Indian Journal Ex perimelllal Biology Vo l. 4 1, Fecru ary 2003, pp. 160- 166

Effect of NaCI on leaf salt secretion and antioxidative enzyme level in roots of a mangrove, Aegiceras corniculatum

Suj atarani Mi shra & A B Das*

Nat ional Institute for Plalll Biodi versity Conse rvati on and Research, C/O Regional Plant Resouree Ce nt er, Nayapall i, Bhubaneswar 75 1 015 , Indi a

ReceiFed 4 Jllll e 2002; revised 20 Novell/ber 2002

Short -term sa lt (NaCI) treatment on Aegiceras comiclI/arllll/ in roots and leaves showed no ehange in fresh and dry wei ght of leaves, roots and leaf area. There was no significant change in total so luble root protein . photosy nthetic pi gments of leaves and spectra l charac teristics o f thylakoids. However, the activity of anti oxidati ve enzy mes (cata lase. ascorbate per­ox idase and gua iacol perox idase) in roots decreased by 72 , 58 and 80% respectively after 96 hI' of tleatment (300 mM of NaCl ). Secreti on of sa lts from the leaf salt glands and salt acc um ul ati on on upper sur face of the leaves were qua nti lied that revealed linear increase of sa lt scc retion of leaf wit h increase in peri od of salt treatment. It was concluded that loss of activi­ties of anti oxidati ve enzymes at high sal t treatment. cau sed lea f senescence in spi te of hi gh ra tes o f salt secretion by Ae­giceras com iclI /a llllll .

Plant growth is limited by salt stress . Salt stress re­duces leaf water potential, causes ion imbalance or disturbances in ion homeostas is and ioni c s tressl ~ and hence, growth su ppression is directly related to total concentration of soluble salts or osmotic potent ial of the med ium in which the plant grows ll. Short- term effect is mainl y after a few hour or withi n day and long-term effect is after several days of ex posure of plant roots to sa lt22

. To cope with sa lt stress, pl ants respond with physiological and biochemi cal changes l3. These changes aim at retenti on of water in spite of hi gh external osmoticum and maintenance of photosynthetic acti vity 13. Accumul ati on of low mo­lecul ar weight compounds such as glyc ine betaine, sugar alcohols and proline aim at balanci ng water po­tenti al when plant exposed to high salinity. In addi tion to these, sa lt stress triggers various antiox idati ve de­fenses in pl ants invari ably including superoxide di s-

. 10 15 19 mutase, ascorbate peroxtdase and catalase . . Mangroves grow in sheltered intertidal zones at in­

terface between land and sea in tropical and subtropi­cal regions and exposed to varying degree of salinity. Mangrove fo res t forms unique zones according to gradient salinity in the mangrove ecosystems. Man­groves are divided into two distinct categories on the basis of their sa it management strategies . One is

*Correspondent author Phone: +9 1-674-2553845 Fax: +9 1-674-2550274 E- mail : a_b_das@ hotmail.com

secretors whi ch have salt g lands or salt hair to secrete excess salt and the other is non-sec retors which lack sueh features to exc lude the salt. Aegiceras cornicula­tum of the family Myrsi naceae is a secretor as well as excluderl7 species and exclude 90-97% of salt through ultrafil tration35

.

Mangroves of Indi a constitute 7% of world 's man­grove vegetati on (spreads over 6740sq km). In Orissa, though mangrove areas are small (215 sq km), but the species density is very high and max imum number of spec ies (62 out of 64) reported from India occurs in Bhitarkan ika, Orissa5

. These mangroves of coastal Ori ssa have been extensively damaged during 1999 due to cyc lone, but their conservation is qu ite immi­nent. Aegiceras cornicl/latul11 is a characteristic ele­ment of minor mangroves occurring in Mahanad i delta and they often fo und in a soc iation with other major mangroves like Rhizophora and Ceriops de­calldra)5 . A. comiculatum has no aeri al roots like Bruguiera parv!j1ora , o!1l y has undergrou nd spreading hori zontal roots. These roots pass through shallow depth of substratum. This horizontal root _ystem often exposed to the air. All these exposed surface roots serve the respiratory function , but their soil holding capacity and function are much more signi ficant in the frequ ent tidal inundated deltaic regions27.

High salinity intertidal mangrove -habitat imposes restriction on photosynthetic rate of mangrove leaves by high water deficit and low stomata l conductance2

.

A. corniculatul11 is a sensitive species to higher salinity

M ISHRA & DAS ~ EFFECT OF NaCi ON AEGICERAS CORNICULATUM 161

compared to sympatric species A vicennia marilla , also a secretor has lower water use efficiency and higher leaf area/plant mass rati03 and show decline in photo­synthetic rate4

. The salt secreti on through salt glands increases with increasing salinity. This plant also shows no change in growth even at hi ghest level of heavy metals36

. Aegiceras can however, survive and grow under fairly non-saline conditions . The observa­tions on A. corniculatum are the results from long­term studies3

.4, but short-term studies are rather scanty, although studies on a sympatric species A. marina are there8

. Here, we have examined the effect of short-term ex posure to hi gh salt (salt shock) on biomass, leaf salt secretion , photosynthetic pigments of leaves and thylakoids, spectral characteristics of thylakoids and anti ox idative enzymes like catalase (CAT), gui acol perox idase (POX), ascorbate peroxi­dase (APX) activity of roots.

Materials and Methods Plant material and growth conditions- Propagules

of Aegiceras corniculatum (L.) Blanco were collected from mangrove forests of Bhitarkanika (l atitude 20°4' to 200 8'N; longitude 86°45' to 87°50 'E) Orissa, India and was raised in the net house. Plants were watered with non-saline and non-bracki sh water. Three months old pl ants were taken and washed thoroughly with tap water to remove soils from root system, washed in di stilled water and then transferred to half­strength Hoagland's medium l6 for hydroponic cu lture. These cu ltures were aerated for 24 hr by ai r bubblers. The experiment was performed under controlled tem­perature (22°±2°C) day/night); under a photon flu x density of 300 !lmole m'2s' l with a photoperi od of 14 hr and 80% RH. Plants were harvested, leaves and roots were used for biochemical determination and measurement of biomass. Plants onl y in Hoagland 's mediu m were designated as control and those in the aforesaid medium containing NaCI (3 00mM) as treated plants. Effect of short-term exposure to salin­ity on biomass was determined by measurin g fresh and dry weight of leaves and roots, foliar area. Enzy­matic activ ity was measured at an interval of 24 hr.

Measurement of bioJ/lass - Aegicears cornicula­tum is a salt-sensitive spec ies. In a pilot experiment, we grew nursery-raised three month-old plants at di f­ferent concentrations of NaCI (l00, L50, 200, 250 and 300 mM) for 30 days supplemented with Hoagland's medium (Fig. 1). The plants could survive in NaCI (300 mM) supplemented medium for 10 days, while at lower concentrations, the plan ts survived for more

than 4 weeks. At 300mM aCI concentration, plants showed sign of senescence after 10 days under pre­sent ex perimental conditions. We, therefore, analyze the effect of short-term exposure to NaCI (300 mM) on A. corn iculatulI1 . After 96 hI' of NaCI (300 mM) treatment, roots and leaves were washed with tap wa­ter to remove surface salt and other materials, dried with blotting paper, weighed and then dried in an oven to constant weight at 80°C. Area of leaves was measured graphically.

Extraction of enzymes- Roots were cut (2c m) from lower end and washed properly with dist il led water to remove surface particles, if any, then blotted and weighed. They were homogenized under ice-cold condition in a ch illed pestle and mortar with a grind­ing medium [(3 ml , buffer g' l fresh weight of roots) contain ing 100 mM of phosphate buffer (PH 8); 1 mM, ethylenediaminetetraacetic acid (EDT A); 10mM sod ium ascorbate, 0.11 %, mercaptoethano!; and 10%, polyvinylpolypyrrol idone. Homogenate was centri ­fuged at 10,000g fo r 10 min and supernatant was taken for assay of catalase (CAT) , guaiacol peroxi­dase (POX) and ascorbate perox idase (APX). All as­says were done at room temperature.

Assay of enzyme activity- Activity of catalase was determined by monitoring the disappearance of H20 2 at 240 nm (molar absorption coefficient , 39.4 mM' 1 cm'l) following the procedure as described earlier6.lo . The assay mixture contained 50 mM of phosphate buffer (PH 7) and 20 mM of H20 2. The ac tivity of per­oxidase and ascorbate perox idase was determined fol­lowing the procedure as described25 and modified ear­lier28 . The assay mixture for general peroxidase con­tained 50 mM of phosphate buffer (pH 6), 0.8 mM, EDT A; 1 mM, H20 2; and 0 mM, guaiacol. For ascor­bate perox idase, assay medium was same as that for guaiacol perox idase except using ascorbate as elec­tron donor. Guaiacol peroxidase was measured by monitoring the increase in absorbance at 470 nm (ab­sorpti on coefficient 26.6 mM' lcnf l) due to tetra­guai acol formatio n. Oxidation of ascorbate was fol­lowed by decrease in absorbance at 290 nm (absorp­tion coefficient 2.8 mM lcm'I ).

Protein extraction and analysis - Root proteins were extracted fo llowing TCA-acetone method of Damerval9 and total soluble root proteins were re­solved in SDS-PAGE following the method of Laemmlil R.

Protein estimation - Protein concentrat ion was evaluated by the method as described earlier7 using bovine serum albumin as standard.

162 I DIAN J EXP SIOL, FEBRUARY 2003

Nal ive PAGE and aCli viry slaining of al1lioxidant enZYlI/e.\' - Nati ve polyacry lam ide gel electrophoresis (p AG E) was performed at 4 cC fo r catalase (CAT) , and perox idase (POX) using Laemmli IX buffer sys­tems with ail exception that SDS was absent fro m all buffers. Samples were mixed with 10% glycero l (vi \') and 0.25%, bromophenol blue before loading onto the gels. For each lane, SjJ.g of plotein ex tract was ap­plied. The gels were run at constant voltage of 200 V at 4°C in Bio-Rad (USA) Mini protean II electropho­res is apparatus until bromophenol blue marker dye had swept through most of the ge!.

Polyacrylamide ge l (7 5%) containing 0.5% so luble starch was prepared for CAT activity stai ning, the gels were stained34 and visualized fo r CAT act ivity. Im mediately afte r electrophores is, the gel was incu­bated in a wlution con tain ing sod iu m thiosl1lphate ( 18 111M) and 0.5% H20 2 (w/v) for 30 sec at room tem­perature. The gel was rinsed with di sti iled water and nooded with potassium iodide so lution (90 mM) acidified with 0.5% glacial aceti c acid. egative bands of CAT en zymes were appeared in blue back grou nd of the ge l.

Activity of POX in the ge ls was visuai ized on PAGE (7.5 %) according to stai ning procedure of Scandali os.lo. The gels were incubated at room tem­perature in the staining solution containing 49 mg 0-dianisidine, 29 mg ~-naphtho l , 20 ml acetone. 10 ml of 0.1 M, Tris-acetate buffer (pH 4), 0.3ml of 30% H20 2 and 70 ml water. Brick red colour bands ap­peared on gel, the reaction was arrested by i mmersi ng the gel in to a large vo lume of aceti c ac id 0%) for lOmin after Staining of bands sufficiently.

Thy!akoids is('/alioll - Thylakoids were isolated followi ng the procedure of Nakatani and Barberc6

Leaves were homogenized in chilled mortar and pes­tle using ice-cold grinding buffer (leaf to buffer rati o J '8) containing 15 111M tricine (pH 7.5), 5 mM MgCl], 400 mM sNbilOI and homogenate was fi Itered thro~l gh

<1 layers of nylon and 4 layers of cheese cloth . and the resulting slurry was centrifu ged at 2000g for 5min. The pellet was wa:,hed twice in a medium contain in g 10 ITIM tricine (pH 7.5), 10 tnM NaCI, and 5 mM MgCl2 at 2000g fo r 5min and finally the pellet was suspended in a buffer containing 20 mNi , Hepes (pH 7), i 0 InM, NaC I; 5 mM, MgCI2; and 100mM, sorbi­tol. Chlorophy ll and carotenoids were determi ned as mentioned in follow ing para.

ChlorolJ/7yll alld carolenoids delerlllinalion ­Chlorophyll and carotenoids were ex tracted by ho­mogenizing fl'esh leaves (0. 1 g) in 100% methanol

(2 ml). After centrifugation for IOmin at 2000g, ch lorophy ll s and carotenoids content were determined spectrophotometri cally follow ing the procedure de­scribed earli er20 (Chi a ()lg/m l)= 15 .65xA666- 7.34x A r,s.1 ; eh I h ()lg/ml) = 27 .05 X A(5) -I 1.21 x A666 ; Caro­tenoids ()lg/ml )=r I 000 x Al7o-(2 .86 x Ch i 0)- 129.2 x Ch i bl.

Salt secrelion rale - Salt secretion rate was meas­ured under culture room condit ions (see plant materi­als and growth cond itions). The leaves were washed with distilled water followed by blotting wi th a paper t.oweling just pi'ior to onset of photoperiod. Secreted sa lt (NaCl) was weighed at an interval of 24 hr. After 24 hI', the leaves were rinsed with distilled water and again blotted with paper toweli ng].

Results and Discussion Sa!1 secrelio!l- Aegiceras cornicII!allll/1 secretes

salt on the upper surface of the leaves. The secreted salt constitutes of number of ions32

, but the secretion consists mainly of NaCI JI . It has been reponed th at salts are accumu lated during day and removed during the ni ght and therefore, there is an increase in sal t secretion at night. In thi ~; way, mineral content of the pl ant is regul ated and compartmented in the leaves that co ntained the sa lts during the day are emptied during the ni ght to be available for a new supply in the foliowing day2'1. Salt secretion is ve ry effective in maintaining salt balance at low to moderate sa lin ity and ineffect ive at high salinit/ 4

• At moderate salinity , salt secretion increases with increasi ng sa linit/ I. Plants of /1. comiclI!afllll/ were raised in different concentrations of NaC I ( 100, 150, 200, 250 and 300 InM) supplemented with Hog land's solution (rig. 1). The plant could sLirvive in 300 mM of NaCi for 10 Jays. With increase in salt treatment period of A. cor­

!liell!allll/l, ihe rate of salt secretion increased linearl y. After 24 hI' of aCI (300 mM) treatment. 2.95)..lll1ole cm C of ~a\t secreted a11(1 after 96 hr sal t secretion in­creased to 13.732 ).llnole CITI ·

2 wi th a secretion rate of O.135)..l1l10Ie cm C h( 1 (Fig. 2). No salt was found to be deposi ted un lower surface of the leaves. These results are different from that of Ball '\ may be due to light intens ity and hu midity.

Spectra! characteristics 0/ thy!akoids- There wa, not much change in spectral characteristic; of thy la­koids upon salt treatment (Fig. 3). The spectrum showed sli ght clecrease in intensity and broadening of peak at 680nm in salt treated thylako ids. Some of the cross over pnints were observed at 695 .5 and 665 nm after normali zation of spectrum at 68011m.

MISHRA & DAS: EFFECT OF NaCI ON AEGICERAS CORNICULATUM 163

Measurement of photosynthetic pigments, proteins (lnd biomass- Effect of NaCI (300 mM) on the fresh weight, dry weight and leaf area of control and treated plants has been shown in Table I. Biomass did not change significantly after 96 hr of salt treatment. Fresh weight to dry weight ratio remained same (-4.0) and leaf area to dry weight ratio remain unal­tered. Photosynthetic pigments (ChI a, ChI band ca­rotenoids) did not change significantly upon high salt treatment. Total soluble root proteins were measured at an interval of 24 hr and it was found that there was not appreciable change in the protein concentration (Fig. 4). SDS-PAGE analysis of proteins of salt­treated ancl control roots also showed no change in protein profile (Fig. 6) that ranges from - 14 to 94kOa. This is expected as there was no significant change in fres h wt and dry wt during the experimental period.

Eflecf of Noel on activity oj antioxidant enzymes ­Root is the pri mary sensor of !>al t stress effect. In re­sponse to salinity , number of antioxidant enzymes increase or decrease invariably including catalase, peroxidase and ascorbate peroxidase 10. Therefore, we

examined effect of high NaCl on three antioxidant enzymes, catalase (CAT); guaiacol peroxidase (GPX) ; and ascorbate peroxidase (APX), in roots. There was an inhibition of activity of these antioxidant enzymes. Activity of CAT and GPX was inhibited more than APX. Activity of these enzy mes decreased by about

(I)

0 E ::1. - C. - I

ctI en

15

10

5

0

24 48

Hour 72 96

Fig. 2 -Salt secretion on the leaf of A. comiculatu.'1l in control and treated plants. Secreted salt was measured at intervals of 24 hr and amount of salt expressed in leaf area bas is. Data arc mean of two independent experiments.

Table I - Effect of NaCltreatment on pl ant growth parameters. Samples were t3-lcen after 96 hours. Data arc mean of two independent experiments. Each val ue represents mean of 5 replicates

RUOi

Leaf

Fresh wtlDry wt gg- I

C T

4.5 ± O.6 4 .37

±0.12

4.17 ± 0.1 2 4.05

± 0.27

Leaf area/Dry wt cm2 mg-1

C T

0.093 ±O.O

0.12 4 ±O. 12

Photosynthetic pigments mg g- Idry wt Ch la Chlb Carotenoids

C T C T C T

1.250 ±O.IO

1.285 ± O.II

0.650 ±0.13

0.640 ± .15

0.130 ±.0.04

-1

0_125

±0.05

Fig. I - (a) - Hydroponic culture of Aegiceras comiculatum ex posed to different concentrations of NaCi exposure for long term; (b) ­Three months old nursery grown plants were taken for hydroponic in Hoagland's medium in a culture room.

164 INDIAN J EXP BIOL, FEBRUARY 2003

1.8 .--------------------------~

1.5

\ \

\

\, 680 nm ~ .+,

558~---.._ 1\\ .~ .. '-~nm\ 1

694 .

0.7 L-______ ~ ______ _L ______ _L ______ ~ ________ L_ ______ ~ ______ _L ______ ~

350 400 500 600 700 750

Wavelength[mn] Fig. 3 - Room temperature absorption spectra of thylakoid membranes isolated from control and NaCltreated A. eorniell/alu lII . Thylako­ids equivalent to 6)1g Chi mrl were suspended in a buffer contai ning 100 mM, sorbitol; 10 mM, NaCI; 5 mM, MgCI2 ; and 20 mM, Hepes (PH 7). The dOlled li ne and the solid line graph represent the spectra of control and salt-treatcd samples rcspectivel ~' .

1.5

c 0 .5 .~ ... o '"' Q..

o o 24 48

Hours 72 96

Fig. 4 - Effect of high NaCl (300mM) on total soluble root pro­tein of A. eomieu/nlUm. Protein concentration was measured at an interval of 24 he Data given arc mean of two independent ex­periments.

90% of environmental control (Fig. 5). Activity of APX, CAT, and GPX decreased by 58, 72 and 80% respectively as compared to that of hydroponic con­trol (Fig. 5 inset). Staining of peroxidase (POX) and catalase (CAT) showed presence of 4 isoforms of peroxidase (POX 1, 2, 3 and 4) and two of catalase (CAT 1 and 2) in roots and there was also decrease in activity of these enzymes in salt treated roots (Fig. 7a, b). Guaiacol peroxidase activity is a general measure

1.2 -r--------;=::::::;r=====;_ -+-CAT

0.8

~ 0.6 '!> .~

0.4

i 0.2

0

0 24 48 72 96 Hours

Fig. 5 -Salt induced change in acti vity of antioxidant enzymes of roots. Activity of enzymes was measured at an interval of 24 hr. Data arc mean of two independent experiments. Activ ity of catalase (CAn, ascorbate peroxidase (APX) and guaiacol perox idase (GPX) expressed in )1mole min·lmg protein·l. Insert shows decrease in enzyme activity of salt treated and hydroponic control roots.

of peroxidase activity and its decrease reflects the loss of ability of plants to sustain ox idative damage by salt shock .

Decrease in acti vity of enzymes upon salt treatment was also reported in roots of glycophytes22 and

MISHRA & DAS: EFFECT OF NaCI ON AEGICERAS CORNICULATUM 165

M

66JJ>- -

43JJ>- ......

29JJ>- .....

2lJ.l>- ...-.

6

C T I •

POX 1>-

POX 3:>-l ....

c T

C T

Fig. 6- SDS-PAGE showing the protein patterns of control and salt-treated roots of A. corniculalum. Root proteins were extracted arter 96 hr of salt treatment I Lane M-Molccul ar mass markers in kDa; Lane C - Contro l; Lane T-salt-treated] .

Fig. 7 - Effect o f NaCI on antioxidant enzymes extracted from roots of Aegiceras cornicu falum. Enzy mes were acti vity stained followi ng standard procedure. (a) Activity staining of peroxidase; and (b) Catal ase. ILane C -control; and Lane T - salt treated]

halophytes t2• Ashiara et al. t examined seven enzymes

from the leaves of secretor mangrove A. marina and fo und that six of the enzymes has decrease in their activity as NaCI concentration increases. All seven enzymes have inhibition at 500 mM of NaCI. There­fore, present results are in agreement with earlier re­sults on mangroves. Although the antioxidative en­zyme, superoxide dismutase (SOD), plays important role against oxidative stress33

, the leve ls of SOD in the present study was not measured.

It was concluded from the present study that the high salt concentration (~300 rnM) suppressed activ­ity of antioxidative enzymes without losing the ability to secrete salt. This immediate loss of activity of anti­oxidative enzymes might induce leaf senescence after short term salt exposure.

Acknowledgement The authors are thankful to Council of Scientific &

Industrial Research, New-Delhi for financial sup-

ported (grant No. #38IEMR-II) and to the Director, Regional Plant Resource Center, Bhubaneswar for facilities .

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