Aquatic Botany 68 (2000) 15–28 Physiological and biochemical responses to salt stress in the mangrove, Bruguiera gymnorrhiza Taro Takemura a , Nobutaka Hanagata a,∗ , Koichi Sugihara a , Shigeyuki Baba b , Isao Karube a , Zvy Dubinsky a,c a RCAST Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan b College of Agriculture, University of Ryukyus, Okinawa 903-01, Japan c Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Received 27 July 1998; received in revised form 29 March 2000; accepted 20 April 2000 Abstract Physiological and biochemical responses induced by salt stress were studied in laboratory-grown young plants ofthemangrove, Bruguieragymnorrhiza.Thegrowthratesandleafareas were highest in the culture with125mM NaCl. Transpirationratesshoweda diel periodicity when the plants were placed in water, but the oscillatory cycles disappeared for plants placed in higher salt concentration (250–500 mM NaCl). The transfer of plants from water to any higher salinity resulted in an immedi- ate increase in transpiration. Both the steady-state rates of transpiration and light-saturated rates of photosynthesis decreased as the salt concentration was increased. The activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase, showed an immediate increase after the plants were transferred from water to high salinity, reaching in 10 days five and eight times those of initial activities, respectively.The activities of these two enzymes were not affected by salt concentrations up to 1000 mM NaCl, twice that of seawater. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bruguiera gymnorrhiza; Catalase; Mangrove; Photosynthesis; Salt; Superoxide dismutase; Transpiration 1. Introduction Mangroves form unique communities in tropical coastal regions and tidal lowlands. They are considered an ecologically essential component in protecting adjacent land from wave and storm erosion (Banijbatana, 1957; Savage, 1972) while preventing terrigenous nutrients from affecting nearby reefs (Dubinsky and Stambler, 1996). ∗ Corresponding author. Tel.: +81-3-5452-5320; fax: +81-3-5452-5320. E-mail address: hanagata@bio.rcast.u-tokyo.ac.jp (N. Hanagata) 0304-3770/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0304-3770(00)00106-6 16 T. Takemura et al./ Aquatic Botany 68 (2000) 15–28 The most striking feature of mangroves is their ability to tolerate NaCl to seawater level (500 mM). From the physiological aspects, the effects of salinity on the photosynthesis of mangroves have been studied to some extent, mostly in relation to transpiration and stomatal conductance. Ball and Farquhar (1984a,b) found that unlike in the salt-sensitive Aegiceras corniculatum, in the most salt tolerant mangrove, Avicennia marina, photosynthesis rates were barely affected by salinity. The depression of carbon assimilation that was observed in A. corniculatum was attributable to a reduction in stomatal opening. The likely sequence of events was thought to be increased salinity, water stress, stomatal closure, decrease in intracellular CO 2 , and decrease in photosynthesis. The linear relation between photosyn- thesis and transpiration rates, the latter being a good proxy for stomatal opening, was shown in all species studied but with slopes reflecting their various salt tolerances. By increasing CO 2 supply, it was demonstrated that high salinities depress photosynthesis directly, prob- ably through partial inhibition of the activity of RUBISCO (Kotmire and Bhosale, 1985; Nazaenko, 1992), in addition to the stomatal-closure-mediated effects. Since plant growth amounts to the balance sheet of photosynthesis gains after the de- duction of respiratory losses, it is to be expected that whatever effects salinity has on these processes will be reflected in the growth rate of the plant integrated over time. Any stress exerted on an organism increases maintenance costs, as reflected in its respiration. High temperatures, excessively high light, drought, disease and high salinities were all shown to activate ‘heat shock protein’ related genes (Morimoto, 1991; McKersie and Leshem, 1994; Lichtenthaler, 1996) and to elicit an increase in plant respiration. The available data on the response of mangroves to salinity are no exception. In the few available studies, high salinities were shown to increase respiration, and in the case of A. corniculatum, reduce photosynthesis. Fukushimaetal. (1997)foundthatin thesalttolerant mangroveA. marinaoxygendemand for respiration increased under high salinities. Using 14 C labeling they demonstrated that under high salinity sucrose was diverted from macromolecule biosynthesis to respiration. Burchett et al. (1989) also found a salt induced increase in respiration in both A. marina and A. corniculatum, with a higher increase in the more salt-sensitive A. corniculatum. Net photosynthesis, P N , equals the residuum of gross photosynthesis, P G , once the res- piration, R, is subtracted. Therefore, when only net photosynthesis or its corroboratory growth is measured, there is no way to attribute the effects of salinity on photosynthesis or respiration separately. Ball and Pidsley (1995) found that the salt related decrease in daily dry-weight increment was more marked in the salt-sensitive Sonneratia lanceolata than in the hardier Sonneratia alba. One of the biochemical mechanisms by which mangroves counter the high osmolarity of salt is an accumulation of compatible solutes. In a survey of 23 mangrove species from northern Queensland, Popp et al. (1985) found that pinitol and mannitol were the most common compatible solutes. They also found proline in Xylocarpus species, and methy- lated quaternary ammonium compounds in two Avicennia species, in Acanthus ilicifolius, Heritiera littoralis and Hibiscus tiliaceus, whereas other species had small carbohydrates as the dominant osmoregulating compounds. Glycinebetaine, the most common compatible solute, has also been found in A. marina (Ashihara et al., 1997). The active oxygen species including superoxide, hydrogen peroxide and hydroxyl free radicals can be induced by various environmental stresses such as extreme temperatures, T. Takemura et al./ Aquatic Botany 68 (2000) 15–28 17 herbicides, drought and nutrient stress (Monk et al., 1989; Scandalios, 1993; Hernández et al., 1993a,b, 1995). It is known that higher plants resist active oxygen species by in- creasing the activities of antioxidant enzymes. Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide and oxygen (Fridovich, 1986). Hydrogen peroxide is decomposed by catalase and peroxidase (Fridovich, 1986; Salin, 1991). How- ever, information about the effect of salt stress on active oxygen metabolism is insufficient. In this paper, we present the effect of salt stress on growth, transpiration, photosynthesis, and activities of antioxidant enzymes using laboratory-cultured Bruguiera gymnorrhiza,a common mangrove in Okinawa, Japan. 2. Materials and methods 2.1. Source of plants and growth Seeds of B. gymnorrhiza were collected from Iriomote Island, Okinawa, Japan, at the end of May in 1997 and June in 1999. Fifteen seeds of B. gymnorrhiza were planted in each culture pot (15 cm in diameter and 14 cm in height) with vermiculite. The pots were irrigated every 2 days with water contain- ing 0, 125, 250 and 500mM NaCl. Salt concentration in each culture pot was checked once every month, either by analyzing water samples by inductively coupled plasma emission spectrometry (ICP) on a Optima 3000XL (Perkin-Elmer, USA) or directly in the pot, with a conductivity meter, and adjusted whenever needed. Liquid fertilizer was first added into the culture pots after the cotyledons had developed, and then supplied every month. These pots were placed in a culture room at 25 ◦ C with 12 h photoperiod. The photon irradiance at the leaflevelwasapproximately150 ␮mol m −2 s −1 .Lightwasalwaysmeasuredas scalarirradi- ancewithBiosphericalInstruments(SanDiego, CA)QSL-100 mequippedwitha4␲ sensor. The height of each plant and its leaf area were measured after 4 months. The height was averaged for all plants in the pot, and the leaf areas were averaged for eight to nine leaves of the same age. 2.2. Transpiration Youngplantsculturedinthe controlled environmentalchamberwithwater for4–6months were transferred withrootsintactintoaflaskfilledwithwater containing thedesiredconcen- tration of NaCl (0, 125, 250 or 500 mM). The flask was filled completely, avoiding any air bubbles, and connected to a 5ml graduated cylinder. The water in the cylinder was covered with 5 mm layer of oil to prevent evaporation, and its level was monitored over 140 h. These experiments were conducted in the culture room at 25 ◦ C, and a 12 h photoperiod. Water was added as needed to prevent the effects of hydrostatic pressure. Results were normalized to leaf area. 2.3. Photosynthesis and respiration One leaf of an intact plant in one of the culture pots was enclosed in a flow-through 157 cm 3 glass cuvette (2 cm in height, 10 cm in diameter). The cuvette was connected to 18 T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 an infrared gas analyzer (Model RI-550A, RIKEN, Japan) through a series of gas-wash with bottles filled with anhydrous CaSO 4 and CoCl 2 as an indicator (Indicating Drierite, from W.A. Hammond, Xenia, OH, USA), in order to remove any humidity from the air. The internal air pump in the gas analyzer provided a flow of fresh air, which was monitored with a flow meter and adjusted to the rate of 1 l min −1 , flushing the cuvette ca. every 10 s. Carbon dioxide concentration in the dry air from the chamber was constantly recorded, and its uptake or evolution rate was then calculated from the flow rate, and normalized to leaf area. Each leaf was exposed for about 30min to each of a series of photon irradiances between 0 and 2000 ␮mol m −2 s −1 (4␲ sensor). For each salt concentration (0, 125, 250 and 500 mM NaCl), a photosynthesis versus irradiance relationship (P versus I curve) was established. Light was provided from a slide projector with a quartz-halogen lamp. The appropriate irradiance was obtained by changing the distance between the projector and the leaf. Dark respiration measurements were continued until the temperature of the chamber and airflow rates were stabilized, typically for 20–30 min. At each irradiance, measurement was continued until a constant rate of CO 2 evolution (in the dark) or uptake (in the light) was established which usually required ca. 5min. 2.4. Sodium concentration in sap and leaves A culture pot with 11 young plants grown for 4–6 months in water was used. The water in the culture pot was discarded and was replaced with water containing 500 mM NaCl. After 1, 2, 3, 9, 14 and 16 days, a plant was taken from the pot and washed with water. The root and leaves of the plant were removed, and the stems were cut into several segments. Each segment was separated into xylem and cortex. These tissues were put into tubes and centrifuged at 1300×g for 1 h to obtain 25–65 ␮l of sap from xylem and cortex of two to three segments of 10cm in length. One or two fresh leaves removed from the plant were homogenized in 2–3 ml of distilled water, using a mortar and pestle. The homogenate was centrifuged at 1500×g for 20 min to obtain supernatant. Samples of 20 ␮l from both the sap and supernatant were diluted to 50 ml with distilled water. Sodium concentrations in the sap and in the supernatant of the leaf homogenate were determined using ICP emission spectrometry on a Optima 3000XL (Perkin-Elmer, USA). 2.5. Enzyme assays One or two freshly collected leaves were weighed, and then homogenized in a chilled mortar and pestle in 1ml g −1 fresh weight of 50mM phosphate buffer (pH 7.0) containing 2 mM MgCl 2 , 1 mM EDTA and 0.1% (v/v) 2-mercaptoethanol. The resulting slurry was homogenized again with a URC 24 R (Ingenierbüro M. Zipperer Staufen, Germany) ho- mogenizer. The homogenate was centrifuged at 1500×g for 20 min at 4 ◦ C. The protein in the supernatant was precipitated in a 30–90% ammonium sulfate fraction, redissolved in the same buffer and desalted with Sephadex G-25 (Pharmacia Biotech, USA). The protein concentration for enzyme assay was measured with the Bio-Rad protein assay kit (Bio-Rad Laboratories, USA) using bovine serum albumin (BSA) as standard. The desalted protein was then used for all the enzyme activity measurements. T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 19 All enzyme activitymeasurementsweredone in vitro. The changes in activitiesovertime, following the transfer of the plants to the experimental salt solutions were determined. The SOD and catalase were assayed using the modified method of Elstner and Heupel (1976) and Ganschow and Schike (1969), respectively, and these results were then normalized to total protein in the sample. To investigate the effect of salt on enzyme activity, protein extracted from the same leaf was divided into five reaction mixtures containing 0, 250, 500, 1000 and 2000 mM NaCl in 3 ml total volume. 3. Results 3.1. Growth, leaf area and chlorophyll content Table 1shows the effect of salt concentration on the plant height, leaf area and chlorophyll (chl) content of B. gymnorrhiza. Plant height was greatest in the 125 mM NaCl solution, followed by 250mM. The leaf area was similar at 0, 125 and 250 mM NaCl, but at 500mM NaCl it was reduced by approximately half. Areal chl a content in leaves increased with NaCl concentration in the culture, while chl b content did not change in any of the salt treatments. 3.2. Transpiration rate Transpiration rates of B. gymnorrhiza, whose roots were immersed in water without NaCl, showed a strong diel periodicity, with daytime rates 5.4 higher than night time ones, ignoring the first light–dark cycle, in which the rate during the light period was lower than that of the dark one (Fig. 1). Such regular oscillatory cycles of the transpiration rate were also observed in the plant whose roots were placed in the 125mM NaCl solution, although their amplitude was considerably lower (light:dark=1.94). When the plants were placed at higher salt concentrations, the oscillations were dampened. In the plant in the 250 mM NaCl solution, the dampening of the oscillations began after the first cycle, and following two Table 1 The effect of salt concentration on plant height, leaf area and chlorophyll content in the leaves of Bruguiera gymnorrhiza seedling grown for 4 months NaCl concentration (mM) Plant height (cm) a Leaf area (cm 2 ) b Chlorophyll content (␮gcm −2 ) c Chl a Chl b 0 12.5±1.8 20.1±1.9 30.3±1.2 14.8±1.0 125 15.1±2.6 20.5±2.1 32.1±1.1 16.1±1.1 250 14.2±2.1 19.1±1.3 37.2±1.4 16.2±1.2 500 9.7±1.6 10.3±1.7 48.1±1.8 16.4±1.3 a Values were calculated for 9–13 plants in each condition. b Values were calculated for eight to nine leaves in each condition. c Chlorophyll content was averaged for eight to nine leaves in each condition. 20 T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 Fig. 1. Transpiration rates of Bruguiera gymnorrhiza seedlings kept in media of different salinities under 12 h photoperiod. Open bars and shaded bars represent light and dark periods, respectively. Results are means±1 S.D. of four determinations using different plants from the same treatment. strongly attenuated cycles, they disappeared completely. At that steady state transpiration continued at a very low rate with no detectable oscillations. In the 500mM NaCl plant, the oscillations were irregular. In the first light period, the transpiration rates of the plant whose roots potted into 125, 250 and 500 mM NaCl solutions were higher than those of the plant in the distilled water. T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 21 3.3. Photosynthesis rate Photosynthesis was adversely affected by salt. The light-saturated rates of photosynthesis (P max ) decreased, as salt in the culture medium was raised from 0 to 500 mM salt (Fig. 2). Fig. 2. The effect of salinity on the net (a) and gross (b) photosynthesis vs. irradiance relationships. NaCl concen- trations: ( ᭺)0mM;(ᮀ) 125 mM; () 250 mM and (᭛) 500mM. Resultsare means±1S.D. of fivedeterminations using different leaves. 22 T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 Fig. 3. Time course of salt accumulation in different tissues of Bruguiera gymnorrhiza seedlings during 16 days following their transfer from water to a 500mM NaCl solution. ( ᭺) Stem xylem; (ᮀ) stem cortex; () root xylem and ( ᭹) leaf. Results are means±1 S.D. of five determinations using different plants. In the plant cultured in water, the net photosynthesis rate was almost linear up to about 400 ␮mol m −2 s −1 , and saturated above 450␮mol m −2 s −1 . Also, the saturation irradiance (I k ) of photosynthesis in the plants cultured in 125, 250 and 500 mM NaCl solutions were 330, 280 and 210 ␮mol m −2 s −1 , respectively, reflecting the correspondingly decreasing P max values. The error bars clearly suggest that at least the differences between the fresh- water and full seawater treated plants were significant in all photosynthetic parameters, initial slope, I k and P max . The responses to intermediate salinities were significantly differ- ent from either freshwater or seawater at some, not all, values of the P versus I relationship. Compensation points and also dark respiration rate (4.54±0.36 ␮mol O 2 m −2 leaf s −1 )of the plants cultured in water were higher than those of cultures in any of the NaCl solu- tions (e.g. 2.98±0.38 ␮mol O 2 m −2 leaf s −1 for the seawater NaCl concentration). During the 5–10 min exposures required for our photosynthetic rate measurements, there was no evidence of photoinhibition up to 2000–2500␮molm −2 s −1 at any salt concentration. 3.4. Sodium concentration in sap and leaves Bruguiera gymnorrhiza grown in distilled water for 4–6 months was transferred into culture pots with 500 mM NaCl solution, and time courses of the changes in sodium con- centrations in the sap of stem xylem, stem cortex, root xylem and leaves were followed, as shownin Fig.3.Sodiumconcentrationin leavesincreasedrapidly,andreached asteady-state value in 3 days. Sodium concentration in the sap of stem xylem, stem cortex and root xylem increased up to 350–380 mM in 9 days. 3.5. Effect of NaCl on activities of enzymes There was no significant inhibition of the activities of SOD and catalase by in vitro concentration of NaCl up to 500 and 1000 mM, respectively (Fig. 4). T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 23 Fig. 4.The effectof salinity onin vitro activitiesof superoxide dismutase(a) and catalase(b). Results aremeans±1 S.D. of four determinations using leaf extract prepared from different plants from the same treatment. Activity unit of SOD is represented by the level of 50% inhibition of nitrite formation from hydroxylamine in the presence of xanthineoxidase. A steep increase in the activity of SOD began immediately following the transfer of the plant grown in water into a 500mM NaCl solution (Fig. 5). The SOD and catalase activities of leaves increased up to 8.1 and 4.9 times, respectively, and those of controls in 9 days. These enzyme activities increased until they leveled-off after 9 days, although the sodium concentration in leaves had already reached steady state values within 3 days (Fig. 3). 4. Discussion Bruguiera gymnorrhiza showed clear stunting of plants grown under seawater levels of NaCl (500 mM). The reduction in biomass accretion was evident from the reduction in leaf 24 T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 Fig. 5. Time course of the response of superoxide dismutase (a) and catalase (b) in 16 days following the transfer of Bruguiera gymnorrhiza seedlings from water to a 500 mM NaCl solution. Results are means±S.D. of four determinations using leaf extract prepared from different plants with the same treatment. Activity unit of SOD as in Fig. 4. growth and leaf area, although the increase in the dry-weight to leaf area had offset some of that decrease, as may be seen in the apparent increase in chl a content. Mangrove species differ in their growth-response to salinity. While the least salt tolerant species such as S. lanceolata showed clear preference for low salinities, with growth peaking between 0 and 5% of seawater concentrations, the more salt tolerant S. alba grew at the fastest rate at considerably higher concentrations, peaking at 25% of seawater. Furthermore, growth of the more salt tolerant species continued, albeit much reduced, even at salinities equal to those of seawater, and was clearly sub optimal in fresh water (Ball and Pidsley, 1995). [...]... that are salt- sensitive the problem is solved in mangroves in ways similar to those found in other halophytes or salt tolerant species The salt ions that contribute to the cell osmoticum are confined to the vacuole, thereby being kept apart from the sensitive enzymes Such compartmentalization was described in the halophyte A gmelini by Matoh et al (1987) It is likely that in mangroves all of these strategies... by Ball and Pidsley (1995) Kawamitsu et al (1995) also reported a depression of photosynthesis (Pmax ) in the Okinawa mangrove species Kandelia candel, B gymnorrhiza and Rhizophora stylosa in response to increased salinities The decrease in Pmax is attributable to restriction of CO2 access through the stomata, an interpretation strongly supported by the linear proportionality of photosynthesis and transpiration... an effect of salinity, such responses are evident in the data of Ball and Farquhar (1984b), Kotmire and Bhosale (1985) and Kawamitsu et al (1995) The decrease in the rate of photosynthesis under low light cannot be due to reduced stomatal access of CO2 , since under limiting light, there would be enough CO2 in ux to match the photon flux We were unable to find such major differences in salt concentration... internal salt concentrations to stabilize which demonstrates the close coupling of these two processes Such a response is likely to be of great ecological advantage under the fluctuating salinity regimes common in mangal communities exposed to the tidal and riverine interactions and seasonal changes in rainfall Matoh et al (1987) showed that Atriplex gmelini positively took up salt from roots to balance the. .. concentration increased, and only one of them, aldolase was stimulated to peak activity by 250 mM salt All seven enzymes were strongly inhibited at 500 mM NaCl They also detected glycinebetaine as compatible solute, and suggested that salt probably appears to be concentrated in vacuole, and glycinebetaine accumulated in the cytoplasm In our study, at 500 mM NaCl, the activity of SOD extracted from the halophyte... two mangrove species, Aegiceras corniculatum and Avicennia marina, to long term salinity and humidity conditions Plant Physiol 74, 1–6 Ball, M.C., Farquhar, G.D., 1984b Photosynthetic and stomata responses of the grey mangrove Avicennia marina to transient salinity conditions Plant Physiol 74, 7–11 Ball, M.C., Pidsley, S.M., 1995 Growth responses to salinity in relation to distribution of two mangrove. .. transpiration and leaf conductance (Kawamitsu et al., 1995; Ball, 1988) In our study, the integrated photosynthesis rates at the different salinities were related by similar ratios to their daytime rates of transpiration However, this explanation cannot be applied to the reduction in the initial slope of the photosynthesis versus irradiance curves Although in our data we were unable to find significant changes in. .. few interesting features The transfer of B gymnorrhiza from water to any higher salinity, resulted in an immediate increase in transpiration We suggest that this is a response aimed at increasing the internal salt concentration and eventually balance the increased external osmoticum The time needed to establish a steady-state rate of transpiration was on the same order as that required by the internal... are combined; salt ions are kept in the vacuole, whereas betain, glycerol and any other compatible solutes which do not adversely affect enzyme activity as well as the susceptible enzymes themselves are all in the cytosol Superoxide dismutase and catalase showed an immediate induction upon exposure of the whole plant to NaCl solution Interestingly, the response of these two enzymes differed, since catalase... marina and their effects on the activities of enzymes Z Naturforsh 52c, 433–440 T Takemura et al / Aquatic Botany 68 (2000) 15–28 27 Ball, M.C., 1988 Salinity tolerance in the mangroves Aegiceras corniculatum and Avicennia marina I Water use in relation to growth, carbon partitioning and salt balance Aust J Plant Physiol 15, 447–464 Ball, M.C., Farquhar, G.D., 1984a Photosynthetic and stomatal responses . in stomatal opening. The likely sequence of events was thought to be increased salinity, water stress, stomatal closure, decrease in intracellular CO 2 , and decrease in photosynthesis. The linear. respectively, and these results were then normalized to total protein in the sample. To investigate the effect of salt on enzyme activity, protein extracted from the same leaf was divided into five reaction. 15–28 The most striking feature of mangroves is their ability to tolerate NaCl to seawater level (500 mM). From the physiological aspects, the effects of salinity on the photosynthesis of mangroves