IMPACT OF SORBITOL- INDUCED OSMOTIC STRESS ON SOME BIOCHEMICAL TRAITS OF POTATO IN VITRO

Abstract

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM). The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

Headnote

ABSTRACT

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM). The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

Key words: proline, MDA, H2O2, osmotic stress, sorbitol, potato.

INTRODUCTION

Potato (Solanum tuberosum L.) is often considered as a drought sensitive crop and its sustainable production is threatened due to frequent drought episodes. Potato is of great economic value and ranks as the fourth most important food crop in the world (11). Potatoes are grown in over 125 countries and more than a billion people worldwide consume them on a daily basis (24). Potato represents an excellent source of nutrients including carbohydrates, proteins, vitamin C, several forms of vitamin B, and minerals (6). Drought stress considerably decreases potato yield, making water availability a limiting factor in the production of this crop (27), so one of the great challenges for the next decade is to mitigate any effect of climate change on crop production with a main focus being to maintain crop production levels with reduced availability of water. In the Mediterranean regions during the heat- free periods of the year, potato yields will go down dramatically as the suitable periods become shorter and higher evaporative demands will minimize water use efficiently causing a remarkable decline in tuber potato yield in the temperate environments (14). Globally, for potato, drought will decrease potential potato yield by 18-32% in the projected period of 2040-2069 (16). Plants respond to limiting water availability through a complex series of adaptive changes often accompanied by deleterious pleiotropic effects (17). Water stress reduces plant growth through a reduction in photosynthesis, mainly caused by a stomatal limitation (28). This decrease in the photosynthetic rate under water deficit conditions leads to an increase in the production of reactive oxygen species (ROS) (9). ROS can alter the normal functioning of plants due to the damage caused to lipids, proteins, nucleic acids, photosynthetic pigments and enzymes (18). Biochemical responses of potato to drought are complex with levels of antioxidants showing increases, decreases, or no effect, depending on the genotype and kind of antioxidant (36). Plants adapt to drought conditions either by decreasing water loss or by maintaining water uptake. Osmotic adjustment (OA) results in an increase in solutes in plant cells leading to a lowered osmotic potential, which in turn can improve cell hydration, help maintain cell turgor in leaf tissue, maintain metabolic processes and thus enhance plant growth and yield under drought conditions (33). Studies in potato have demonstrated that drought stress leads to accumulation of osmotically active solutes including proline. The principal damage caused by ROS during water stress is lipid peroxidation, which decreases the stability of cellular membranes and increases their permeability, thereby modifying cellular metabolism (38). In order to overcome oxidative stress, plants have developed enzymatic and non-enzymatic antioxidants (8). Proline serves as an ROS scavenger and is used as a non-enzymatic antioxidant to counteract the damaging effect of different ROS members helps in water stress survival through osmoprotection (34). Ascorbic acid (AsA) is a naturally occurring compound with antioxidant activity that plays a pivotal role in plant cell adaptation (1). The generation of H2O2 is increased due to a wide variety of stresses, and some authors have suggested that H2O2 is a key factor mediating the phenomena of acclimation and cross-tolerance (26). It plays a dual role in plants: at low concentrations, it acts as an acclamatory signal, triggering tolerance to various stresses (10). Therefore, biochemical and physiological behavior of the plants, even at early developmental stage under osmotic stress could provide information on their capacity to tolerate drought stress. Resistance to water stress by conventional methods takes time, is costly and labor-intensive. In vitro tissue culture allowed a deeper understanding of the physiology and biochemistry of plants grown under unfavorable environmental (5). The most widely used method for the selection of genotypes tolerant to abiotic stress is the in vitro selection pressure technique (29). In vitro simulation drought was made to identify varieties with optimum tolerance at drought (3). In Syria, potato is cultivated in highly mountainous areas with few or no available water, suggesting that this crop is often subjected to drought stress conditions, so it is of great importance to evaluate the response of the available genetic material for drought stress to identify the most drought adaptive genotypes and drought-associated traits. The objective of this study is to evaluate the response of eleven potato genotypes to sorbitol-induced osmotic stress In vitro based on some biochemical traits.

MATERIALS AND METHODS

Plant material and site of experimentation: The experiment was conducted at the laboratories of Plant Biotechnology, Faculty of Agriculture, Damascus University, during the growing season 2017 - 2018, in order to evaluate the response of eleven potato genotypes (Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta) to osmotic stress In Vitro, using sorbitol as a stimulating agent, based on some biochemical traits.

Treatments: In this study, to induce in vitro water stress, sorbitol was used with the basic medium MS, considered control. Osmotic stress was imposed through the addition of different concentrations of sorbitol (C6Hi4O6) to the growth media (0, 50, 110, 220, 330 and 440 mM), which equivalent to osmotic potentials (- 0.82, -1.09, - 1.44, - 1.79 and - 2.14 Mpa respectively). Sorbitol was used to exert a water deficiency in the nutrient medium necessary for growth and development of the plantlet with the purpose of cause changes of growth, similar to those produced by the drying of the soil.

Methodology: The genotypes were first cultivated in pots under greenhouse conditions. Explants from each genotype were grown in vitro in test tubes containing 13.5 ml of solid Murashige and Skoog (25) medium, supplemented with 30 g l-1 sucrose and 7 g l-1 plant agar. The pH of the medium was adjusted to 5.8 before autoclaving for 20 min at 120°C. Then, 1.5-2.0 cm long stem cuttings with one or two axillary buds were prepared by excluding the basal and apical portions of the plantlets, to be evaluated for osmotic stress tolerance. The experiment was laid out as a randomized complete design in a factorial arrangement and replicated four times in two rounds. The experimental materials were cultured for 30 days at a constant temperature of 22 ± 2°C, 16/8 hours light/dark photoperiod with a photosynthetically active photon flux density of approximately 35 pmol m-2 s-1.

Investigated traits

Membrane permeability: Solutes leakage (SL) was measured as described by Leopold et al., (20). Plant material (0.5 g) was washed with deionized water, placed in tubes with 10 ml of deionized water and left on a shaker for 3 h at 25°C. Subsequently, the initial absorbance of the solution (A1) was determined at an optical density of 273 nm. Samples were then boiled in a boiling bath at 100 °C for 20 min and the final absorbance of the solution (A2) was determined at the same optical density. The percentage of the solutes leakage was measured according to the following formula: SL (%) = (A1/A2) x 100

Proline content: Free proline content was measured by the method of Bates et al. (4). Plant tissue (100 mg) was homogenized in 3% (w/v) sulphosalycylic acid and centrifuged at 700 ' g for 3 min. After addition of ninhydrin reagent, mixtures were heated at 100 °C for 1 h and cooled in an ice-bath. The chromophore obtained was extracted from liquid phase with toluene and the absorbance of organic layer was read at 520 nm. Proline concentration was determined from calibration curve using LProline as standard and expressed as pg (proline) g-1 fresh weight.

MDA and H2O2 content: Lipid peroxidation was determined by estimating the amount of malondialdehyde (MDA) content using the thiobarbituric acid method described by Heath and Packer (15). The crude extracts were mixed with 0.25% (w/v) thiobarbituric acid solution containing 10% (w/v) trichloroacetic acid, heated at 95 °C for 30 min and the reaction was stopped in an ice bath. The cooled mixtures were centrifuged at 10000 ' g for 10 min and the MDA content calculated from the absorbance at 532 nm (correction was done by subtracting the absorbance at 600 nm for non-specific turbidity) by using extinction coefficient of 155 mM-1cm-1. H2O2 was extracted from 100 mg fresh tissue in ice-cold acetone and estimated according to the method of Mukherjee and Choudhuri (23). After addition of titanyl-sulphate and conc.NH4OH solution, the formed peroxide-titanium precipitate was dissolved in 2M H2SO4 and absorbance of the mixtures read at 415 nm. The H2O2 content was calculated from a standard curve and expressed as nmol (H2O2) g-1 fresh weight.

Ascorbate content: Ascorbate was isolated by extraction with 6% trichloroacetic acid from 100 mg plant tissue, following the method of Mukherjee and Choudhuri (23). Extracts were mixed with 2% dinitrophenyl hydrazyne followed by the addition of 1 drop of 10% thiourea solution and boiled for 15 min in a water bath. After adding 80% (w/v) H2SO4 (in an ice-bath), the absorbance of the mixtures containing hydrazone complex was read at 530 nm. Ascorbate concentration was determined using calibration curve and expressed as mmol (ascorbate) g-1 fresh weight.

RESULTS AND DISCUSSION

Membrane permeability (solutes leakage %): Results showed significant differences (P<0.01) in the solutes leakage through the cytoplasmic membranes among genotypes and the osmotic stress levels and their interaction. Solutes leakage increased significantly and proportionately with the increment of sorbitol in the growth media, where it was significantly the highest at the highest concentration of sorbitol (82.39%), while it was the lowest in the control (without sorbitol) (39.01%). The percentage of the solutes leakage was significantly higher in the genotype Colomba (93.91%), while it was significantly lower in the genotype Fabulla (28.10%), followed by Toronto, Alison and Barcelona (39.73, 40.51 and 47.41% respectively) (Table, 1). This increase in the solutes leakage could be due to the peroxidation of lipids caused by an increase in ROS, as has been reported for plants such as tomato (Lycopersicon esculentum Mill.) (32), and potato (9).

Proline content (^g g-1 fresh wt.): Results showed significant differences (P<0.01) in the proline content among genotypes and the osmotic stress levels and their interaction. The proline content in the leaves increased significantly and roportionately with the increment of sorbitol in the growth media. The free amino acid proline content in the leaves was significantly higher in the highest sorbitol concentration in the nutritive media (440 mM) ( 31.87pg g-1 fresh wt.) and decreased proportionally and significantly with decreasing the osmotic stress intensity, where it was the lowest in the control (without sorbitol) ( 11.34pg g-1 fresh wt.). Proline content was significantly higher in the genotype Fabulla ( 32.37pg g-1 fresh wt.), followed with the genotype Nectar ( 26.77pg g-1 fresh wt.), while it was significantly lower in the genotype Barcelona ( 17.52pg g-1 fresh wt.) (table, 2). In water-deficit conditions, proline retains osmotic potential (22) and redox balance of cells (37), scavenges free radicals and ROS as an antioxidant, protects macromolecules from denaturation as a chemical chaperone (31). Under stressful conditions, proline is considered as a nitrogen and carbon provider after rehydration (2), source of energy (35). Proline can protect plants from stress through different mechanisms,

MDA content (nmol g-1 fresh wt.): Results revealed significant differences (P<0.01) in the MDA content in the leaves among genotypes and the osmotic stress levels and their interaction. MDA content was including osmotic adjustment, detoxification of ROS, protection of membrane integrity, and stabilization of proteins/enzymes (19). In general, Proline accumulation improves stress tolerances without disrupting cellular structure. When the cellular water content decreases, proline and other compatible solutes can act as water substitutes to stabilize cellular structure through hydrophilic interactions and hydrogen bonding.

significantly higher at the two osmotic stress levels 330 and 440 mM sorbitol without significant differences between them (44.27 and 43.87 nmol g-1 fresh wt. respectively), while it was significantly lower in the control treatment (without sorbitol) (24.88 nmol g-1 fresh wt.). MDA content was significantly higher in the genotype Malice ( 74.87nmol g-1 fresh wt.), followed by Nazca ( 48.83nmol g-1 fresh wt.), while it was significantly lower in the genotype Barcelona ( 17.71nmol g-1 fresh wt.0 followed by the genotypes Colomba, Toronto and Spunta without significant Differences among them (20.31, 20.40 and 20.76 nmol g fresh wt. respectively) (table, 3). It can be noticed that the genotypes which could maintain the cytoplasmic stability accumulated the least quantity of MDA, such as Barcelona (17.71 nmol g-1 fresh wt.), followed with the genotypes Colomba, Toronto and Spunta (20.31, 20.40and 20.76 nmol g-1 fresh wt. respectively), indicating the relevance of estimating MDA to assess the membranes integrity and the capacity of the genotypes to recover growth as aconsequence.

H2O2 content (ц mol g-1 fresh wt.): Results revealed significant differences (P<0.01) in the H2O2 content in the leaves among genotypes and the osmotic stress levels and their interaction. H2O2 content was significantly higher at the osmotic stress level 440 mM sorbitol (60.667 gmol g-1 fresh wt.), while it was significantly lower in the control treatment (without sorbitol) and the lowest osmotic stress level (55 mM sorbitol) without significant differences between them (26.19 and 26.36 gmol g-1 fresh wt. respectively). H2O2 content was significantly higher in the genotype Malice ( 72.92gmol g-1 fresh wt.), while it was significantly lower in the genotype Toronto ( 15.35 gmol g-1 fresh wt.). (table, 4). H2O2 content increased with increasing the sorbitol level in the growth medium, particularly at the osmotic stress level 220 mM and the higher concentrations. Generally, H2O2 is produced in response to many abiotic stresses, suggested that H2O2 is a key factor mediating the phenomena of acclimation and cross-tolerance (26). Recent investigations have revealed that H2O2 is a central component of the signal transduction cascade involved in plant adaptation to a changing environment (26). Results showed that the genotype Malice produced the highest concentration of H2O2, so it was more susceptible to osmotic stress conditions, due to formation of more amount of ROS, where the MDA level in such genotype was significantly higher ( 74.87nmol g-1 fresh wt.), indicating a higher level of lipid peroxidation. On the other hand, the concentration of H2O2 was significantly lower in the two genotypes Toronto and Colomba ( 15.35and 16.92umol g-1 fresh wt. respectively), but unfortunately the percentage of solutes leakage was significantly higher in the genotype Colomba (93.91%), while it was significantly lower in the genotype Toronto (39.73%), indicating that the denaturation of the cytoplasmic membranes in the genotype Colomba was not due to the ROS rather than the indirect effect of osmotic stress on the membrane proteins.=

Ascorbate content (pg g-1 fresh wt. ): The ascorbic acid (AsA) content was significantly higher at the osmotic stress level 110 mM sorbitol ( 485.30pg g-1 fresh wt.), followed by the osmotic level 55 mM sorbitol ( 472.50pg g-1 fresh wt.), while it was significantly lower at the sorbitol concentration 440 mM ( 286.40 pg g-1 fresh wt.), followed by the two sorbitol concentrations 220 and 330 mM ( 345.50and 347.20pg g-1 fresh wt. respectively). Increasing the osmotic stress intensity in the growth medium above 220 mM was found to inhibit the biosynthesis of ascorbic acid, which in turns explains the detrimental effects of higher levels of osmotic stress on all the investigated traits. Generally, ascorbic acid plays a pivotal role in improving the capacity of the plant cells to endure abiotic stresses (7). The concentration of ascorbic acid was significantly higher in the genotype Toronto ( 614.8pg g-1 fresh wt.), while it was significantly lower in the genotype Colomba ( 63.45 pg g-1 fresh wt.) (table, 5). AsA is known to play a role in response to oxidative stress, although the regulatory molecular mechanism of AsA synthesis has not been yet well understood. Plants have four major H2O2scavenging pathways. Two of these pathways, the water-water cycle and the ascorbateglutathione cycle (21), are related to AsA, which is known to have roles in plant stress responses (39). In addition, AsA also has a role in ROS detoxification, as an antioxidant and H2O2-scavenger in plant cell to avoid accumulation of ROS under stress conditions (12). In our study, the increased AsA content in some genotypes with increased osmotic stress levels suggests its importance in improving tolerance to osmotic stresses, which in turn promotes the scavenging of excess H2O2. According to Puthur (30), the ascorbic acid could react directly with hydroxyl radicals, superoxide, and singlet oxygen and thus provides protection by scavenging free radicals. The genotypic variation in the H2O2 concentration is partially attributed to to the variability in the AsA content, where the genotypes which synthesized significantly higher amount of AsA, such as Toronto, Nictar and Barcelona (614.80, 544.40and 460.70pg g-1 fresh wt. respectively) produced significantly lower H2O2 (15.35, 20.17and 35.72pmol g-1 fresh wt. respectively), indicating the importance of AsA in detoxifying the ROS, especially in potato (12).

Conclusions

In vitro screening of potato genotypes for osmotic stress tolerance evaluated in the current study demonstrated that increased osmotic stress levels due to sorbitol treatment in the growth medium resulted in an increased of the solutes leakage, proline content, ascorbic acid, MDA and H2O2 with observed genotypic variation among the tested potato genotypes. However, some of the genotypes were observed to have tolerance to the osmotic stress, indicating the possibility to select genotypes that could adapt to drought in field conditions. Generally, that the in vitro method could be particularly helpful to screen a large number of plant genotypes within a short period of time. However, the effectiveness of in vitro screening should be further tested under field conditions on promising potato genotypes for better root yield and quality production capacity under different moisture stress regimes. Therefore, evaluating the potato genotypes that showed in vitro osmotic stress tolerance further under moisture-stressed conditions in the field may validate the result and could be a step in the direction to develop potato varieties that can be cultivated in drought-prone areas in Syria in particular and temperate environments in general. In vitro screening of a large number of genotypes for osmotic stress can be carried out by adding sorbitol to the Murashige and Skoog (MS) medium to reduce the osmotic potential (25). Such assays can identify genotypes based on osmotic stress tolerance, are less costly, less time consuming than field trials, and easier to reproduce (13).

Sidebar

*Received:11/9/2019, Accepted:30/12/2019

References

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