Physiological and biochemical responses to salt stress in the mangrove pptx

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: (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).
T. Takemura et al. /Aquatic Botany 68 (2000) 15–28 25
Our transpiration data reveal a few interesting features. The transfer of B. gymnorrhiza
from water to any higher salinity,resulted in an immediate increase in transpiration. We sug-
gest 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 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 increased external osmoticum. They also revealed that sodium was ac-
cumulated into the vacuoles, suggesting that the vacuoles are the major site for intercellular
compartmentalization of NaCl.
The rates of light-saturated gross photosynthesis (P
max
) were nearly twice as high in B.
gymnorrhiza plants when in fresh water than in seawater. These differences are in the same
range as those reported by Ball and Pidsley (1995). Kawamitsu et al. (1995) also reported

a depression of photosynthesis (P
max
) in the Okinawa mangrove species Kandelia candel,
B. gymnorrhiza and Rhizophora stylosa in response to increased salinities. The decrease
in P
max
is attributable to restriction of CO
2
access through the stomata, an interpretation
strongly supported by the linear proportionality of photosynthesis and transpiration and leaf
conductance (Kawamitsu et al., 1995; Ball, 1988). In our study, the integrated photosyn-
thesis 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 this parameter, as 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 CO
2
, since under limiting light, there would be enough CO
2
influx to
match the photon flux.
We were unable to find such major differences in salt concentration between xylem sap
and medium as those reported by Scholander and co-workers (Scholander et al., 1962;
Scholander, 1968). As seen in Fig. 3, salt concentrations in sap from different plant parts
were always lower than in the medium. One would expect, according to Scholander, that
this mangrove would be able to exclude ∼99% of salts, and to have salt concentrations in
its sap on the order of 1% that of seawater, however, these were never nearly as low.
The effects of NaCl on two enzymes, SOD and catalase were examined. Our in vitro

experiments showed that these enzymes retained full activity at least up to seawater salt
levels. These enzymes differed in their response from the seven enzymes from the leaves of
the secreter mangrove A. marina, studied by Ashihara et al. (1997), six of which decreased
in activityasNaClconcentrationincreased,andonlyoneofthem,aldolasewasstimulatedto
peak activity by 250mM 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 spinach
was also reduced to about 30% that of the control (data not shown). Thiyagarajah et al.
(1996) compared the response to salt of various cytoplasmatic enzymes to that of ones
26 T. Takemura et al. /Aquatic Botany 68 (2000) 15–28
located in the cell wall. In their study they found that the wall enzymes of both halophytes
and glycophytes are far more salt tolerant than cytoplasmatic ones. For most enzymes 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 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
wholeplantto NaClsolution.Interestingly,the responseofthese twoenzymesdiffered,since
catalaserespondedsimilarlyto 500 mM ofeithersaltorsucrose, whereas theactivityofSOD
decreased by 50% when exposed to sucrose (data not shown). This illustrates the distinct
mechanisms, which govern the response of these two enzymes to salt; SOD is controlled by
the salt, while catalase seems to respond to the osmoticum regardless of its chemical nature.
Fadzillaet al.(1997)reported thattheactivitiesof Mn-SODandCu/Zn-SOD ofsalt-sensitive
Oryza sativa were significantly increased by NaCl stress, but the activities of ascorbate
peroxidase and catalase were not changed. They also indicated that glutathion reductase
activity was immediately increased by NaCl stress, however, extensive increase of lipid

peroxidationwasnotobserved.InNicotianatabacum,NaCl stressinduced2–3-foldincrease
in activitiesofFe-SOD,Cu/Zn-SOD,ascorbateperoxidase,dehydroascorbatereductaseand
glutathionreductase(Van-Campet al.,1996).Thereisno doubtthatexposuretohigh salinity
incurs water stress, which has been demonstrated to elicit different antioxidative defenses
in plants, invariably including SOD, ascorbate peroxidase and catalase (Del Longo et al.,
1993; Hernández et al., 1993a,b, 1995; Gosset et al., 1994, 1996; Larson, 1995). Our results
show that mangroves respond to NaCl by a steep increase in the antioxidant enzymes SOD
and catalase, and that the activitiesof these enzymes were not affectedbyhigh concentration
of NaCl.
Our study was conducted using laboratory grown materials. All the material plants were
grown under the same nutrient regimes and controlled irradiance and temperature condi-
tions. Since the photon irradiance was only 150 ␮mol m
−2
s
−1
, the responses obtained in
this study are limited to the low irradiance conditions common inside the thick mangal
where many of the seedlings develop.
Acknowledgements
An endowed chair in Environmental Biotechnology funded by the EBARA Corporation,
Japan, supported this work.
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