Fitociencia

 

Betacyanin and antioxidant system in tolerance to salt stress in Alternanthera philoxeroides

 

Betacianinas y el sistema de antioxidantes en la tolerancia al estrés salino de Alternanthera philoxeroides

 

Márcia Vaz Ribeiro, Sidnei Deuner, Letícia Carvalho Benitez, Andersom Milech Einhardt, José A. Peters, Eugenia J. Bolacel Braga

 

Universidade Federal de Pelotas (UFPel), Campus Universitário s/n, Caixa Postal 354-96010900. Capão do Leão - RS, Brasil. (jacirabraga@hotmail.com).

 

Received: April, 2013.
Approved: February, 2014.

 

Abstract

Salinity is a limiting factor fot growth and development, since it affects several physiological processes in plants. This trial aimed to assess the levels of photosynthetic pigments, betacyanins, lipid peroxidation and activity of antioxidant enzymes in Alternanthera philoxeroides under salt stress. Plants originated from an in vitro culture were acclimatized and irrigated with sodium chloride (0, 200 and 400 mM) for 30 d. The experimental design was completely randomized with a 3×2 factorial arrangement of treatments: three concentrations of NaCl and two plant tissues (stems and leaves). The experimental data was subjected to an analysis of variance and means where compared using Tukey test (p≤0.05). The levels of chlorophylls a, chlorophylls b and total carotenoids presented similar responses, decreasing their values according to salt concentrations, while the chlorophyll a/b ratio of the plants that were submitted to the highest salt concentration presented a significant increase when compared to the control. Higher levels of betacyanin were observed on stems, when compared to the leaves, in the highest salt concentrations. On the leaves, there was a significant increase of lipid peroxidation and superoxide dismutase activity, whereas roots showed an increase of the enzymes catalase and ascorbate peroxidase. Salt stress caused greater degradation in the photosynthetic pigments, increment of betacyanin synthesis in stems, and damage to the cell membranes of the leaves. However, the increase of antioxidant enzymes activity stimulated the protective system against oxidative stress. It is concluded that Alternanthera philoxeroides (Mart.) Griseb. plants present reduction in levels of photosynthetic pigments, increased synthesis of betacyanins and increased activity of antioxidant enzymes in leaves and roots when exposed to salt stress.

Key words: Amaranthaceae, alligator weed, sodium chloride, photosynthetic pigments, antioxidant enzymes.

 

Resumen

La salinidad es un factor limitante para el crecimiento y el desarrollo, ya que afecta varios procesos fisiológicos en las plantas. Este estudio tuvo el objetivo de evaluar los niveles de pigmentos fotosintéticos, betacianinas, peroxidación lipídica y actividad de enzimas antioxidantes en Alternanthera philoxeroides bajo estrés salino. Las plantas se originaron de un cultivo in vitro y se aclimataron e irrigaron con cloruro de sodio (0, 200 y 400 mM) durante 30 d. El diseño experimental fue completamente aleatorio con un arreglo factorial 3×2 de los tratamientos: tres concentraciones de NaCl y dos tejidos vegetales (tallos y hojas). Los datos experimentales se sometieron a un análisis de varianza y las medias se compararon usando la prueba de Tukey (p≤0.05). Los niveles de clorofilas a, clorofilas b y carotenoides totales presentaron respuestas similares, disminuyeron sus valores con las concentraciones de sal, mientras que la relación de clorofila a/b de las plantas que se expusieron a la concentración mayor de sal presentó un incremento significativo respecto al control. Niveles mayores de betacianinas se observaron en los tallos, en comparación con las hojas, para las concentraciones de sal más altas. En las hojas hubo un aumento significativo de peroxidación lipídica y de actividad de superóxido dismutasa, mientras que las raíces mostraron un aumento en las enzimas catalasa y ascorbato peroxidasa. El estrés salino causó degradación mayor de los pigmentos fotosintéticos, un aumento en la síntesis de betacianinas en los tallos, y daño a las membranas celulares de las hojas. Sin embargo, el incremento de actividad de las enzimas antioxidantes estimuló el sistema protector contra el estrés oxidativo. Se concluye que las plantas de Alternanthera philoxeroides (Mart.) Griseb. presentan una reducción en los niveles de pigmentos fotosintéticos, mayor síntesis de betacianinas y mayor actividad de enzimas antioxidantes en las hojas y raíces cuando están expuestas a estrés salino.

Palabras clave: Amaranthaceae, hierba de caimán, cloruro de sodio, pigmentos fotosintéticos, enzimas antioxidantes.

 

INTRODUCTION

Salinity is one of the most important factors among abiotic stress limiting plant growth and yield, because it affects almost all physiological and biochemical processes, reducing the yield (Flowers, 2004). High salt concentrations induce water stress (hydric deficit), causing cellular and ionic imbalance, due to the toxicity of these ions, and promoting osmotic stress (Mandhania et al., 2006; Khan and Panda, 2008). Specific effects of salt stress on plants' metabolism have been linked to the accumulation of toxic ions such as Na⁺ and Cl^- (Khan and Panda, 2008) and to the depletion of K⁺ and Ca²⁺ (Sumithra et al., 2006). The primary consequence of salt stress is ionic imbalance and cellular hyperosmotic stress, whereas the secondary consequence is the increased production of reactive oxygen species (ROS) (Panda and Khan, 2009): superoxide radicals (O₂^•-), hydrogen peroxide (H₂O₂), hydroxyl radical (OH^•) and singlet oxygen (¹O₂).

Plants, in order to protect their cell membranes and organelles from the damaging effects of these radicals, activate an antioxidant defense system consisting of superoxide dismutase (SOD-EC 1.15.1.1.), catalase (CAT-EC 1.11.1.6) and ascorbate peroxidase (APX-EC 1.11.1.1) that efficiently reduce ROS under normal circumstances. But if the complete reduction does not occur under conditions of increased production, the result can be a state of stress leading to oxidation of lipids, proteins and DNA. These events can trigger inactivation of cellular components, accelerating the process of cell death (Buckner et al., 2000). In this sense, the antioxidant defense system plays a key role towards the acquisition of tolerance. Leaf pigments such as chlorophylls and carotenoids can also be used as indicative parameters of stress, when plants are cultivated under high salt concentrations. Depending on the species under study, cultivar, exposure time and salt concentration, such stress may affect the absorption of nutrients that constitute the chlorophyll molecule and the formation of other photosynthetic pigments, therefore hampering photosynthesis itself (Santos, 2004).

The species Alternanthera philoxeroides (Mart.) Griseb., which belongs to the Amaranthaceae family is known as alligator-weed. These plants are composed of flavonoids, saponins and betalains; betalains are natural N-heterocyclic pigments soluble in water that is divided into two structural groups: the betaxanthins (yellow) and betacyanins (red to red purple). Betacyanins are widely used as an additive to food and drugs, being an nontoxic natural dye (Azeredo, 2009; Volp et al., 2009; Perotti et al., 2010). Moreover, betalains play important roles in stress physiology, acting as ROS (resultant from abiotic stresses) detoxifiers, as it was observed in Beta vulgaris subsp. vulgaris (Sepúlveda-Jiménez et al., 2004) and abiotic stresses induced by UV radiation on Mesembryanthemum crystallinum (Vogt et al., 1999). Native to South America this species is mainly found in pioneer formations, "restingas", usually in recent sandy terrains (Blum, 2008). It is classified as a perennial species that grows abundantly in different ecosystems, from aquatic environments to those extremely dry, such as dunes (Gao et al., 2007).

Given the above, this study aimed to investigate the effect of salinity on chlorophyll contents, total carotenoids, betacyanins, lipid peroxidation and antioxidant enzymes in plants of A. philoxeroides in order to obtain information about the biochemical mechanisms that allow it to grow and develop in salty environments.

 

MATERIALS AND METHODS

The study was conducted in the Laboratory of Tissue Culture Plants at the Universidade Federal de Pelotas, Brazil, in 2011. A. philoxeroides plants cultivated in vitro were taken to a greenhouse and acclimatized in expanded polystyrene trays containing washed sand as substrate. The plants were kept 10 d in a moist chamber with irrigation (microsprinkler); then plants were removed from the humid chamber and transferred to plastic pots (2 L capacity) containing the same substrate and maintained in a greenhouse. The plants received 100 mL of Hoagland complete nutritive solution (Hoagland and Arnon, 1938) every two days during 10 d; afterwards the application of 100 mL of NaCl per pot was initiated, at concentrations of 0, 200 and 400 mM, at intervals of four consecutive days, interspersed with water, during 30 d and then leaves, stems and roots were collected for biochemical analyses.

The photosynthetic pigments were extracted by maceration of leaves (approximately 200 mg) in acetone 80 %. The chlorophylls (Chla, Chlb and total - Chlt) and carotenoids were evaluated using the method described by Lichtenthaler (1987) with some modifications. From the values obtained for the chlorophylls, the ratio of chlorophyll a/b was calculated. To quantify betacyanin, approximately 200 mg of leaves and stems were macerated in 5 mL of distilled water followed by centrifugation at 13 632 g (4 °C) for 25 min, following the methods described by Cai et al. (1998). Betacyanin values were expressed as a function of the amarantine content.

Lipid peroxidation was determined by quantification of thiobarbituric acid reactive species (TBARS) as described by Buege and Aust (1978). Two hundred milligrams of foliar and root tissue were macerated in liquid N₂ adding 20 % polyvinylpolypyrrolidone (PVPP) and homogenized in trichloroacetic acid (TCA) 0.1 % (w/v). The homogenate was centrifuged at 10 000 g for 10 min and an aliquot of 250 μL of the supernatant was added to 1 mL of the reaction medium composed of thiobarbituric acid (TBA) 0.5 % (w/v) and trichloroacetic acid (TCA) 10 % (w/v). The mixture was then incubated in a water bath at 95 °C for 30 min. The reaction was stopped using rapid cooling on ice and the readings carried out in a spectrophotometer at 535 nm and 600 nm. The concentration of the complex Malonaldehyde (MDA)/thiobarbituric acid reactive species (TBARS) was calculated using the extinction coefficient of 1.55 mM^-1 cm^-1.

The activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) was determined in leaves and roots. The leaves used for this analysis were formed after the onset of the induction treatment. Thus, approximately 400 mg of fresh tissue (leaves and roots) were soaked and macerated in liquid N₂ added of 20 % PVPP and homogenized in 1.5 mL of extraction buffer containing 100 mM potassium phosphate (pH 7.0), 0.1 mM EDTA and 10 mM ascorbic acid. The homogenate was centrifuged at 13 000 g for 10 min at 4 °C and the supernatant collected for enzyme activity measurement.

The SOD activity was assessed by the ability of the enzyme to inhibit the photo-reduction of the nitroblue tetrazolium (NBT) (Giannopolitis and Ries, 1977) in a reaction medium comprising 100 mM potassium phosphate (pH 7.8), 14 mM methionine, 0.1 μM EDTA, 75 μM NBT and 2 μM riboflavin. Readings were taken at 560 nm, and one unit of SOD was considered to be the amount of enzyme capable of inhibiting by 50 % NBT photo-reduction under the tested conditions. CAT activity was determined according to the protocol described by Azevedo et al. (1998) with some modifications. Its activity was monitored by the decrease in absorbance at 240 nm during 2 min in the reaction medium incubated at 28 °C, containing 100 mM potassium phosphate buffer (pH 7.0) and 12.5 mM H₂O₂. The plant extract was added prior to reading in the spectrophotometer. The APX activity was quantified following Nakano and Asada (1981), by monitoring the ascorbate oxidation rates at 290 nm during 2 min. The reaction medium incubated at 28 °C contained 100 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid and 0.1 mM H₂O₂, and also of the plant extracts added at the time of reading in the spectrophotometer.

The experimental design was completely randomized with a 3×2 factorial arrangement of treatments: three concentrations of NaCl and two plant tissues. For analyses of betacyanins the stem and leaf were used and for the enzymes analysis, roots and leaf. Leaves removed from each treatment (NaCl concentration) were used for the analyses of pigments. There were three repetitions and four plants per plot were the experimental unit. The experimental data was subjected to an analysis of variance and means where compared using Tukey test (p≤ 0.05).

 

RESULTS AND DISCUSSION

The contents of pigments showed significant differences between treatment (NaCl concentrations) and control (Table 1).

Salt stress significantly reduced the levels of chlorophyll; however, among the saline treatments (200 and 400 mM NaCl), no difference was found. This response indicates that in the presence of high salt concentration plants are unable to synthesis chlorophyll or alternatively that a greater degradation of such pigments occurs. Both chlorophyll a and b were reduced by NaCl, reflecting the highest ratio of Chl a/b in the 400 mM NaCl treatment, which differed significantly from control. Salt stress also negatively affected the carotenoids content, with a significant reduction in the levels of these pigments in plants subjected to 400 mM NaCl when compared to control.

The plant's ability to grow and adapt to different environments is related to its reproductive efficiency, which is associated to the levels of chlorophyll. The content of chlorophylls in the leaves is used to estimate the photosynthetic potential of the plants, through its direct connection with the absorption and transfer of light energy and growth and adaptation to various environments (Almeida et al., 2004). In plants, oxidative stress and signs of senescence include loss of chlorophyll, which lead to a progressive reduction in photosynthetic capacity (Thompson et al., 1987). The smaller content of chlorophylls in plants subjected to salt stress can be explained by a decrease in its synthesis or higher degradation of these pigments, because during the process of degradation of chlorophyll Chlb is converted to the Chla (Fang et al., 1998), which may explain the increase in the ratio of Chl a/b, observed in this experiment. Panda and Khan (2009) also observed a reduction in chlorophyll and carotenoids in Vigna radiata plants after 24 h of salt stress. Santos (2004) noted that Helianthus annuus (L.) plants when exposed to salt stress displayed a strong inhibition of 5-aminolevulinate (ALA) synthesis, molecule precursor of chlorophyll. Besides, the enzyme chlorophyllase is stimulated to synthesize chlorophyll in the first days of moderate stress (Santos, 2004). However, such increased activity is strongly inhibited by high salt concentration.

Salt stress also leads to an increase of free radicals in the chloroplasts and consequently to the destruction of chlorophyll molecules, resulting in reduced photosynthetic ability and growth. Singlet oxygen atoms (¹O₂) and superoxide radicals (O₂^•-) possess the ability to break the double bonds of unsaturated fatty acids and chlorophylls, thereby damaging the chloroplast membranes systems and photosynthetic reaction centers (Zhang et al., 2003); this may result in the release of chlorophyll from the thylakoids. In this situation, the chlorophylls must be degraded as soon as possible to prevent cellular damage due to its photodynamic action (Takamiya et al., 2000). If the chlorophyll degradation does not occur efficiently, it can be followed by an increase in the ROS production to an extent that the antioxidant system is no longer capable detoxify it, thus requiring the activation of other antioxidant systems (Dolatabadian et al., 2008).

Carotenoids are pigments related to cell protection against photo-oxidative damage, especially the xanthophylls cycle (violaxanthin, antheraxanthin and zeaxanthin) (Garcia-Plazaola and Becerril, 1999; Mittler, 2002). In the present study the decrease in total carotenoids content agrees with the work performed by Pinheiro et al. (2006) in Ricinus communis (L.) plants, which also showed a reduction in the content of carotenoids by NaCl stress. Considering that the increase in the salt concentration was accompanied by decreases in the synthesis of chlorophylls and in the levels of total carotenoids, we suggest that A. philoxeroides plants possess a low capacity to absorb and transfer of light energy, as well as a thermal dissipation (in the form of heat) via the xanthophylls cycle. This loss of capacity for thermal dissipation associated with an accumulation of ATP and NADPH in the stroma, may result in the direct reaction of electrons arising from the photochemical phase of photosynthesis with molecular oxygen (O₂), resulting in a reversal of one of its spins, making it highly reactive (¹O₂) (Pinheiro et al., 2006).

In relation to betacyanins, there was a significant interaction between tissue and NaCl concentration, and the highest levels were observed in the stem (in relation to leaves) in all NaCl concentrations tested (Figure 1A). In leaves, no significant difference between treatments was found. However, in the stem, the increase in salt concentration induced a higher synthesis of betacyanins (increase of approximately 80 % in the treatment with 400 mM NaCl compared to control). These results indicate that the stem is the main tissue for the synthesis of the pigment, since in the leaves its contents were low and did not differ among the treatments. The increased synthesis of betacyanins observed in this study is consistent with the results obtained by Wang et al. (2008a) with Suaeda salsa plants, which belongs to the Chenopodiaceae family. These results suggest that the betacyanins may function as osmolytes in the defense of physiological processes against abiotic stress, by modulating the pool of amino acids.

As already mentioned, salt stress can lead to increased production of ROS, which can damage cell membranes due to lipid peroxidation. This behavior was evident in this study, with a significant interaction between tissue and NaCl concentration (Figure 1B). Thus, lipid peroxidation, represented by the increased production of malondialdehyde (MDA), to the leaves, was significantly higher in plants treated with 400 mM NaCl as compared to control (51.17 % increase). But in the roots there were no significant differences, indicating the greatest effect of stress on the shoots of A. philoxeroides. According to Savoure et al. (1999), the salt stress induces ions loss, and causes injuries that compromise the integrity of the membrane, which will be affected by ROS formed during photosynthesis or respiration. As lipid peroxidation is directly linked to oxidative stress, it is an indicative of increased stress (Hernandez et al., 2000).

Salt stress also increases the H₂O₂ content, and therefore lipid peroxidation, by interrupting membrane permeability or inducing oxidative stress in plant tissues (Dolatabadian et al., 2008; Panda and Khan, 2009; Deuner et al., 2011a). Increased levels of H₂O₂ is related to the activity of SOD, the first enzyme acting in the antioxidant defense system through dismutation of the O₂^•- radical to H₂O₂ (Sinha and Saxena, 2006). This compound must be eliminated in the sequence of enzymatic reactions by CAT and APX. Thus, as noted in the contents of betacyanin (Figure 1A) and lipid peroxidation (Figure 1B), A. philoxeroides also presents significant interaction between tissues and the salt concentrations tested for the SOD activity (Figure 2A). The highest level of stress (400 mM NaCl) increased activity of this enzyme in leaves and roots, compared to control (89.41 to 18.31 %). There was a significant difference between tissues, comparing control and 200 mM NaCl with higher values in the roots. This might justify its lower increase compared to leaves, since with 400 mM NaCl no differences were detected between tissues. For CAT there were no differences between treatments. Besides, in roots, the saline stress induced a higher activity of this enzyme, with a significant increase 75 % for 400 mM NaCl compared to the control (Figure 2B).

APX displayed similar behavior considering the saline treatments, i.e. did not differ in the leaves and being significantly higher in the roots of plants in 400 mM NaCl (increase of 43.90 %) (Figure 2C). Regarding the differences between tissues (leaf and root), the highest activity was observed in roots in all treatments. Abiotic stresses, such as high salt concentrations, damage plant cells, directly or indirectly through the formation of ROS. To protect their membranes and organelles from the damaging effects of these radicals, plants must activate their antioxidant defense system. Besides, Wang et al. (2008b) described increases in the activity of antioxidant enzyme as a function of salt stress in Iris lactea Pall. var. chinensis, Dolatabadian et al. (2008) in canola plants, which were also reported by Azooz et al. (2009) in different maize cultivars, Carneiro et al. (2011) in sunflower seedlings and Deuner et al. (2011a) in Vigna unguiculata (L.) genotypes. A higher activity of SOD, CAT and APX was observed by Silva et al. (2011) in seeds and seedlings of two rice cultivars when irradiated with gamma rays and Co⁶⁰ and Adamski et al. (2012) in plants of Ipomoea batatas (L.) subjected to high concentrations of iron in the culture medium.

Thus, joint analysis of the evaluated variables indicates that the reduction in levels of chlorophyll and carotenoids (Table 1) as a function of salinity stress, may have resulted in lower protection of the leaves, enhancing the formation of ROS, since plants ability to dissipate energy as heat or fluorescence may have been reduced. Moreover, electrons may have been incorporated into O₂, leading to formation of the O₂^•- radical. Thus, the increase in SOD activity was faster in leaves (Figure 2A) for treatment with 400 mM NaCl, generating H₂O₂, which is also a potentially damaging ROS. Its further removal was not evidenced by the activity of CAT (Figure 2B) and APX (Figure 2C). These enzymes belong to two different classes of detoxification enzymes due to their different affinities for the H₂O₂ (APX in the magnitude of μM and CAT in the magnitude of mM). Therefore, while APX would be responsible for the fine modulation of ROS signaling, the CAT would be responsible for removal of ROS excess generated during stress (Mittler, 2002). In the present study an increase in the activity of these enzymes in the leaves was not detected, which may justify the increased lipid peroxidation (Figure 1B) in plants subjected to 400 mM NaCl. Besides, in the roots, the increase in SOD activity as a function of salinity was accompanied by increases in CAT and APX activity, effectively eliminating the ROS and not leading to lipid peroxidation in this tissue.

 

CONCLUSIONS

Alternanthera philoxeroides (Mart.) Griseb. plants present reduction in levels of photosynthetic pigments when exposed to salt stress. However, the increased synthesis of betacyanins in the stem and increased activity of antioxidant enzymes in leaves and roots is thought to be the mechanism of tolerance that allows it to grow and develop in environments with high salt concentrations.

 

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