Spermidine Modify Antioxidant Activity in Cucumber Exposed to Salinity Stress

Abstract

The effects of short-term 48 h long NaCl-stress and spermidine level modification on polyamines level and antioxidant status in cucumber (Cucumis sativus cv. Dar) leaves were investigated. Seedlings kept in nutrient solutions treated with 50 mM NaCl for 48 h exhibited reduced relative water content and accumulation of free polyamines, especially spermidine. Salinity stress caused an increase in superoxide radicals and hydrogen peroxide generation during the salinity-induced increase in antioxidant enzyme activities. Spermidine application before stress resulted in a marked increase in spermidine and spermine contents in the leaves of salt-stressed cucumber seedlings. Additionally, increased spermidine/spermine level mobilised the antioxidant enzyme’s activity and limited reactive oxygen species content. Polyamine synthesis inhibitor (MGBG) slightly decreased spermidine and spermine levels during salinity and reversed the antioxidant activity mobilisation. These results showed that Spd modifications significantly improved PAs, enhancing salinity stress tolerance by detoxifying ROS. Our findings determined the implication of PAs for improving the salinity tolerance of important vegetable species.

1. Introduction

Similarly to drought, temperature, and other kinds of abiotic stress, salinity enhances the synthesis of polyamines (PAs), low-weight molecules with aliphatic structures, two amino groups, and one or more internal amino groups, found in prokaryotic and eukaryotic organisms [1,2]. Diamine putrescine (Put), triamine spermidine (Spd), and tetramine spermine (Spm) are the most common PAs in plants.

Drought and salt stress are two major abiotic stress factors in agriculture, decreasing water potential in plants. Recent studies suggested that PAs protect plants from abiotic stresses, such as osmotic stress, salinity, drought, heat, chilling, and oxidative stress [3,4,5,6,7]. Studies on the relationship between PAs and water stress focused on drought resistance [8]. Polyamines can regulate the size of the K-channel and the size of pores in the plasma membrane of guard cells, regulating pore opening and closing and controlling water loss in leaves [9]. The exogenous application of PAs can induce the biosynthesis of osmotic substances, such as free amino acids, soluble sugars, and proline, which alleviates the impact of drought stress on plants [10]. Soil salinity is one of modern agriculture’s most important global problems that negatively affect crop productivity [9,10,11]. Legocka and Kluk [12] found that both osmotic and salt stresses trigger organ-specific changes in PA levels. A high concentration of salts causes osmotic, ionic, and associated secondary stresses in plants [11,13,14,15,16]. There are complex plant responses to these stresses, which Zhu [2,13,14,15,16] grouped into three general categories: homeostasis, detoxification, and growth control. Researchers suggest that PAs control the growth of cells exposed to salt stress. Zapata et al. [14,15,16] studied the effect of salinity on PA levels in different species of plants treated with 100–150 mM NaCl. They found that the Put level decreased in saline-stressed plants, whereas the Spd and Spm levels increased. Free spermidine was the most abundant PA in Arabidopsis. Similarly to the free spermine level, its level increased with the salt concentration, thus supporting the hypothesis that these PAs have a specific role in the response and tolerance to salt stress of Arabidopsis thaliana [2]. Zhao et al. [17] examined the influence of a wide range of NaCl concentrations (0–300 mM) on PA levels in barley seedlings. They found that the PA content increased dose-dependent to the salt concentration. During the salt stress, there were high spermidine and spermine levels in salt-tolerant rice cultivars but low putrescine levels in salt-sensitive cultivars [18]. The authors suggested that Spd could be significantly active in plant stress signal transduction and thus activate the stress tolerance mechanism [7,19,20,21].

In salt-sensitive wheat cultivars, putrescine accumulation is more pronounced while spermidine content decreases [22]. The sudden increase in Put, Spd, and Spm concentrations was higher, earlier, and more pronounced in the more sensitive species (pepper and lettuce) than in the less sensitive ones (spinach and beetroot) [23].

Although PA accumulation is considered a general response to abiotic stress factors, the close relationship between PA accumulation and protection remains unclear [24]. Researchers observed different PA levels depending on the species, tissue, time, and conditions of experiments [20,23,24]. The dynamic regulation of PA levels is important to induce responses to stress through a complex interplay among signalling molecules such as H₂O₂, NO, Ca²⁺, and ABA, which have been revealed to be essential components of PA signalling. NO, a well-known signalling molecule implicated in many biological processes and stress responses, is a component of PA signalling [25]. To understand the role of PAs in stress, it is necessary to modulate their level by exogenous application, overexpression of PA biosynthetic genes, or PA synthesis inhibitors [20,26].

The experiment was carried out on cucumber, an important and widespread vegetable species. Because of its origin, it has high heat and water requirements, typical of glycophytes, and cucumber is sensitive to soil salinisation and water deficits. Apart from that, it is a valuable model plant in genetic and biotechnological studies. Cucumis sativus cv. Dar was chosen for our experiment because, as an open-pollinated plant, it easily propagates generatively and vegetatively [27,28].

This study aimed to investigate how 48 h exposure to NaCl stress and modification of the PAs level influenced the ROS level and antioxidative activity in cucumber seedlings. The PA level was modified through exogenous spermidine treatment or MGBG (methylglyoxal bis(guanylhydrazone), which is a potent inhibitor of S-adenosylmethionine decarboxylase (SAMDC).

2. Materials and Methods

Plant Material and Treatment

Cucumber seeds (Cucumis sativus cv. Dar) were sown in Petri dishes to germinate for two days. Then, they developed for ten days in a germination chamber. The seedlings were transferred into one-litre containers, five plants in each, and grown hydroponically in aerated Hoagland solutions under the following conditions: fluorescent light intensity—120 μmol m⁻² s⁻¹; photon flux density—400–700 nm supplied by Osram LUMILUX L18/840 lamps; photoperiod—14/10 h; temperature—25/20 °C day/night; and relative humidity—60–70%. The PAR intensity was measured with a phytophotometer FF-01 (Sonopan, Białystok, Poland). The seedlings were divided into three groups at the third fully expanded leaf stage. Their roots were immersed in 1 mM K-phosphate buffer (pH 5.8)—the first group; in buffered Spd solutions concentrated at 0.1 mM and 1.0 mM—the second group; or in 0.1 mM buffered solution of MGBG—the third group. They were kept under these conditions for 24 h. The solutions were continuously aerated. After the treatment, the plants from the three groups were exposed to 24 and 48 h stress in 50 mM NaCl dissolved in Hoagland solutions. The salt concentration was chosen on the base of initial experiments. The control plants of all three treatment groups, so called respective control, were kept in aerated Hoagland solutions. The third and fourth fully expanded leaves were used as plant material for analyses. The scheme of obtaining plant material for estimations is shown below (Scheme 1).

The relative water content (RWC), indicating the leaf water content during dehydration, was estimated with the Weatherley method [29] and calculated according to the following formula: RWC = [(fresh weight − dry weight)/(weight at full turgor − dry weight)] × 100%.

The content of free polyamines was measured with the method described by Flores and Galston [30] with some modifications. Plant material was homogenised in 5% HClO₄ (0.25 g/mL w/v) and was allowed to stand in an ice bath for 1 h. Samples were then centrifuged at 48,000× g for 1 h, and PAs contained in the supernatant were subjected to benzoylation under alkaline conditions. The benzoyl PA derivates were extracted by diethyl ether, and the last was dissolved in methanol. The content of benzoyl polyamine derivatives was measured using high performance liquid chromatography (Varian ProStar HPLC System, Varian Inc., Walnut Creek, CA, USA), using a Discovery C18 (25 cm × 2.1 mm × 5 µm) column (Supelco Inc., Bellefonte, PA, USA) at 254 nm in a 64% (v/v) methanol/water solution. The applied standards were Put, Spd, and Spm in the form of hydrochlorides (Sigma, St. Louis, MO, USA). The results were expressed as nmol of particular polyamine per 1 g dry weight (DW).

The antioxidative enzyme activity was measured spectrophotometrically (Jasco V-530 UV–VIS). Guaiacol peroxidase (GPX, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and superoxide dismutase (SOD, EC 1.15.1.1) activities were calculated based on the protein level determined using the method described by Bradford [31].

The GPX activity measurement was based on the method described by Hammerschmidt et al. [32]. The reaction mixture contained 0.1 M K-phosphate buffer (pH 7.4), extract, 3.4 mM guaiacol, and 0.9 mM H₂O₂. Guaiacol dehydrogenation was measured at a wavelength of 480 nm. The absorbance increase was expressed as units per minute per mg of protein. One unit of the enzyme activity caused an increase in absorbance of 0.1 per min.

The CAT activity was assayed by the method described by Aebi [33]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0) and 10.5 mM H₂O₂. The activity was measured by following the decomposition of H₂O₂ at 240 nm and expressed as μg H₂O₂ per mg protein per min.

The APX activity was measured with the method described by Asada [34] and Smirnoff and Colombe [35]. The reaction mixture contained 50 mM K-phosphate buffer (pH 7.0), 5 mM sodium ascorbate, enzyme extract, and 1 mM H₂O₂. The activity was determined by following the H₂O₂-dependent decomposition of ascorbate at 285 nm and expressed in nkat per mg protein per min. Enzyme activity was calculated using the molar extinction coefficient of 2.9 mM⁻¹·cm⁻¹.

The SOD activity was estimated according to the method described by Beauchamp and Fridovich [36], which measures inhibition in the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. The 3 mL reaction mixture contained 0.05 M Na-phosphate buffer (pH 7.8), 0.1 mM EDTA, 97 mM L-methionine, 2 mM NBT, enzyme extract, and 120 mM riboflavin. The reaction was carried out for 10 min under a fluorescent lamp. One unit of activity was estimated as the quantity of enzyme reducing absorbance to 50% of that of tubes lacking the enzyme. Total enzyme activity was expressed in units per µg protein.

Hydrogen peroxide concentration. The concentration of H₂O₂ was measured with a titanium reagent, according to the method described by Becana et al. [37]. This reagent was made daily by mixing 1:1 (v/v) 0.6 mM 4-(2-pyridylazo)resorcinol (disodium salt) (Sigma) and 0.6 mM potassium titanium oxalate (BDH Chemicals Ltd., Dubai, United Arab Emirates) and was maintained in ice until use. The decrease in A₅₀₈ against distilled H₂O was followed and taken at the minimal value. Blanks were obtained by making 25 or 100 μL 5% TCA to 2 mL with buffer. The contents of H₂O₂ were determined from differences of A₅₀₈ between samples and blanks, using H₂O₂ (30%, Merck, Rahway, NJ, USA) (5–50 mM) as a standard, and expressed as nmol H₂O₂ per g dry weight.

Superoxide anion. The detection of O₂^•− was based on its ability to reduce NBT [38]. Samples were immersed in 3 mL of 0.01 M potassium phosphate buffer (pH 7.8) containing 0.05% NBT and 10 mM sodium azide and then incubated at room temperature for 1 h. Then, 2 mL of extract was incubated at 80 °C for 15 min and cooled. Optical density was measured at 580 nm, and O₂^•− content was expressed as an increase in absorbance A₅₈₀ per g of dry weight.

Statistical analyses were performed, and the data were presented as a mean ± standard deviation. The experimental data were subjected to an analysis of variance and one-way ANOVA, and significant differences between the means were determined by Tukey’s multiple range test on the base of repeated measurements (n = 5) using STATISTICA 13.3 package (Stat-Soft Inc., Tulsa, OK, USA). The data that differed significantly from the respective control sample were marked with asterisks * p < 0.05, ** p < 0.01. Correlation analysis was carried out to verify the hypothesis about the effects of stress intensity—RWC interaction for polyamine—spermidine accumulation, as well as the hypothesis about spermidine level influence on antioxidant enzyme activities and ROS accumulation. The correlation coefficient based on five replications between spermidine accumulation and estimated parameters is presented in

Table 1. The correlation coefficient between spermidine accumulation and antioxidant enzyme activities and ROS accumulation.

Traits	Significance on the Level α = 0.05	Correlation Coefficient (r)
guaiacol peroxidase activity	significant	0.69
catalase activity	significant	0.27
ascorbate peroxidase activity	ns	0.11
superoxide dismutase activity	significant	0.24
relative water content	significant	−0.46
hydrogen peroxide content	significant	−0.17
superoxide anion	significant	−0.14

.

3. Results

RWC. The salt stress caused a water deficit and decreased the RWC in the leaves of the cucumber seedlings (Figure 1). At the end of the continuous 48 h stress period, the leaf water content dropped to 71% in the seedlings treated with 50 mM NaCl, to 64% and 54% in the seedlings treated with 0.1 and 1.0 mM Spd, respectively, and then subjected to the 50 mM NaCl stress and to 55% in the plants treated with 0.1 mM MGBG before the NaCl stress. During the experiment, the water content in the well-watered control seedlings in all groups exceeded 90%.

POLYAMINES. The salt stress-induced PA accumulation (Figure 2a) in the cucumber leaves. The accumulation of spermidine and spermine after 24 h of the stress was observed. After 48 h, the PA levels were lower, similarly to the control plants. Putrescine content (Figure 2b) did not increase after 24 h. After 48 h, its level was lower than in the control plants. Spermidine (Figure 2c) was the most abundant PAs under analysis—the accumulation only took place after 24 h of the stress. The highest spermine concentration (Figure 2d) was also observed after 24 h. The exogenous application of spermidine (0.1 mM) increased spermidine and spermine levels, especially after 24 h stress duration. The putrescine level did not change. The PA synthesis inhibitor—MGBG slightly limited the stress-induced polyamine accumulation. During the entire experiment (48 h), there were constant PA levels in the control plants, so we did not include these data in the diagram. Exogenous spermidine slightly increased the spermidine and spermine levels, but MGBG decreased them.

ENZYME ANTIOXIDATIVE ACTIVITY. The NaCl stress generally increased the enzyme antioxidative activity (Figure 3) in the cucumber leaves after 24 and 48 h. The guaiacol peroxidase activity (Figure 3a) increased in the plants treated with 50 mM NaCl. Spermidine caused an additional increase in the plants treated with 0.1 mM after 24 h and 1.0 mM after 48 h exposure to the stress. The correlation coefficient was positive and significant (Table 1). After 24 and 48 h, the polyamine synthesis inhibitor reduced the salt-stress-inducing activity. The treatment of the control plants with Spd or MGBG slightly increased the guaiacol peroxidase activity at the start after 24 and 48 h. The salt stress also increased the catalase activity (Figure 3b) in the cucumber leaves. Before exposure to the stress, the spermidine treatment resulted in a higher increase after 24 h and a lower one after 48 h. The catalase activity remained at the respective time-control level in the plants with reduced polyamine content after 24 and 48 h. The Spd treatment slightly increased the catalase activity in the plants not exposed to the stress. In contrast, MGBG treatment caused the activity to remain at a similar level at the start of the exposure to the stress after 24 and 48 h. Generally, the correlation coefficient between spermidine level and catalase activity was low but statistically significant (Table 1). NaCl stress slightly increased the ascorbate peroxidase activity (Figure 3c) in the cucumber leaves. There were no differences between the samples after 24 and 48 h. Neither the Spd nor the MGBG treatment influenced ascorbate peroxidase activity in the stressed seedlings, and the positive correlation coefficient was statistically insignificant (Table 1). By contrast, both treatments increased the enzyme activity in the control plants at the start, after 24 h, and 48 h of exposure to the stress. Similarly, the salt stress slightly changed the superoxide dismutase activity (Figure 3d). The spermidine treatment additionally increased the activity of this enzyme, especially in the 0.1 mM variant after 48 h of the stress. The spermidine treatment slightly increased the superoxide dismutase activity in the control plants after 24 and 48 h—the correlation coefficient was low but statistically significant (Table 1).

ROS. In general, salt stress increased the formation of reactive oxygen species (Figure 4) in the cucumber leaves after 24 and 48 h. The hydrogen peroxide content (Figure 4a) was over two times higher than in the control plants after 24 and 48 h exposure to the stress. Exogenous spermidine limited the formation of H₂O₂ to the same level as in the respective non-stressed plants; the correlation coefficient was negative and statistically significant (Table 1). The polyamine synthesis inhibitor also limited the stress-induced formation of H₂O₂, but its level was higher than in the Spd-treated plants. The spermidine treatment slightly increased the hydrogen peroxide level in the control plants at the start, after 24 and 48 h. The MGBG treatment generally did not change the formation of this ROS. The NaCl stress increased the formation of the superoxide anion by 30% and 40%, compared with the respective control plants after 24 and 48 h. The spermidine treatment limited the stress-induced superoxide formation; the negative correlation coefficient was low but statistically significant (Table 1).

The MGBG treatment significantly increased the superoxide anion formation—it almost doubled.

4. Discussion

Salinity and drought are major abiotic stresses in agriculture. Salinity is one of the main stress factors, which greatly inhibits the growth and decreases the yield of crops. More than 6% of the world’s arable land is affected by salt [39]. Soil salinity has become one of the most important environmental issues of the 21st century. Salinity impairs plants’ growth and development, causing water stress and an excessive uptake of ions such as sodium, chloride, and nutritional imbalance [11,40]. Salt stress has been widely reported to induce PA accumulation [23,26,41]. In our study, the salt stress resulted in the accumulation of PAs (especially spermidine and spermine but not putrescine) in the cucumber leaves. Studies on various plants showed that exogenous PAs, especially spermidine, alleviated the effect of NaCl stress [42,43,44,45]. Our study showed that the content of O₂^•− and H₂O₂ increased under the short-term salinity stress, but exogenous Spd alleviated the deleterious effect of NaCl. This result is similar to the findings of studies on rice [46], bermuda grass [47], calendula [48], and bluegrass [44]. Salinity is usually accompanied by oxidative stress due to the intensive generation of ROS [9]. Wu et al. [41] observed that polyamine oxidase-mediated PA oxidation was the main source of H₂O₂ in salt-stressed cucumber roots; 1,8-diaminooctane—a specific polyamine oxidase inhibitor—was used to examine the relationship. Li et al. [49] reported that H₂O₂ signalling played an important role in PA-regulated tolerance to water stress in white clover. Spd oxidised by polyamine oxidase in the apoplast generates H₂O₂, which induces tolerance responses in tobacco [50]. Various detailed studies on the regulation of plant stress tolerance by PAs suggested PAs should be regarded as compounds involved in a complex signalling system rather than simple protective molecules [42,51,52,53].

An experiment on cucumber roots showed that Spd added to salinised nutrient solutions alleviated the effects of salinity [43]. Verma and Mishra [53] suggested that the ROS scavenging system is an essential element of the stress protection mechanism. A more effective enzyme scavenging activity correlates closely with salinity tolerance [54]. PAs may protect plasma membrane against damages caused by stress conditions—prevented from activation of NADPH oxidase, protease, and RNAse [47]. Research showed that the salt-tolerant potato could be better designed by breeding a high-yield and tolerant cultivar, which would have a better protective mechanism, increasing the activity of antioxidative enzymes detoxifying ROS [55]. In our study, the NaCl stress increased the antioxidative enzyme activity in cucumber leaves. The activity of guaiacol peroxidase and catalase increased significantly, but stress had little effect on the ascorbate peroxidase and superoxide dismutase activities. The spermidine treatment additionally increased antioxidative enzyme activities. The correlation coefficient was positive and statistically significant for GPX, CAT, and SOD. Wu et al. [41] suggested that a stronger response to salt stress can be elicited by endogenous and especially by exogenous Spd. There were marked changes in the activity of antioxidant enzymes—SOD, POX, and CAT—in cucumber roots treated with NaCl, especially when NaCl was combined with Spd. The activity of POX and SOD increased 8 h after the NaCl treatment, whereas CAT activity increased 4 h later. An increase became significant for gene expression levels after 2 h. A further increase was noted in salt-stressed plants treated with Spd—an almost two times more significant increase after 24 h than the levels in the plants exposed to the stress only.

The polyamine synthesis inhibitor—MGBG—slightly limited the stress-induced polyamine accumulation. Under lower PAs, especially the spermidine level, the MGBG treatment significantly increased the formation of superoxide anion—an almost double increase was noted. In contrast, the stress-induced H₂O₂ formation was slightly limited. The polyamine synthesis inhibitor generally reduced the salt-stress-induced antioxidative capacity of guaiacol peroxidase, catalase, and superoxide dismutase, but there were minor changes in ascorbate peroxidase activity. The comparison of the effects of the short-term salt stress, i.e., the formation of superoxide with the antioxidative enzyme activity under changed Spd level, suggests that Spd suppressed the production of free radicals, mitigating oxidative stress. The negative and statistically significant correlation confirmed the hypothesis that polyamine spermidine was at least partially able to alleviate the deleterious effect of NaCl stress in cucumber leaves. This result is consistent with the findings of studies on Bermuda grass [47], calendula [48], rice [46], tomato [56], pepper seedlings [57], and cucumber roots [40,43]. The effect of exogenous Spd was more pronounced in the salinity-sensitive cucumber variety than in the salinity-tolerant cucumber roots [43]. The authors suggested that the salt-tolerant cucumber variety had a higher Spd level and less stress-induced damage. However, the recovery by exogenous Spd was more effective in the stress-sensitive variety, which had a lower Spd level [43]. Therefore, based on the antioxidative system in cucumber seedlings, our study investigated the relationship between ROS formation and the alleviative effects of Spd on injuries caused by NaCl stress. The MGBG treatment generally did not change the formation of this ROS. The experiment’s results on cucumber leaves with modified Spd levels support the statement that Spd could be involved in the tolerance of short-term salinity stress.