCl inhibited this process [
The preference for Na+ accumulation over K+ in plant tissues at low NaCl concentrations in the medium may be due to different hydration of these ions in water solutions. Hydration is currently viewed not as a process of binding a certain number of water molecules of the solution to ions, but as the effect of ions on translational motion of water molecules [
The results and considerations stated above give reasons to conclude that some processes in the halophyte probably occur only in the presence of sodium, and these functions of sodium cannot be fully fulfilled by potassium.
In previous studies of ion-exchange properties of the cell wall in seablite and spinach, we have shown that cell walls are a compartment which can hold 10% to 20% of cations absorbed by the plant [23,24]. Cell walls of roots and shoots show different capacity in different conditions of salt nutrition during plant growth [
Cl− is an element necessary for all plants, but the amount of this ion that is needed for plant nutrition is rather low and reaches only 3 - 10 µmol/g dry weight. However, all plants accumulate chloride in concentrations higher than the required level. The reasons for this phenomenon are yet unknown [
Upon an increase in NaCl concentration in the medium (CNaCl) in the medium, chloride content increases in both leaves and roots of the halophyte (
Nitrate is another anion that plays an important physiological role. In tissues of the halophyte, the functional relation between nitrate content and salinity level has a distinct minimum at 250 mM NaCl in the nutrition medium (
Data on protein and total nitrogen content in tissues of the halophyte (Figures 5(a) and (b)) show that an increase in CNaCl in the medium leads to an increase in nitrogen content per unit of protein mass (
In the glycophyte, upon an increase in NaCl concentration (from 0.5 to 150 mM) nitrate content decreases in leaves and does not change much in roots (
Upon an increase in salinity level, the amount of nitrogen per unit of protein mass increases in seablite and decreases in spinach (
It is widely known that in response to stress, various proteins are synthesized in plant cells: Regulatory and protective proteins, enzymes that carry out cell metabolism under stress, including hydrolases that are response
Note: The ratio of total nitrogen and protein content was calculated by dividing the average total nitrogen content (mg nitrogen per 1 g tissue dry weight) by the average protein content (mg protein per 1 g tissue dry weight). Presented are average values and their relative errors.
ble for degrading damaged macromolecules [
In response to salt stress cells of many plants accumulate proline [
The data obtained in this work show that in response to an increase in NaCl concentration to 250 mM, proline content increases in roots and leaves of the glycophyte (
In seablite, an increase in NaCl concentration from 0.5 to 250 mM leads to a two-fold decrease in proline content in both roots and leaves; further increase in salinity level to 750 mM leads to an increase in this parameter in both organs: five-fold in roots and two-fold in leaves (
Numerous data from biochemistry, physiology and molecular biology provide a good understanding for the structure and dynamics of the processes of adaptation to salinity on the cellular level [4,6,28]. However, they do not explain the diversity of reactions in whole organisms from different ecological groups. Studies on the physiological functions in the whole plant conducted previously [9,10], as well as data presented in this work on the accumulation and distribution of Na+, Cl−, K+ and ions, growth and biochemical parameters, show that the development of adaptation reactions in plants in the conditions of high salinity is determined by morphofunctional systems and their interaction.
The work was supported by the Russian Foundation for Basic Research (grants No. 04-04-49379-a and 08- 04-01398-a).