Salinity Tolerance of Avicennia officinalis L. (Acanthaceae) from Gujarat Coasts of India

8

Salinity Tolerance of Avicennia officinalis L. (Acanthaceae)

from Gujarat Coasts of India

Ashish Dahyabhai Patel, Kishor Lalcheta, Sarvajeet Singh Gill, and Narendra Tuteja

Abstract

Greenhouse experiments were conducted to assess the effects of soil salinity onAvicennia officinalis L. (Acanthaceae) of Gujarat at different salinity levels (0.2, 5.4,10.3, 15.4, 20, 25.6, 30, and 34 ppt). Growth and physiological characteristics weremonitored over the subsequent 6 months. Total dry weight of plant tissuesincreased up to 5.1 ppt, but decreased at high salt concentrations. Organic soluteconcentration, such as soluble sugars, proline, and glycine betaine, decreased withan increase in salinity concentration up to 5.1 ppt, but increased with a furtherincrease in salinity (above 5.1 ppt). There was an increase in total chlorophyll anddecrease in total free amino acids and protein oxidation up to 5.1 ppt, after that itshowed the reverse trend with a further increase in salinity. Hydrogen peroxide(H2O2) continuously increased with an increase in salinity stress. Membraneleakage and lipid peroxidation decreased at 5.1 ppt, but increased with a furtherincrease in salinity levels. The activity of antioxidant enzymes, such as superoxidedismutase, catalase, ascorbate peroxidase, and glutathione reductase, decreased at5.1 ppt, but increased with a further increase in salinity levels. The overall resultsuggests that A. officinalis has a remarkably high degree of salinity tolerance, andshows an optimal growth and high activity of reactive oxygen species-scavengingantioxidant enzymes when soil water salinity was 5.1 ppt.

8.1

Introduction

Salinity is one of the major abiotic stresses and a key factor affecting plantproduction and growth in many arid and semi-arid areas throughout the world[1–3]. Salt stress affects plants in many ways by water deficit, ion toxicity, nutrientimbalance, and oxidative stress, which can further result in cellular damage orplant death [4,5]. Salinity tolerance by plants is a complex phenomenon thatinvolves many physiological, biochemical, and molecular processes [5,6]. Man-

189

Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

groves are woody plants that grow in intertidal zones with high salinity [7,8]. Theseplants can tolerate 200– 400mM or high-salinity stress, are able to survive andcomplete their life cycle under such natural conditions [9,10], and display a rangeof adaptive responses to their specific habitat, including salt exclusion by rootultrafiltration [11], salt secretion (elimination of substance not metabolicallychanged) via glands [12,9], and ion accumulation of organic acids as osmoticato counter the toxic effects of salinity stress [13]. Further, mangroves, like otherhalophytes, decrease their water and osmotic potential to maintain leaf turgor athigh salinity concentrations [14,15]. The level of salinity required for optimalgrowth varies from 10% to 50% sea water [15,16–18] and a decline in growth occurswith a further increase in salinity.One of the biochemical plant responses to salt stress is synthesis and

accumulation of low-molecular-weight organic compounds (compatible osmo-lytes) in the cytosol and organelles [19,20]. A major function of compatibleosmolytes has been reported to provide osmotic stress protection by allowingosmotic adjustment to counteract higher inorganic salts in the vacuole and rootmedium [21–23]. Salinity stress enhances the generation of reactive oxygenspecies (ROS) such as superoxide (O2

��), hydrogen peroxide (H2O2), and thehydroxyl radical (OH�) in plant tissues. Increased ROS concentration causesoxidative damage to plant tissues in various ways (e.g., decrease in chlorophyllcontent, damage to cell membrane, protein oxidation, strand breaks in nucleicacids, etc.), leading finally to cell death [24–27]. The degree of stress injuries toplants is reflected by membrane permeability [28]. Furthermore, ROS levelsabove threshold cause lipid peroxidation (LPO) in both cellular and organellemembranes, which not only directly affects cellular functioning, but alsoaggravates the oxidation stress through the production of lipid-derived radicals[27]. It is very important to select and characterize some salt-tolerant plants toidentify their antioxidative defense mechanism. Recently, some studies showeda correlation between salt tolerance mechanisms and antioxidative defensemechanisms in halophytic plants like Centaurea tuzgoluensis [29], Plantagomaritima [30], and Cakila maritime [31].Along the coasts of the Arabian Sea in Western Gujarat (India), mangroves are

common, but the stands are repeatedly decimated and fragmented [32]. Avicenniaofficinalis is the most common species extending from the semi-arid (near-arid)region of Saurashtra to the saline desert of Kutch. This plant species is also one ofthe most salt tolerant and has the ability to grow in coarser substrates [33]. A.officinalis also contains a salt tolerance mechanism by which it may able to regulatesalt concentration and water conservation at the whole-plant level via aquaporins[34]. Also, for the rehabilitation of ecologically degraded areas that may be hyper-saline, it is prudent to use A. officinalis to fill up the breaking coverage. Artificialregeneration of A. officinalis in the vacant areas of Sundarban in India was tried onan experimental basis [33]. It is assumed that mangroves growing in WesternGujarat have acclimated to arid conditions and can tolerate high salinity [32]. Thus,the present study was designed to investigate salt avoidance or salt tolerancemechanisms of A. officinalis of Gujarat at increasing salinity levels, by biochemical

190 8 Salinity Tolerance of Avicennia officinalis L. (Acanthaceae) from Gujarat Coasts of India

profiling in plant tissues. Such studies have been lacking up to now for mangrovesof arid regions.

8.2

Materials and Methods

8.2.1

Plant Material and Study Area

Seeds of A. officinalis were collected on 14 June 2011 from the Jamnagar coast (22� 270

N latitude, 70� 070 E longitude) of the Saurashtra region of Western Gujarat with thehelp of Gujarat Forest Department. The whole experiment was performed in agreenhouse of the botanical garden of Saurashtra University, Rajkot, Gujarat, India(22� 180 N latitude, 70� 560 E longitude). For establishment and growth of seedlings,black-cotton soil (vertisol) from the top 15 cm of soil was collected from a nearbyagricultural field. This soil is a clayey loam; it contains 19.6% sand, 20.3% silt, and0.1% clay by weight of dry soil. The available soil water between wilting coefficient andfield capacity ranged from 18.3% to 35.0%. The total organic carbon content was 1.3%and pH was 7.2. The salinity of the soil was 0.2 ppt. Nitrogen, phosphorus, potassium,calcium, and sodium concentrations were 0.15%, 0.05%, 0.03%, 0.05%, and 0.002%by weight of dry soil, respectively. This soil is fertile and used for intensive agriculture.Physical and chemical properties of the soil were reported earlier [35].

8.2.2

Salinization of Soil

Surface soil was collected, air dried, and passed through a 2-mmmesh. Eight lots ofsoil, each of 100 kg, were separately spread, about 50mm thick, over polyethylenesheets. Sodium chloride (NaCl) amounting to 0, 875, 1703, 2531, 3171, 3954, 4609,and 5484 g was then thoroughly mixed with the soil to give interstitial soil watersalinities of 0.2, 5.4, 10.3, 15.4, 20, 25.6, 30, and 34 ppt, respectively. Formeasurement of soil salinity, a soil suspension was prepared in distilled water witha 1 : 2 soil : water ratio [15]. The soil suspension was shaken vigorously and allowedto stand overnight. After that the conductivity of the soil suspension was measuredwith a conductivity meter (Systronics; Model 304). Control soil had a conductivityof 0.2 ppt. In the rainy season at Jamnagar coast the sea water salinity varies from28.8 to 30.7 ppt, which is favorable for seedling establishment. Therefore, thesalinity level in the present study was not levied above 34 ppt.

8.2.3

Plant Establishment

Thirty polyethylene bags (20.5 cm wide and 41 cm long) were each filled with 5 kgof soil for each level of soil salinity. Tap water was added to the soil in each bag until

8.2 Materials and Methods 191

the water level was 2 cm above the soil surface. Bags were then kept under naturaltemperature and light in an uncontrolled greenhouse. Ten seeds were gentlypressed to a depth of about 10–15mm in each bag on 15 June 2011. Tap water wasadded daily to compensate for evapotranspiration loss. Plant establishment wasrecorded daily over 40 days.

8.2.4

Plant Growth

Two seedlings that established first were left in each of 30 bags at each level ofsalinity and others were uprooted as they appeared. Seedlings exhibited emergenceof the second leaf after 9–30 days; later at higher salinity levels. Following theemergence of the second leaf, one seedling having better vigor was allowed to growin each bag and another seedling was further uprooted. Thus, 30 replicatesfactorialized with eight grades of soil (0.2, 5.4, 10.3, 15.4, 20, 25.6, 30, and 34 ppt)were prepared. This gave a total of 240 bags, which were arranged in 30randomized blocks. Seedlings were watered on alternate days to maintain thewater level above the soil surface and the experiment was terminated after6 months. Twenty plants grown at each salinity level were then washed with tapwater to remove soil particles that adhered to roots. Dry weights of leaves, stems,and roots for each plant were determined. The remaining 10 plants were used forbiochemical analyses.

8.2.5

Organic Solutes (Soluble Sugars, Proline, and Glycine Betaine)

Total soluble sugar content was estimated by the phenol sulfuric method [36]. A100-mg tissue sample was hydrolyzed by keeping it in a boiling water bath for 3 hwith 5ml of 2.5N HCl and then neutralized with solid sodium carbonate until theeffervescence ceased. The volume was made up to 100ml and centrifuged.Supernatant aliquots of 0.1 and 0.2ml was taken in separate test tubes and made to1ml. Then, 1ml of phenol solution followed by 5ml of 96% sulfuric acid wereadded to each test tube, shaken well, and placed in a water bath at 25–30 �C for20min. Chromophore was read at 490 nm. The amount of total carbohydrate wascalculated using the standard curve of glucose.Proline content was determined according to [37] with some modifications.

Proline was extracted from 500mg of plant tissues by grinding in 10ml of 3%sulfosalicyclic acid and the mixture was then centrifuged at 10 000� g for 10min.An aliquot of 2ml of supernatant was taken in a test tube to which an equal volumeof freshly prepared ninhydrin solution was added. Tubes were incubated for 30minin a water bath at 90 �C. After incubation, the reaction was terminated in an ice bath.Then, the reaction mixture was extracted with 5ml toluene with continuous stirringfor 15min. The tubes were allowed to stand for 20min in the dark for the separationof the supernatant of the toluene and aqueous phases. The toluene phase was thencarefully collected into a test tube and absorbance was measured at 520 nm. The


concentration of proline was calculated from a standard curve using the followingequation: (mg proline in extract/111.5)/g of sample¼mmol g�1 of fresh tissue.Quaternary ammonium compounds were extracted and measured as glycine

betaine equivalents [38]. Dried and finely ground lyophilized samples weremechanically shaken with 20ml of deionized water for 24h at 25 �C. Samples werethen filtered and the filtrates were diluted (1 : 1) with 2N sulfuric acid. Aliquots(0.5ml) were taken into centrifuge tubes and cooled in ice water for 1h. Then cold KI-I2 reagent (15.7 g of iodine and 20 g of potassium iodide dissolved in 100ml ofdistilled water) (20ml) was added and reactants gently stirred. The reaction mixturewas stored at 4 �C for 16h followed by centrifugation at 10 000 rpm for 15min at 0 �C.Supernatant was carefully removed and the periodide crystals suspended in 9ml of1,2-dichloroethane. The absorbance was measured at 365nm after 2 h. Glycine betaine(50–200 gml�1) in 1N H2SO4 was used as standard. The results were expressed asmillimole glycine betaine per kilogram tissue water in the leaf and root samples.

8.2.6

Chlorophyll Content, Total Free Amino Acids, Hydrogen Peroxide (H2O2),

and Protein Oxidation

Chlorophyll content was estimated according to [39]. Chlorophyll was extractedwith 80% (v/v) chilled acetone. The absorbance for chlorophyll B at 645 nm and forchlorophyll A at 663 nm was recorded. Total chlorophyll was calculated using:

Total chlorophyll ¼ ½fð20:2� A645Þ þ ð8:02� A663Þg�=1000�W � V ;

whereW is the fresh weight of the material and V is the extraction volume.The estimation of total free amino acids was performed by [40]. An aliquot of

plant tissue extracts was made up to 1ml with distilled water and 1ml of ninhydrinreagent was added to it. The tubes were placed in a boiling water bath for 20min.Then the tubes were cooled and 5ml of diluents added to each tube. Theabsorbance was then measured at 570 nm.Hydrogen peroxide content was determined according to modified method of

[41]. Plant tissue (300mg) was homogenized in 3ml of 1% w/v trichloroaceticacid (TCA). The homogenate was centrifuged at 10 000� g for 10min at 4 �C.Subsequently, 0.75ml of supernatant was added to 0.75ml of 10mM potassiumphosphate buffer (pH 7.0) and 1.5ml KI. The H2O2 concentration of supernatantwas evaluated by comparing its absorbance at 390 nm to a standard calibrationcurve. The concentration of H2O2 was calculated from a standard curve of H2O2

and concentration was expressed as mmol g�1 fresh weight.Protein carbonylation content was measured according to the method of Levine

et al. [42]. Samples containing at least 0.5mg protein were incubated with 0.3% w/vTriton X-100 for 20min and thereafter centrifuged at 2000� g for 10min. A 200-mlaliquot of supernatant was mixed with 300 ml of 10mM DNPH in 2M HCl. Proteinwas precipitated with 10% w/v TCA after 1 h incubation at room temperature.Protein pellets were washed with 500 ml of ethanol : ethylacetate (1 : 1) 3 times anddissolved in 6M guanidine hydrochloride in 20mM potassium phosphate buffer

8.2 Materials and Methods 193

(pH 2.3). Absorbance was taken at 370 nm and recovery of protein was alsomeasured at 280 nm for each tissue. A molar extinction coefficient of 22 000M�1

cm�1 was used for the calculation of the carbonyl content.

8.2.7

Membrane Permeability and Lipid Peroxidation

Electrolyte leakage rate (ELR) was measured with some modifications in themethod of Lutts et al. [28]. Samples of 100mg of fresh tissues were cut into smallpieces and placed into a boiling tube containing 20ml of deionized water and theelectrical conductivity (EC1) of the bathing solution was determined. The tubeswere incubated at 30 �C for 3 h and then the electrical conductivity (EC2) wasmeasured. Later, the test tubes containing samples were autoclaved at 121 �C for20min to release all electrolytes and finally cooled, and the electrical conductivity(EC3) was determined. The ELR can be calculated as:

ELR% ¼ ðEC2 � EC1=EC3Þ � 100;

LPO was measured as the amount of malondialdehyde (MDA) determined by theTBA reaction as described by Heath and Packer [43] with some modifications.Samples of 200mg of leaf tissue were homogenized nearly in 2ml of 1% TCA andthen centrifuged at 10 000� g for 15min. One milliliter of the supernatant aliquot,2ml of 20% w/v TCA, and 2ml of 0.5% TBA were mixed, and incubated for 30minat 95 �C followed by quick transfer in an ice bath for termination of the reaction.The absorbance was measured at 532 nm after centrifugation at 10 000� g for5min. The value at 532 nm was subtracted from the value of non-specificabsorbance at 600 nm. MDA content was calculated from the extinction coefficientat 155mM�1 cm�1 and defined as mmol g�1 fresh weight MDA.

8.2.8

Antioxidant Enzymes

Tissue extractions were prepared for the analysis by homogenizing 200mg of plantmaterial in 2ml of 0.2M potassium phosphate buffer (pH 7.8 with 0.1mM EDTA).The homogenate was centrifuged at 15 000� g for 20min at 4 �C. Afterwards,tissue extract was stored in �20 �C and used within 48 h to determine differentantioxidant enzymatic activity.Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured using a

modified NBT (nitro blue tetrazolium) method described by Beyer and Fridovich[44]. The 2-ml assay reaction mixture containing 50mM phosphate buffer (pH 7.8),2mM EDTA, 9.9mM L-methionine, 55 mM NBT, and 0.025% Triton-X100 wastaken in a test tube. Then 40ml of diluted (�2) plant sample and 20 ml of 1mMriboflavin were added, and the reaction was initiated by illuminating the sampleunder a 15-W fluorescent tube. During the 10-min exposure, the test tubes wereplaced in a box lined with aluminum foil that was at a distance of approximately12 cm from the light source. Duplicate tubes with the same reaction mixture were


also kept in the dark and used as blanks. One unit of SOD was defined as theamount of enzyme per milligram of protein sample causing 50% inhibition of therate of NBTreduction at 560 nm.Catalase (CAT; EC 1.11.1.6) activity was determined according to Aebi and Lester

[45]. The 3-ml assay mixture contained 2ml tissue extract (diluted 200 times in 50mMpotassium phosphate buffer, pH 7.0) and 10mM H2O2. The decomposition of H2O2

was followed as a decrease in absorbance at 240nm. The extinction coefficient ofH2O2 (40mM�1 cm�1 at 240nm) was used to calculate the enzyme activity.Ascorbate peroxidase (APX; EC 1.11.1.11) was measured using method of Nakano

and Asada [46]. The 1-ml assay mixture contained 50mM potassium phosphatebuffer (pH 7.0), 0.5mM ascorbate, 0.5mM H2O2, and 10ml of tissue extract. H2O2

was added last to initiate the reaction and the decrease in absorbance was recordedfor 3min. APX activity was determined from the decrease in absorbance at 290nmdue to oxidation of ascorbate in the reaction. The extinction coefficient of 2.8mM�1

cm�1 for reduced ascorbate was used in calculating the enzyme activity of APX.Glutathione reductase (EC 1.8.1.7) activity was assayed according to Smith et al.

[47]. A 10-ml aliquot of tissue extract was used in the assay along with 0.75mMDTNB (Ellman’s reagent; 5,50-dithio-bis-(2-nitrobenzoic acid)), 0.1mM NADPH,and 1mMGSSG in a total of 1ml assay volume. GSSGwas added last to initiate thereaction and the increase in absorbance was recorded for 3min. The increase inabsorbance at 412 nm was measured when DTNB was reduced to TNB by GSH inthe reaction. The extinction coefficient of TNB (14.15mM�1 cm�1) was used tocalculate the activity of glutathione reductase.

8.2.9

Statistical Analyses

Data recorded for total dry weight and biochemical characteristic were analyzed byone-way ANOVA to assess the effect of salinity levels on plant growth with the helpof SPSS software. A second-order polynomial model was fitted to the data for theexamined parameters and increasing soil salinity using:

Y ¼ aþ b1X þ b2X2;

where Y is the examined parameter, X is the soil salinity, a is the y-intercept, and b1and b2 are constants.

8.3

Results

8.3.1

Total Dry Weight of Plants

Total dry weight of plants (shootsþ roots) significantly increased (p< 0.01) untilsalinity levels increased up to 5.1 ppt, but decreased at higher salinity levels,although never below that of control plants (Figure 8.1)

8.3 Results 195

8.3.2

Organic Solutes (Soluble Sugars, Proline, and Glycine Betaine)

Soluble sugars in plant tissues (leaves, stems, and roots) decreased up to salinity of5.1 ppt, but at higher salinity levels significantly (p< 0.05) increased to valueshigher than those of controls (Figure 8.2a). Tissues, according to their soluble sugarcontent, can be arranged in the decreasing order of leaves, roots, and stems. Therewas a quadratic relationship between soluble sugar content in different tissues andsalt concentration (r2¼ 0.503, 0.495, and 0.465, p< 0.01 for leaves, roots, andstems, respectively). Concentrations of proline and glycine betaine showed a trendsimilar to that of total sugar content (Figure 8.2b and c). Salinity significantlyincreased the concentration of proline (p< 0.05) and glycine betaine (p< 0.01) inleaves, stems, and roots. Tissues, according to their proline and glycine betainecontent, can be arranged in the decreasing order of leaves, roots, and stems. Therewas a quadratic relationship between proline content in different tissues and saltconcentration (r2¼ 0.508, 0.562, and 0.471, p< 0.01 for leaves, roots, and stems,respectively). A quadratic relationship was also obtained between glycine betainecontent in different tissues and salt concentration (r2¼ 0.683, 0.736, and 0.672,p< 0.01 for leaves, roots, and stems, respectively).

8.3.3

Chlorophyll Content, Total Free Amino Acids, H2O2, and Protein Oxidation

Chlorophyll content in plant tissues increased up to the salinity of 5.1 ppt, but athigher salinities significantly (p< 0.05) declined, reaching values lower than thoseof controls (Figure 8.3a). Tissues, according to their total chlorophyll content, canbe arranged in the decreasing order of leaves, stems, and roots. There was aquadratic relationship between chlorophyll content in different tissues and salt

1600

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Real optimum curve with estimated values

Optimum curve with observed value

0343028.62015.410.35.10.2

Salinity (ppt)

Figure 8.1 Effect of increasing soil salinity on total dry weight of A. officinalis plants after 6months

growthfromseeds.Afittedquadraticpolynomialcurve(Y¼ 805.96þ 425.64X� 53.93X2, r2¼ 0.690,

p< 0.010) is shown.


concentration (r2¼�0.867, �0.874, and �0.928, p< 0.01 for leaves, stems, androots, respectively). Total free amino acids in plant tissues decreased up to salinitiesof 5.1 ppt, but at higher salinities significantly increased (p< 0.01) to values similarto those of controls (Figure 8.3b). Tissues, according to their total free amino acidscontent, can be arranged in the decreasing order of leaves, roots, and stems. Therewas a quadratic relationship between total free amino acids content in differenttissues and salt concentration (r2¼ 0.615, 0.615, and 0.479, p< 0.01 for leaves,roots, and stems, respectively). H2O2 concentration in plant tissues significantlyincreased (p< 0.01) with the increase in salinity (Figure 8.3c). Tissues, according totheir H2O2 content, can be arranged in the decreasing order of leaves, roots, and

0

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Figure 8.2 Effect of increasing soil salinity concentration on (a) soluble sugars, (b) proline, and (c)

glycine betaine ofA. officinalis after 6months growth from seeds. Error bars represent SE. FW, fresh

weight; DW, dry weight.

8.3 Results 197

stems. There was a positive relationship between H2O2 content in different tissuesand salt concentration (r2¼ 0.967, 0.985, and 0.977, p< 0.01 for leaves, roots, andstems, respectively). Protein oxidation in plant tissues declined up to the salinity of5.1 ppt, but, with increasing salinity, increased to values higher than those of controls(Figure 8.3d). Tissues, according to their protein oxidation, can be arranged in thedecreasing order of leaves, roots, and stems. There was a quadratic relationshipbetween protein oxidation content in different tissues and salt concentration(r2¼ 0.568, 0.635, and 0.685, p< 0.01 for leaves, roots, and stems, respectively).

8.3.4

Lipid Peroxidation and Membrane Permeability

LPO in plant tissues significantly decreased (p< 0.05) up to salinities of 5.1 ppt,but, at higher salinities, increased to values similar to those of controls

0

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40

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H2O

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(µM

g-1

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)

Salinity (ppt)

(c)

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mol

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Salinity (ppt)

(d)

0

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0.6

0.8

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1.2

1.4

1.6T

otal

Chl

orop

hyll

(µg

g-1 D

W)

(a)

Figure 8.3 Effect of increasing soil salinity on (a) total chlorophyll, (b) total free amino acids (c),

H2O2,and(d)proteinoxidationofA.officinalisafter6monthsgrowthfromseeds.Errorbarsrepresent

SE. DW, dry weight.


(Figure 8.4a). Tissues, according to their LPO, can be arranged in the decreasingorder of leaves, roots, and stems. There was a quadratic relationship between LPOin different tissues and salt concentration (r2¼ 0.655, 0.624, and 0.600, p< 0.01 forleaves, roots, and stems, respectively). Membrane permeability in plant tissuesshowed a trend similar to that of LPO (Figure 8.4b). Tissues, according to theirmembrane permeability, can be arranged in the decreasing order of leaves, roots,and stems. There was a quadratic relationship between membrane permeability indifferent tissues and salt concentration (r2¼ 0.518, 0.559, and 0.510, p< 0.01 forleaves, roots, and stems, respectively).

8.3.5

Antioxidant Enzymes (SOD, CAT, APX, and Glutathione Reductase)

Antioxidant enzymes activities exhibited a similar trend in response to salinity.There was a decline in activities of antioxidant enzymes up to the salinity of 5.1 ppt,but at higher salinities, activity significantly increased for SOD, CAT, and APX(p< 0.05), and glutathione reductase (p< 0.01) to levels similar to those of controltissues (Figure 8.5a–d). Tissues, according to their antioxidant enzymes activities,can be arranged in the decreasing order of leaves, roots, and stems. There was a

0

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100E

lect

olyt

e le

akag

e%

(a) Leaves

Stems

Roots

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30

0.2 5.1 10.3 15.4 20 28.6 30 34

MD

A (

µm

ol/g

m F

W)

Salinity (ppt)

(b)

Figure8.4 Effectof increasingsoilsalinityon(a)membranepermeabilityand(b)LPOofA.officinalis

after 6 months growth from seeds. Error bars represent SE. FW, fresh weight.

8.3 Results 199

quadratic relationship between antioxidant enzymes activities in different planttissues and salt concentration (SOD, r2¼ 0.544, 0.599, and 0.380; CAT, r2¼ 0.518,0.588, and 0.669; APX, r2¼ 0.636, 0.419, and 0.475, and glutathione reductase,r2¼ 0.548, 0.594, and 0.585, p< 0.01 for leaves, roots, and stems, respectively).

8.4

Discussion

Growth of young A. officinalis plants was stimulated by low salinity and was optimalat 5.1 ppt. Similar results have also been reported for other halophytes [15,48–52].Soil salinity at 5.1 ppt approximately equals 17% rainy season sea water at theJamanagar coast. Optimum growth of seedlings was obtained at 50% sea water for

0

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Uni

t ac

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pro

tein

Salinity (ppt)

(d)

Figure 8.5 Effect of increasing soil salinity on (a) SOD, (b) CAT, (c) APX, and (d) glutathione

reductase of A. officinalis after 6 months growth from seeds. Error bars represent SE.


A. marina from Sundarban [53], Ceriops tagal and Rhizophora mucronata fromPakistan [54], and Sonneratia alba from Australia [18]. Other studies have reportedlower optimal around 25% sea water for A. marina and Rhizophora stylosa[16,17,55]. As a result, A. officinalis growing along the semi-arid and arid coasts ofGujarat in India has considerable salinity tolerance. Under natural conditions inGujarat, seedlings of A. officinalis establish during the rainy season when thesalinity level of sea water is somewhat diluted.Under salt stress conditions plants accumulate sugars, proline, and glycine

betaine in cytosol for osmotic protection. The accumulation of soluble carbohy-drates in plants has been widely reported as a response to salinity or drought,despite a significant decrease in the net CO2 accumulation rate [56]. According toCram [57], of the various organic osmotica, sugars contribute up to 50% of the totalosmotic potential in glycophytes subjected to saline conditions. Proline regulatesthe accumulation of usable N, is osmotically very active [58], contributes tomembrane stability [59–62], and mitigates the effect of NaCl on cell membranedisruption [63]. Proline is also found to protect enzyme and/or protein degradationby oxidative damage in saline conditions [64]. Even at supraoptimal levels, prolinedoes not suppress enzyme activity. Some authors [65,66] are of the view that prolinemay act as signaling/regulatory molecule able to activate multiple responses thatare components of the adaptation process. In several plant species, a positivecorrelation between leaf osmotic potential and glycine betaine has been observed[67,68]. The quaternary ammonium compounds are now known to have an osmoticeffect on cells [67]. In the present study, a decrease of soluble sugars, proline, andglycine betaine was recorded at the optimum growth stage at the salinity of 5.1 ppt.It appears that Naþ might have been used for osmotic adjustment and/orconcentrations of organic osmotica could have been diluted because of the rapidgrowth rate of A. officinalis. Moreover, above this salinity level the concentration ofthese organic compounds increased when plant growth decreased in response toincreasing salinity.Total chlorophyll content in tissues of A. officinalis decreased with an increase

in soil salinity. Similar findings have been reported in different crop plants (e.g.alfalfa [69], sunflower [70], wheat [71], and Brassica napus [72]). The decrease intotal chlorophyll of this A. officinalis might have been due to a salt-inducedincrease in activity of the chlorophyll-degrading enzyme, chlorophylase [73],and/or destruction of chloroplast structure and the instability of pigment proteincomplexes [74]. An increase in total chlorophyll at 5.1 ppt can be attributed todilution of Naþ and Cl� in tissue due to the rapid growth of plants at thissalinity level. Total free amino acids increased in plants in response to saltstress. Total free amino acids in leaves are reported to be higher in salt-tolerantthan in salt-sensitive lines of sunflower [70], safflower [75], Eruca sativa [58], andLens culinaris [76]. The decline in total free amino acids in tissues at optimumplant growth at 5.1 ppt can again be accounted for by the dilution effect. It isreported that H2O2 plays a dual role in plants: at low concentrations, it acts as asignal molecule involved in acclamatory signaling, triggering tolerance tovarious biotic and abiotic stresses; at high concentrations, by activating various

8.4 Discussion 201

antioxidative enzymes [77], which leads to programmed cell death [78]. H2O2 hasalso been known to act as a key regulator in a broad range of physiologicalprocesses, such as senescence [79], photorespiration and photosynthesis [80],stomatal movement [81], cell cycle [82], and growth and development [83]. Moreimportantly, H2O2 is found to increase under saline conditions in many salt-tolerant plant species [84,85] and can be connected to the induction of variousSOD isoenzymes [86]. In the current study, an increase in dry weight content at5.1 ppt shows the increase in H2O2 level at 5.1 ppt significantly.Protein oxidation is regarded to as a covalent modification of a protein induced by

ROS or by products of oxidative stress. Most types of protein oxidations areeventually irreversible, whereas a few involving sulfur-containing amino acids arereversible [87]. Protein carbonylation is widely used as a marker of proteinoxidation [88,89]. It has been found that various stresses lead to the carbonylationof proteins in tissues [27]. Reduction in growth of A. officinalis at salinities above5.1 ppt can be due to an increase in protein oxidation with increasing salinity.The peroxidation of lipids is considered as one of the most damaging process

known to occur in every living organism and the extent of LPO has been seen as ameans to assess the severity of induced oxidative stress due to salt and degree ofplant sensitivity [90]. When ROS levels increase above the threshold level then LPOtakes place in cellular as well as in organelle membranes. Therefore, LPO does notdirectly affect normal cellular metabolism and other functions, but by productionof lipid-derived radicals it generates the oxidative stress condition [91]. In the initialstage it changes metal complexes of Fe and Cu. OH� plays a crucial role in theinitiation of LPO by abstraction of a hydrogen molecule in the polyunsaturated fattyacid (PUFA) residue. Oxygen is added in an aerobic environment in lipid radicalsfor the production of ROO�. After that ROO� can further propagate theperoxidation chain reaction by abstracting a hydrogen atom from adjacent side-chains of PUFAs. This further results in the formation of lipid alkoxyl radicals,aldehydes (MDA), alkanes, lipid epoxides, and alcohols [92,93]. LPO mainly affectsthe functionality of membranes. It increases membrane permeability, whichproduces leakiness in the membrane.The metalloenzyme SOD is a very important enzymatic antioxidant. SOD is

found in all aerobic organisms in which it is localized to all subcellularcompartments where ROS is produced [94]. Under various stress conditions, ROSgenerates toxic effects inside the cell. SOD was proposed to be the first line ofdefense against these adverse conditions. It alleviates ROS and increases tolerancein plant tissues. The superoxide anion (O2

��) is reduced to H2O2 and furtheroxidized to O2 by SOD. This decreases the risk of OH� formation. The reaction isalso known as a Haber–Weiss-type reaction and is 10 000-fold faster thanspontaneous dismutation. SOD is found in association with different metalcofactors, which generate copper/zinc (Cu/Zn-SOD), manganese (Mn-SOD) andiron (Fe-SOD), and are localized in different cellular compartments [95]. It is widelyobserved that under saline environments different isoenzymes of SOD exhibitdifferent intensities [96]. Induction of these types of isoenzymes is well understoodin the halophyte Gypsophila oblanceolata [85].


CATs have the potential to directly dismutate H2O2 into H2O and O2, which isvital for ROS detoxification during stressed conditions. This tetrameric heme-containing enzyme has the capacity to convert 6 million molecules of H2O2 intoH2O and O2 in 1min. The H2O2 generated during b-oxidation, photorespiration,and catabolism of purine in peroxisomes is also removed by CAT [27]. Despitemany reports, CAT activity under salinity stress still remains unclear. According to[27] and [97], CAT activity increased under high salinity stress; however, Sabra et al.[98] reported a decrease in CATactivity.APX is one of the most important ROS-scavenging enzymes and plays a crucial

role in protecting cells from stress conditions [94]. The mechanism of scavengingROS by APX involves ascorbate–reduced glutathione (ASH-GSH) cycles and water–water generation in which ASH acts as an electron donor. APX has different tissue-specific isoforms: thylakoid (tAPX), glyoxisome membrane (gmAPX), chloroplaststromal (sAPX), and cytosolic (cAPX) [80]. Due to the higher affinity towards itssubstrate (H2O2) as compare to POD and CAT, it plays a key role in ROS defense.Many reports suggested induced expression of APX in plant tissues duringdifferent stress conditions [5].Glutathione reductase is a flavo-protein oxidoreductase class enzyme, and is

found in both prokaryotes and eukaryotes [99]. Glutathione reductase utilizes theASH–GSH cycle for its mechanism; however, unlike APX, it neutralizes ROS bymaintaining the reduced status of GSH [94]. Glutathione reductase is foundpredominantly in chloroplasts of all plant tissues, but some is also found inmitochondria and the cytosol in smaller quantities [100,101]. The main mechanismof catalysis of glutathione reductase is reduction of GSH, which is involved inmany antioxidative processes and regulatory metabolic pathways. In plants,glutathione reductase is NADPH dependent and acts on the disulfide bond ofGSSG (oxidized), and thus plays a very important role in GSH (reduced) poolmaintenance [102,103]. Oxidized GSSG has two GSH bound by a disulfide linkagethat are converted into GSH (reduced) by the activity of glutathione reductase.Glutathione reductase is mainly an oxidative stress-released enzyme, while GSHparticipates in the ASH–GSH cycle and sulfhydryl (SH) group maintenance. It alsoacts as a substrate for GSTs [102]. According to Sekman et al. [85], the increasedactivity of glutathione reductase and GSH plays a crucial role in determining thetolerance of a plant under various stresses. The results suggest that the increasedconcentration of antioxidant enzymes (SOD, CAT, APX, and glutathione reductase)with an increase in salinity may elicit the ability of A. officinalis to endure andsurvive under the high salinity of Arabian seawater.

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Salinity Tolerance of Avicennia officinalis L. (Acanthaceae) from Gujarat Coasts of India

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