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Aquatic Botany 88 (2008) 303310 www.elsevier.com/locate/aquabot

Effect of NaCl on photosynthesis, salt accumulation and ion compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorhiza
Niya Li a,c, Shaoliang Chen a,b,*, Xiaoyang Zhou a, Chunyan Li a, Jie Shao a, ttermann d, Andrea Polle d Ruigang Wang a, Eberhard Fritz d, Aloys Hu
a

College of Biological Sciences and Technology (Box 162), Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants (Ministry of Education), Beijing Forestry University, Beijing 100083, PR China b Key Laboratory of Biological Resources Protection and Utilization in Hubei Province, Hubei Institute for Nationalities, Enshi 445000, PR China c Department of Biology, Hainan Normal University, Haikou 571158, PR China d r Forstbotanik, Georg-August-Universita t Go ttingen, Bu sgenweg 2, 37077 Go ttingen, Germany Institut fu Received 29 August 2006; received in revised form 22 November 2007; accepted 3 December 2007 Available online 8 December 2007

Abstract In a 4-week study, we investigated the effects of increasing soil NaCl (100400 mM) on photosynthesis, salt uptake and transport, and intracellular compartmentation of Na+ and Cl in 1-year-old seedlings of Kandelia candel (L.) Druce and Bruguiera gymnorhiza (L.) Savigny. Increasing NaCl stress signicantly elevated Na+ and Cl in root and shoot tissues (stem + leaf) of both species, but B. gymnorhiza showed a rapid Na+ accumulation upon the initiation of salt stress and leaves contained 90% more Na+ and 40% more Cl than K. candel at the end of experiment. Net photosynthetic rate (Pn) declined with increasing salinity, and the most marked reduction occurred after exposure of mangrove seedlings to a severe salinity, 400 mM NaCl. However, the inhibitory effects of severe stress varied with species: Pn decreased by 80% in K. candel whereas in B. gymnorhiza the decline was 60%. The quantum yield (AQY) and carboxylation efciency (CE) response to severe salinity showed a trend similar to Pn, in which a lesser reduction of AQYand CE was observed in B. gymnorhiza (3335%), as compared to K. candel (4352%). X-ray microanalysis of leaf mesophyll cells showed evidence of distinct vacuolar compartmentation of Na+ in K. candel but Cl in B. gymnorhiza after seedlings were subjected to 100 mM NaCl for 7 d. Moreover, Na+ within cell wall, cytoplasm, vacuole and chloroplast remained 2372% lower in stressed B. gymnorhiza as compared to K. candel. In conclusion, B. gymnorhiza exhibited effective salt exclusion from chloroplasts although increasing salt stress caused a rapid and higher build up of Na+ and Cl in the leaves. We suggest that the salt-induced Pn reduction in the two mangrove species is correlated with the ability to exclude Na+ and Cl from the chloroplast, rather than with the bulk leaf salt concentration. # 2007 Elsevier B.V. All rights reserved.
Keywords: Bruguiera gymnorhiza; Kandelia candel; Mangrove; Salt stress; X-ray microanalysis; Ion compartmentation

1. Introduction Mangrove forest forms an unique community in tropical coastal regions and tidal lowlands (Takemura et al., 2000). The most striking feature of mangroves is the ability to tolerate the seawater level of NaCl (ca. 500 mM). Mangroves maintain cell turgor by accumulating inorganic ions in the
* Corresponding author at: College of Biological Sciences and Technology (Box 162), Beijing Forestry University, Qinghua East-Road 35, Haidian District, Beijing 100083, PR China. Tel.: +86 10 62338129; fax: +86 10 62336164. E-mail address: Lschen@bjfu.edu.cn (S. Chen). 0304-3770/$ see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.12.003

vacuole and organic compounds in the cytoplasm (Jennings, 1976; Popp, 1984). Moreover, some mangrove species have developed morphological alternations to adapt to saline environment, e.g. salt glands, leaf succulence (Tomlinson, 1986). Bruguiera gymnorhiza (L.) Savigny and Kandelia candel (L.) Druce are two major mangrove species along southern China coastlines. These two species have no salt glands, but hypocotyls are considered as an additional lter to restrict salt transport to shoot in B. gymnorhiza (Lawton et al., 1981). Scholander (1968) and Paliyavuth et al. (2004) found that non-secretors, e.g. B. gymnorhiza, excluded 99% of the salt by root ultraltration. However, a high level of sodium in

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xylem sap of B. gymnorhiza was reported by Takemura et al. (2000). These results suggest that the capacity for salt exclusion in mangroves likely depends on the concentration of NaCl in the external solutions and the duration of salt exposure. However, the mechanism for salt uptake and transport regulation has not been compared in B. gymnorhiza and K. candel. Salinity reduced leaf photosynthesis in mangrove plants (Ball and Farquhar, 1984; Werner and Stelzer, 1990; Parida et al., 2003). Hui et al. (2004) conclude that the inhibition of photosynthesis in Lycium barbarum (a non-mangrove) after the onset of salt stress is mainly caused by temporary stomatal limitations, whereas non-stomatal limitations contribute to the reduction in photosynthesis when salinity is of longer duration. It is suggested that salt-induced structure alternations of mesophyll or chloroplasts limited CO2 assimilation in both mangroves and crops (Kao and Tsai, 1999; Mitsuya et al., 2000; Parida et al., 2004). Parida et al. (2003) found that a 45-d NaCl stress (400 mM) caused structural alterations and functional loss in thylakoid membranes. In a recent study, Parida et al. (2004) showed that high salinity reduced the diffusion of CO2 to the chloroplast of B. parviora, resulting from stomatal closure and changes in mesophyll structure. Salt-induced Na+ accumulation in leaves may decrease the stability of PSII functions and inhibit photosynthetic electron transport (Muranaka et al., 2002). However, salt compartmentation in chloroplasts and the effects of salt ions on light and dark reaction needs to be investigated. Two typical non-secretor mangroves B. gymnorhiza and K. candel were used in this study. We attempted to evaluate genotypic differences in salt uptake and transport control. The effects of increasing NaCl level on photosynthesis were compared in the two species. Furthermore, the inhibition of leaf salt on photosynthesis, and the relevance to intracellular salt compartmentation in mesophyll cells were discussed. 2. Materials and methods 2.1. Plant materials Propagules of K. candel (L.) Druce and B. gymnorhiza (L.) Savigny were obtained from Dongzhai Harbor in Hainan Province of China (latitude 198 510 N and longitude 1108240 E). Propagules were collected from the surface of soil or seawater during the ebb tide. Single hypocotyl was planted in individual pots (15 cm in diameter and 18 cm in height) containing sand and placed in a greenhouse at Beijing Forestry University, China. Seedlings were raised from March to August under non-saline conditions. The photosynthetically active radiation (PAR) was 400 800 mmol m2 s1 and relative humidity was maintained at 6070%. The pots were fertilized with 1 L 50% (v/v) Hoaglands nutrient solution every 14 d. Salt treatment was started when the 4th pair of leaves came out from the apex of the growing shoot (August).

2.2. Salt treatment NaCl treatment started from 100 mM and increased stepwise by weekly 100 mM, reaching 400 mM in the fourth week. Control plants were kept well-watered with no addition of NaCl. Destructive harvests were made after 7, 14, 21 and 28 d of exposure to the initial saline treatment. All sampled materials (root, leaf, stem, hypocotyl) were oven-dried (65 8C for 6 d) to obtain the dry weight. Dried samples were ground into powder and stored for mineral analysis. Three replicated seedlings per treatment were harvested at each sampling time. 2.3. Gas exchange measurements Gas exchange measurements were carried out after 7, 14, 21, and 28 d of salt treatment. Diel courses of net photosynthetic rate (Pn), unit transpiration rate (Tr) and stomatal conductance (Gs) of upper mature leaves were measured with a CIRAS-2 portable photosynthesis system (PP Systems Ltd., UK) under natural conditions (PAR was 4001200 mmol m2 s1 and air temperature was 2035 8C). PnPAR curves were measured over a range of PAR (from 1500 to 20 mmol m2 s1) supplemented with dysprosium lamps. Four to ve stable values were recorded from different plants at each irradiance. Measurements of leaf gas exchange as a function of CO2 concentrations (PnCi curves) were carried out with an ADC gas diluter (GD-602-GC), which produced a series of intermediate CO2 concentration from a single cylinder containing 1000 mmol mol1 CO2. Externally supplied CO2 concentration decreased stepwise by 100 mmol mol1 CO2 (10000 mmol mol1) and the irradiance was maintained at saturating value of 1000 mmol m2 s1 which was obtained from PnPAR curves. 2.4. Ions analysis of plant tissues 2.4.1. Sodium Duplicated 0.3 g dried samples were digested with 98% H2SO4 supplemented with H2O2 (30%, v/v) and Na+ concentrations were measured by atomic absorption spectrophotometer (Chen et al., 2001). 2.4.2. Chloride Samples of equal mass were extracted with 1N HNO3 as described in Storey (1995) and Cl determined by a modied method of silver titration as described in Chen et al. (2001). Briey, excess AgNO3 solution was used to precipitate the Cl of aqueous extracts and excess Ag+ was estimated by NH4SCN titration. NH4Fe(SO4)2 was used as a colour indicator for isoionic point determination. Cl concentration was calculated using the following formula: Cl mmol g1 Dry Wt NAgNO3 V 1 NNH4 SCN V 2 Dry Wt

where NAgNO3 and NNH4 SCN are the concentrations (mol L1) of AgNO3 and NH4SCN solutions, Dry Wt is the dry mass (g) of

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plant tissue, V1 is the total volume (mL) of AgNO3 solution in Cl extracts, and V2 is the volume (mL) of NH4SCN solution used for excess Ag+ precipitation. 2.5. Sample preparation for X-ray microanalysis At the rst harvest time (7 d), leaf segments of ca 3 mm long and 12 mm wide were cut along the central vein and placed into aluminum sample holders and rapidly frozen in a 2:1 mixture of propane: isopentane at the temperature of liquid nitrogen. Standard procedures required for sample preparation, i.e. freeze-drying, inltration and polymerization were followed as described in Fritz (1989) and Chen et al. (2002, 2003). 2.6. X-ray microanalysis Polymerised leaf samples were cut into 1 mm thick sections using a dry glass knife with an ultramicrotome. The slices were mounted in copper grids (mesh 50), coated with carbon and stored over silica gel until analysis. Sections were examined with a HITACHI-H800 transmission electron microscope assembled with an EDAX-9100 energy-disperse X-ray analyzer. The accelerating voltage was 150 kV with the take-off angle of 258. The counting time for all analyses was 60 s. The data were expressed as counts per second (cps) of an element peak after manual tting of the background. Probe measurements were made in mesophyll cells and following cell compartments were measured: cell walls, cytoplasm, chloroplasts and vacuoles. Seven measurements were taken from each leaf section. 2.7. Data analysis Data were subjected to ANOVA and signicant differences between means were determined by Duncans multiple-range test. Unless otherwise stated, differences were considered statistically signicant when p < 0.05. 3. Results 3.1. Gas exchange response to salt stress Net photosynthetic rate (Pn) in control plants of the two mangroves uctuated over the observation period, 10:00 18:00 h, exhibiting peaking values at 10:0011:00 h and 14:0016:00 h, respectively (Fig. 1). Stomatal conductance (Gs) followed the same trend of Pn, but unit transpiration rate (Tr) exhibited peaking value at 12:0013:00 h (Fig. 1). Increasing NaCl decreased gas exchange in both species, and Pn, Gs and Tr reached the minimum at 400 mM NaCl (Fig. 1). However, the inhibitory effects of severe salinity were more pronounced in K. candel, e.g. mean value of Pn decreased by 80% in K. candel, whereas a 60% reduction was observed in B. gymnorhiza (Fig. 1). 3.2. Pn versus PAR and Pn versus Ci relationships Compared with control K. candel plants, non-salinised B. gymnorhiza plants typically had a higher apparent quantum

yield (AQY), CO2 compensation point (CCP) and carboxylation efciency (CE), but the light compensation point (LCP) was lower (Table 1). After exposure of seedlings to salt solution, LCP and CCP increased concomitantly with increasing salt levels, and the increase was paralleled by decreases in AQYand CE in the two species (Table 1). The inhibition of light and dark reaction varied with species, especially under high saline conditions, e.g. 400 mM NaCl. (i) LCP and CCP in salinised B. gymnorhiza increased to 34 mmol m2 s1 photon and 202 mmol mol1 CO2, while they reached 65 mmol m2 s1 photon and 215 mmol mol1 CO2, respectively, in stressed K. candel (Table 1). (ii) AQY and CE in B. gymnorhiza decreased by 3335%, but in K. candel the extent of decline was 43-52% (Table 1). 3.3. Salt ion concentrations in roots, leaves, stem and hypocotyls 3.3.1. Sodium Under non-saline conditions, K. candel contained 0.1 0.2 mmol g1 Dry Wt Na+ in roots and leaves, 0.3 0.4 mmol g1 Dry Wt in hypocotyls and stem, respectively (Fig. 2). And an even higher level of tissue Na+ was recorded in control B. gymnorhiza (Fig. 2). External NaCl signicantly increased Na+ in root and shoot (stem and leaves) of both species, except hypocotyls, where Na+ remained at control levels during 4 weeks of salt exposure (Fig. 2). Differences in the pattern of Na+ accumulation are apparent in the two mangroves. Root and shoot Na+ in K. candel gradually increased corresponded with the increase in salinity and with the duration of exposure, although Na+ in root and leaf remained unchanged at 100 mM NaCl (Fig. 2). Unlike K. candel, Na+ in root and shoot rapidly increased after exposure of B. gymnorhiza seedlings to 100 mM NaCl (Fig. 2). Moreover, stressed B. gymnorhiza leaves accumulated 90% more Na+ than K. candel at the end of experiment (Fig. 2). 3.3.2. Chloride Tissue Cl varied from 0.2 to 0.6 mmol g1 Dry Wt in control plants of K. candel and B. gymnorhiza (Fig. 3). NaClinduced Cl concentration occurred in all measured tissues of both species with the exception of hypocotyl in B. gymnorhiza (Fig. 3). Root and leaf Cl showed a tendency to increase with increasing salinity in both species, but a more marked Cl accumulation took place in B. gymnorhiza at the end of experiment (Fig. 3). The pattern of Cl changes in woody tissues of K. candel differs from that in B. gymnorhiza. Hypocotyl Cl in K. candel rapidly increased from 0.18 to 0.55 mmol g1 Dry Wt after the onset of salt stress, but there was no corresponding change in B. gymnorhiza (Fig. 3). A converse trend was observed in stem where Cl increased more rapidly in B. gymnorhiza than in K. candel (Fig. 3). 3.4. Ion distribution within leaf mesophyll cells In mesophyll cells of control K. candel leaves, Na+ and Cl concentrations within the apoplast (cell wall), ranging from 40

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Fig. 1. Effects of increasing NaCl on diel course of net photosynthetic rate (Pn), stomatal conductance (Gs) and unit transpiration rate (Tr) in leaves of K. candel and B. gymnorhiza. Each point is the mean of three plants and bars represent the standard error of the mean.

to 45 cps were higher than that in the vacuole and symplast, 23 39 cps (Table 2). Compared with K. candel, control B. gymnorhiza exhibited higher Cl within cell compartments, but Na+ fractions were lower (Table 2). Salt stress (100 mM NaCl, 7d) caused a signicant rise of Na+ and Cl in mesophyll cells of both species, but there

were species-specic differences in the pattern of salt compartmentation (Table 2). Vacuolar Na+ compartmentation was clearly seen in K. candel, in which the fraction of Na+ remained higher in the vacuole than in the cytoplasm and chloroplast (Table 2). In contrast, there was no evidence of Cl compartmentation in the vacuole (Table 2). As distinct

Table 1 Effects of increasing NaCl on PnPAR and PnCi curves in leaves of K. candel and B. gymnorhiza seedlings (LCP: light compensation point; AQY: apparent quantum yield; CCP: CO2 compensation point; CE: carboxylation efciency) [NaCl] (mM) K. candel LCP (mmol m2 s1) 0 100 200 300 400 34.1 3.9 40.9 2.5 37.7 5.1 48.4 4.3 65.3 6.8 AQY (mol mol1) 0.016 0.001 0.014 0.001 0.012 0.001 0.010 0.001 0.008 0.001 CCP (mmol mol1) 78.6 6.5 126.8 8.6 134.7 11.2 165.9 9.5 214.5 13.8 CE (mol m2 s1) 0.020 0.002 0.015 0.001 0.013 0.001 0.013 0.001 0.011 0.001 B. gymnorhiza LCP (mmol m2 s1) 25.0 3.2 28.0 1.9 28.9 4.0 29.4 5.9 34.1 3.9 AQY (mol mol1) 0.024 0.003 0.020 0.001 0.020 0.001 0.017 0.001 0.016 0.001 CCP (mmol mol1) 110.5 10.1 131.2 7.9 148.2 11.3 168.7 9.2 201.7 12.9 CE (mol m2 s1) 0.025 0.001 0.023 0.001 0.021 0.001 0.019 0.001 0.016 0.001

Each value is the mean of three plants and four to ve measurements were taken from each plant at each designed irradiance or CO2 concentration.

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Fig. 2. Effects of increasing NaCl on Na+ concentrations in leaves, stem, hypocotyls and roots of K. candel and B. gymnorhiza. NaCl treatment started from 100 mM and increased stepwise by weekly 100 mM, reaching 400 mM in the fourth week. Control plants were kept well-watered with no addition of NaCl and four points on each control line represent mean values at weeks 1, 2, 3 and 4, respectively, after the initiation of experiment. Each point is the mean of three plants and bars represent the standard error of the mean.

from K. candel, B. gymnorhiza showed vacuolar compartmentation of Cl, relative to Na+ (Table 2). Noteworthy, B. gymnorhiza exhibited apparent Na+ and Cl exclusion from mesophyll cells. Stressed B. gymnorhiza remained 23 72% lower in Na+ in all measured cell compartments, cell wall, cytoplasm, vacuole and chloroplast, as compared to K. candel (Table 2). Likewise, a lesser extent of Cl increase occurred in the apoplast and symplast of B. gymnorhiza (Table 2).

4. Discussion 4.1. Salt uptake and transport in mangroves under salt stress In control plants of the two species, Na+ and Cl in roots, leaves and stem presumably originated from hypocotyls since propagules were collected from the surface of soil or seawater in coastal habitats of mangrove forest. Mangrove propagules

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can absorb sodium and chloride ions very quickly when they contact seawater. Increasing NaCl signicantly elevated Na+ and Cl in root and shoot of seedlings, but the pattern of salt accumulation revealed striking differences between the two mangroves. Compared with K. candel, a more rapid increase in Na+ concentration occurred in leaf and root of B. gymnorhiza upon the initiation of salt stress (100 mM NaCl) and leaf salt accumulated to a higher level at the end of salt exposure

(400 mM NaCl). The marked exclusion of Na+ and Cl from leaf tissue of K. candel presumably resulted from the root ultraltration. Mallery and Teas (1984) found that nearly all of the incorporated 36Cl was still located within the roots of Rhizophora mangle and only 0.2% had passed into the leaves after 48 h of stress; similarly, leaves remained totally free of the 22 Na+ isotope although 79% of 22Na+ had translocated to the stem and the hypocotyls.

Fig. 3. Effects of increasing NaCl on Cl concentrations in leaves, stem, hypocotyls and roots of K. candel and B. gymnorhiza. NaCl treatment started from 100 mM and increased stepwise by weekly 100 mM, reaching 400 mM in the fourth week. Control plants were kept well-watered with no addition of NaCl and four points on each control line represent mean values at weeks 1, 2, 3 and 4, respectively, after the initiation of experiment. Each point is the mean of three plants and bars represent the standard error of the mean.

N. Li et al. / Aquatic Botany 88 (2008) 303310 Table 2 Intracellular salt compartmentation in mesophyll cells of K. candel and B. gymnhorhiza Compartment [NaCl] (mM) K. candel Na (cps) Cell wall Cytoplasm Vacuole Chloroplast 0 100 0 100 0 100 0 100 45.4 3.0 106.1 9.2 25.6 1.6 41.3 2.8 39.2 3.3 102.7 9.9 22.7 2.1 35.4 3.0
+

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B. gymnorhiza Cl (cps) 40.2 3.5 82.3 5.1 34.6 2.7 112.6 10.1 35.6 1.8 82.0 4.1 30.0 2.9 114.8 10.8

Na+ (cps) 23.4 2.5 33.4 3.4 15.3 1.2 27.8 1.7 20.3 1.9 28.4 1.9 12.5 1.2 27.4 3.3

Cl (cps) 82.4 3.2 110.2 12.8 50.3 1.2 81.8 4.0 79.8 4.6 105.8 9.8 44.1 2.9 72.3 3.5

Each value is the mean of three plants and seven measurements (for each compartment) were taken from each leaf section.

4.2. Ion compartmentation within leaf cells and the relevance to photosynthesis X-ray microanalysis of leaf mesophyll cells showed that the fractions of Na+ and Cl in cell compartments increased in both species after exposure to 100 mM NaCl for 7 d, but there were signicant differences in the pattern of vacuolar salt compartmentation. K. candel had a greater ability to compartmentalise Na+, relative to Cl, into vacuoles, while B. gymnorhiza was efcient at vacuolar Cl compartmentation. The pattern of Na+ compartmentation in K. candel is similar to B. sexangula, in which Na+ concentration in the vacuole was enhanced by the treatment with 50 mM NaCl (Tanaka et al., 2000). Compartmentalising NaCl into the vacuole is likely to depend on Na+/H+ antiporter systems (Blumwald and Poole, 1985; Garbarino and Dupont, 1988), H+-coupled Cl antiport (Schumaker and Sze, 1987) or ion channels (Pantoja et al., 1989; Maathuis and Prins, 1990). Ion compartmentation in the vacuole would limit excessive salt accumulation in the symplast, thus protecting salt-sensitive enzymes in the cytoplasm and chloroplasts. Hence, the ability to maintain lower Na+ and Cl in the symplast may be an underlying determinant of the tolerance of mangroves. The decline of quantum yield (AQY), carboxylation efciency (CE) and net photosynthetic rate (Pn) in the two species gradually increased corresponded with the increase in leaf Na+ and Cl, implying that salt build up in leaves had detrimental effects on light and dark reactions. Compared with B. gymnorhiza, the inhibitory effect was more pronounced in K. candel although leaf Na+ and Cl accumulated to a lesser extent under severe salinity, suggesting that variations in the reduction of photosynthesis are correlated with the ability to exclude Na+ and Cl from the chloroplast, rather than with the bulk leaf salt concentration. Leaf cells of K. candel showed a lower capacity to exclude Na+ and Cl from the chloroplast, as compared to B. gymnorhiza. Higher Na+ and Cl content in the chloroplast would inhibit PSII functions, photosynthetic electron transport and the activity of ribulose bisphosphate carboxylase oxygenase (Rubisco). Similarly, photosynthesis was severely reduced in a salt-sensitive poplar with lower capacity to exclude salt from chloroplasts (Wang et al., 2007).

In conclusion, seedlings of the two tested mangroves could survive 400 mM salt although leaf photosynthesis was signicantly restricted. However, differences for salt adaptation are apparent: 1. At the whole-plant level, K. candel exhibited a higher capacity to restrict salt uptake and subsequent accumulation under increasing salt stress. 2. NaCl-treated B. gymnorhiza had a higher capacity to exclude salts from mesophyll cells although salt treatment caused a rapid and marked build up of Na+ and Cl in leaves. The lesser reduction in photosynthesis could be a consequence of salt exclusion from mesophyll chloroplasts.

Acknowledgements The research was supported jointly by the Alexander von Humboldt-Stiftung/Foundation (Germany), German Science Foundation through Poplar Research Group Germany (PRG), the key project of National Natural Science Foundation of China (30430430), the HI-TECH Research and Development Program of China (863 Program, 2006AA10Z131), a Foundation for the Author of National Excellent Doctoral Dissertation of PR China (200152), the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institution of MOE, PRC (2002-323) and Natural Science Foundation of Hainan Province (30408). We thank Dr. Jinchi Zhou for valuable help with ion analysis. References
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