Photosynthetic and physiological responses of Kandelia candel L. Druce seedlings to duration of tidal immersion in artificial seawater

Environmental and Experimental Botany 54 (2005) 256–266

Photosynthetic and physiological responses ofKandelia candelL.Druce seedlings to duration of tidal immersion in artificial seawater

Chen Luzhena,b, Wang Wenqinga,b,∗, Lin Penga,b

a School of Life Sciences, Xiamen University, Xiamen, 361005 Fujian, PR Chinab Research Centre for Wetlands and Ecological Engineering, Xiamen University, Xiamen, 361005 Fujian, PR China

Accepted 13 September 2004

Abstract

The present study demonstrates the influence of the duration of periodical waterlogging with artificial seawater on the photo-synthetic and physiological responses ofKandelia candelL. Druce seedlings, the pre-dominant species of subtropical mangrovesin China. Artificial tidal fluctuations applied here closely mimicked the twice daily tidal inundation which mangroves experiencein the field. All the seedlings were immersed in artificial seawater during 70-day cultivation. Similar trends with increasing dura-tion of immersion occurred in photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration,where significant decreases occurred only in long time treatments of 10 or 12 h. Water used efficiency and chlorophyll contentsshowed lower in medium periods and higher in long periods of immersion. This indicates that the increase in pigment contentsof leaves was ineffective in promotingPn under long time immersion. Light saturation points under short time waterlogging(0–4 h) occurred at light intensities of 800–1000�mol/m2/s, and at around 400�mol/m2/s in long time treatments (8–12 h). Longp d oxidasea under longt treatments,r cumulationi re inducedbc tion ofc©

K

fndical

0

eriods of tidal immersion therefore significantly inhibited photosynthesis of mature leaves. Alcohol dehydrogenase anctivity in roots both increased under longer immersion periods, suggesting that roots are sensitive to anaerobiosis

erm waterlogging. The activities of peroxidase and superoxide dismutase in mature leaves increased in 8 h and 10 hespectively. The content of malondialdehyde in mature leaves increased under long time treatments. Abscisic acid acn mature leaves also had a sharp increase from 8 h to 12 h inundation. Even though the anti-oxidative enzymes wey waterlogging, this was not sufficient to protect the seedlings from senescence. The results suggested thatK. candelseedlingsompletely tolerated tidal immersion by seawater up to about 8 h in each cycle, which matches the natural distribuK.andelin inter-tidal zones of China.2004 Elsevier B.V. All rights reserved.

eywords:Mangroves; Semidiurnal tide; Artificial tide; Photosynthesis; Abscisic acid (ABA)

∗ Corresponding author. Tel.: +86 592 2181431;ax: +86 592 2188846.E-mail address:[email protected] (W. Wenqing).

1. Introduction

Mangroves are inter-tidal forests of tropical asubtropical regions. They are adapted to period

098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.envexpbot.2004.09.004

C. Luzhen et al. / Environmental and Experimental Botany 54 (2005) 256–266 257

waterlogging. Due to enclosure for aquaculture andjetty construction, some/many mid-tidal zones ofmangroves have been damaged. Mangroves face thechallenge of subsiding plantable tidal flats in China(Fan and Li, 1997; Mo and Fan, 2001). Furthermore,the rise of sea level accompanying the rapid changesof climate (Field, 1995; Ellison and Farnsworth, 1997)also prolongs the period of waterlogging of mangrovesat high tide. Consequently, the growth of naturalmangroves and the survival rate of mangrove plantingshave been greatly influenced, and this is of greatconcern to scientists. Under waterlogged conditions,root respiration rate and oxidase activity declined(McKee, 1996; Ye et al., 2003). Contents of pigmentsin leaves changed (Ye et al., 2003); stomatal closure,water uptake reduced, transpiration rate (Tr) and pho-tosynthetic rate (Pn) declined (Naidoo, 1984; Ellisonand Farnsworth, 1997; Pezeshki et al., 1997). Thoughthe transpiration rate (Tr) and stomatal conductance(gs) were decreased, the water used efficiency (WUE)appeared lower and less variable under waterlogging(Naidoo et al., 1997). Activities of enzymes processingreactive oxygen species, such as superoxide dismutase(SOD) and peroxidase (POD) activities, increaseswith prolonged waterlogging (Ye et al., 2003). Theplant growth regulator, abscisic acid (ABA) wasgreatly induced by waterlogging (Kozlowski, 1984;Guan, 1996; Li and Zhou, 1996), and inactivatedthe PSII complex (Ahmed et al., 2002). No reportsabout the relation between ABA and waterlogging inm gicala lingsw

am (1 ndEb andi thes yi -d lyf d toa edd ura-t ort .,1

an urgent need to ascertain the suitable for this species(Fan and Li, 1997; Chen et al., 2001).

In the current study, we focussed on the physiolog-ical responses and resistance ofK. candelseedlingsto periodical waterlogging. We hypothesized thatK.candelseedlings are tolerant of waterlogging becausethe distribution ofK. candelseedlings in the inter-tidal zones was consistent with this. A former studyhas found out that the maximum period of waterlog-ging that allows growth ofK. candelseedlings was 8 h(Chen et al., 2004). We also aimed to seek out the corre-lation between physiological responses and the growthof K. candelseedlings under periodical waterlogging.To achieve this, we used equipment, which simulatedthe tidal waterlogging at different levels on the tidalflats. In the field work of Watson, the Western Malayanmangrove forest was divided into five gradients of tidalinundation classes, and the frequency of flooding permonth was the basis of his inundation classes (Watson,1928). In our simulation experiment, we simplifiedthe conditions, and averaged the inundation time fora semidiurnal tidal cycle into seven classes, accordingto the length of waterlogging during a tidal cycle at dif-ferent locations in the inter-tidal zones. Twelve hourstreatment meant that seedlings were waterlogged all thetime in a tidal cycle (12 h), which denoted the water-logged time at the lowest tidal level, while 0 h treatmentequalled the highest tidal level where seedlings werenot inundated. All plants were inundated twice dailyexcept treatmentG (0 h). In this paper, we present datas zymea ffer-e ure.

2

2

id-i nks(I ghp eri teri er-c ook5 re,

angroves have been published. These physiolond photosynthetic responses of mangrove seedould indicate that they tolerate waterlogging.Kandelia candel(L.) Druce, Rhizophoraceae, is

ajor mangrove species of the eastern groupLin,999), occurring only along the coastlines of Asia aast–Pacific Archipelagos (Lin, 1999). K. candelcane found in all mangrove associations in China,

t is the major mangrove species for forestation onoutheast coast of China (Lin, 1988). It occurs naturalln mid-to-high tidal zones (Lin, 1999). Under field conitions, one-year-oldK. candelseedlings are usual

ully covered in seawater at high tide, and exposeir at low tide. The duration of seedling fully coverepends on their location in the tidal zone. The d

ion of periodical waterlogging is a limiting factor fhe survival of seedlings (Liu, 1995; Komiyama et al996; Chen et al., 2001; Kitaya et al., 2002). There is

howing changes in rates of leaf gas exchange, enctivities and hormone levels accompanying the dint periodical waterlogging times after 70 days cult

. Materials and methods

.1. Experimental design

A set of artificial-tidal tanks simulating the semurnal tide were constructed from seven plastic ta65 cm× 50 cm× 50 cm) arranged as shown inFig. 1.t took 2 h to fill a tank with artificial seawater throuipes. After Tank A was full, the water flowed ov

nto Tank B, and so on. After Tank F was full, all wan Tanks A, B, C, D, E and F was unloaded by timontrolled valves at the bottom of each tank. It tmin to let the water drain out of the tank. Therefo

258 C. Luzhen et al. / Environmental and Experimental Botany 54 (2005) 256–266

Fig. 1. Arrangement of artificial tidal tanks. It takes 2 h to fill each tank with artificial seawater through the pipe. After Tank A was full itoverflowed to fill Tank B, and so on. When Tank F was full, all water in Tanks A, B, C, D, E and F was unloaded by timer-controlled valves atthe bottom of each tank. Therefore, the inundated periods of tanks from A to G were 12, 10, 8, 6, 4, 2 and 0 h per-tidal cycle, respectively. Therewere 2 tide cycles every day.

Tank A was full of water for 12 h per tidal cycle, and theinundation periods in the other tanks were 10, 8, 6, 4, 2and 0 h, respectively. There were two tide cycles everyday. There were four pots in each tank, each 25 cm talland 25 cm in diameter, with a small hole at the bottomto allow rapid drainage while water in the tanks wasdrained away. Each pot was filled with washed riversand (diameter = 1 mm). Three sets of equipment actedas three replicates. Tanks were arranged by randomizedblock design in the same greenhouse and the arrange-ment of tanks was changed randomly every week.

2.2. Materials and growing conditions

Healthy and mature hypocotyls ofK. candelwerecollected from Jiulong River Estuary in Fugong Town,Longhai County, Fujian Province of China (24◦29′N,117◦55′E). The average seawater salinity there was17‰ (Lin, 1988, 1999). Five hypocotyls were plantedin each pot at random. The seedlings were periodicallysubmerged under artificial seawater, as inFig. 1, witha salinity of 15‰ (seawater from the west coast ofXiamen of 22–28‰ in salinity was diluted by tapwater). Tap water was added daily to compensatefor evaporation losses and the seawater was renewedweekly. All seedlings were grown in a greenhousewith air temperature of 27–32◦C, and under naturalsunlight for 70 days (28 April, 2003–3 July, 2003).Seedlings were flooded at ‘high tide’ to a maximumdepth of 60 cm above the bottom of the tanks, anda ill hs the

second leaf blades had matured. All the analyses wereperformed on the second leaf blades from the base.

2.3. Gas exchange measurements and contents ofpigments in mature leaves

Photosynthesis and transpiration of mature leaveswere measured using a portable photosynthesis system(Model CIRAS-1, UK). Before the measurement, wa-ter was drained from all the tanks. Three to five matureleaves for each replicate were chosen for the measure-ment of photosynthetic rate (Pn), transpiration rate (Tr),stomatal conductance (gs) and intercellular CO2 con-centration (Ci ). Mean values ofPn, Tr, gs andCi foreach replicate were used for statistics. Gas exchangemeasurements were made on entire and fully expandedleaves and were carried out between 10 a.m. and 12 p.m.in natural sunlight of 800–1000�mol/m2/s photosyn-thetically active radiation and in the temperature range29–32◦C. Water used efficiency (WUE) was calculatedfrom the ratioPn/Tr. Light-response curves were ob-tained using the same portable photosynthesis systemequipped with artificial light. The measurements werecarried out at 10 levels of illumination intensity, 1400,1200, 1000, 800, 600, 400, 200, 100, 50 and 0�molphotons/m2/s, in the sequence from the highest to thelowest values.

For the measurement of pigment contents, about0.1 g of mature leaves were cut and immersed in amixture of ethanol: acetone: HO = 4.5:4.5:1 for 3d( wasm

t ‘low tide’ the water was very slightly below soevel as showed inFig. 1. After 70 days culture, eaceedling had two to three pairs of leaves and

2ays, protected from light by the method ofZhang1990). The absorbance at 645 nm and 663 nmeasured.


2.4. Physiological analysis

2.4.1. Alcohol dehydrogenase (ADH) activity ofroots

The methods used for ADH extraction and activ-ity determination were those ofLiu et al. (1991), andKato-Noguchi and Saito (2000), with some modifi-cations. Fresh roots were homogenized in four vol-umes of ice-cold solution containing 100 mM Tris–HCl(pH 8.0), 2 M KCl, 20 mM EDTA, and 1.0% (m/v)polyvinyl pyrrolidone (PVP) to neutralize the effect ofphenol from mangrove tissues. The homogenate wascentrifuged at 12,000×g and 4◦C for 20 min and thesupernatant was used for ADH assay. Activity of ADHwas measured by monitoring ethanol-dependent NAD+

reduction at 340 nm in a spectrophotometer. The reac-tion mixture with a final volume of 3.0 ml, contained2.7 ml of 50 mol/l Gly-NaOH buffer (pH 9.0), 0.1 mlNAD+ solution (2 mg NAD+), 0.1 ml enzyme extractand 0.1 ml 97% ethanol. The absorbance at 340 nmwas measured at 1 min intervals for 3 min. An increaseof 0.01 absorbance units per minute equalled one unitof ADH activity. The activity of ADH was expressedas units per mg protein. Protein was determined us-ing Coomassie Brilliant Blue G-250 (Bradford, 1976),using bovine serum albumin (BSA) as a standard.

2.4.2. Root oxidase activityRoot oxidase activity was determined according to

Zhang (1990). One to two grams of fresh fine rootsw heyw ureon 5f ast lus1 nd1 t atr l ofd wasm tento dasean

2mi-

n

(2000)with a little modification. 0.5 g of mature leaveswere collected and ground in a mortar with 5 mlof ice-cold 62.5 mM phosphate buffer (pH 7.8) with1.0% (m/v) polyvinyl pyrrolidone (PVP). The ho-mogenates were centrifuged at 4◦C and 12,000×gfor 20 min. The supernatant was stored at 4◦C andthen used for assays of enzymatic activities and proteincontent.

Superoxide dismutase (SOD) activity was mea-sured as the amount of inhibition of photo-reductionof nitroblue tetrazolium (NBT). The reaction mix-ture, with a final volume of 3.0 ml, contained 1.5 ml62.5 mM phosphate buffer, 0.3 ml 20�M riboflavin,0.3 ml 130 mM methionine, 0.3 ml 100�M Na2EDTA,0.3 ml 750�M NBT, 0.05 ml enzyme extract and0.25 ml deionized water. Phosphate buffer was sub-stituted for enzyme to determine the maximumphoto-reduction of NBT. The reaction was carriedout in 75�mol/m2/s illumination for 20 min. Oneunit of SOD activity was calculated as that inhibit-ing the maximum photo-reduction of NBT by 50%at 560 nm of optical density,k measured with aspectrophotometer.

For the measurement of peroxidase (POD) ac-tivity, 2.9 ml of 100 mM phosphate buffer + 20 mMguaiacol, pH 7.0, was mixed with 0.1 ml of enzymeextract and allowed to stand at room temperature for3 min. Twenty microliters of 2% hydrogen peroxidewas added to activate the reaction. The absorbanceat 470 nm was measured at 1 min intervals for5 unitsp asea

d asu ord-i in(

de-t -z oroa lu-t asc2 erer de-s

C

ere washed with distilled water and blotted dry. Tere then immersed in 50 ml of solution (1:1 mixtf 0.1 mM phosphate buffer at pH 7.0 and 40 ppm�-aphthylamine) and placed on a shaker table at 2◦C

or 5 h. Before and after shaking, 2 ml of solution waken out and added to 10 ml of distilled water pml 1% sulfanilic acid (w/v in 30% acetic acid) aml 100 ppm sodium nitrite. The mixture was kep

oom temperature for 5 min, diluted by adding 6 mistilled water, and then the absorbance at 510 nmeasured. During the incubation period, the conf �-naphthylamine decreases, due to the root oxictivity. Root oxidase activity was expressed as�g �-aphthylamine/h/mg FW.

.4.3. Lipid peroxidation of leaf membranesThe methods of extraction and activity deter

ation were those ofLiu and Zhang (1994)and Li

min, and an increase of 0.01 absorbanceer minute was equated to one unit of peroxidctivity.

The activities of SOD and POD was expressenit per mg protein. Protein was determined acc

ng toBradford (1976)by using bovine serum albumBSA) as a standard.

Malondialdehyde (MDA) of mature leaves wasermined according toLi (2000). Two microliters enyme extracts were incubated with 10% (w/v) trichlcetic acid (TCA) plus 0.6% thibarbituric acid so

ion in boiling water for 10 min. Then the mixture wentrifuged at room temperature and 10,000×g for0 min. Absorbances in 450, 532 and 600 nm wecorded. The MDA content was calculated ascribed byLi (2000), as follows:

(�M) = 6.45 (A532 − A600) − 0.56A450


2.5. Endogenous ABA content of mature leaves

The endogenous ABA in mature leaves was deter-mined using the ELISA method according toWu et al.(1988).

3. Data analysis

Mean and standard deviation (S.D.) values of threereplicates were calculated. Data for leaf gas exchange;physiological responses and ABA concentration wereanalyzed by one-way ANOVA usingF- and t-tests.The Student–Newman–Keuls univariate comparisonmethod was used to analyze the difference among thelight-response curves of seven treatments. The illumi-nation effects on each treatment were analyzed by one-way ANOVA using at-test.

3.1. Gas exchange measurement and pigmentcontents

With increasing duration of waterlogging,Pn of ma-ture leaves declined progressively (Fig. 2, Table 1).Maximum Pn occurred in the 0 h and 2 h treat-ments, 23.89± 1.13 and 23.78± 1.44�mol/m2/s, re-spectively. Transpiration rate (Tr) of 0–8 h treatmentswere not significantly different (P> 0.05) (Fig. 2). Wa-terlogging periods of up to 8 h did not affectTr, but10 h and 12 h immersions reducedTr by 20.5% and4l0 ent( -s umoc thc sion(

weree sf sec ent(d thelow na-

tion exceeded 400�mol/m2/s. At low light levels(0–200�mol/m2/s),Pn was considerably higher in alltreatments. The maximumPn under 200�mol/m2/sillumination occurred in the 4 h treatment, at8.75± 0.92�mol/m2/s (Figs. 4 and 5).

Significant changes in photosynthetic pigments ofChl.a, Chl.b and total chlorophyll (Chl.) were approx-imately uniform and all followed a U-shape (Fig. 6andTable 1). The amount of Chl.a, Chl.b and Chl. was

Fig. 2. Pn, Tr, gs, Ci , and WUE of mature leaves ofKandeliacandelseedlings under periodical waterlogging. One-way ANOVAshowed significant differences between effects of treatments onPn(*** P< 0.001),Tr (*** P< 0.001),gs (*** P= 0.005),Ci (*** P= 0.003)and WUE (*P= 0.027).

7.0%, respectively. Stomatal conductance (gs) fol-owed the same trend asPn (Fig. 2). But gs under theh treatment was lower than that of the 2 h treatm

*** P< 0.001). Maximumgs occurred with 2 h immerion, and was 162.4% higher than that of the minimf 12 h treatment (** P= 0.003). Intercellular CO2 con-entration (Ci ) and water use efficiency (WUE) bohanged significant over the range 0–12 h immerFig. 2andTable 1).

The photo-response curves of mature leavesxpressed byPn in different illumination rangerom 0–1400�mol/m2/s. The trend of photo-responurves in each treatment was significantly differTable 2. and Fig. 3). Photosynthetic rate (Pn) un-er short time treatments was much higher, and

ight saturation point occurred at 800–1000�mol/m2/sf illumination. Otherwise, in 8–12 h treatments,Pnas low and increased smoothly when illumi


Table 1Effect of immersion in seawater on photosynthetic and physiological activities inKandelia candelseedlings

Parameter Immersion time (h) One-way ANVOA

0 2 4 6 8 10 12

Pn (�mol/m2/s) 23.1± 2.0 23.5± 1.8 19.9± 0.8 19.3± 0.8 17.9± 0.4 16.6± 0.3 13.7± 2.0 20.2***

Tr (mmol/m2/s) 5.8± 0.32 6.0± 0.4 6.6± 0.9 5.9± 0.3 5.5± 0.3 4.8± 0.6 3.2± 0.3 16.8***

gs (mol/m2/s) 0.36± 0.03 0.38± 0.07 0.43± 0.04 0.38± 0.03 0.37± 0.01 0.32± 0.02 0.27± 0.04 5.32**

Ci (ppm) 246± 45 244± 9 245± 10 229± 7 212± 9 202± 17 174± 12 5.70**

WUE (�mol/mmol) 3.96± 0.21 3.93± 0.52 3.07± 0.31 3.27± 0.21 3.24± 0.21 3.30± 0.29 4.28± 0.79 3.524*

ADH (U/mg protein) 3.55± 0.69 6.78± 1.06 8.75± 1.29 6.15± 1.46 7.53± 0.26 6.63± 0.44 4.51± 0.36 7.49**

Root oxidase-activity(�g/h/g FW)

13.4± 2.1 24.0± 1.8 27.3± 4.3 21.3± 3.1 20.5± 6.1 17.0± 1.8 15.6± 3.3 5.25**

POD (U/mg protein) 901± 50 605± 46 587± 67 587± 103 894± 70 920± 105 1159± 315 7.98**

SOD (U/mg protein) 71.4± 6.4 67.9± 10.1 62.5± 3.5 73.7± 7.1 72.6± 9.4 102.8± 11.0 140.0± 25.2 13.7***

MDA (�mol/g FW) 3.65± 0.10 2.13± 0.29 2.77± 0.22 2.61± 0.18 3.18± 0.31 3.36± 0.18 3.54± 0.53 7.21**

Chl.a (mg/g FW) 1.39± 0.11 1.24± 0.21 0.59± 0.13 0.62± 0.14 0.85± 0.12 0.76± 0.05 1.30± 0.142 15.7***

Chl.b (mg/g FW) 0.436± 0.033 0.283± 0.071 0.197± 0.049 0.199± 0.044 0.272± 0.041 0.262± 0.014 0.417± 0.048 12.5***

Chl. (mg/g FW) 1.84± 0.15 1.65± 0.29 0.77± 0.18 0.83± 0.19 1.13± 0.16 1.03± 0.07 1.36± 0.35 10.3**

ABA (fmol/g FW) 851 ± 68 753± 49 726± 17 717± 15 854± 113 1233± 102 1669± 77 48.1***

Values are means of three replicates±S.D.F-values are given and significance is shown as:* 0.05,** 0.01 and*** 0.001.

Table 2Results of ANOVA (F-values) for the illumination effects onPn of Kandelia candelseedlings in different waterlogging times

Parameter Sources of variation for univariatecomparison method

One-way ANOVA: illuminationeffects on seven treatments

Illumination (I) Treatment (T) I×T 0 h 2 h 4 h 6 h 8 h 10 h 12 h

Pn (�mol/m2/s) 745*** 88.8*** 7.83*** 603*** 1318*** 496*** 525*** 795*** 162*** 82.2***

F-values are given and significance is shown as:* 0.05,** 0.01 and*** 0.001.

lower in 4 h and 6 h treatments and higher in 0 h and12 h treatments.

3.2. Physiological responses

Activities of all the enzymes exhibited significantchanges with the prolonging of periodical waterlog-

Fig. 3. The photo-response of mature leaves ofKandelia candelseedlings to periodical waterlogging. Analysis by Student–Newman–Keuls univariate comparison method showed no significant differ-ence among treatments (*** P< 0.001).

ging time (Figs. 4 and 5and Table 1). The max-imum ADH activity occurred in the 4 h treatment,87.45± 12.89 U/mg protein. ADH activities under 0 hand 2 h treatments were less and were 59.5% and 22.5%lower than that of the maximum (4 h), respectively(** P= 0.001;P= 0.067). When the periodical water-logging time was longer than 4 h, ADH activity de-creased up to 12 h treatment showing a reduction by94.0% of the 4 h treatment (** P= 0.002).

Root oxidase activity increased at first and then fellsignificantly when the periodical waterlogging timewas longer than 4 h (Fig. 4 and Table 1). The max-imum root oxidase activity was observed in the 4 htreatment at 27.27± 4.34�g/h/g FW, approximatelytwice the lowest value in the 0 h and 12 h treatments(** P= 0.001;** P= 0.002). The decrease of root oxi-dase activity from 4 h treatment to 8 h treatment wasnot significant (P= 0.056;P= 0.053).

Changes in SOD activity of mature leaves is shownin Fig. 5andTable 1. There were insignificant changes


Fig. 4. Activities of ADH and oxidase in roots ofKandelia can-delseedlings under periodical waterlogging. One-way ANOVA testshowed significant differences between effects of treatments on ADH(** P= 0.009) and root oxidase activity (** P= 0.009).

Fig. 5. Activities of SOD and POD and content of MDA in matureleaves ofKandelia candelseedlings under periodical waterlogging.One-way ANOVA test showed significant difference between effectsof treatments on SOD (*** P< 0.001), POD (** P= 0.002) and MDA(** P= 0.003).

Fig. 6. Contents of Chl.a, Chl.b, and total Chl in mature leavesof Kandelia candelseedlings under periodical waterlogging. One-way ANOVA test showed significant difference between effects oftreatments on Chl.a (*** P< 0.001), Chl.b (*** P< 0.001) and Chl.(** P= 0.002).

among the treatments from 0–8 h (P> 0.05). SOD ac-tivity was remarkably activated when periodical wa-terlogging time was prolonged to 10 h and 12 h, being164.4% and 223.9% of the 8 h treatment, respectively(*P= 0.013;*P= 0.04).

Peroxidase (POD) activity of mature leaves fol-lowed the same trend as SOD (Fig. 5 and Table 1).Insignificant differences were found between the 2–6 htreatments (P> 0.05) and between the 8–12 h treat-ments (P> 0.05). POD activity at 8 h was 152.1% ofthat of the 6 h treatment (*P= 0.019).

The content of MDA also changed similarly to thatof POD and SOD (Fig. 5andTable 1). A sharp fall oc-curred in the 2 h treatment (** P= 0.001), but the contentrose significantly when periodical waterlogging timewas longer than 2 h (** P= 0.003).

3.3. Content of ABA in leaves

The content of endogenous ABA in mature leaveswas highest in the 12 h treatment followed by 10 h, 8 hand 6 h, showing a tendency for longer immersion topromote the secretion of ABA (Fig. 7 and Table 1).No significantly changes of endogenous ABA contentsoccurred between 0–8 h treatments (P> 0.05), but therewas a sharp increase in the 10 h and 12 h treatments,which were 144.3% and 195.3% higher than that of 8 htreatment, respectively (*** P= 0.001;*** P< 0.001).

4

theri ved

. Discussion

Mangroves are well adapted to waterlogging ein anatomy or in physiology. Parallel study has pro


Fig. 7. ABA accumulation in mature leaves ofKandelia candelseedlings under periodical waterlogging. One-way ANOVA testshowed significant difference between effects of treatments on ABA(*** P< 0.001).

thatK. candelseedlings grown in different waterlog-ging duration when fully covered by artificial seawaterand growth was reduced with long immersion. The gasexchange characteristics and physiological responsesin current results have proved further thatK. candelseedlings also have strong tolerance to long time wa-terlogging.

4.1. Photo-response to waterlogging

Due to stomatal closure and ribulose bisphosphatecarboxylase/oxygenase (RUBISCO) activity being in-hibited under waterlogging, photosynthetic rate (Pn)declined in mangroves (Ellison and Farnsworth, 1997;Pezeshki et al., 1997). Though the transpiration rate(Tr) and stomatal conductance (gs) were also reduced,the water used efficiency (WUE) appeared lower andless variable under waterlogging (Naidoo et al., 1997).The chlorophyll contents also changed (Ellison andFarnsworth, 1997; Ye et al., 2003). In the present study,similarly, Pn of mature leaves was maximum undershort time inundation, and declined progressively withthe prolonging of periodical waterlogging. Transpira-tion rate (Tr) did not significantly differ between 0 hand 8 h treatments, which lead to a reduction of WUE(Pn/Tr). However,Tr declined sharply when inundationperiods were longer than 8 h, which resulted in a riseof WUE under 10–12 h inundations. Changes ofgs andCi indicate that waterlogging inhibitedPn andTr byclosing the stomata and reducing the intercellular COc pho-t unto lls t the

increase of pigment contents ofK. candelseedlingsunder long inundations was ineffective in promotingPn.

The photo-response curves also showed the inhibi-tion of Pn in long time treatments, indicating that thecarbon assimilation rates were reduced. Data here wereconsistent with the effects of inundation on growth ina parallel study (Chen et al., 2004) where the biomassand area of mature leaves were reduced under long timetreatments. Similar conclusions about carbon assimi-lation in mature leaves have also been reached for theother mangroves speciesAvicennia germinans, Lagun-cularia racemosaandRhizophora mangle(Pezeshkiet al., 1989), Avicennia marina(Naidoo et al., 1997)andR. mangle(Ellison and Farnsworth, 1997) underprolonged waterlogging.

4.2. Physiological response to waterlogging

It has been reported that youngA. marinaseedlingswithout pneumatophores had a continuum of gas-spaces throughout leaves, stem, hypocotyls and roots(Ashord and Allaways, 1995). For a one-year-oldA.marinaseedling, oxygen in the root system might bereduced to zero, if flooding lasted more than 3.5 h(Hovenden et al., 1995). As indicated by alcohol dehy-drogenase (ADH), the anaerobic respiration increasedin A. germinansseedlings when subjected to hypoxiafor 96 h (McKee and Mendelssohn, 1987). Duringwaterlogging, roots may eventually use up all theiri ero-b oge-n tes(p thiss ent,a timei se oft Hc s ofK ec-t ingt tionr ta iesr gings otso ity

2oncentration. On the other hand, the contents ofosynthetic pigments of Chl.a, Chl.b and total amof chlorophyll (Chl.), which changed uniformly, ahowed a U-shaped response. We concluded tha

nternally stored air and then begin to respire anaically, ethanol accumulates and alcohol dehydrase (ADH) activity increases in other hydrophyPazeshki et al., 1993; Akhtar et al., 1998). ADH helpedlants avoid the damage caused by ethanol. Intudy ADH activity was the highest in the 4 h treatmnd was less in long time treatments. Roots in long

nundation were damaged, as shown by the decreahe root’s oxidase activity (Fig. 4). This suggested ADould not detoxify that ethanol accumulated in root. candelseedlings during long time inundations eff

ively. This result agrees with previous studies showhat hypoxia leads to a significant decline in respiraate and oxidase activity of roots (McKee, 1996; Ye el., 2003). Changes in ADH and root oxidase activiteported here suggest that long time waterlogignificantly inhibited the normal metabolism in rof K. candel seedlings. We conclude that toxic


caused by anaerobiosis was the major inhibiter to rootsunder waterlogging (Liu, 1992; Guan, 1996).

Lipid peroxidation is an indicator of oxidative dam-age under environmental stress, (Guan, 1996). Underprolonged waterlogging, superoxide dismutase (SOD)and peroxidase (POD) activities increased inK. can-del, as have previously been reported for other hygro-phytes (Guan, 1996; Ye et al., 2003). Malondialdehyde(MDA) is a major product of lipid peroxidation, and itsincrease reflects damage to leaves under waterlogging(Guan, 1996). The observation of significant increasesin SOD and POD, and the contents of MDA in matureleaves indicated thatK. candelseedlings have resis-tance to oxidation damage due to waterlogging, whichagrees with the results ofYe et al. (2003).

Abscisic acid (ABA) is one of the most importanthormones and indicators of stress caused by flooding(Kozlowski, 1984; Guan, 1996). It is produced in ma-ture or aged organism and transported to the younger.The accumulation of ABA in mature leaves ofK. can-delseedlings was higher under inundations longer than8 h, which suggests that ABA was induced by longerwaterlogging. ABA stimulates stomatal closure, whichleads to the decreased ings,Pn andTr of mature leaves(Guan, 1996; Li and Zhou, 1996). The observed in-crease in ABA reported here is probably the cause ofthe photosynthetic response we reported. ABA is alsoknown to induce the growth of aerial roots (Guan, 1996;Li and Zhou, 1996), which was also observed in the par-allel study (Chen et al., 2004). Senescence is caused bywf tivee nt top

4 s

ls n bysn n 8 hl e asr DHa thei ert nds ciesi

The increase of tidal immersion time caused bythe subsidence of plantable tidal flats influences sur-vival rates in mangroves (re)-forestation (Liu, 1995;Komiyama, 1996;Chen et al., 2001; Kitaya et al.,2002) by inhibiting the growth of seedlings (Ye et al.,2003; Chen et al., 2004), and by causing other phys-iological changes, even resulting in stunting or deathof mangroves (Zhang et al., 1997). A parallel studyChen et al. (2004)showed thatK. candelseedlings hadstrong tolerance of periodical waterlogging and that 8 hwas the maximum, beyond which growth was reduced.Combined with the photosynthetic and physiologicalresponses, this supports the conclusion that regular im-mersion in seawater for more than 8 h significantly re-duces carbon assimilation and speeds up senescence ofseedling leaves. Despite the detrimental effects of longterm waterlogging, the induction of anti-oxidation en-zymes indicates thatK. candelseedlings have someresistance to waterlogging.

The survival rate ofK. candelseedlings during af-forestation is reduced by zero waterlogging and longtime waterlogging (Chen et al., 2004). Action shouldbe taken to raise the tidal flats and shorten waterloggingtime in mangrove restoration. Our results provide the-oretical guidance for tidal level selection in mangroverehabilitation.

5. Conclusion

e ofp on-s ight rover thea Ther thei ho-t ls reat-e nda-t adsti re-d ls aterf int

aterlogging (Kozlowski, 1984; Guan, 1996). It there-ore seems that even the induction of anti-oxidanzymes under long time treatments is not sufficierotect the seedlings from senescence.

.3. Waterlogging tolerance of K. candel seedling

Previous studies have established thatK. candeeedlings have a strong resistance to tidal inundatioeawater (Ye et al., 2003; Chen et al., 2004). Data hereevertheless showed that immersion of more tha

ead to inhibition of photosynthesis to senescenceflected by hormone production in leaves and to Activity declining in roots. These results matched

mmersion times thatK. candelseedlings tolerate undheir natural distribution in mid-to-high tidal zones aome in mid-to-low tidal zone as a pioneering spen the field (Lin, 1988, 1999).

Mangroves are threatened by the subsidenclantable tidal flats, by rising sea level and the cequent increase of duration of waterlogging by hides. In order to undertake the most effective mangehabilitation there is an urgent need to determineppropriate tidal levels for mangrove seedlings.esults of this simulation experiment clearly shownfluenced of periodical waterlogging time on the posynthetic and physiological responses onK. candeeedlings, and we concluded that seedlings are thned by long periods of immersion in seawater. Inu

ion by sea water for more than 8 h in a tidal cycle leo increased ABA accumulation and decreasedPn, andnduction of anti-oxidative enzymes in leaves, anduced ADH and oxidase activity in roots.K. candeeedlings were tolerant of tidal immersion in sea wor up to 8 h, which matches its natural distributionhe inter-tidal zones of China.


Acknowledgements

The project was supported by National Natu-ral Science Fund of China (NNSFC) under con-tract No. 30200031, Doctorate Program Foundation ofthe Education Ministry of China under contact No.20030384007, and Item of Science and Technologyin Xiamen, China under contact No. 3502Z20021046.We greatly appreciate Prof. W.J. Cram for his com-ments and advices on phraseology and expression onthe manuscript. We also thank Dr. Chen C.P., Dr. ZhangY.H., and Wang L., Jie Z.L., Chi M.J., for their as-sistance in seedlings cultivation and measurement. Weare grateful to two anonymous reviewers for their com-ments on this work.

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