Physiological and biochemical responses to salt stress in the mangrove pptx

Transpiration rates showed a 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.

Physiological and biochemical responses to salt

stress in the mangrove, Bruguiera gymnorrhiza

Taro Takemuraa, Nobutaka Hanagataa,∗, Koichi Sugiharaa, Shigeyuki Babab, Isao Karubea, Zvy Dubinskya,c

aRCAST Research Center for Advanced Science and Technology, The University of Tokyo,

4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

bCollege of Agriculture, University of Ryukyus, Okinawa 903-01, Japan

cFaculty 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 of the mangrove, Bruguiera gymnorrhiza The growth rates and leaf areas were highest

in the culture with 125 mM NaCl Transpiration rates showed a 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-stimmedi-ate rimmedi-ates of transpiration and light-saturimmedi-ated rimmedi-ates 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: S 0 3 0 4 - 3 7 7 0 ( 0 0 ) 0 0 1 0 6 - 6

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 CO2, 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

CO2supply, 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

Fukushima et al (1997) found that in the salt tolerant mangrove A marina oxygen demand

for respiration increased under high salinities Using14C 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, PN, equals the residuum of gross photosynthesis, PG, 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,

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 500 mM 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

leaf level was approximately 150␮mol m−2s−1 Light was always measured as scalar

irradi-ance with Biospherical Instruments (San Diego, CA) QSL-100 m equipped with a 4␲ 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

Young plants cultured in the controlled environmental chamber with water for 4–6 months were transferred with roots intact into a flask filled with water containing the desired concen-tration of NaCl (0, 125, 250 or 500 mM) The flask was filled completely, avoiding any air bubbles, and connected to a 5 ml 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 cm3glass cuvette (2 cm in height, 10 cm in diameter) The cuvette was connected to

an infrared gas analyzer (Model RI-550A, RIKEN, Japan) through a series of gas-wash with bottles filled with anhydrous CaSO4and CoCl2as 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 30 min to each of a series of photon irradiances between 0 and 2000␮mol m−2s−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 CO2evolution (in the dark) or uptake (in the light) was established which usually required ca 5 min

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 10 cm 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 1 ml g−1fresh weight of 50 mM phosphate buffer (pH 7.0) containing

2 mM MgCl2, 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

All enzyme activity measurements were done in vitro The changes in activities over time, 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 1 shows 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 250 mM The leaf area was similar at 0, 125 and 250 mM NaCl, but at 500 mM

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 125 mM 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 ( ␮g cm −2)c

Chl a Chl b

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.

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 500 mM 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

3.3 Photosynthesis rate

Photosynthesis was adversely affected by salt The light-saturated rates of photosynthesis

(Pmax) 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: (䊊) 0 mM; (䊐) 125 mM; ( 4) 250 mM and (䉫) 500 mM Results are means ±1 S.D of five determinations

using different leaves.

Fig 3 Time course of salt accumulation in different tissues of Bruguiera gymnorrhiza seedlings during 16 days

following their transfer from water to a 500 mM NaCl solution (䊊) Stem xylem; (䊐) stem cortex; ( 4) root xylem

and (䊉) 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−2s−1, and saturated above 450␮mol m−2s−1 Also, the saturation irradiance

(Ik) of photosynthesis in the plants cultured in 125, 250 and 500 mM NaCl solutions were

330, 280 and 210␮mol m−2s−1, respectively, reflecting the correspondingly decreasing

Pmaxvalues The error bars clearly suggest that at least the differences between the fresh-water and full seafresh-water treated plants were significant in all photosynthetic parameters,

initial slope, Ikand Pmax 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 O2m−2leaf 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 O2m−2leaf s−1for 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␮mol m−2s−1at 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 shown in Fig 3 Sodium concentration in leaves increased rapidly, and reached a steady-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)

Fig 4 The effect of salinity on in vitro activities of superoxide dismutase (a) and catalase (b) Results are means ±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 500 mM 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

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)