Divergences in morphological changes and antioxidant responses in salt-tolerant and salt-sensitive rice seedlings after salt stress

Plant Physiology and Biochemistry 70 (2013) 325e335

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Research article

Divergences in morphological changes and antioxidant responses in salt-tolerant and salt-sensitive rice seedlings after salt stress Min Hee Lee a, d,1, Eun Ju Cho a,1, Seung Gon Wi b, Hyoungwoo Bae a, Ji Eun Kim a, Jae-Young Cho c, Sungbeom Lee a, Jin-Hong Kim a, Byung Yeoup Chung a, * a

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Gunmgu-gil, Jeongeup-si, Jeollabuk-do 580-185, Republic of Korea Bio-Energy Research Institute, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Applied Life Science, Chonbuk National University, Jeonju 561-756, Republic of Korea d Rice Breeding and Cultivation Division, National Institute of Crop Science, 457 Pyeongdong-ro, Iksan, Jeollabuk-Do 570-080, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 November 2012 Accepted 28 May 2013 Available online 13 June 2013

Salinization plays a primary role in soil degradation and reduced agricultural productivity. We observed that salt stress reversed photosynthesis and reactive oxygen scavenging responses in leaves or roots of two rice cultivars, a salt-tolerant cultivar Pokkali and a salt-sensitive cultivar IR-29. Salt treatment (100 mM NaCl) on IR-29 decreased the maximum photochemical efficiency (Fv/Fm) and the photochemical quenching coefficient (qP), thereby inhibiting photosynthetic activity. By contrast, the salt treatment on Pokkali had the converse effect on Fv/Fm and qP, while increasing the nonphotochemical quenching coefficient (NPQ), thereby favoring photosynthetic activity. Notably, chloroplast or root cells in Pokkali maintained their ultrastructures largely intact under the salt stress, but, IR-29 showed severe disintegration of existing grana stacks, increase of plastoglobuli, and swelling of thylakoidal membranes in addition to collapsed vascular region in adventitious roots. Pokkali is known to have higher hydrogen peroxide (H2O2)scavenging enzyme activities in non-treated seedlings, including ascorbate peroxidase, catalase, and peroxidase activities. However, these enzymatic activities were induced to a greater extent in IR-29 by the salt stress. While the level of endogenous H2O2 was lower in Pokkali than in IR-29, it was reversed upon the salt treatment. Nevertheless, the decreased amount of H2O2 in IR-29 upon the salt stress didn’t result in a high scavenging activity of total cell extracts for H2O2, as well as O2
 and
OH species. The present study suggests that the tolerance to the moderate salinity in Pokkali derives largely from the constitutively maintained antioxidant enzymatic activities as well as the induced antioxidant enzyme system. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Antioxidative enzyme Oryza sativa L. Photosynthesis Reactive oxygen species Salt stress

1. Introduction Salinity of soil or irrigation water is one of the major abiotic stresses that severely affects crop production worldwide [1]. Salinization plays a primary role in soil degradation, affecting up to 20% of irrigated land and 2.1% of dryland agriculture globally [2]. Salinity effects are more apparent in arid and semi-arid regions,

Abbreviations: AA, ascorbic acid; APX, ascorbate peroxidase; CAT, catalase; CO2, carbon dioxide; Chl, achlorophyll a; Chl, bchlorophyll b; H2O2, hydrogen peroxide;
OH, hydroxyl radical; MS, Murashige and Skoog; NPQ, nonphotochemical quenching; HO2
, perhydroxyl radical; POD, peroxidase; PS II, photosystem II; ROS, reactive oxygen species; O2
, superoxide anion; 1O2, singlet oxygen; qP, photochemical quenching coefficient; SOD, superoxide dismutase; TEM, transmission electron microscopy; tChl, total chlorophylls; tCar, total carotenoids. * Corresponding author. Tel.: þ82 63 570 3331; fax: þ82 63 570 3339. E-mail address: [email protected] (B.Y. Chung). 1 These authors contributed equally to this work. 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.05.047

where limited rainfall, high evapotranspiration, and high temperature (associated with poor water and soil management) contribute to the salinization of soil and greatly affect agricultural production [3]. High salt concentrations in soil cause various cooperative events that negatively impact agricultural production, such as delays in plant growth and development [4], inhibition of enzymatic activities, and reductions in photosynthetic rates [5]. Therefore, investigators are aiming to understand the mechanisms by which plants respond and adapt to such stresses. Plant responses to salt stress have generally been conducted using anatomical, ecological, physiological, and molecular approaches [6e8] in relation to regulatory mechanisms of ionic and osmotic homeostasis. Although several prior reports of morphological and biochemical responses of plants have displayed different salt sensitivities, few anatomical or ultrastructural studies in salttolerant and salt-sensitive rice cultivars under conditions of salt stress have been reported.

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It has been reported that the ionic and osmotic effects of salinity cause growth impairment in plants [9,10]. Moreover, salt stress, like other abiotic stresses, results in oxidative stress through an increase in reactive oxygen species (ROS), such as the superoxide radical (O2
), hydrogen peroxide (H2O2), and the hydroxyl radical (
OH). These ROS are highly reactive, altering normal cellular metabolism through oxidative damage to lipids, proteins, and nucleic acids [11]. To mitigate the ROS-mediated oxidative damage, plants have developed a complex antioxidative defense system, including low-molecular mass antioxidants e such as ascorbate, reduced glutathione, tocopherol, carotenoids, and flavonoids e as well as antioxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) [12,13]. Therefore, the complex antioxidative defense system develops with a concomitant increase in ROS. In addition, salt adversely affects the metabolism of plants, resulting in substantial modifications in plant gene expression. These modifications may lead to the accumulation or depletion of certain metabolites, resulting in an imbalance in the levels of cellular proteins, which may increase, decrease, appear, or disappear after salt treatment [14]. A previous study showed that antioxidant responses under conditions of salt stress in salt-tolerant Pokkali rice plants included slight increases in SOD activity and decreases in POD activity, with virtually unchanged lipid peroxidation, electrolyte leakage, and Naþ accumulation against salt-sensitive rice varieties Hitomebore and IR-28 [15]. Comparative investigations of the leaves and roots of the saltsensitive IR-29 cultivar and the salt-tolerant Pokkali cultivar could result in comprehensive morphological, biochemical, and photochemical insights into response mechanisms induced by salt stress in rice plants. The results of this particular study may provide valuable information regarding how rice plants are damaged by salt stress and how they cope with the stress. Therefore, we investigated the effects of varying salt stress on the activities of various antioxidant (iso)enzymes analysis, antioxidants, scavenging capacity of ROS, and the morphological changes in leaves and roots of two rice cultivars, IR-29 and Pokkali.

Table 1 The effects of salt stress on the content of pigments and chlorophyll-fluorescence parameters in two rice cultivars. Rice seedlings were cultivated in half MS liquid medium for 3 weeks and were exposed salt stress by adding 100 mM NaCl to the hydroponic solution for 7 days. Fv/Fm, the maximum photochemical efficiency of PS II; qP, photochemical quenching coefficient; NPQ, nonphotochemical quenching coefficient. Each value represents the mean of three replications  S.E. n ¼ 9.

2. Results

2.2. Effects of salt stress on cellular structures of IR-29 and Pokkali

2.1. The effects of salt stress on seedling growth and photosynthesis of rice cultivars IR-29 and Pokkali

While physiological effects of salt stress on the two rice cultivars have been reported [15], the changes in cellular ultrastructures under the stress have not yet been examined. This study examined the ultrastructural changes in IR-29 and Pokkali in response to salt stress by light microscopy and transmission electron microscopy (TEM) (Figs. 2 and 3). The ultrastructural alteration of chloroplasts in Pokkali was not conspicuous (Fig. 2C and D), whereas the thylakoidal membranes in IR-29 were severely disorganized by swelling and curling (Fig. 2A and B). The chloroplasts in the control IR-29 were located at mesophyll and parenchyma cells, containing large starch grains (Fig. 2A), while those in the salt-treated IR-29 showed a noticeable increase of plastoglobuli (shown by red arrows in Fig. 2B). When cross-sections of the segment at 10 mm from the tips of adventitious roots were imaged by bright-field microscopy, it was observed that the vascular cylinder region of the salt-treated IR-29 had entirely collapsed (Fig. 3F), which included the central metaxylem vessel, compared with that of the control plant (Fig. 3E). Consistent with this finding, disintegrated root cell structures were only observed in salt-treated IR-29 via TEM analysis (shown by red arrows in Fig. 2F). After the salt treatment, cortex cellular membranes were disintegrated, resulting in the release of cell organelles. The ultrastructural components of root cells were nearly the same between the control (Fig. 2G) and salt-treated Pokkali (Fig. 2H). These results indicated that leaf and root cells of IR-29 were more sensitive to the salt stress than those of Pokkali.

Salt stress is known to cause severe inhibition on plant growth and development [16]. A high concentration of salt in soil may induce three additional major types of stress: ionic, osmotic, and oxidative stress [17]. Salt stress also results in a considerable reduction in the fresh and dry weights of leaves, stems, and roots [18e20]. During 7 days of salt treatment (100 mM NaCl), saltsensitive IR-29 showed early symptoms of wilting within 48 h after the treatment and the severity of the symptoms was directly related to the consecutive exposure to salinity, eventually leading to death. However, the newly developed leaves of the plant remained pale green until the end of the treatment. In contrast, salt-tolerant Pokkali did not develop wilting or any visible symptoms in response to the salt treatment up to 1 week after the treatment. The effects of salt stress on photosynthetic pigment contents and photosynthetic activities in the two cultivars were measured at 7 days after the salt treatment. IR-29 showed that decreased chlorophyll content upon the salt stress, but increased in carotenoid content. In contrast, total chlorophyll and total carotenoid content in Pokkali remained unchanged by the salt stress (Table 1). The maximum photochemical efficiency (variable fluorescence/ maximal fluorescence, Fv/Fm) of photosystem II (PS II) has been

Cultivars

IR-29

NaCl (mM) Total chlorophyll Total carotenoids Chlorophyll a/b Fv/Fm qP NPQ Protein (leaves) Protein (roots)

0

Pokkali

3.11  0.03

100 2.46  0.19

0.87  0.08 3.19  0.11 0.794 0.598 0.609 7.930

   

0.004 0.019 0.097 0.423

0.439  0.027

4.19  0.05

100 4.29  0.10

1.19  0.06

1.37  0.04

1.29  0.04

3.34  0.12

3.13  0.08

3.39  0.05

0.736 0.428 0.539 2.808

   

0.029 0.032 0.045 0.085

0.190  0.014

0

0.740 0.378 0.379 5.272

   

0.018 0.004 0.067 0.386

0.580  0.022

0.837 0.465 1.020 4.957

   

0.004 0.019 0.066 0.383

0.561  0.032

demonstrated as a reliable chlorophyll fluorescence parameter by measuring photosynthetic rates under stress conditions [21]. When the two rice cultivars were exposed to salt stress conditions for 7 days, the Fv/Fm of treated IR-29 was slightly lower than that of the control (Table 1). By contrast, Pokkali demonstrated an increase in Fv/Fm under conditions of salt stress. In addition, other parameters, including the photochemical quenching coefficient (qP) and nonphotochemical quenching coefficient (NPQ), were also reduced in IR-29 but increased in Pokkali by the salt stress (Table 1). For protein contents under the salt stress condition, IR-29 showed a significant decrease in protein contents both leaves and roots, while Pokkali showed a slight decrease in leaves but negligible change in roots. Our findings revealed that the salt stress inhibited photosynthetic capacities or growth of IR-29, but, elevated photosynthetic activities in Pokkali, as described in the earlier study [15], suggesting that Pokkali was more resistant to the salt stress than IR-29.

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salt-treated IR-29 were likely to be explained by putative APX enzyme(s) shown in Fig. 4B, particularly APX7 we named. Moreover, IR-29 in leaves exhibited markedly increased total CAT activity up to 184%, but, Pokkali in leaves rather had the reduced activity by 21% (Fig. 4C). Likewise, the salt treatment of the leaves of IR-29 also markedly increased the total CAT activity up to 184%, while only slightly reducing (21%) the activity of Pokkali (Fig. 4C). The increase in CAT activity in the salt-treated IR-29 leaves would partly be attributable to the strong induction of low-molecular weight CAT isozymes (Fig. 4D). By contrast, the CAT activities in roots of both cultivars were rarely affected by the salt treatment. Finally, the salt treatment also increased POD activities in both cultivars, albeit to a greater extent in IR-29 than in Pokkali (Fig. 4E). The salt treatment of the leaves and roots of IR-29 increased the total POD activity up to 244% and 316%, respectively, while increasing POD activity in Pokkali up to only 129% and 120%, respectively. Gel assays for detecting POD activity revealed that the abundance of lowmolecular weight POD isozymes after salt treatment increased in the leaves and roots of IR-29, along with the total POD activity (Fig. 4F). Unlike H2O2-scavenging enzymes (e.g. APX, CAT and POD), the salt treatment significantly reduced the ascorbate content in leaves of IR-29, but neither in roots of IR-29 nor in leaves and roots of Pokkali (Fig. 5A). After the salt treatment, the ratio of reduced:oxidized ascorbate was reduced in both IR-29 and Pokkali (Fig. 5B). However, the decrease in ratio was far more conspicuous in IR-29 than in Pokkali, especially in the leaves. Since ascorbate is oxidized in H2O2-scavenging reaction by APX, the decrease in the ratio would likely be explained by the increase of APX activity. 2.4. Changes in H2O2 content and ROS-scavenging capacity in IR-29 and Pokkali after the salt treatment

Fig. 1. The effect of the salt stress on the two rice cultivars, IR-29 (salt-sensitive) and Pokkali (salt-tolerant). Rice seedlings were cultivated in 1/2 MS liquid medium for 3 weeks, followed by salt treatment (100 mM NaCl) in the culture medium for 7 days. A photo was taken at 3 days after the salt treatment. Non-treated plant are used as controls. Bar ¼ 5 cm.

2.3. Effects of salt stress on H2O2-scavenging enzymatic activity and antioxidant content It was observed that salt stress differentially damaged to or structurally altered the morphology of photosynthetic apparatus and its capacity between the two cultivars (Figs. 1 and 2). The photosynthetic apparatus generates various ROS, especially H2O2, a key tissue-damaging agent [22]. To further investigate the mechanism of action of the salt stress, we compared the anti-oxidation capacities of the two cultivars based on their H2O2-scavenging abilities. Specifically, APX, CAT, and POD activity and ascorbate content in the control and salt-treated plants of IR-29 or Pokkali were considered. Salt treatment on the leaves and roots of IR-29 elicited significant increase of total APX activity up to 238% and 122%, respectively, in comparison with those of the control plants. In contrast, Pokkali showed slight decrease of total APX activity by 17% in leaves, but, 8% increase of total APX activity in roots in comparison with those of the control plants, although the amplitude was negligible (Fig. 4A). The increased APX activities of the

Hydrogen peroxide content is a good indicator for evaluating the ROS-scavenging capacity of plants under oxidative stress [23]. After salt treatment of IR-29 and Pokkali seedlings, the estimated H2O2 content was observed to be reduced in the leaves and roots of IR-29 but increased in the leaves and roots of Pokkali, displaying a more distinct pattern in the roots (Fig. 6). The low H2O2 content in IR-29 after the salt treatment could be partially accounted for by the increased H2O2-scavenging activity, as shown in Figs. 4 and 5. Therefore, the H2O2-scavenging capacity of total extracts from the leaves or roots of IR-29 and Pokkali was estimated and compared with each another. On the basis of fresh weight, leaf or root extracts of IR-29 after the salt treatment showed a significant reduction in scavenging activities for
OH and 1O2 or H2O2 and
OH, respectively (Fig. 7). By contrast, salt treatment did not substantially change the aforementioned ROS-scavenging capacities in Pokkali. These results suggested that salt treatment affects the ROSscavenging systems in IR-29 and Pokkali cultivars differentially. 3. Discussion 3.1. Contrasting responses of IR-29 and Pokkali to moderate salt stress When plants are grown under conditions of salt stress, which leads to reduced growth and productivity (as shown in Fig. 1), photosynthesis is particularly reduced by severe impairments in photosynthetic activities and the photosynthetic apparatus, the degree of which depends on the varieties of species and genus [15,24e26]. In Table 1, the content of photosynthetic pigments after the salt treatment was significantly decreased by 20% in IR-29, but marginal changes in Pokkali. Since chlorophyll content correlates directly with the growth and development of the plant [27], the decrease in chlorophyll content as well as protein content suggested

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Fig. 2. The observation of morphological changes in leaves (A, B, C, D) and roots (E, F, G, H) of two rice cultivars (IR-29 and Pokkali) by transmission electron microscope (TEM) after 7 days of treatment with 100 mM NaCl. A and E, IR-29 control; B and F, IR-29 with 100 mM NaCl treatment; C and G, Pokkali control; D and H, Pokkali with 100 mM NaCl treatment. Bar ¼ 1 mm.

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329

Fig. 3. The light micrographs showing the morphological changes in leaves (AeD) and roots (EeH) of two rice cultivars (IR-29 and Pokkali) after 7 days of treatment with 100 mM NaCl. A and E, IR-29 control; B and F, IR-29 treated with 100 mM NaCl; C and G, Pokkali control; D and H, Pokkali treated with 100 mM NaCl. Bar ¼ 100 mm.

substantial damage to the photosynthetic mechanism in IR-29, as shown previously in salt-treated rice, sorghum and maize plants [28e30]. Our results showed that the salt treatment with 100 mM NaCl significantly decreased photosynthetic activity in IR-29, as indicated by Fv/Fm and qP values (Table 1). By contrast, the higher Fv/Fm and qP values were examined in Pokkali after the salt treatment, likely resulting from the higher chlorophyll content or the chlorophyll a/b ratio (Table 1). Our findings in total chlorophyll content was comparable with the observation by Singh et al., where HBC-19, another salt-sensitive rice cultivar, showed 30% reduction of total chlorophyll content after 7 days of the treatment (12 dS m1 NaCl z 132 mM NaCl) [31]. The observations presented by Tiwari et al. strongly supported our measurements in changes of a photosynthetic parameter such as Fv/Fm in the presence of salinity in IR-29 and IR-8, although they observed 30% of decrease in Pokkali

rather than 10% of increase measured in our examinations. The difference would be derived from the materials they employed e the suspension of isolated chloroplasts instead of a leaf disc [32]. The decline of the photosynthetic rate in salt-treated plants has been associated with a series of reductions in water potential, stomatal conductance, carbon dioxide (CO2) availability, and CO2 assimilation, resulting in drastic reduction of the photosynthetic electron transport chain in chloroplasts [33,34]. Among the thylakoid components constituting the photosynthetic electron transport chain, PS II is a relatively sensitive component with regard to salt stress [35]. Accordingly, the salt stress-induced increase of NPQ in Pokkali is likely expected to provide with improved protection for high photosynthetic activity during the salt treatment (Table 1). Since stimulation of photosynthesis in Ceriops roxburghiana and Alhagi pseudalhagi (Bieb.) by low salt concentrations has been

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Fig. 4. The effects of the salt stress on the antioxidant enzyme activities on the two rice cultivars: IR-29 (salt-sensitive) and Pokkali (salt-tolerant). After 7 days of treatment with 100 mM NaCl, anti-oxidation capacities were compared based on their H2O2-scavenging abilities. (A) Ascorbate peroxidase (APX) activity. (B) In-gel assay for APX isozymes. (C) Catalase (CAT) activity. (D) In-gel assay for CAT isozymes. (E) Peroxidase (POD) activity, and (F) In-gel assay for POD isozymes. ‘þ’ and ‘’ indicated the presence and the absence of salt treatment of 100 mM NaCl, respectively. Each value represents mean  standard error (S.E.) from three independent experiments.

reported [36,37], salt treatment with 100 mM NaCl would stimulate photosynthesis in Pokkali. It has been reported that salt stress causes swelling of thylakoid membranes, an increase of plastoglobuli, a decrease of starch levels, and/or an absence of grana reported in pea, sweet potato, potato, tomato, and eucalyptus [18,38e42]. These phenomena are consistent with those observed in chloroplasts of salt-treated IR-29 (Fig. 2B). By contrast, Pokkali maintained stacked grana with no swelling of thylakoid membranes (Fig. 2C and D). Therefore, the ultrastructure of the photosynthetic apparatus in Pokkali appears

to be well-maintained under moderate salt stress conditions, suggesting that Pokkali is better equipped with more advanced defense mechanisms against salt stress than IR-29 (especially in the maintenance of chloroplast ultrastructure and photosynthetic capacity). In practice, the increased NPQ in salt-treated Pokkali may provide a good standard in investigating how salt-tolerant cultivars cope with the elevated threats of reactive oxygen species (ROS) under conditions of salt stress. The protective role of NPQ is associated with the scavenging of singlet oxygen species as well as the down-regulation of PS II activity.

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Fig. 5. Changes of ascorbate contents in the leaves and roots of IR-29 and Pokkali under the salt stress. (A) Ascorbic acid (AA) contents. (B) reduced/oxidized AA ratio. ‘þ’ and ‘’ indicated the presence and the absence of salt treatment of 100 mM NaCl, respectively. Each value represents mean  S.E. from three independent experiments.

Fig. 7. Changes of ROS-scavenging activity in the leaves and roots of two rice cultivars after the salt treatment. (A) Hydrogen peroxide-scavenging capacity. (B) Hydroxyl radical-scavenging capacity. (C) Singlet oxygen-scavenging activity. ‘þ’ and ‘’ indicated the presence and the absence of salt treatment of 100 mM NaCl, respectively. Each value represents mean  S.E. from three independent experiments.

3.2. Relationship between H2O2 scavenging activity and salt tolerance in rice seedlings

Fig. 6. Changes of hydrogen peroxide in the leaves and roots of two rice cultivars after the salt treatment. ‘þ’ and ‘’ indicated the presence and the absence of salt treatment of 100 mM NaCl, respectively. Each value represents mean  S.E. from three independent experiments.

Water deficits imposed by salt stress lead to the formation of ROS, such as the superoxide radical (O2
), hydrogen peroxide (H2O2), the hydroxyl radical (
OH) [43], and singlet oxygen (1O2) [44]. The importance of H2O2 and 1O2 scavenging has been emphasized with regard to photosystem damage under conditions of oxidative stress [22]. Although genetic differences in salt tolerance among plants are not necessarily due to differences in the

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ability to detoxify ROS [45], many comparative studies using salttolerant and salt-sensitive genotypes have correlated salt tolerance to an increase in the activity of antioxidant enzymes [10,46e 49]. Therefore, the high photosynthetic activity and the intact chloroplast ultrastructure in salt treated Pokkali (shown in Table 1 and Fig. 2) may be associated with the high scavenging activities for H2O2 and 1O2 (Fig. 4). The higher control levels of APX, CAT, and POD activities in Pokkali compared to IR-29 may have contributed to the more efficient scavenging of H2O2 during the salt treatment. In Fig. 6, the lower control level of endogenous H2O2 in Pokkali than IR-29 is also consistent with this presumption. Although the enzymatic activities of Pokkali did not increase to the extent of those of IR-29, the H2O2 content of Pokkali after the salt treatment was significantly higher than that of IR-29. This finding indicates that the amount of H2O2 formation in Pokkali is greater than that of scavenged H2O2 due to the active physiological activity of Pokkali. These results were comparable with a previous report by Senadheera et al., where accumulation of H2O2 in the roots of FL478 (salt-tolerant rice cultivar) was significantly higher than that of IR29 (salt-sensitive rice cultivar) in response to moderate salt stress (50 mM NaCl) for 12 days [50]. High enzymatic activity and severe photosystem damage are simultaneously observed during the process of aging and stress as well, because the induction of common stress- and damage-induced enzymes. Moreover, the overexpression of several antioxidant enzymes has been reported to improve salt tolerance [51,52]. High levels of antioxidant enzymatic activities have been associated with salt sensitivity as well as salt tolerance. Thus, ROSscavenging as a whole may be relatively less important for plants that generate low ROS under conditions of salt stress [53]. Along with the salt stress-induced damages occurring in IR-29 (shown in Table 1 and Fig. 2), the high increases in APX, CAT, and POD activities after the salt treatment are thought to be induced by oxidative stress-mediated signaling in association with salt sensitivity rather than salt tolerance (Fig. 4). Specifically, low-molecular weight antioxidant isozymes could contribute to increased enzymatic activities in the scavenging of H2O2 in salt stress-treated IR-29. The increased enzymatic activities may partially explain the reason for decreased levels of endogenous H2O2 in salt treated IR-29 (Fig. 6). However, the overall reduction in physiological activity such as photosynthesis is more likely to be a reason for the low H2O2 level in IR-29 after salt stress, while the reverse may be true for Pokkali. Since the reduced form of ascorbate can be oxidized either by serving as a substrate for APX to toxify H2O2 [54] or by direct reaction with O2
 [55], the substantially decreased reduced/oxidized ratio of ascorbate may be associated with the higher initial H2O2 level observed in IR-29 than in Pokkali upon salt stress (Figs. 5 and 6). By contrast, no significant decreases in total content and reduced/oxidized ratio of ascorbate may support the fact that both leaves and roots of Pokkali are hardly damaged under conditions of 100 mM NaCl treatment. Although each antioxidant enzyme performs a specific function and its activity is assigned to a specific role in ROS detoxification, efficient antioxidant activity does not necessarily translate into the strong up-regulation of the full complement of antioxidant enzymes and vice versa [53]. Similarly, Fig. 7 indicated that the high induction of APX, CAT, and POD activities in IR-29 by the salt stress could never result in high scavenging activity of total cell extracts for H2O2 as well as O2
 and
OH. This finding suggests that the entire antioxidant capacity of the extract could be partially damaged in IR-29 after the salt treatment (despite the induction of antioxidant enzymes), while being maintained in a relatively constant manner in Pokkali. Moreover, the actual efficiency of H2O2 scavenging is likely to be associated with basal or control levels to a greater extent than the induced levels of antioxidant enzymatic activities.

Together, we revealed that two rice cultivars, IR-29 (salt-sensitive) and Pokkali (salt-tolerant), undergo differing morphological and biochemical changes under conditions of salt stress. Although treatment with 100 mM NaCl caused structural damage in chloroplasts and reduction of photosynthetic activity in IR-29, the identical treatment improved photosynthetic activity in Pokkali without inducing any notable physiological damage. The chloroplast ultrastructure in the salt stress-treated Pokkali was hardly damaged, despite increased endogenous levels of H2O2. The high enzymatic activities for H2O2 scavenging were substantially induced in IR-29 by 100 mM NaCl but never resulted in enhancement of actual antioxidant capacity, implying that the increased enzymatic activities in IR29 are likely associated with salt sensitivity more than salt tolerance. The present study suggests that the tolerance to the moderate salinity in Pokkali derives largely from the basal level of antioxidant enzymatic activities constitutively maintained as well as the induced antioxidant system upon salt treatment (Fig. 4). Nevertheless, it would be of great interest to examine the physiological symptoms, antioxidant enzymatic activities and the change of ultrastructures in Pokkali upon the higher salt concentrations beyond 100 mM NaCl, together with elucidating the mechanism of dynamic regulation in membrane homeostasis during the salt stress. 4. Materials and methods 4.1. Plant materials and salinity treatments Two types of rice (Oryza sativa L.) cultivars such as Pokkali (salttolerant) and IR-29 (salt-sensitive) were obtained from International Rice Research Institute (IRRI, Los Baños, Philippines). Rice seeds were surface-sterilized with 2.5% sodium hypochlorite solution and rinsed with distilled water. The seeds were then transferred to sterile moist filter paper after swelling them in distilled water at 30  C for 6 h. The seeds were placed in glass petridish for germination at 30  C for 72 h in the dark. Uniformly germinated seeds were selected and cultivated in holes of Styrofoam boards with 0.5 Murashige and Skoog (MS) medium under following conditions: 30  C/25  C day and night temperature, relative humidity 70e80%, and a light intensity of 700e 800 mmol m2 s1 over a 12 h photoperiod (in the semi-controlled plant greenhouse at the Advanced Radiation Technology Institute (ARTI)). The nutrient solution was aerated continuously and replaced every two days. For salt treatment, 100 mM NaCl was added to the culture medium. Rice seedlings were exposed to salt stress by adding 100 mM NaCl to the culture medium at 3-week-old plant. Plants were harvested at 7 days after the treatment for analysis. All experiments were repeated at least three times. 4.2. Measurement of chlorophyll and carotenoid contents Leaf tissues of Pokkali and IR-29 were ground with liquid nitrogen, and then 1 ml 100% acetone was added for the extraction of chlorophyll and carotenoid. After vigorous vortex at 4  C for 1 h in darkness, cell debris were removed by centrifugation at 15,000 g at 4  C for 15 min. Concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophylls (tChl), and total carotenoids (tCar) were calculated by the equations of Lichtenthaler as follows: Chl a ¼ 11.24  A661.6  2.04  A644.8; Chl b ¼ 21.13  A644.8  4.19  A661.6; tChl ¼ 18.09  A644.8 þ 7.05  A661.6; and tCar ¼ (1000  A470  1.90  Chl a-63.14  Chl b)/214 [56]. 4.3. Chlorophyll fluorescence analysis Chlorophyll (Chl) fluorescence was measured using an IMAGINGPAM (Walz, Effeltrich, Germany). Readings were taken 5-mm-diameter leaf disks after dark-adaptation on water-soaked filter papers for

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15 min at room temperature. Variable fluorescence (Fv) was calculated by subtracting initial Chl fluorescence (Fo) from the maximum yield of fluorescence (Fm). The ratio of Fv/Fm represents the maximum photochemical efficiency of photosystem II (PS II) [57]. The parameter for non-photochemical quenching (NPQ) was measured by analyzing Chl fluorescence quenching with the same fluorometer. 4.4. Light microscopy and transmission electron microscope Leaves and roots were cut into 2 segments of 1 mm each and placed immediately in a freshly prepared the mixture of 0.2% (w/v) glutaraldehyde and 4% (w/v) paraformaldehyde in 50 mM sodium phosphate buffer (pH 7.4). The segments were then de-gassed and fixed under vacuum for 4 h at room temperature. After washing in the same buffer, they were post-fixed in 1% (w/v) osmium tetroxide (in the same buffer) for 1 h. After fixation, the samples were rinsed three with same buffer for 15 min. Rapid dehydration was accomplished by placing the sample in 50% ethanol for 30 min, followed by changes in 70%, 90%, 95%, 100% ethanol (30 min each). The specimens were then placed in low-viscosity epoxy resin [58]. Ultrathin sections (80 nm) were mounted on uncoated nickel grids (300 mesh) and sequentially stained with uranyl acetate and lead citrate before being examined at 80 kV under a transmission electron microscope (J-1010, JEOL, Japan). For transmission electron microscope, the dehydrated samples were placed in 25% London Resin White (LR-white, London Resin Co., London, UK) resin with ethanol for 45 min in the room temperature, followed by changes in 50% and 75% LR white with ethanol for 45 min. The samples were subsequently left rotating in pure resin overnight at room temperature, and then samples were placed in fresh resin and, after 4 h, molded in fresh LR White and polymerized an aerobically at 60  C in gelatin capsules for 48 h [59]. Sections were cut at a thickness of 2 mm and placed on warm glasssubbed slides containing drops of double-distilled water. Also, serial thin sections cut with glass knife using a EM UC6 ultramicrotome (Leica, Wetzlar, Germany). For observation, the samples were stained by periodic acid Schiff’s stain (PAS) solution on slides and washed with water three times and then cover slips were placed. Images were obtained through a light microscope (Zeiss, Oberkochen, Germany) using a CCD camera. 4.5. Assay for antioxidant enzymes For determination of antioxidant enzyme activities, 0.5 g of rice leaves were homogenized with ice cold 50 mM KPi (pH 7.0) containing 0.1 mM EDTA, 1% (w/v) polyvinyl-pyrrolidone (PVP) and 0.5% (v/v) Triton X-100 at 4  C, and, for that of APX activity, leaves were homogenized in 100 mM NaPi (pH 7.0) containing 5 mM ascorbate and 1 mM EDTA. The homogenate was filtered through four layers of cheesecloth and centrifuged at 14,000 g for 15 min at 4  C. The supernatant was collected for determination of antioxidant enzyme activities, and stored at 80  C for further analyses. Protein concentration was determined according to Bradford method, using BSA as a standard [60]. 4.5.1. APX activity APX (EC 1.11.1.11) activity was determined according to the method of Nakano and Asada. The concentration of oxidized ascorbate was calculated using extinction coefficient (¼2.8 mM1 cm1). One unit of APX was defined as 1 mM/ml ascorbate oxidized per minute [61]. 4.5.2. Native PAGE assay for APX APX isozymes were detected by the procedure described by Mittler and Zilinskas [62]. APX isozymes were loaded on 10% (w/v)

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non-denaturing polyacrylamide gel and the electrophoresis was done at 10 mA for 20 min, and subsequently at 30 mA for 90 min at 4  C. After electrophoresis, APX was stained with 2.45 mM NBT (nitroblue tetrazolium) and 2 mM H2O2. 4.5.3. CAT activity CAT (EC 1.11.1.6) activity was assayed by measuring the initial H2O2-scavenging rate. Extinction coefficient for H2O2 at 240 nm was 40 mM1 cm1 [63]. 4.5.4. Native PAGE assay for CAT CAT isozymes were separated on 10% non-denaturating polyacrylamide gel and were visualized by 1% (w/v) ferric chloride and 1% (w/v) potassium ferricyanide, as described by Fath et al. [64]. 4.5.5. POD activity POD (EC 1.11.1.7) activity was assayed according to the method described by Kwak et al. using pyrogallol as a substrate [65]. The molar extinction coefficient of purpurogallin is 2.47 mM1 cm1. 4.5.6. Native PAGE assay for POD The separation and staining of POD isozymes were done as described by Huh et al. [66]. POD isozymes were separated on 10% (w/v) non-denaturing polyacrylamide gel and the electrophoresis was done at 10 mA for 20 min, and subsequently at 30 mA for 90 min at 4  C. After electrophoresis, POD was stained with 1% (v/v) benzidine and 1.5% (v/v) H2O2. 4.6. Measurements of reduced, oxidized, and total ascorbic acid contents Measurements of reduced, oxidized, and total ascorbic acid (AA) contents of rice leaves and roots were conducted by a colorimetric method by Gillespie and Ainsworth [67]. AA was determined by the use of a calibration curve of known quantities of ascorbate (0e 1.5 mM), and expressed as mM AA/g FW. 4.7. Measurement of H2O2 contents The changes in the concentration of H2O2 in the medium were detected by an increase in Amplex Red fluorescence using excitation and emission wavelengths of 585 and 550 nm, respectively. The concentration of commercial 30% (v/v) H2O2 solution was calculated using light absorbance at 240 nm using E240 mM ¼ 43.6 cm1; the stock solution was diluted to 100 mM with dH2O and used for calibration immediately [68]. 4.8. ROS scavenging effects One gram of leaves were collected and immediately frozen in liquid nitrogen. Samples were crudely extracted with 0.1 M potassium phosphate buffer (pH 7.0). The extract was centrifuged at 300 g for 5 min at 4  C to remove cell debris. For assay of H2O2 scavenging effect, the supernatant was mixed with 20 mM H2O2 (1:1, v/v) and incubated at 25  C for 10 min. After centrifugation at 15,000 g for 5 min at 4  C, the supernatant was further incubated at 25  C with 50 mM 3,30 -diaminobenzidine (DAB) for 30 min to quantify the remaining H2O2. Finally, polymerization products of DAB were estimated by obtaining the absorbance at 485 nm. H2O2 scavenging efficiency was expressed as percentages of [DA485 (Blank)  A485 (Sample)]/DA485 (Blank) [69]. Singlet oxygen (1O2) was generated in riboflavin-sensitized photo-inactivation of lysozyme in the presence of high light. The supernatant was mixed with 0.1 mM riboflavin and 50 mM TEMP. EPR spectra were measured within 30 min after treatment of high

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light (PPFD of 800 mol m2 s1) with an EPR spectrometer (JESFA200, JEOL Ltd., Tokyo, Japan) working at the X band. Spectra were recorded at room temperature with 9.4 GHz microwave frequency, 10 mW microwave power, 2.0 mT modulation amplitude, 100 kHz modulation frequency and 2.0 amplification. The amount of trapped 1O2 was obtained as the area of EPR absorption spectra (double integral of measured spectra). Production of hydroxyl radical (
OH) under Fenton reaction (H2O2 þ Fe2þ) was assayed by EPR spectroscopy with a specific chemical probe, ethanol/a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) [70]. EPR spectra were measured within 1 min after reaction with an EPR spectrometer working at the X band. Spectra were recorded at room temperature with 9.4 GHz microwave frequency, 10 mW microwave power, 0.2 mT modulation amplitude, 100 kHz modulation frequency and 3.0 amplification. The amount of trapped
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