Physiological adaptability of three mangrove species to salt stress ppt

Net photosynthesis rate, stomata con-ductance and transpiration rate of leaves decreased and soluble sugar content in leaves increased, with salt concentration in all three mangrove sp

Volume 27, Issue 6, June 2007

Online English edition of the Chinese language journal

Cite this article as: Acta Ecologica Sinica, 2007, 27(6), 2208−2214

Received date: 2006-12-04; Accepted date: 2007-03-16

*Corresponding author E-mail: chenguizhu@yeah.net

Copyright © 2007, Ecological Society of China Published by Elsevier BV All rights reserved

RESEARCH PAPER

Physiological adaptability of three mangrove species to salt stress

Liao Yan, Chen Guizhu*

School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China

Abstract: The impact of salinity on three arboreal mangrove plants, Sonneratia apetala (Sa), S caseolaris (Sc) and Rhizophora

stylosa (Rs), was studied The three mangrove species were treated with different salinity levels over a three-month period The

re-sponse and adaptation of these three mangrove species to salinity were shown to be different Net photosynthesis rate, stomata

con-ductance and transpiration rate of leaves decreased and soluble sugar content in leaves increased, with salt concentration in all three

mangrove species The malondial dehyde (MDA) content in stems and leaves of Sa and Sc somewhat decreased when the salinity was lower than 10, but rapidly increased with increasing salt concentration The MDA content in stems and leaves of Rs increased

only when salinity was greater than 40 No changes were observed in the MDA content of roots in the three mangrove species The

adaptabilities of Sa and Sc to salt tolerance were limited The more salt tolerant the mangrove Rs, the more likely the free oxygen

radicals were eliminated through the increase in activity of superoxide dismutase (SOD) Results of this experiment identified

salin-ity levels best suited for the growth and metabolism of the species, which provides information necessary for maintaining mangrove forestation along the South China coast

Key Words: Sonneratia apetala; S caseolaris; Rhizophora stylosa; salt stress; net photosynthesis rate; stomata conductance;

tran-spiration rate; soluble sugar; membrane peroxidation; SOD

Mangroves, a kind of xylophyte, form unique communities

in tropical and subtropical regions and tidal lowlands

Ecol-ogically, they play an essential role in protecting adjacent land

from wave and storm erosion[1,2] and providing habitat and

food for fish, prawns, crabs, shellfish and birds They can also

filter surface water from inland, and reduce pollutants in

off-shore waters[3]

Mangrove plants generally grow in a special environment

with certain salinity and consequently have their special

adap-tation system, different from land plants and freshwater plants

It is generally thought that mangrove plants grow in certain

salinity conditions and the adaptation of different species to

salinity is different[4,5] Above or below certain salinity level,

their growth will be inhibited and even death can occur Under

salt stress, that is, beyond appropriate salinity, either low or

high, the flexibilities and physiological reactions of different

mangrove species are dissimilar The major physiological

characteristics, such as photosynthesis, protein synthesis and

energy metabolism, are also more or less affected[6]

Sonneratia apetala (Sa), S caseolaris (Sc) and Rhizophora stylosa (Rs) are three dominant mangrove species on the coastland of South China Of them, Sa and Sc were introduced

from Bengal, and have become the most important species in afforestation along coastal areas In this article, research on the physiological reaction of the three mangrove species with various treatments of salinity has been carried out and the relationship between different physiological reactions is ex-plored, which helps to reveal the effect of salinity changes on the three species and provides a reference index for the choice

of salt tolerant species

1 Materials and methods

1.1 Materials

Seedlings of Sa, Sc and Rs were collected from the

man-grove nature reserve (19°51'N, 110°24'E), Dongzhai Gulf in Hainan Island, China, at the end of January 2006, and all the

seedlings were one-year-old with similar size Seventy-two

seedlings of each mangrove species were planted in plastic

pots with 60 kg clay and every pot contained nine seedlings

They were irrigated daily for three months with six liters of

brine, whose salinity varied from 0 to 50, that is, 0, 5, 10, 15,

20, 30, 40 and 50 Water was emptied every night Every

sev-eral days water was replenished to keep the salinity stable

They were placed in natural light Every 30 days, brine was

replaced by water with the same salinity The apparatus (Orion

4 star) was used to analyze the water quality The total time

period was 90 days

1.2 Methods

Salt scale: use water to dissolve sea salt, and use WYY35T

salinometer to rectify salinity

Net photosynthesis rate, stomata conductance and

transpira-tion rate were measured with the help of Li-6200 portable

photosynthesis apparatus (made in USA) The mature, stable

leaves were chosen as research objects and measured, with

temperature controlled between (20 ± 2)℃, light quantum flux

between 600–800 μmol/(m2·s), relative humidity between 60%

± 5%, and CO2 density 3.6 × 104

The total soluble sugar content was measured using the

anthrone colorimetry[7] The extracts were gained by referring

to Lin’s method[8]

Protein content was measured according to Bradford’s

method[9] Coomassie brilliant blue G250 dye and colorimetry

were used at the wavelength of 595 nm Bovine serum

albu-min (BSA) was used as standard protein Protein content was

expressed by mg/g fresh weight

The upper lucid extracts served as the materials for

meas-uring the activity of superoxide dismutase (SOD) and the

content of malondial dehyde (MDA) The above operations

were carried out in a temperature between 0–4℃ Heath’s

methods were used as reference in the measurement of

mem-brane peroxidation function, and MDA was used as index of

membrane peroxidation function[10] MDA content was

calcu-lated according to the MDA mole extinction coefficient △ε

(532–600 nm) = 155 μmol–1·cm–1 and was expressed by mmol/

established was used to measure the activities of SOD By

suppressing reducing agent NBT, 50% was taken as an

en-zyme activity unit, and the enen-zyme activity was expressed by

unit/mg protein

The measurements of all indices were repeated at least

thrice

2 Results and analysis

2.1 Effects of salinity on leaf net photosynthesis rate,

stomata conductance and transpiration rate of Sa, Sc and

Rs

The indices of leaf net photosynthesis rate, stomata

con-ductance and transpiration rate of the three mangrove species

were measured at the same time (9 a.m., April 27, 2006) It was found that photosynthesis was inhibited by high salinity

Net photosynthesis rate and stomata conductance of Sa and Sc

decreased as salinity in the culture medium rose from 0 to 40

(Figs 1 and 2) When salinity was above 15, the decreasing tendency was obvious Net photosynthesis rate of Rs increased

appreciably in the beginning (< 20) and decreased subse-quently (> 20), but the changes tended to be minor Stomata

conductance of Rs increased to 10, the highest level in salinity,

but the overall changes were not obvious In high salinity, in all three mangrove species, the metabolic flow with the out-side slowed down, which was shown by the fact that net pho-tosynthesis rate and stomata conductance decreased and these plants completed metabolism by accumulating internal CO2

According to the changing tendency, in low salinity (< 15), the

net photosynthesis rate and stomata conductance of Sa and Sc

were relatively stable, which showed the adaptation of the two species to low salinity Net photosynthesis rate and stomata

conductance of Rs rose to the highest level in salinity, 20 and

15, or even higher salinity, they did not change visibly, which

was possibly related to the salt rejection in the roots of Rs, that

is, salt did not get to the leaves and consequently had less

ef-fect on Rs than on Sa and Sc

Transpiration rate of Sa and Sc decreased with an increase

in salinity Transpiration rate of Sc decreased sharply in low salinity, but transpiration rate of Rs kept steady (Fig 3) This

tendency was similar to that of the effects of salinity on

sto-Fig 1 Effect of salinity on leaf net photosynthesis rate of Sa, Sc

and Rs

Fig 2 Effect of salinity on leaf stomata conductance of Sa, Sc and

Rs

mata conductance (Fig 2), which indicated that the decrease

of stomata conductance affected transpiration rate to some

extent, and enhanced the mangrove’s function of conserving

water in high salinity Rs stability of transpiration rate proved

that metabolism was kept at a stable level

2.2 Effects of salinity on total soluble sugar content in

leaves of Sa, Sc and Rs

Through cultivating three mangrove species with various

salt concentrations for three months, it was discovered that

total soluble sugar content in leaves of Sa, Sc and Rs all tended

to rise with increase in salinity Total soluble sugar content in

leaves of Sa and Sc increased by a larger margin in high

salin-ity (> 30) than in low salinsalin-ity (< 30) For Rs, total soluble

sugar content increased steadily (Fig 4)

This result indicated that water scarcity stress enhanced as salinity increased (from 0 to 40) To control the balance of ions in the vacuole, the cells accumulated a type of low, com-patible molecular compound, which took the place of water as

a solvent in biochemical reactions and protected cell structure and water circulation[12] As was shown in the experiment, with the salinity above 30, soluble sugar content in leaves of

Sa and Sc increased, thus improving the permeability and

maintaining a balance of water metabolism[13] However,

solu-ble sugar content in leaves of Rs was always at a low level, which could have resulted from the salt rejection of Rs, that is,

it was difficult for the salt to get to the leaves, and the total soluble sugar content did not change a lot Or it could be ex-plained as the accumulation of other compatible solutes, such

as praline[14,15], glycine[16,17] and polyol[18,19] 2.3 Effects of salinity on membrane peroxidation and

activities of SOD of roots, stems and leaves of Sa, Sc and

Rs

It was discovered that membrane peroxidation was affected

remarkably by salt stress in leaves and stems of Sa, Sc and Rs MDA content in leaves and stems of Sa and Sc decreased

slightly in low salinity (< 10) and then increased rapidly (Figs

5 and 7), which was reverse to the activities of SOD (Figs 6

Fig 3 Effect of salinity on leaf transpiration rate of Sa, Sc and Rs

Fig 4 Effect of salinity on soluble sugar content in leaves of Sa,

Sc and Rs

Fig 5 Effect of salinity on leaf membrance peroxidation in leaves,

stems and roots of Sa

Fig 6 Effect of salinity on SOD activity in leaves, stems and roots

of Sa

Fig 7 Effect of salinity on leaf membrance peroxidation in leaves,

stems and roots of Sc

and 8) It was thus evident that, although membrane

peroxida-tion was affected remarkably in leaves and stems of Sa, Sc, no

more SOD was generated, which might be related to the types

of the plants or possibly to the fact that Sa and Sc made other

peroxisome or catalase protect membrane peroxidation from

being destroyed with the help of active oxygen free radicals

Dissimilar to the changing tendency of salinity effects on

MDA content in leaves and stems of Sa and Sc, MDA content

of Rs decreased in low salinity (< 40) and increased only

when salinity was higher than 30 (Fig 9) It was because high

salinity affected the metabolism system balance of active

oxygen, increased the content of active oxygen, and conse-quently the membrane structure was destroyed by the super-oxide anion free radicals, and then the persuper-oxide was produced,

for instance For Rs, under high salinity stress, SOD was more

active (Fig 10) and the changing tendency was reverse to that

of the salinity effect on the MDA content (pertinence

coeffi-cient R2 = 0.893) It showed that Rs mainly depended on SOD

to remove the active oxygen free radicals and SOD activity in leaves and steams directly affected the MDA content When salinity was above 40, the membrane protection system was destroyed, the SOD activity decreased and the MDA content increased

Changes in MDA content and activities of SOD in roots of three mangrove species showed that they were insensitive to salinity The reason is that the mangrove plant roots are sub-mersed in seawater perennially and the roots don’t have enough oxygen to absorb, and therefore mangrove plants have developed a root metabolism system, which has adapted to the special environment This kind of root metabolism system has possessed a special physiological adaptation mechanism, which can be explained by the relatively small changes in the mem-brane peroxidation of roots in high salinity

To summarize, when salinity is higher than 40, the

man-grove species Rs can have some adaptations or endurance to

salt stress, the function of membrane protection system is im-proved in the stems and leaves or almost kept at a high level in the roots, and therefore, the damage that various free radicals cause to membranes is kept at a small degree, which protects the normal cell function and ensures the seedlings with a normal growth When the salinity becomes much higher, the function

of the membrane protection system decreases, various free radicals gradually cause great damage to the membrane, and membrane peroxidation becomes obvious All this causes a disturbance in the normal metabolism, a weakness in cell function, and a decrease in adaptation and endurance to salin-ity stress It can be concluded that under salt stress, in

seed-lings of Rs, free radicals do cause damage to membrane,

namely, membrane peroxidation and protection resulted from SOD or other systems influence each other In comparison

with Rs, Sa and Sc have a little bad salt tolerance, and SOD

does not have strong protection ability for membrane

3 Discussion

The physiological characteristics of the three mangrove species were greatly affected with various salt treatments Its

overall tendency was that changes in Sa and Sc were more obvious than in Rs, which proved that Rs adaptability to high salinity was much stronger Sa and Sc could survive in high

salinity, but high salinity deeply influenced their growth and metabolism

In the field of phytophysiology, many studies have been carried out on the effects of salinity on physiology of

man-Fig 8 Effect of salinity on SOD activity in leaves, stem and roots

of Sc

Fig 9 Effect of salinity on leaf membrance peroxidation in leaves,

stems and roots of Rs

Fig 10 Effect of salinity on SOD activity in leaves, stems and

roots of Rs

grove species, and also on effects of salinity on stomata

con-ductance and transpiration Kotmire[20] and Nazaenko[21] found

that high salinity inhibited photosynthesis by closing the

stoma and partially suppressing RUBISCO’s activity High

temperature, intense light, drought and high salinity proved to

be able to stimulate genes related to heat shock proteins in

plants and in mangrove plants, too Several studies showed

that salt stress could weaken photosynthesis of mangrove

plants[22–24] There were also studies that with salt

concentra-tion, photosynthesis is not weakened and even in low salinity,

photosynthesis is enhanced[25,26] All the difference is mainly

related to the species, and the salt stress exerts different effects

on different species

The reasons for the decrease of photosynthesis rate are

mainly as follows: first, dehydration in cell membrane

de-creases permeability to CO2; second, it is because of salt

toxic-ity; third, closure of the stoma causes the decrease of CO2;

fourth, salt concentration quickens senescence; fifth, changes

in cell structure cause changes in enzyme activity[27] Decrease

in the photosynthesis rate also results from the decrease of

stomata conductance, which causes the lack of CO2, necessary

for carboxyl reaction[28] Closure of the stoma reduces water

evaporation, affects photosynthesis of the chloroplast and

im-pacts energy transformation system, thereby changing the

activity of the chloroplast To what extent the closure of the

stoma affects the photosynthesis rate depends on the partial

pressure of CO2 in the lamina However a study reported that

under salt stress, photosynthesis decreases because stoma

opening is not being inhibited[27] Because of enhancing the

transfer resistance to CO2 and decreasing the efficiency for

RUBISCO, stoma opening is not affected According to the

feedback of other salt-induced reactions, inhibiting the

me-tabolism processes of some carbons can also decrease

photo-synthesis[29]

Another mechanism that mangrove plants overcame under

high permeable pressure, in an environment with high salinity,

was accumulating a compatible solute Popp and other

re-searchers[30] studied 23 types of mangrove plants and

discov-ered that pinitol and mannitol were the most common

com-patible solute They also discovered proline in Xylocarpus

plants, methyl quaternary ammonium compounds in two kinds

of Avicennia plants, Acanthus ilicifolius and Heritiera

lit-toralis, and also in Hibiscus tiliaceus As a compatible solute,

glycine was discovered in Avicennia marina[31]

As is shown by experiment results in this article, when

sa-linity is higher than 30, total soluble sugar content in leaves of

Sa and Sc increases, thus improving the permeability and

maintaining a balance of water metabolism However, total

soluble sugar content in leaves of Rs is always at a low level

and this could result from the salt rejection of Rs, so salt does

not have a lot of effect on total soluble sugar content

Other-wise, it could be explained as the accumulation of other

com-patible solutes

Active oxygen includes superoxide, hydrogen peroxide and hydroxyl, which can be induced by extreme environment stress, such as extreme temperature, herbicide, drought and nutrient stress Some higher plants resist active oxygen by improving the activity of the antioxidizing enzyme SOD catalyzes the transformation of superoxide to hydrogen per-oxide[32,33], and then hydrogen peroxide is decomposed by the hydrogen peroxide enzyme and peroxide enzyme[34] However, there are very few reports about effects of salt stress on the active oxygen mechanism Superoxide toxicology holds that

on the one hand, active oxygen free radicals can cause oxida-tion of membrane and damage the membrane structure and normal cell physiology, but on the other hand, a membrane protection system also exists and slows the damage[35] This membrane protection system is in fact an anti-oxidation sys-tem, which generates different antioxidizing enzymes includ-ing the important SOD SOD can effectively eliminate the active oxygen free radicals and prevent the membrane from damaging by oxidation[36]

The research of Zhao[37] indicated that MDA content of the plant increased with the increase of salt concentration, whereas the activity of SOD and ATP enzyme reduced with the increase of salt concentration, which proved the existence

of a damage mechanism of free radicals to terraneous plants This research showed that the activities of SOD were in-versely correlated to the MDA content in organs of salt

toler-ant mangrove pltoler-ants Sa, Sc and Rs (Figs 5–10) The overall tendency in Sa and Sc was that membrane peroxidation

in-creased with the increase of salt concentration and the

activi-ties of SOD decreased at the same time In Rs, when salinity

was above 40, the damage of membrane peroxidation became obvious

Acknowledgements

The project was financially supported by UNEP GEF “Re-versing Environmental degradation trends in the South China Sea and Gulf of Thailand” Program (UNEP/GEF/SCS/Chi/ MoU 2d)

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