Ascorbic Acid and Salicylic Acid Mitigate NaCl Stressin Caralluma tuberculata Calli

Riaz Ur Rehman & Muhammad Zia &

Bilal Haider Abbasi & Gang Lu &

Muhammad Fayyaz Chaudhary

Received: 2 February 2014 /Accepted: 24 March 2014# Springer Science+Business Media New York 2014

Abstract Plants exposed to salt stress undergo biochemical and morphological changes evenat cellular level. Such changes also include activation of antioxidant enzymes to scavengereactive oxygen species, while morphological changes are determined as deformation ofmembranes and organelles. Present investigation substantiates this phenomenon forCaralluma tuberculata calli when exposed to NaCl stress at different concentrations.Elevated levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbateperoxidase (APX), and glutathione reductase (GR) in NaCl-stressed calli dwindled uponapplication of non-enzymatic antioxidants; ascorbic acid (AA) and salicylic acid (SA).Many fold increased enzymes concentrations trimmed down even below as present in thecontrol calli. Electron microscopic images accentuated several cellular changes upon NaClstress such as plasmolysed plasma membrane, disruption of nuclear membrane, increasednumbers of nucleoli, alteration in shape and lamellar membrane system in plastid, andincreased number of plastoglobuli. The cells retrieved their normal structure upon exposureto non-enzymatic antioxidants. The results of the present experiments conclude that NaClaggravate oxidative molecules that eventually alleviate antioxidant enzymatic system.Furthermore, the salt stress knocked down by applying ascorbic acid and salicylic acidmanifested by normal enzyme level and restoration of cellular structure.

Keywords Antioxidant enzymes .Caralluma tuberculata calli . NaCl stress . ROS .

Ultra-structure

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0890-6

R. U. RehmanHorticulture and Floriculture Institute, Government of Punjab, Rawalpindi, Pakistan

M. Zia (*) : B. H. AbbasiDepartment of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 45320e-mail: ziachaudhary@gmail.com

G. LuCollege of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

M. F. ChaudharyPreston Institute of Nanoscience and Technology, Preston University, Islamabad, Pakistan

AbbreviationsAA Ascorbic acidAPX Ascorbate peroxidaseCAT CatalaseFWGR Fresh weight growth rateGR Glutathione reductaseROS Reactive oxygen speciesSA Salicylic acidSOD Superoxide dismutasePOD Peroxidase

Introduction

Salinity is among the sternest factors influencing crop efficiency, even in well-watered soils.Considerable changes in water balance and ionic form result damage at molecular level andseverely affect the growth in stressed plants. Consequently, the plant tissues die and death ofplant may occur in severe saline conditions [1]. Such stresses result in interference of growthand metabolism by triggering secondary responses like the production of highly reactiveoxygen species (ROS).

The production of ROS such as the hydrogen peroxide (H2O2), the superoxide radical(O−2), and the hydroxyl radical (OH−1) are critical; however, enzymatic or non-enzymaticROS-scavenging systems in plants efficiently wipe out these hazardous components. ROS,mainly hydrogen peroxide (H2O2), also act as important signal in both biotic and abiotic stressresponses [2]. The major antioxidant enzymes are superoxide dismutase (SOD) catalyzing thedismutation of O−2 to H2O2; catalase (CAT) that dismutase H2O2 to oxygen and water; andascorbate peroxidase (APX) that reduces H2O2 to water by utilizing ascorbate as particularelectron donor. Moreover, other enzymes involved are glutathione reductase (GR),monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathi-one peroxidase (GPX), and glutathione-D-transferase, which are significant in protecting cellagainst oxidative stress [3].

In salt-affected cell/plants, biochemical as well as physiological changes occur i.e., dehy-dration at cellular level, swelling and structural collapse of membranes, disorder of the outerchloroplast envelope, thinning of partitions, adhesion within the grana, decrease in chloroplastvolume [4–6], swelling of thylakoids at earlier stage [7, 8], and deformation of other organ-elles. Such physiological changes have been observed both in salt-sensitive and salt-adaptivecell lines. Osmoregulation mechanism is a complex process; however, the adaptive capacity tomaintain membrane integrity during a long period of water deficit may be an essentialbiological trait for drought tolerance.

Salicylic acid (SA) and ascorbic acid (AA) are small antioxidant molecules, which arewater soluble and act as a principal substrate in non-enzymatic detoxification of hydro-gen peroxide in the cyclic pathway. Consistent findings have reported the valuable effectof ascorbic acid application used exogenously in improving the adverse effects ongrowth due to salt stress [9]. Salicylic acid also intervenes the oxidative rupture thatcauses death of the cells in the oversensitive reaction and proceeds as signal to developcomplete internal resistance [10]. It also plays an important role in many abiotic stressesto survive the plants against these pressures [11]. However, unexpectedly, little is knownabout the role of these antioxidative compounds in callus stress adaptation. The aims ofthe present study were to investigate the antioxidant enzyme status in the callus ofCaralluma tuberculata, under NaCl stress, alleviation of NaCl-stress by ascorbic acid

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and salicylic acid, and to investigate the intracellular changes resulted by the stress incallus tissues of C. tuberculata.

Materials and Methods

Plant Material and Explant Preparation

The plant material of C. tuberculata used for the study was obtained from the local market ofQuetta (Balochistan, Pakistan) and was identified by Prof. Dr. Mir Ajab Khan, Department ofPlant Sciences, Quaid-i-Azam University Islamabad, Pakistan. The plant material brought tolab was multiplied in earthen pots in greenhouse for continuous supply of explants.

The methodology to produce callus was adopted as described by Rehman et al. [12]. Indetail, before starting the experiment, the plants collected from the earthen pots were washedunder running tap water for 30 min to remove all adhering contaminants following washingwith 0.2 % liquid detergent (Triton X-100) for about 15 min. Thereafter, the plants were rinsedwith distilled water and treated with bevistin (a fungicide) for 30 min followed by rinsing withwater. These plantlets were now treated with 0.1 % HgCl2 solution for 10 min followed by a5×5 min rinsing with sterilized distilled water under aseptic conditions. Thereafter, the shoottip portion (∼10 mm long) of the plants was isolated aseptically and cultured on MS mediumcontaining different concentrations of plant growth regulators.

Culture Media and Culture Conditions

The MS medium [13] supplemented with 4.44 μM 6-benzyl amino purine (BAP)+9.04 μM2,4-dichloro-phenoxy acetic acid (2,4-D) along with 9.08×10−3 μM thidiazuron (TDZ) wasused to induce callus from shoot tip explants of C. tuberculata. Sucrose (3 %) was added as acarbon source, and pH was adjusted at 5.7±0.1 using 0.1 N KOH or HCl. The media wassolidified with 0.7 % noble agar (Merck) and autoclaved at 121 °C under pressure of103.42 kPA for 20 min. All the cultures were maintained in culture room at 25±2 °C under4 ft long 40 W tubes (Philips) and incandescent bulb (25 W) at 3,500 lx intensity ofillumination using 16 h light photoperiod.

After 28 days of initiation of calli, small pieces (approx. 1 g) were transferred on plantgrowth regulators supplemented MS medium (as described above) along with differentconcentrations of NaCl (100–300 mM) for 15 days. To analyze the effect of stress alleviators,calli were transferred on MS medium containing 300 mM NaCl with ascorbic acid (AA 100and 200 μM) and salicylic acid (SA 100 and 200 μM) for 15 days. The weight of callusmeasured before and after the application of NaCl alone and in combination of antioxidantsand the change in fresh weight were calculated in percentage.

Determination of Antioxidant Activities

For determination of antioxidant activities, callus was ground in chilled mortar and pestle withhomogenization buffer. The homogenized callus was centrifuged at 10,000g for 20 min at4 °C. Supernatant was used to determine the activity of SOD, POD, APX, CAT, and GR aswell as protein contents.

Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by using the photochemicalNBT method [14]. The samples (0.5 g) were homogenized in 5.0 ml extraction bufferconsisting of phosphate 50.0 mM, pH 7.8. The assay mixture (3.0 ml) contained 50.0 mM

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phosphate buffer (pH 7.8), 1.0 μM EDTA, 26.0 mM L-methionine, 750.0 μM NBT, and20.0 μM riboflavin. The photoreduction (formation of purple formazan) of NBTwas measuredat 560 nm through spectrophotometer, and an inhibition curve was made against differentvolumes of extract. One unit of SOD is defined as the volume of extract present in reactionmixture that causes inhibition of the photoreduction of NBT by 50 %.

Volume of 3.0 ml guaiacol was used as a substrate to measure the peroxidase (POD; EC1.11.1.7) activity. A reaction mixture was constituted by mixing of 1 % guaiacol, 0.4 % H2O2,50.0 mM potassium phosphate buffer (pH 6.1), and enzyme extract. Guaiacol oxidized andincrease in absorbance was measured at 470 nm through spectrophotometer. Activity of theenzyme was found at 25±2 °C in micromolar of guaiacol oxidized per minute per gram freshweight [15].

The assay for ascorbate peroxidase (APX; EC 1.11.1.11) activity was carried out accordingto the method of Nakano and Asada [15]. In a reaction mixture (3.0 ml) containing 100.0 μLenzyme extract, 100.0 mM phosphate (pH 7), 0.3 mM ascorbic acid, 0.1 mM EDTA-Na2, and0.06 mM H2O2. In this reaction mixture, H2O2 was added, and after 30 s of this addition, thechange in absorption was recorded through spectrophotometer at 290 nm.

Assay to find catalase (CAT; EC 1.11.1.6) activity was done by the method of Cakmak andMarschner [16]. In this assay, 25.0 mM buffer of potassium phosphate containing 0.1 mMEDTA (pH 7.0) was mixed with 10.0 mM H2O2 and the enzyme extract. Within 1 min ofmixing the enzyme extract, the reduction in absorbance of H2O2 (E=39.4 mM−1 cm−1) wasrecorded at 240 nm on spectrophotometer.

Assay of glutathione reductase (GR; EC 1.6.4.2) was followed by the method of Foyer andHalliwell [17]. Reduction in absorbance was monitored at 340 nm through spectrophotometer.This reduction in absorbance was recorded due to oxidation of NADPH (E=6.2 mM−1 cm−1).The reaction was carried out by mixing 25.0 mM buffer of potassium phosphate. This bufferwas formulated at pH 7.8 by the addition of 0.2 mM EDTA. Enzyme aliquot was added andabsorbance was recorded.

The measurement of concentration of soluble protein was done by following the method ofBradford [18]. In this assay, bovine serum albumin was used as standard. Stable dye–albumincomplex is the base of this assay. The stable dye–albumin could be measured at 590 nmspectrophotometrically. A dye which is known as Coomassie brilliant blue G-250 was weighed0.01 % (w/v) and was mixed together with ethanol 4.7 % (w/v) and 8.5 % (w/v) phosphoricacid to make protein-dye reagent.

Transmission Electron microscopy of Treated Calli

The callus treated with NaCl and alleviated by ascorbic acid (AA) and salicylic acid (SA) for15 day were selected for fixation. Callus (2–3 mm2) was fixed in 2.5 % glutaraldehyde (v/v) atroom temperature in 0.1 M sodium phosphate buffer (pH 7.4) and then rinsed three times withsame sodium phosphate buffer. The washed callus samples were post fixed in 1 %osmium(VIII) oxide (OsO4) for 1 h. After 1 h, the samples were again washed three timeswith 0.1 M sodium phosphate buffer. The three rinses were given in a way that there should be10 min difference in each rinse. After washing, the samples were dried for 15–20 min intervalin a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) and in the end step 20 min inabsolute acetone. The samples were then penetrated and implanted in Spurr’s resin for wholenight. The specimen was heated at 70 °C for 9 h to prepare very slim cuttings(80 nm) of the specimens. Copper grids were used to mount these ultra-thin speci-mens for screening in the transmission electron microscope (JEOL TEM-1230EX) atan accelerating voltage of 60.0 kV.

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Data Analysis

Percent variation for growth protein content and antioxidant enzymes was calculated asfollows:

% variation for NaCl stressð Þ ¼ value for treated calli−untreated callið Þ=untreated callið Þ � 100

% variation for mitigantsð Þ ¼ value of treated calli−calli at 300 mM NaClð Þ=calli at 300 mM NaClð Þ � 100

All the experiments were performed in triplicate, and the results are presented as mean±standard error. The values were analyzed by LSD test with P<0.05.

Results and Discussion

Growth Characteristics of Calli, Protein Content, and Antioxidant Enzymes

The calli of C. tuberculata responded quickly to salt stress, and a linear reduction in the weightof calli and total soluble protein was recorded. The NaCl stress considerably enhanced thelevels of antioxidant enzymes. However, application of antioxidant molecules (salicylic acid orascorbic acid) mitigated the callous salt effects; increase in fresh weight and reduction inantioxidant enzymes was observed.

Application of 100 mM NaCl in callus culture media reduced fresh weight up to 18.3 %,and this reduction rose up to 67.4 % at 300 mM NaCl as compared with control (Table 1).Total soluble protein contents also reduced up to 35.7 % at 200 mM NaCl; however, thisreduction was less (14.2 %) in 300 mM NaCl-treated calli. Several studies on different plantspecies have reported similar growth inhibition kinetics upon exposure of cultured cells to highlevels of NaCl i.e., Suaeda nudiflora [19], Nitraria tangutorum, and Oryza sativa callus [20].Visually, it was also observed that the C. tuberculata calli generated in the presence of NaClwas smaller, harder, and desiccated. Upon salt stress due to cellular dehydration, the packedcell volume decreases following inflammation and structural collapse [21]. Proteins involve inosmotic balance and signaling pathway specifically express on salt stress, reducing the solubleprotein contents [20, 22]. Such variations, reduction in weight, and protein content have beenreported in both salt stressed and salt adaptive calli [23].

Table 1 Effects of NaCl and antioxidant treatments on fresh weight (g) and total soluble protein (mg/g FW) ofCaralluma tubarculata calli. The results are presented as average±standard deviation of triplicate values

Treatment Fresh weight (g) Total soluble protein (mg/g FW)

Control 19.5±1.1 bc 1.4±0.3 d

NaCl 100 mM 15.9±0.8 cd 1.1±0.2 e

NaCl 200 mM 10.5±1.0 d 0.9±0.18 f

NaCl 300 mM 6.4±0.7 e 1.2±0.3 e

NaCl 300 mM AA 100 μM 17.2±1.2 c 1.5±0.2 d

NaCl 300 mM+AA 200 μM 20.7±1.6 b 2.3±0.5 b

NaCl 300 mM+SA 100 μM 19.1±0.9 bc 2.1±0.3 c

NaCl 300 mM+SA 200 μM 23.1±0.7 a 3.7±0.5 a

Means followed by same small letters are not significantly different by the LSD test at P≤0.05AA ascorbic acid, SA salicylic acid

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Addition of salicylic acid (SA) and ascorbic acid (AA) antagonized the negative effects of NaClstress on callus growth. AA and SA at higher concentration (200 μM) also showed a growthpromoting effect as it gave the callus growth rate even higher than that of unstressed calli (control).The FW increased up to 223 and 260 % when calli were cultured in presence of 200 μMAA andSA, respectively, in addition with 300 mMNaCl (Table 1). These non-enzymatic antioxidants (SAand AA) are important for plant growth and development along with antioxidant capacity [24, 25].Salicylic acid also works as signal due to which plants develop internal resistance against biotic andabiotic stresses [10]. A research on chick pea indicated that the additional ascorbic acid (4.0 mM)gave strength to the stem and roots and improved fresh and dry biomass of salt-stressed plants [26].Exogenous application of AA also modulates salt-stressed undesired effects on growth, celldivision, and cell enlargement [9]. NaCl stress had negative effect on total soluble protein contents,which showed significant reduction in stressed callus. However, compared to control as well asstressed calli, a significant increase in protein contents was observed with the addition of antiox-idants. SA proved comparatively better for protein contents of C. tuberculata callus showing thehighest value of protein contents (3.7 mg/g FW; 208 % increase as compared with 300 mMNaCl-stressed calli) at 200 μM. The non-enzymatic antioxidants provide shield against oxidative burstand also stimulate biomass accumulation, increasing fresh and dry weight [27, 28]. Therefore,appropriate concentration is important for optimum results.

The calli grown in the presence of NaCl varied antioxidant enzymes response. As theconcentration of NaCl in the culture media increased, a boost in peroxidase, ascorbateperoxidase, and catalase activities were observed in the calli. A maximum increase of 134.4,123.5, and 153.5 % was observed in peroxidase, ascorbate peroxidase, and catalase, respec-tively, in the calli grown at 300 mM NaCl concentration (Fig. 1). Concentrations of theseenzymes decreased when AA and SA were also applied in combination with 300 mM NaCl.The reduction was more pronounced by applying 200 μM as compared with 100 μM. It wasalso observed that ascorbate peroxidase reduced at high rate (75–82 %) as compared withperoxidase (26–84 %) and catalase (43–61 %). The figure also shows that the reduction inascorbate peroxidase was consistent irrespective to type and concentration of mitigant.

Enhanced concentration of salt increased the POD activity as compared to control.Application of 300 mM NaCl in the culture media increased POD activity up to 134 %. Toreduce injurious effect of NaCl, SA functioned better as compared with AA and higherconcentration was optimum. It was observed that application of 200 μM SA or AA decreasedthe POD activity below the level present in control calli (Fig. 1). In salinized cells ofS. nudiflora and cotton, NaCl-induced enhancement of POD activity to decompose H2O2

produced (Cherian and Reddy 2003; Lin and Kao 1999). Increase in POD activity confers salttolerance ability in plant species and protection against oxidative stress [29, 30].

Ascorbate peroxidase (APX) reflected a gradual rise in its activity in response to enhanced NaClconcentrations, and at 300mMNaCl, a fourfold increase in APX activity was observed as comparedwith control. However, application of SA and AA decreased APX activity five to six times ascompared with control (300 mM NaCl). It was observed that decrease in APX activity was notdependent on the type of antioxidant and concentration. Stimulation of APX indicates that theenzyme has a critical role in plant cells dissimulating H2O2 produced during O−2 scavenging [31].Such variations have already been observed in pea [32], cotton [33], and rice [34]. However, level ofAPX is determined by salt concentration, time of stress, type of tissue, and age of plant [35]. Ascorbic

Fig. 1 Effects of salinity (NaCl, 0–300 mM) and antioxidants (salicylic acid and ascorbic acid; 100 and 200 μM)on SOD, POD, APX, CAT, and GR activities in Caralluma tubarculata calli. Data are the mean±SD of threereplicates. Small lettersmarked on each bar are not significantly different by the LSD test at P≤0.05. Control calli(CC),. Ascorbic acid (AA), salicylic acid (SA)

b

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acid plays a vital role in mediating the undesired affects of salt on plant metabolism and growth.These mitigating effects are attributed by stimulation of reaction by enzymes [36] along with thestabilization and protection of organs responsible for photosynthesis from damage due to oxidation[37]. The results also show that AA and SA reduced the POD levelmany times and diminished toxiceffects of NaCl. However, type and concentration of mitigant did not affect at higher extinct.

Catalase also takes away H2O2 into water and oxygen produced inside the cell [38], and thisreaction takes place at higher extent in biotic and abiotic stress conditions [39]. An overallincrease in CATactivity was observed whenC. tubarcaulata calli was subcultured in the presenceof NaCl. A threefold increase (153 %) in CAT activity was calculated on 300 mM NaClconcentration as compared with control. Submission of AA and SA reduced the CAT activityand trimmed down CAT level approximately equal to control calli (Fig. 1). Statistically, not muchdifference was observed between both non-enzymatic antioxidants and concentrations.

In case of SOD and GR, an increase in activities was observed at 100 and 200 mM NaCl.Further increase in NaCl concentration (300 mM) decreased the enzyme values (Fig. 1). Anincrease (54 %) in GR activity was observed in stressed calli (200 mM) as compared to control,while at 300 mM NaCl, the activity was equal to control. In comparison, much increase in SODactivity was not observed at 200 mM NaCl stress (16 % increase); however, 26 % decrease inSOD activity was observed in calli regenerated at 300mMNaCl. Application of AA and SA (100

Fig. 2 Electron micrographs of Caralluma tuberculata calli describing modifications in cell wall, cell mem-brane, and vauoles: a control, b exposed to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid(200 μM), d exposed to 300 mM NaCl+salicylic acid (200 μM). Cell wall (CW), cell membrane (CM),mitochondria (Mt), plastids (P), vacuole (Vac), plastid (P)

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and 200 μM) increased SOD activity in the stressed calli as compared with control (300 mMNaCl); however, in case of GR increase was observed at 100 μM AA and 200 μM SA. It wasobserved that both non-oxidants did not much favored SOD to mitigate injurious effects of NaCl.An increase in SOD activity due to salt stress has been documented in Buddleja parviflora andBruguiera gymnorrhiza [40], Avicennia marina [41], and Rhizophora stylosa [42], and suchincrease has also been reported in cotton, tomatoes, and pea genotypes which are salinity tolerant[6, 35, 43]. The roles of GR and glutathione in the H2O2 scavenging in plant cells have been wellestablished in Halliwell–Asada pathway [44]. GR catalyzes the rate limiting the last step ofascorbate-glutathione pathway. Reduced glutathione is a very efficient scavenger of ROS as it is apowerful reductant. Non-enzymatic antioxidants like reduced ascorbate and glutathione scav-enged superoxide radicals generated in plants. However, the APX and glutathione reductaseexhibit enhanced activities [42]. The results also show that in C. tuberculata calli, GR plays amajor role to fight against oxidative molecules as compared with SOD.

Ultra-Structural Modifications of Calli Upon Salt and Antioxidant Treatments

Ultra-structural observations of Caralluma cell revealed modifications in NaCl-treated cells,and these modifications were more obvious on the cell wall, nucleus, and plastids. In the cells

Fig. 3 Electron micrographs of Caralluma tuberculata calli describing variations in nucleus a control, b exposedto 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid (200 μM), d exposed to 300 mM NaCl+salicylic acid (200 μM). Cell wall (CW), nucleus (N), nucleolus (Nu)

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of the untreated callus (control), the cytoplasm looked granular and thick with severalsubcellular organelles. These cells possessed a thick cell wall, continuous and smooth cellmembranes, a central vacuole, a well-shaped nucleus, and few numbers of mitochondria andplastids (Figs. 2, 3, and 4).

A considerable reduction in the cell wall thickness with increased vacuolization was evidentunder NaCl stress (Fig. 2). Plasma membrane was plasmolysed and was either absent orinsignificant; however, disruption of nuclear membrane and increased numbers of nucleoliwere some of the other obvious changes observed in the nucleus of NaCl-treated cells (Fig. 3).The plastids of NaCl-treated cells showed alteration in shape and in lamellar membrane systemwith increased number of plastoglobuli (Fig. 4). In case of AA treatment along with NaCl, thecell wall was relatively better in shape, with slight shrinkage of cytoplasmic and increasednumber of mitochondria. Addition of AA improved the shape of plastids and nucleus. Thelamellar membrane was more compact although high amount of plastoglobuli was present.However, SA proved better, where the cell wall was almost fully recovered and numbers ofvacuoles were reduced. Both AA and SA recovered damaged to nucleus and plastid. The

Fig. 4 Electron micrographs of Caralluma tuberculata calli describing variations in plastids: a control, bexposed to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid (200 μM), d exposed to 300 mMNaCl+salicylic acid (200 μM). Cell wall (CW), cell membrane (CM), plastids (P), plastoglobuli (PG), lamellarmembrane (LM)

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shape of plastid restored, and it turned into elongated with reduced plastoglobuli, whilelamellar membrane became compacted again. The number of plastids remained constant,and no change was observed in any treatment. Electron microscopy had been used to assessdamages at the ultra-structural and tissue levels to make the foundation of examinationmacroscopically on which the damage rating is based [45]. Many reports describe variationsin cellular structure due to salt stress e.g., alteration in the cell wall [46] reduced thickness inthe cell wall [47], increased number of micro bodies and mitochondria [48], swelling ofthylakoid [49], etc. It has been postulated that increase in salt concentration inducesenhanced F-ATPase activity by increase in mitochondria number to provide excessiveenergy supply for osmotic adjustment [50]. While plastids are considered to be at highrisk by oxidative stress due to electron flux, elevated levels of oxygen might be thereasons for swelling of plastids [51], break down of thylakoid membrane, and highernumber of plastoglobuli.

Conclusions

In conclusion, the higher activities of SOD, CAT, GR, POD, and APX in response to salinitystress play an important role in salt tolerance in the calli of C. tuberculata. The physiologicaleffects at cellular level include cell membrane damage, disruption of nuclear membrane,variation in nucleoli number, and deformation of plastids. The antioxidant molecules (SAand AA) successfully mitigated salt toxicity and improved the growth of C. tuberculata callirevealed by normal distribution of antioxidant enzymes and revival of cellular structure.

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