Effects of exogenous abscisic acid on antioxidant system of cotton seedlings under salinity conditions

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Effects of exogenous abscisic acid on antioxidant system of cotton seedlings under salinity conditions

Karomat M. Kuldoshova, Ali A. Akhunov, Nigora R. Khashimova

Sadykov Institute of Bioorganic Chemistry

of the Academy of Sciences of Uzbekistan

(Tashkent, Uzbekistan)

The effect of exogenous abscisic acid (ABA) on the indicators of antioxidant activity and salt tolerance of two cotton (Gossypium hirsutum L.) cultivars, namely salt-tolerant Gulistan and saltsensitive C-4727, was compared. The results revealed high levels of proline and higher activity of ascorbate peroxidase and superoxide dismutase for the salt-tolerant variety Gulistan as compared to the salt stress-sensitive variety C-4727. The treatment of plants with ABA promoted an increase in the indicators of antioxidant activity. A conclusion was made about the important role of ABA in salt tolerance of cotton.

Key words: Gossypium hirsutum, salinity, abscisic acid, superoxide dismutase, ascorbate peroxidase, proline

ВПЛИВ ЕКЗОГЕННОЇ АБСЦИЗОВОЇ КИСЛОТИ НА АНТИОКСИДАНТНУ СИСТЕМУ БАВОВНИКА ЗА УМОВ ЗАСОЛЕННЯ

К. М. Кулдошова, А. А. Ахунов, Н. Р. Хашимова

Інститут біоорганічної хімії ім. А.С. Садикова

Академії наук Узбекистану (Ташкент, Узбекистан)

Порівнювали вплив екзогенної абсцизової кислоти (АБК) на показники антиоксидантної активності і солестійкість двох сортів бавовника (Gossypium hirsutum L.): Гулістан (солестійкий) і С-4727 (чутливий до засолення). Виявлено вищі показники вмісту проліну і активності аскорбатпероксидази та супероксидисмутази у солестійкого сорту Гулістан порівняно з чутливим до сольового стресу сортом C-4727. Обробка рослин АБК сприяла підвищенню показників антиоксидантної активності. Зроблено висновок про важливу роль АБК у солестійкості бавовника.

Ключові слова: Gossypium hirsutum, засолення, абсцизовая кислота, супероксиддисмутаза, аскорбатпероксидази, пролін

ВЛИЯНИЕ ЭКЗОГЕННОЙ АБСЦИЗОВОЙ КИСЛОТЫ НА АНТИОКСИДАНТНУЮ СИСТЕМУ ХЛОПЧАТНИКА В УСЛОВИЯХ ЗАСОЛЕНИЯ

К. М. Кулдошова, А. А. Ахунов, Н. Р. Хашимова

Институт биоорганической химии им. А.С. Садыкова Академии наук Узбекистана (Ташкент, Узбекистан)

Сравнивали влияние экзогенной абсцизовой кислоты (АБК) на показатели антиоксидантной активности и солеустойчивость двух сортов хлопчатника (Gossypium hirsutum L.): Гулистан (солеустойчивый) и С-4727 (чувствительный к засолению). Обнаружены более высокие показатели содержания пролина и более высокая активность аскорбатпероксидазы и супероксид- дисмутазы у солеустойчивого сорта Гулистан по сравнению с чувствительным к солевому стрессу сортом C-4727. Обработка растений АБК способствовала повышению показателей антиоксидантной активности. Сделано заключение о важной роли АБК в солеустойчивости хлопчатника.

Ключевые слова: Gossypium hirsutum, засоление, абсцизовая кислота, супероксиддисмутаза, аскорбатпероксидаза, пролин

Soil salinity is a major environmental limitation to world agriculture affecting 800 million hectares of land throughout the world. This is over 6 % of the world's total land area, affected either by salinity (397 million hectares) or associated condition of sodicity (434 million hectares) (Munns, 2005). Salt stress alters various biochemical and physiological responses in plants and thus affects almost all plant processes including photosynthesis, growth, and development (Iqbal et al., 2006). Studies on salinity stress will be a stride towards the urgent need of developing crop varieties possessing higher growth rate and yield in salt - affected environments.

Involvement of plant hormones in the resistance of cotton plant to salt stress is one of significant points that require deeper insight. Phytohormones, as the main components of the plant regulatory system, play a key role not only in plant growth and morphogenetic processes, but also in adaptive reactions associated with exposure to unfavorable factors.

To improve the adverse outcome of salinity stress on a plant growth, diverse phytohormones are extensively used. In this context, abscisic acid (ABA), a plant hormone, is considered as an important agent in the mechanisms of resistance and adaptation in plants against salt stress conditions (Bakhsh, 2011). ABA was also found involved in several other physiological processes, such as sto- matal closure, embryo morphogenesis, development of seeds, synthesis of storage proteins and lipids (Rock, Quatrano, 1995), as well as germination, leaf senescence, and defense against pathogens (Richardson et al., 1987). On the other hand, ABA acts as a mediator in controlling adaptive plant responses to environmental stresses. It has been well documented that endogenous ABA accumulates in plants under abiotic stresses (Xiong et al., 2002). The mechanisms of induction of ABA accumulation and transmission of its signal in response to water deficiency have been studied in the most detail (Kolupaev, Karpets, 2019). Additionally, exogenous application of ABA enhances the tolerance of plants to various stresses including cold, heat, drought, heavy metals, anoxia, and other environmental factors (Ahmad et al., 2010). These and other findings suggest that ABA has great agronomic potential for improving the stress tolerance of important crops. Furthermore, enzymes of antioxidant system play considerable role in the resistance of cotton plant to stress factors, and the research remains urgent in biology of cotton plant. Antioxidant state of the plant is identified by the balance between prooxidant and antioxidant reactions occurring in cells. Characterization of antioxidant system functions is important to evaluate how the plant adapts to environmental changes (Mittal et al., 2012).

Our study aimed at comparing the effects of exogenous ABA on salt tolerance in two cotton cultivars, to name a salt tolerant (Gulistan) and a salt sensitive one (C-4727).

METHODS

Plant materials and stress treatment. 7-day- old seedlings of two cotton (Gossypium hirsutum L.) cultivars with varying degrees of salt tolerance, to name a salt-tolerant (Gulistan) and a saltsensitive one (C-4727), were used in this study. The seedlings were grown with tap water to be subsequently subjected to salt stress by exposing the samples to 1% and 4% NaCl solutions for 1 hour and for 24 hours. In the experiment 10^-7 M concentration of exogenous ABA was used. Thus, the seedlings were used for further analyses.

Enzyme determination. About 200 mg of seedlings of both varieties were homogenized in 5 ml of 50 mM phosphate buffer (pH 7.8) containing 1% polyvinylpyrrolidone (PVP), 1 mM ascorbic acid, and 1 mM phenylmethylsulfonyl fluoride (PMSF) as described by Moran et al. (1994). After centrifugation at 7000 g for 15 min at 4-8°C the supernatant was dialyzed against the same extraction buffer to be used as an enzyme extract.

Ascorbate peroxidase (APX) was assayed according to Nakano and Asada (Nakano, Asada, 1981). To a reaction mixture containing 1 ml 50 mM phosphate buffer (pH 7.0), 0.2 mM ascorbic acid, 0.2 mM ethylenediaminetetraacetic acid (EDTA), and the enzyme, 20 mM H2O2 was added. The absorbance was recorded at 290 nm on UV - Vis spectrophotometer (ELICO, India) (extinction coefficient of 2.8 mM^-1 cm^-1 at 30 s intervals up to 7 min). Correction was made for the low non- enzymatic oxidation of ascorbic acid by H2O2. Specific activity of the enzyme was expressed as units/mg protein.

Superoxide dismutase (SOD) activity was determined as described by Giannopolitis and Ries (1977). Assays were carried out on a rotating plate under illumination. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of p-nitroblue tetrazoli- um chloride reduction at 560 nm.

Total protein determination. To measure the activity of antioxidative enzymes, such as APX and SOD, to make a standard curve and to read the absorbance, quantitation of total protein content of samples was performed by Lowry et al. (1951) using bovine serum albumin (BSA; Sigma-Aldrich, USA)

Determination of proline. The method of Bates et al. (1973) was used for determination of proline. About 500 mg of leaf tissue was homogenized in 5 ml of 3% aqueous sulphosalicylic acid. The homogenate was centrifuged at 7000 g for 15 min. In a test tube, 1 ml of the extract was mixed with 2 ml of acid-ninhydrin containing 1.25 g of ninhydrin in 30 ml of glacial acetic acid and 20 ml of 6 M phosphoric acid. Subsequently, 2 ml of glacial acetic acid was added, and the mixture was heated for an hour at 100°C. Later, 4 ml of toluene was used to extract the reaction mixture. Further, the reaction mixture was vortexed for 20-25 sec. The chromophore-containing toluene was aspirated from the aqueous phase, and the absorbance of the toluene layer was measured at 520 nm with toluene as blank. Values were expressed as micrograms per gram fresh weight.

Statistical analysis. The results were statistically analyzed using the Graph Pad Prism 8. Values are expressed as mean ± SD for three biological replicates.

RESULTS AND DISCUSSION

To assess the activity of antioxidant systems of salt-tolerant (Gulistan) and salt-sensitive (C4727) cotton cultivars, we determined the effects of main enzymes of antioxidant protective system of the plants above, such as APX and SOD. The response of this system to salinization is known to depend on the variety and type of plant, its physiological state, as well as on the level and duration of stress (Miguel et al., 2006). In our study, we managed to identify the direct correlation between the level of antioxidant system induction and the degree of plant resistance to salinization.

APX is a key enzyme in the ascorbateglutathione cycle playing a critical role in H2O2 scavenging (Chawla et al., 2012). APX uses ascorbate as an electron donor to reduce H2O2 to water. Our findings showed that the resistant cultivars had much higher APX level in 1 hour (32.23 units) as compared to sensitive cultivar C-4727 (20.6 units) (Table). There was no appreciable change in APX activity in the tolerant variety, but the sensitive one demonstrated APX increase at both levels of salinity stress. The 4% NaCl solution enhanced APX activity in C-4727, but it was much lower than in the tolerant cultivar. These results are supported by Benavides et al. (2000), who reported on more significant elevation of the APX levels in salt-tolerant potato clones than the one in the salt-sensitive clone. Similarly, Meneguzzo et al. (1999) observed a much greater increase in APX activity in saltsensitive cultivar than in the salt-tolerant cultivar of wheat exposed to NaCl stress. With increase in salt stress, APX activity also increased in cotton (Meloni et al., 2002), wheat (Heidari, Mesri, 2008) and sea rocket (Greenway, Munns, 1980) suggesting that high level of APX and/or salt-induced increase in APX activity could impart tolerance by detoxifying H2O2 generated upon exposure of plants to saline conditions.

Effect of exogenous ABA on the activities of APX and SOD in seedlings of two cotton varieties

In our study the activity of APX in the seedlings of salt-tolerant variety of cotton plant was significantly higher in samples treated with 1% NaCl + ABA, as compared with the ones treated with NaCl (39.43 units versus 32.23 units). The control sample exhibited significantly lower APX activity (27.6 units) (Table).

Our results suggest that oxidative stress can play a key role in the salt tolerance of the seedlings of a cotton cultivar under the salt stress; our salt - tolerant cultivar, Gulistan, turned out to have more efficient antioxidant protection promoting resistance to the oxidative stress.

As to the SOD activity, it increased progre s- sively with increase in salinity in the salt -resistant cultivar, but receded in the sensitive one (Table). After 1 and 24 hours in 1% NaCl condition, the level of SOD activity was also significantly higher in the salt-tolerant cultivar than the one in the saltsensitive, 5.53 and 5.1 units in Gulistan, and 3.0 and 3.55 units in C-4727, respectively. After 24 hours in experimental samples of 4% NaCl + ABA the increase of the enzyme activity was observed in both cultivars. Possibly, the SOD activity increase allows reducing lipid peroxidation under higher salt stress. These observations are in agreement with those reported earlier in Solanum tuberosum (Benavides et al., 2000), Brassica juncea (Chawla et al., 2012), and Najas graminia (Rout, Shaw,

2001) where the salt-tolerant cultivars had higher level of the enzyme activity, as compared to that in the salt-sensitive ones. Similar to the present findings, the SOD activity increased in the salt-tolerant cultivars, but reduced in the salt-sensitive cultivar of cotton (Meloni et al., 2002), wheat (Mandhania et al., 2006) and Catharanthus roseus (Jaleel, 2009). Contrarily, salinity has been reported to stimulate the SOD activity in both salt-tolerant and salt-sensitive cultivars of Brassica juncea with higher level in the salt-tolerant ones (Chawla et al., 2012). Increase in the SOD activity upon salinization in leaves of all the salt-tolerant cultivars could accelerate dismutation of superoxide ions generated upon salt treatment, which may allow this variety to survive under oxidative stress (Meloni et al., 2002. In the salt-sensitive cultivar, reduction in the SOD activity would limit its metabolic capacity to withstand oxidative stress.

Proline performs a protective function against salinity stress in plants. It acts as a compatible osmolyte, enzyme protectant, free radical scavenger, cell redox balancer, cytosolic pH buffer, and stabilizer for subcellular structures to bring about salinity tolerance. Exogenous ABA inhibited the rate of synthesis of proline or stimulated its degradation intensity in saline conditions (Nikolaeva et al., 2015). The accumulation of proline under salinity stress was reported in Pisum sativum, Brassica juncea and Triticum aestivum.

Effect of exogenous ABA on proline content in cotton seedlings under conditions of NaCl salinity.

1 - salt-tolerant Gulistan cotton cultivar; 2 - salt-sensitive C-4727 cotton cultivar; A - control, B -NaCl (1%), C -NaCl (1%) + ABA(10^-7 M) , D - NaCl (4%), E - NaCl (4%) + ABA (10^-7 M).

In our study the exogenous effect of ABA on the concentrations of proline in the seedlings of both varieties of cotton plant was assessed in several experiments (Figure). Considerable increase of the concentrations took place under the effect of 1% NaCl within 1 hour in the resistant variety. Proline could also act as the major source of energy and nitrogen during immediate post-stress metabolism. Accumulating in plants, proline apparently supplies energy for their growth and survival, thereby inducing salt tolerance. Similar findings were reported by Hassine and Lutts (2010). ABA was found to decrease the amount of Pro not only in 1% NaCl solution but also in a solution with higher concentration of the salt (4% NaCl).

Under the effect of ABA, after 1-hour exposure the decrease of proline was observed in both cultivars. After 24 hours, 1 and 4% salinization caused rapid increase of proline levels.

CONCLUSION

Our data support that additional protection of cells in the condition of saline stress happens due to activation of genes of superoxide dismutase, which can also occur in the presence of plant hormones.

Our studies showed that there are significant differences in response of sensitive and resistant cotton varieties to salt stress. Differences in the response to salinization are closely associated with differences in the activity of antioxidant enzymes and the concentrations of proline. The resistance of cotton varieties to salinity is associated with high efficiency of the enzymatic system for neutralizing reactive oxygen intermediates, thus increasing oxidation-reduction homeostasis and preservation of cell components.

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