Effects of NaCl on H2O2 (nmol g−1 fw) levels and O2−• production rates (nmol min−1 mg−1 pro) in leaves and roots of K. obovata and B. gymnorhiza.
The effects of increasing NaCl (100–400 mM) on cellular salt distribution, antioxidant enzymes, and the relevance to reactive oxygen species (ROS) homeostasis were investigated in 1-year-old seedlings of two non-salt secretor mangroves, Kandelia obovata and Bruguiera gymnorhiza. K. obovata accumulated less Na+ and Cl− in root cells and leaf compartments under 400 mM NaCl compared to B. gymnorhiza. However, B. gymnorhiza leaves are notable for preferential accumulation of salt ions in epidermal vacuoles relative to mesophyll vacuoles. Both mangroves upregulated antioxidant enzymes in ASC-GSH cycle to scavenge the salt-elicited ROS in roots and leaves but with different patterns. K. obovata rapidly initiated antioxidant defense to reduce ROS at an early stage of salt stress, whereas B. gymnorhiza maintained a high capacity to detoxify ROS at high saline. Collectively, our results suggest that salinized plants of the two mangroves maintained ROS homeostasis through (i) ROS scavenging by antioxidant enzymes and (ii) limiting ROS production by protective salt compartmentation. In the latter case, an efficient salt exclusion is favorable for K. obovata to reduce the formation of ROS in roots and leaves, while the effective vacuolar salt compartmentation benefited B. gymnorhiza leaves to avoid excessive ROS production in a longer term of increasing salinity.
- Bruguiera gymnorhiza
- Kandelia obovata
- reactive oxygen species
- antioxidant enzymes
- X-ray microanalysis
Mangrove plants form a dominant ecosystem in tropical and subtropical coastlines . Bruguiera gymnorhiza is widely distributed in tropical and subtropical area, from the southeastern coast of Africa through Asia to Australia and the southwestern Pacific . Kandelia obovata is distributed mostly in the transition regions from tropical to subtropical coastlines of southern China, Taiwan, and the southern islands of Japan . Climatic factors affecting the vegetative and reproductive phenology of B. gymnorhiza and K. obovata growing in subtropical regions were assessed in recent years. Temperature, day length, and rainfall are suggested to be the important external controlling factors of leaf initiation in B. gymnorhiza . Leaf litterfall of the subtropical mangrove K. obovata was correlated to monthly day length and maximum wind speed . Flowering of K. obovata was influenced by monthly sunshine hour and monthly mean air temperature . While in B. gymnorhiza, flowering phenophase was linked with rainfall and relative humidity . B. gymnorhiza and K. obovata are two major mangrove species along southern China coastlines. B. gymnorhiza is a frontline species and mostly occurs in high-saline zones compared with K. obovata, which grows in low-saline creeks in mangrove areas .
The most striking feature of mangroves is the capacity to withstand high salinity concentrations [8, 9, 10, 11]. In general, secretor and non-salt secretor mangroves both exhibited a high capacity to maintain Na+ homeostasis under sodium chloride (NaCl) stress [7, 12, 13, 14, 15]. Root flux recordings showed that B. gymnorhiza, K. candel (or K. obovata, non-salt secretors), Aegiceras corniculatum, and Avicennia marina (secretors) retained an obvious Na+ exclusion under NaCl treatment [7, 13, 14, 15, 16]. Hydrogen peroxide (H2O2), nitric oxide (NO), and calcium (Ca2+) mediated Na+/H+ antiport across the PM, thus contributing to control ionic homeostasis in the two non-salt secretor mangrove species . Recently, multiple signaling networks of extracellular ATP (eATP), H2O2, Ca2+, and NO in the mediation of root ion fluxes were established in salt-stressed K. obovata and A. corniculatum . Salt exclusion by roots is the most important salt-tolerant mechanism in woody plants [17, 18, 19, 20, 21, 22] and herbaceous species [23, 24]. Although mangrove roots could effectively exclude salt ions under NaCl stress, Na+ and Cl− taken up by roots would eventually transport to shoots via the transpiration stream during a long-term salt exposure [16, 20, 21, 22]. Jing et al. found that Na+ extrusion capacity in K. candel roots declined with the prolonged duration of salt exposure . As a result, large amount of Na+ accumulated in roots was transported to shoots [12, 16]. Excessive Na+ accumulation in leaves leads to oxidative stress by the production of reactive oxygen species (ROS) in trees [25, 26, 27]. Similarly, salt-induced oxidative stress has been widely shown in herbaceous species [28, 29, 30, 31, 32, 33, 34, 35]. In mitochondria and chloroplasts, superoxide anions (O2−) are generated as a by-product of electron transfer to O2 via photosynthetic and respiratory electron transport chain [36, 37]. The active O2− leads to subsequent formation of H2O2 and hydroxyl radicals (OH•−) through chemical and enzymatic reactions [36, 37]. Salt induced an oxidative stress in chloroplast and mitochondria of pea leaves [28, 29, 30, 34]. In poplars, great buildup of Na+ and Cl− in chloroplasts may directly cause ion toxicity and induce the subsequent oxidative stress [26, 38]. X-ray microanalysis results showed that the inability for the restriction of Na+ entry into the chloroplasts leads to an uncontrolled oxidation in Populus popularis [26, 38]. Salt-resistant plants may maintain ROS homeostasis through limiting ROS production by a protective salt partitioning. Evidence presented elsewhere suggests that NaCl-stressed sorghum plants preferentially partition Cl− into leaf sheaths relative to blades . The preferential accumulation of Cl− in the sheath would lessen the effect of salinity on photosynthetic processes in the leaf blade. Furthermore, X-ray microanalysis of various cell types in leaf sheaths and blades revealed that Cl− was preferentially accumulated in epidermal vacuoles, relative to mesophyll vacuoles in salt-tolerant barley and sorghum [39, 40]. The high Cl− concentration in the leaf blade mesophyll cells of a barley cultivar (cv. Clipper) suggests that the lower salt resistance of this cultivar is directly related to the degree of Cl− exclusion by these cells . Thus, it can be inferred that compartmentalizing salt ions in cell layers of leaf blade would reduce the perturbation of salt on photosynthetic processes in photosynthetically active mesophyll, especially the electron transport processes in chloroplasts. As a result, ROS is less produced [26, 27]. Although salt increased H2O2 in K. candel leaves, the ROS-induced necrotic lesions were not seen during the period of stress . In addition to ROS scavenging by both enzymatic and nonenzymatic antioxidants, it is possible that mangrove plants could attenuate oxidative stress by a reasonable salt compartmentation in cells. However, this needs further investigations, e.g., by X-ray microanalysis, to clarify.
Under salt stress, the antioxidant defense system serves to remove reactive oxygen species (e.g., O2•− and H2O2) in the chloroplast, mitochondria, and cytosol. Superoxide dismutases (SODs) are considered to be the first defense line against O2•− and the reaction product [41, 42]. H2O2 is further detoxified through a reaction catalyzed by an ascorbate-specific peroxidase (APX), glutathione peroxidase (GPX), and catalase (CAT). APX utilizes ascorbate (AsA) as its specific electron donor to reduce H2O2 to water with the concomitant generation of monodehydroascorbate (MDAsA), a univalent oxidant of AsA . CAT, an enzyme that splits hydrogen peroxide to yield oxygen and water, is an important part of the antioxidant defense . GPX efficiently catalyzes the reduction of hydrogen peroxide and organic hydroperoxides by glutathione [45, 46]. In addition to these antioxidant enzymes that can directly scavenge toxic oxygen species, glutathione reductase (GR), which regenerates glutathione (GSH) that has been oxidized during ROS scavenging, is also implicated in redox homeostasis control . The contribution of antioxidant defense to salt tolerance has been confirmed in crop species [32, 33, 48, 49] and woody plants, e.g., poplars [25, 26, 27] and mangroves [8, 10, 50, 51]. Takemura et al. detected an increased activity of SOD and CAT in B. gymnorhiza at high salt . Parida et al. found that the elevation of antioxidant enzymes, APX and guaiacol peroxidase, was able to scavenge salt-induced H2O2 in B. parviflora . Therefore, the capacity for regulating ROS homeostasis serves as one important component for salt tolerance in mangroves.
Analyses of isoforms of antioxidant enzymes showed species differences in antioxidant defense system against salt treatment. Plants generally have three SOD isozymes: Cu/Zn-SOD in the cytosol and chloroplasts, Mn-SOD in mitochondria, and Fe-SOD in chloroplasts . Activity of CuZn-SOD I and CuZn-SOD II, the two dominant SOD proteins in poplar leaves, was not detectable in P. popularis (salt-sensitive) after 16 days of salt stress, while there were no marked inhibitory effects of NaCl on the two SOD isoenzymes in P. euphratica (salt-resistant) during the observation period . Furthermore, genetic differences were found in the timing of APX and CAT response to increasing salinity. Salt treatments increased activity of CAT and APX isoenzymes in the two poplar species, but their activity increased earlier in P. euphratica than in P. popularis . In mangrove, a certain number of SOD isoenzymes (Mn-SOD, Fe-SOD), guaiacol peroxidase isoenzymes, and GR isoenzymes were preferentially elevated by NaCl in B. parviflora . The induction of antioxidant enzymes might be the result of salt-induced gene transcription. Northern blot analysis revealed that the transcript level of cytosolic Cu/Zn-SOD was increased after a few days of NaCl treatment . Similarly, NaCl was shown to increase KcCSD expression in K. candel leaves . Proteomic analysis of K. candel leaves revealed that SOD abundance increased in response to high NaCl at 450–600 mM . Furthermore, overexpression of copper/zinc superoxide dismutase from mangrove K. candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast .
We have previously shown species differences between secretor and non-salt secretor mangroves in root salt exclusion and leaf gas exchange response to salt treatment [7, 12, 15]. The object of this study is to investigate the effect of NaCl on the pattern of cellular salt compartmentation, variations in antioxidant enzymes, and their contributions to ROS (in particular, O2•− and H2O2) homeostasis maintenance in non-salt secretor mangroves.
2. Salt compartmentation and antioxidant defense
2.1. Plant materials and salt treatment
K. obovata hypocotyls developed from fruits turned into mature propagules, which began to drop in March and continued dropping until May . Mature and developing propagules of B. gymnorhiza were found throughout the year, but the abundance of mature propagules was highest in summer and lowest in winter . In early March, 200 of propagules of K. obovata and B. gymnorhiza were obtained from Dongzhai Harbor in Hainan Province of China (latitude 19°51′N and longitude 110°24′E). Propagules were collected from the surface of soil or seawater during the ebb tide. Single hypocotyls were planted in individual pots (15 cm in diameter and 18 cm in height) containing sand and placed in a greenhouse at Beijing Forestry University, Beijing, China (latitude 39°56′N and longitude 116°20′E). The pots were fertilized with 1000 ml half strength Hoagland nutrient solution every 14 d. Seedlings were raised from March to August under nonsaline conditions. The relative humidity was maintained at 60–70%, and photosynthetically active radiation (PAR) varied from 400 to 1200 μmol m−2 s−1. Salt treatment was carried out when the fourth pair of leaves came out from the apex of the growing shoots (mid-August) .
NaCl concentration started from 100 mM and increased stepwise by 100 mM , reaching 400 mM and remained at this salinity until the terminal of experiment. Increasing NaCl saline was applied at day 1 (100 mM), day 3 (200 mM), day 6 (300 mM), and day 10 (400 mM), respectively. Control plants were kept well watered with no addition of NaCl. PAR was 400–1200 μmol m−2 s−1, and air temperature was 20–35°C over the duration of experiment. On day 2, day 5, day 9, and day 14, leaves and roots were sampled for ROS (O2•− and H2O2) determination and total activity measurements of antioxidant enzymes, i.e., superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR). For SOD and CAT isoenzyme analyses, leaves and roots were sampled at day 3, day 6, day 10, and day 15. Three replicated plants per treatment were harvested at each sampling time. At the final harvest time, roots and upper mature leaves were sampled from control and stressed plants and used for X-ray microanalysis.
2.2. O2•− and H2O2 levels in roots and leaves
O2•− production rate was typically higher in roots than in leaves in control plants of the two species (Table 1). High salinity (400 mM NaCl) increased root O2•− production rate by 82 and 83% in K. obovata and B. gymnorhiza, respectively, but the salt-induced rise of O2•− was absent in the two mangroves when NaCl concentration was below 300 mM (Table 1). The same trend was observed in leaves, but the NaCl-induced increase of O2•− was only observed in B. gymnorhiza leaves at 400 mM NaCl (Table 1).
|Treatment||K. obovata||B. gymnorhiza|
|Control||17.63 ± 6.00a||1.16 ± 0.12a||10.04 ± 1.98a||5.77 ± 1.00a||5.93 ± 0.91a||0.61 ± 0.07a||17.50 ± 0.17a||3.44 ± 0.56a|
|NaCl (100 mM)||2.37 ± 0.60b||1.35 ± 0.11a||7.76 ± 2.56a||6.26 ± 0.99a||11.53 ± 4.96a||0.47 ± 0.08a||1.43 ± 1.91b||2.35 ± 0.22a|
|Control||16.48 ± 4.10a||1.11 ± 0.07a||4.95 ± 1.86a||4.03 ± 1.35a||3.33 ± 1.12a||0.50 ± 0.29a||31.43 ± 4.79a||3.63 ± 0.58a|
|NaCl (200 mM)||1.50 ± 0.05b||1.26 ± 0.04a||3.20 ± 1.02a||4.34 ± 1.25a||9.01 ± 4.52a||0.69 ± 0.28a||4.79 ± 1.57b||2.68 ± 0.02a|
|Control||10.97 ± 1.23a||0.67 ± 0.09a||13.10 ± 2.07a||1.54 ± 0.09a||15.93 ± 4.50a||0.48 ± 0.07a||26.44 ± 4.98a||1.81 ± 0.60a|
|NaCl (300 mM)||11.47 ± 5.10a||0.84 ± 0.12a||13.93 ± 2.34a||1.78 ± 0.13a||23.13 ± 1.10a||0.69 ± 0.29a||4.66 ± 0.35b||1.89 ± 0.52a|
|Control||11.60 ± 5.80b||1.76 ± 0.41a||8.05 ± 1.08a||1.69 ± 0.06b||19.12 ± 1.18a||0.71 ± 0.09b||10.39 ± 2.15a||2.65 ± 0.09b|
|NaCl (400 mM)||41.38 ± 8.97a||1.76 ± 0.31a||11.62 ± 3.09a||3.08 ± 0.46a||22.90 ± 3.40a||1.08 ± 0.13a||16.07 ± 3.31a||4.85 ± 0.52a|
Increasing NaCl stress did not significantly elevate root H2O2 levels in either species; rather, a significant reduction of H2O2 was observed in B. gymnorhiza when NaCl saline ranged from 100 to 300 mM (Table 1). An abrupt rise of H2O2 occurred in K. obovata leaves when plants were subjected to 400 mM NaCl, although H2O2 remained less than controls at low salt (100–200 mM, Table 1). However, salinized B. gymnorhiza maintained a H2O2 level similar to control leaves despite of a NaCl increase, from 100 to 400 mM (Table 1).
In general O2•− production and/or H2O2 levels in roots and leaves were enhanced by high salinity (400 mM NaCl) in the two mangrove species, although root and leaf ROS levels were usually downregulated after exposure to a lower salinity (100–200 mM NaCl), e.g., O2− and H2O2 in B. gymnorhiza roots and H2O2 in K. obovata leaves (Table 1). Similarly, NaCl-induced increase of H2O2 was observed in leaves of B. parviflora  and K. candel  under hydroponic conditions. In this study, the moderate ROS increment induced by 400 mM NaCl caused no oxidative burst in both species, suggesting that stressed plants of K. obovata and B. gymnorhiza maintained ROS homeostasis throughout the duration of salt exposure. Our data showed that salt compartmentation and antioxidant enzymes contributed to ROS homeostasis in both species but with different patterns under NaCl stress (see below).
2.3. Salt compartmentation and ROS production
2.3.1. Salt compartmentation within root and leaf cells
In this study, SEM-EDX analysis was performed on cross sections of B. gymnorhiza and K. obovata roots. Na+ and Cl− were detectable in root cells of no-salt controls (Table 2). Under salt conditions, Na+ and Cl− levels significantly increased in the tested structures, i.e., epidermis, exodermis, cortex, endodermis, and stelar parenchyma (Table 2). The long-term salt treatment with increasing NaCl saline (100–400 mM, 15 d) significantly increased the content of salt ions by 0.6–9.6 (Na+) and 0.5–5.1 fold (Cl−), although Na+ and Cl− levels were typically higher in B. gymnorhiza than in K. obovata in all measured structures (Table 2).
|Compartment||Treatment||K. obovata||B. gymnorhiza|
|Epidermis||Control||2.98 ± 0.30b||5.35 ± 1.26b||10.3 ± 4.46b||24.3 ± 1.76b|
|NaCl||7.36 ± 2.19a||32.8 ± 3.94a||16.0 ± 3.82a||44.9 ± 1.96a|
|Exodermis||Control||1.05 ± 0.23b||12.1 ± 2.12b||7.76 ± 1.78b||27.2 ± 4.46b|
|NaCl||11.1 ± 1.24a||23.6 ± 4.72a||23.9 ± 3.33a||51.4 ± 2.14a|
|Cortex||Control||0.70 ± 0.34b||7.71 ± 1.05b||12.6 ± 3.54b||34.2 ± 1.42b|
|NaCl||5.39 ± 1.36a||43.7 ± 2.65a||26.3 ± 3.32a||51.2 ± 3.50a|
|Endodermis||Control||1.39 ± 0.97b||10.2 ± 0.37b||15.8 ± 4.90b||35.9 ± 3.08b|
|NaCl||4.84 ± 0.96a||49.3 ± 1.64a||31.0 ± 5.82a||57.3 ± 5.14a|
|Stelar parenchyma||Control||1.56 ± 0.58b||6.96 ± 1.33b||17.1 ± 2.19b||24.8 ± 2.46b|
|NaCl||5.80 ± 1.12a||38.6 ± 2.51a||35.3 ± 7.89a||48.2 ± 4.51a|
In leaf cells of control plants, TEM-EDX data showed an evident Na+ and Cl− in epidermis, mesophyll, and xylem vessels (leaf vascular bundle), but B. gymnorhiza exhibited 28–195% higher Na+ than K. obovata in all measured cell compartments, such as xylem vessel, epidermal wall and vacuole, mesophyll wall and vacuole, and chloroplast (Table 3). NaCl (400 mM) treatment markedly increased Na+ and Cl− concentrations in the apoplastic space and vacuoles of the two species but with the exception of Cl− in K. obovata (Table 3). In comparison, the fractions of Na+ and Cl− in the xylem vessel, cell wall, and vacuole were 30–196% higher in stressed B. gymnorhiza as compared to K. obovata (Table 3). However, NaCl stress did not significantly increase Na+ and Cl− concentrations in the chloroplast of two mangroves (Table 3).
|Compartment||Treatment||K. obovata||B. gymnorhiza|
|Xylem vessels (leaf vascular bundle)||Control||131 ± 102b||633 ± 48a||309 ± 22b||576 ± 101b|
|NaCl||246 ± 4a||636 ± 101a||729 ± 119a||1069 ± 254a|
|Epidermal wall (abaxial and adaxial)||Control||266 ± 96b||725 ± 181a||403 ± 22b||812 ± 101b|
|NaCl||377 ± 110a||945 ± 71a||840 ± 119a||1305 ± 254a|
|Mesophyll wall (palisade and spongy)||Control||228 ± 35b||669 ± 224a||420 ± 46b||545 ± 67b|
|NaCl||336 ± 27a||926 ± 170a||904 ± 287a||1445 ± 418a|
|Epidermal vacuole (abaxial and adaxial)||Control||75 ± 71b||495 ± 282a||221 ± 42b||646 ± 23b|
|NaCl||183 ± 133a||558 ± 285a||509 ± 125a||1148 ± 121a|
|Mesophyll vacuole (palisade and spongy)||Control||86 ± 14b||510 ± 123a||123 ± 16b||531 ± 33b|
|NaCl||134 ± 31a||634 ± 31a||263 ± 6a||664 ± 3a|
|Chloroplast (palisade and spongy)||Control||103 ± 18a||532 ± 129a||182 ± 69a||640 ± 89a|
|NaCl||141 ± 15a||681 ± 45a||145 ± 57a||494 ± 101a|
Vacuolar compartmentation in mesophyll was clearly seen in stressed B. gymnorhiza, in which the Na+ and Cl− concentrations were higher in the vacuole than in the chloroplast (Table 3). Noteworthy, B. gymnorhiza preferentially accumulated 73–94% higher Na+ and Cl− in vacuoles of epidermal cells as compared to mesophyll vacuoles (Table 3). In contrast to B. gymnorhiza, vacuolar fractions of Na+ and Cl− in stressed K. obovata remained the same as that of chloroplast, and vacuolar Na+ and Cl− in epidermis was similar to that in mesophyll vacuole regardless of treatments (Table 3).
2.3.2. Salt compartmentation and ROS production in roots and leaves
X-ray microanalysis data show that Na+ and Cl− were evident in root and leaf cells of control plants in the two mangroves (Tables 2 and 3), presumably originated from hypocotyls as propagules were collected from the surface of soil or seawater in coastal habitats of mangrove forest. Mangrove propagules absorbed salt ions when they contacted seawater [7, 12]. Na+ and Cl− increased in cell compartments of roots and leaves (Tables 2 and 3). This indicates that the salt ions taken up by roots transported to shoots under NaCl stress [12, 16, 22]. Our data show that there were marked differences in the pattern of salt compartmentation in the two mangroves. K. obovata exhibited a high capacity to exclude NaCl from root and leaf cells, whereas B. gymnorhiza are notable for (1) vacuolar compartmentation in mesophyll cells and (2) preferential accumulation of Na+ and Cl− in epidermal vacuoles, relative to mesophyll vacuoles (Table 3). The ability to extrude Na+ from root cells of K. obovata likely results from an active Na+/H+ antiport driven by H+ pumping activity of PM H+-ATPase [7, 15]. Salt compartmentation in vacuoles likely depends on active transport of salt ions across the tonoplast. Salinity may increase the activity of vacuole H+ pumps, thus making a contribution to the compartmentation of toxic ions into the vacuoles via Na+/H+ antiporter systems [62, 63, 64]. Mimura et al. found that the elevated concentrations of Na+ and Cl− in swelling vacuoles were correlated with the salt-induced activation of tonoplast H+-ATPase in suspension-cultured cells of B. sexangula . We suggest that the two mangrove plants may maintain ROS homeostasis through limiting ROS production by a protective cellular salt compartmentation, in addition to scavenging ROS by antioxidant enzymes in a longer term of increasing salinity (see below). In brief:
Salt exclusion and ROS production in K. obovata: NaCl treatment increased Na+ in the leaf apoplast and vacuole of epidermis and mesophyll, but did not elevate Cl− in K. obovata (Table 3). Moreover, the absolute values of Na+ and Cl− in these measured compartments were lower in K. obovata than in B. gymnorhiza under 400 mM NaCl (Table 3). Result suggests that K. obovata plants had a higher capacity for NaCl exclusion, presumably due to the salt uptake and transport restrictions in roots (Table 2) [7, 12, 15]. Effective salt exclusion is a benefit for K. obovata to reduce ROS production. We have shown that the inability to exclude NaCl favored the formation of O2•− and H2O2, which causes an oxidative burst in leaf cells of a salt-sensitive poplar, P. simonii × (P. pyramidalis × Salix matsudana) (P. popularis cv. ‘35–44’) [26, 27, 38].
Vacuolar salt compartmentation and ROS production in B. gymnorhiza: B. gymnorhiza leaves exhibited a more pronounced salt accumulation than K. obovata (Table 3), resulting from a higher root-to-shoot salt transport . Noteworthy, B. gymnorhiza preferentially accumulated Na+ and Cl− in epidermal vacuoles, instead of mesophyll vacuoles (Table 3). Similar findings were observed in leaf sheaths and blades of sorghum and barley in which Cl− was preferentially accumulated in most cell layers, particularly the adaxial epidermal cells [39, 40]. The evident Cl− exclusion from photosynthetically active mesophyll would lessen the effect of salinity on photosynthetic processes, especially the electron transport in chloroplasts in the mesophyll. Moreover, fractions of Na+ and Cl− remained higher in mesophyll vacuole than in the cytoplasm (Table 3), which may inhibit the enhancement of NaCl on the formation of O2•− and H2O2 in the cytosol, chloroplasts, and mitochondria. NaCl was found to favor the formation of O2•− and H2O2 in chloroplasts and mitochondria of pea cultivars [28, 29]. Accordingly, we hypothesize that the B. gymnorhiza limits ROS production by preferential accumulation of Na+ and Cl− in epidermal vacuoles, as well as vacuolar compartmentation in mesophyll cells.
2.4. Antioxidant enzymes contributed to ROS homeostasis
2.4.1. Activity of antioxidant enzymes in roots and leaves
Under no-salt control conditions, total activity of measured antioxidant enzymes roots and leaves, SOD, APX, CAT, and GR, varied markedly during the observation period (Tables 4 and 5). This was presumably resulted from genetic difference of seedlings and variations in light intensity and air temperature. In our study, natural PAR was 400–1200 μmol m−2 s−1, and air temperature was 20–35°C over the duration of experiment. In general, activities of antioxidant enzymes in roots and leaves were not reduced upon increasing saline (with a few exceptions) but upregulated in both species (Tables 4 and 5). Noteworthy, there were species differences in antioxidant defense to increasing salinity. Activity of each component in the measured antioxidant defense system, SOD, APX, CAT, and GR, drastically increased in K. obovata roots at 300 mM NaCl, while the same trend was observed in B. gymnorhiza roots at 400 mM (Table 4). Furthermore, B. gymnorhiza leaves showed a higher increase of SOD, APX, and CAT at 400 mM NaCl as compared to K. obovata (Table 5). SOD of K. obovata was upregulated after salt exposure, but the response is quite variable in roots and leaves. Root SOD activity was increased by 100 and 300 mM NaCl, while leaf activity was increased by 200 and 400 mM (Tables 4 and 5). SOD activity in roots and leaves of B. gymnorhiza did not increase after exposure to 100–300 mM NaCl (Tables 4 and 5). The variable response of antioxidant enzymes to salt treatment was also seen in GR. It exhibited a marked elevation in B. gymnorhiza leaves at 100 mM NaCl, whereas the steady increase of GR in K. obovata was observed at a salt concentration of 300 mM (root) and 400 mM (leaf) (Tables 4 and 5).
|Treatment||K. obovata||B. gymnorhiza|
|Control||246.9 ± 25.0b||138.1 ± 21.2a||270.0 ± 22.3a||33.7 ± 15.2a||133.3 ± 9.0a||27.8 ± 4.8a||130.2 ± 7.8a||12.9 ± 2.9a|
|NaCl (100 mM)||406.4 ± 21.5a||136.2 ± 11.1a||272.3 ± 22.2a||15.7 ± 3.3a||125.2 ± 7.3a||45.1 ± 19.0a||144.1 ± 18.6a||20.7 ± 3.1a|
|Control||108.0 ± 20.2a||152.9 ± 21.4a||249.7 ± 46.5a||49.9 ± 8.0a||149.0 ± 31.3a||43.3 ± 2.9a||377.1 ± 34.1a||19.1 ± 5.6a|
|NaCl (200 mM)||112.2 ± 61.2a||151.9 ± 27.2a||181.4 ± 15.4a||77.6 ± 12.2a||148.6 ± 17.9a||46.7 ± 10.1a||369.4 ± 94.6a||30.4 ± 13.8a|
|Control||148.4 ± 1.7b||28.9 ± 6.2b||119.8 ± 16.4b||17.4 ± 6.0b||98.4 ± 17.4a||26.4 ± 1.8a||120.8 ± 27.7a||30.0 ± 9.4a|
|NaCl (300 mM)||377.4 ± 32.9a||60.71 ± 8.0a||343.7 ± 96.4a||49.4 ± 11.4a||121.8 ± 28.1a||27.8 ± 1.2a||178.1 ± 47.7a||29.8 ± 6.5a|
|Control||193.8 ± 88.8a||35.1 ± 6.3a||62.2 ± 5.7a||17.9 ± 3.6a||132.6 ± 11.8b||109.0 ± 16.3a||218.1 ± 55.6b||23.7 ± 2.3b|
|NaCl (400 mM)||244.4 ± 64.1a||54.6 ± 18.6a||77.8 ± 4.5a||26.1 ± 0.5a||232.4 ± 19.5a||132.6 ± 1.7a||361.6 ± 80.7a||59.8 ± 15.0a|
|Treatment||K. obovata||B. gymnorhiza|
|Control||49.1 ± 8.6a||107.4 ± 6.5b||62.0 ± 2.2a||16.5 ± 3.1a||38.1 ± 3.0a||69.3 ± 24.7a||58.7 ± 1.1a||5.8 ± 0.6b|
|NaCl (100 mM)||67.5 ± 17.6a||138.9 ± 7.5a||76.4 ± 5.4a||20.0 ± 3.1a||32.6 ± 4.5a||37.5 ± 9.0a||64.9 ± 7.5a||10.4 ± 1.5a|
|Control||61.2 ± 5.9b||223.9 ± 3.3a||102.9 ± 1.9a||25.8 ± 3.4a||52.2 ± 4.2a||88.7 ± 8.2a||181.2 ± 19.5a||22.5 ± 0.2a|
|NaCl (200 mM)||83.8 ± 5.0a||205.7 ± 22.8a||90.7 ± 16.5a||32.0 ± 2.6a||42.2 ± 7.9a||89.1 ± 9.9a||179.0 ± 10.1a||9.7 ± 1.6b|
|Control||66.2 ± 8.6a||135.6 ± 21.8a||83.6 ± 6.4a||22.1 ± 6.4a||32.0 ± 4.9a||118.0 ± 1.4a||130.5 ± 12.4a||18.9 ± 3.3a|
|NaCl (300 mM)||64.4 ± 10.9a||150.5 ± 29.1a||47.3 ± 4.7b||16.0 ± 1.0a||46.4 ± 6.6a||125.2 ± 2.1a||60.4 ± 11.7b||12.9 ± 3.3a|
|Control||52.2 ± 13.9b||331.3 ± 4.0a||111.9 ± 6.4a||19.9 ± 1.2b||30.6 ± 9.4b||75.9 ± 5.1b||116.1 ± 15.7b||9.5 ± 1.4a|
|NaCl (400 mM)||74.8 ± 4.2a||356.6 ± 15.1a||129.8 ± 2.5a||36.8 ± 4.1a||59.7 ± 8.6a||250.7 ± 29.6a||213.9 ± 16.3a||7.4 ± 2.1a|
SOD isoenzymes and CAT isoenzymes in roots and leaves were analyzed by native PAGE. In root extracts, three dominant SOD isoenzymes were detected in K. obovata roots, whereas there were two SOD isoforms in B. gymnorhiza (Figure 1A and B). KCN and H2O2 inhibited the activity of these isoenzymes in the two species, indicating they were CuZn-SOD isoforms (Figure 1A and B). NaCl did not restrict activity of all SOD isoforms in K. obovata roots during the period of salt treatment (Figure 1C), but a marked elevation of CuZn-SODs was observed in B. gymnorhiza at 300–400 mM NaCl (Figure 1D).
Three dominant SOD isoenzymes were detected in control leaves of both species but with different patterns (Figure 2A and B). KCN and H2O2 inhibited activity of two SOD isoenzymes in both genotypes, indicating that these were CuZn-SOD isoforms (Figure 2A and B). Another SOD isoform was defined as Mn-SOD since it was resistant to both inhibitors (Figure 2A and B). Activity of SOD isoenzymes in K. obovata leaves was increased by a lower salt, e.g., Mn-SOD at 100 and 200 mM NaCl and Cu/Zn-SOD1 and Cu/Zn-SOD2 at 200 NaCl mM (Figure 2C). B. gymnorhiza upregulated both Mn-SOD and Cu/Zn-SODs at a higher salt, 300–400 mM NaCl (Figure 2D).
Native PAGE of root extract showed three CAT isoenzymes in K. obovata and two in B. gymnorhiza (Figure 3). Increasing NaCl, from 100 to 400 mM, did not restrict activity of all CAT isoforms in both species, although activity of CAT isoforms in control roots fluctuated over the observation period (Figure 3). Compared with K. obovata, B. gymnorhiza exhibited typically a higher activity of CAT1 and CAT2 regardless of treatments (Figure 3).
Three and four CAT isoenzymes were identified in K. obovata and B. gymnorhiza leaves, respectively (Figure 4). Salt markedly enhanced the activity of CAT2 in K. obovata at 200 mM; however, the enhancement of NaCl on CAT2, CAT3, and CAT4 in B. gymnorhiza was observed at 300–400 mM NaCl (Figure 4).
2.4.2. Salt-elicited antioxidant enzymes contributed to ROS homeostasis
Salt-elicited antioxidant enzymes contributed to ROS homeostasis in the two mangroves but with different patterns. Salinized K. obovata exhibited an early and rapid antioxidative defense as compared to B. gymnorhiza. After exposure to 100–200 mM NaCl, total SOD activity in K. obovata leaves marked increased coincident with the increase of Cu/Zn-SOD1, Cu/Zn-SOD2, and Mn-SOD (Table 5, Figure 2), even though Fe-SOD was not detected as that reported in other mangroves . CAT in K. obovata leaves resembles the trend of SOD, and the increased activity was presumably due to the rise of CAT2 (Table 5, Figure 4). This is inconsistent with a previous report conducted on B. parviflora in which NaCl induced a decrease of CAT activity . In the present study, the coincident increase of CAT with SOD in K. obovata reveals an elevated capacity to detoxify both O2•− and H2O2 that is caused by NaCl, which is required for rapid removal of ROS and thus avoids oxidative damage. Likewise, we found that a salt-resistant Populus species, P. euphratica, was able to enhance active oxygen detoxification by increasing antioxidant enzymes at an early stage of salt stress, thus preventing an oxidative burst . Protein abundance of SOD in K. obovata leaves might increase under a high level of NaCl . Furthermore, Jing et al. showed that NaCl increased KcCSD transcription in K. candel leaves . Thus, it could be inferred that K. obovata would upregulate the gene expression of antioxidant enzymes to deal with a long-term saline stress.
Salt-induced elevation of antioxidant enzymes in B. gymnorhiza was usually found at high salinity. SOD, APX, and CAT in roots and leaves of B. gymnorhiza were all upregulated by 400 mM NaCl (Tables 4 and 5). Native PAGE analyses showed that the elevation of leaf SOD in salinized B. gymnorhiza resulted from the increase of all detected SOD isoforms, Mn-SOD, Cu/Zn-SOD1, and Cu/Zn-SOD2 (Table 5 and Figure 2), whereas the rise of SOD activity in roots was mainly the result of Cu/Zn-SODs (Table 4 and Figure 1). A similar trend was found in salt-stressed B. parviflora in which a significant enhancement of SOD was observed in leaves, mainly due to an increase in Mn-SOD and Fe-SOD2 . NaCl-induced activity of CAT in B. gymnorhiza leaves was due to the increase of CAT2, CAT3, and CAT4 (Table 5 and Figure 4).
Noteworthy, both K. obovata and B. gymnorhiza maintained evident activity of each CAT isoform in root tissues at 400 mM NaCl (Figure 3), showing a constant and stable capacity to control H2O2 levels. This may partly explain the finding that root O2•− increased by 82–83%, whereas there was no corresponding changes in H2O2 when K. obovata and B. gymnorhiza were subjected to 400 mM NaCl (Table 1).
We conclude that both K. obovata and B. gymnorhiza maintained ROS homeostasis as external NaCl saline increased from 100 to 400 mM but via different pathways:
K. obovata restricted the increase of salt influx, which is necessary to avoid abrupt increase of ROS. Moreover, K. obovata was sensitive to lower salt stress and rapidly initiated antioxidant defense to scavenge active oxygen species by, at least in part, components of the ASC-GSH cycle, e.g., SOD, APX, CAT, and GR. The Na+/H+ antiport system and proton pumps, which accelerate the salt exclusion across the plasma membrane, need to be further investigated.
B. gymnorhiza maintained higher capacity to detoxify ROS at high salinity; furthermore, the effective vacuolar salt compartmentation in mesophyll cells and the preferential accumulation of Na+ and Cl− in epidermal vacuoles may benefit B. gymnorhiza plants to reduce ROS production in the mesophyll. Together with antioxidant mechanisms, both enzymatic and nonenzymatic, the critical balance between ROS production and ROS detoxification is remained under salt stress. To elucidate the mechanism underlying the vacuolar compartmentation, critical ion channels and transporters in the vacuolar membranes need to be identified in future investigations.
The research was supported jointly by the National Natural Science Foundation of China (grant nos. 31770643, 31570587, and 31160150), Beijing Natural Science Foundation (grant no. 6182030), the Research Project of the Chinese Ministry of Education (grant no. 113013A), the Program of Introducing Talents of Discipline to Universities (111 Project, grant no. B13007), and the Natural Science Foundation of Hainan Province (grant no. 30408). Ms. Huijuan Zhu, Ms. Yunxia Zhang, Mr. Yong Shi, and Mr. Jie Shao all from Beijing Forestry University are greatly acknowledged for their assistance in electrophoresis and activity measurements of antioxidant enzymes. We thank Ms. Hui Zhang (Beijing Forestry University) for her contribution to the SEM-EDAX analysis.
Conflict of interest
The authors declare that there is no conflict of interest.