34 ascorbic acid and salicylic acid mitigate nacl stress in caralluma tuberculata calli

7/24/2019 34 Ascorbic Acid and Salicylic Acid Mitigate NaCl Stress in Caralluma tuberculata Calli

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Ascorbic Acid and Salicylic Acid Mitigate NaCl Stress

in Caralluma tuberculataCalli

Riaz Ur Rehman & Muhammad Zia &

Bilal Haider Abbasi &Gang Lu &

Muhammad Fayyaz Chaudhary

Received: 2 February 2014 / Accepted: 24 March 2014 /

Published online: 18 April 2014

# Springer Science+Business Media New York 2014

Abstract Plants exposed to salt stress undergo biochemical and morphological changes even

at cellular level. Such changes also include activation of antioxidant enzymes to scavenge

reactive oxygen species, while morphological changes are determined as deformation of

membranes and organelles. Present investigation substantiates this phenomenon for

Caralluma tuberculata calli when exposed to NaCl stress at different concentrations.

Elevated levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate

peroxidase (APX), and glutathione reductase (GR) in NaCl-stressed calli dwindled upon

application of non-enzymatic antioxidants; ascorbic acid (AA) and salicylic acid (SA).

Many fold increased enzymes concentrations trimmed down even below as present in the

control calli. Electron microscopic images accentuated several cellular changes upon NaCl

stress such as plasmolysed plasma membrane, disruption of nuclear membrane, increased

numbers of nucleoli, alteration in shape and lamellar membrane system in plastid, and

increased number of plastoglobuli. The cells retrieved their normal structure upon exposure

to non-enzymatic antioxidants. The results of the present experiments conclude that NaCl

aggravate oxidative molecules that eventually alleviate antioxidant enzymatic system.

Furthermore, the salt stress knocked down by applying ascorbic acid and salicylic acid

manifested by normal enzyme level and restoration of cellular structure.

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

Ultra-structure

Appl Biochem Biotechnol (2014) 173:968979

DOI 10.1007/s12010-014-0890-6

R. U. Rehman

Horticulture and Floriculture Institute, Government of Punjab, Rawalpindi, Pakistan

M. Zia (*) :B. H. AbbasiDepartment of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 45320e-mail: [email protected]

G. Lu

College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

M. F. Chaudhary

Preston Institute of Nanoscience and Technology, Preston University, Islamabad, Pakistan


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Abbreviations

AA Ascorbic acid

APX Ascorbate peroxidase

CAT Catalase

FWGR Fresh weight growth rateGR Glutathione reductase

ROS Reactive oxygen species

SA Salicylic acid

SOD Superoxide dismutase

POD 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 of

plant may occur in severe saline conditions [1]. Such stresses result in interference of growth

and metabolism by triggering secondary responses like the production of highly reactive

oxygen species (ROS).

The production of ROS such as the hydrogen peroxide (H2O2), the superoxide radical

(O2), and the hydroxyl radical (OH1) are critical; however, enzymatic or non-enzymatic

ROS-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 stress

responses [2]. The major antioxidant enzymes are superoxide dismutase (SOD) catalyzing thedismutation of O2 to H2O2; catalase (CAT) that dismutase H2O2 to oxygen and water; and

ascorbate peroxidase (APX) that reduces H2O2 to water by utilizing ascorbate as particular

electron 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 cell

against 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 outer

chloroplast envelope, thinning of partitions, adhesion within the grana, decrease in chloroplast

volume [46], 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-adaptive

cell lines. Osmoregulation mechanism is a complex process; however, the adaptive capacity to

maintain membrane integrity during a long period of water deficit may be an essential

biological trait for drought tolerance.

Salicylic acid (SA) and ascorbic acid (AA) are small antioxidant molecules, which are

water 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 effect

of ascorbic acid application used exogenously in improving the adverse effects on

growth due to salt stress [9]. Salicylic acid also intervenes the oxidative rupture that

causes death of the cells in the oversensitive reaction and proceeds as signal to develop

complete internal resistance [10]. It also plays an important role in many abiotic stresses

to survive the plants against these pressures [11]. However, unexpectedly, little is known

about the role of these antioxidative compounds in callus stress adaptation. The aims of

the present study were to investigate the antioxidant enzyme status in the callus of

Caralluma tuberculata, under NaCl stress, alleviation of NaCl-stress by ascorbic acid

Appl Biochem Biotechnol (2014) 173:968979 969


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and salicylic acid, and to investigate the intracellular changes resulted by the stress in

callus tissues of C. tuberculata.

Materials and Methods

Plant Material and Explant Preparation

The plant material ofC. tuberculataused for the study was obtained from the local market of

Quetta (Balochistan, Pakistan) and was identified by Prof. Dr. Mir Ajab Khan, Department of

Plant Sciences, Quaid-i-Azam University Islamabad, Pakistan. The plant material brought to

lab 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]. In

detail, before starting the experiment, the plants collected from the earthen pots were washed

under running tap water for 30 min to remove all adhering contaminants following washing

with 0.2 % liquid detergent (Triton X-100) for about 15 min. Thereafter, the plants were rinsed

with distilled water and treated with bevistin (a fungicide) for 30 min followed by rinsing with

water. These plantlets were now treated with 0.1 % HgCl2solution for 10 min followed by a

55 min rinsing with sterilized distilled water under aseptic conditions. Thereafter, the shoot

tip portion (10 mm long) of the plants was isolated aseptically and cultured on MS medium

containing different concentrations of plant growth regulators.

Culture Media and Culture Conditions

The MS medium [13] supplemented with 4.44 M 6-benzyl amino purine (BAP)+9.04 M

2,4-dichloro-phenoxy acetic acid (2,4-D) along with 9.08103 M thidiazuron (TDZ) was

used to induce callus from shoot tip explants ofC. tuberculata. Sucrose (3 %) was added as a

carbon source, and pH was adjusted at 5.70.1 using 0.1 N KOH or HCl. The media was

solidified with 0.7 % noble agar (Merck) and autoclaved at 121 C under pressure of

103.42 kPA for 20 min. All the cultures were maintained in culture room at 252 C under

4 ft long 40 W tubes (Philips) and incandescent bulb (25 W) at 3,500 lx intensity of

illumination using 16 h light photoperiod.

After 28 days of initiation of calli, small pieces (approx. 1 g) were transferred on plant

growth regulators supplemented MS medium (as described above) along with differentconcentrations of NaCl (100300 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 100

and 200 M) and salicylic acid (SA 100 and 200 M) for 15 days. The weight of callus

measured before and after the application of NaCl alone and in combination of antioxidants

and 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 at

4 C. Supernatant was used to determine the activity of SOD, POD, APX, CAT, and GR as

well as protein contents.

Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by using the photochemical

NBT method [14]. The samples (0.5 g) were homogenized in 5.0 ml extraction buffer

consisting of phosphate 50.0 mM, pH 7.8. The assay mixture (3.0 ml) contained 50.0 mM

970 Appl Biochem Biotechnol (2014) 173:968979


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phosphate buffer (pH 7.8), 1.0 M EDTA, 26.0 mM L-methionine, 750.0 M NBT, and

20.0 M riboflavin. The photoreduction (formation of purple formazan) of NBT was measured

at 560 nm through spectrophotometer, and an inhibition curve was made against different

volumes of extract. One unit of SOD is defined as the volume of extract present in reaction

mixture 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; EC

1.11.1.7) 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 and

increase in absorbance was measured at 470 nm through spectrophotometer. Activity of the

enzyme was found at 252 C in micromolar of guaiacol oxidized per minute per gram fresh

weight [15].

The assay for ascorbate peroxidase (APX; EC 1.11.1.11) activity was carried out according

to the method of Nakano and Asada [15]. In a reaction mixture (3.0 ml) containing 100.0 L

enzyme extract, 100.0 mM phosphate (pH 7), 0.3 mM ascorbic acid, 0.1 mM EDTA-Na2, and

0.06 mM H2O2. In this reaction mixture, H2O2was added, and after 30 s of this addition, the

change 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 and

Marschner [16]. In this assay, 25.0 mM buffer of potassium phosphate containing 0.1 mM

EDTA (pH 7.0) was mixed with 10.0 mM H2O2 and the enzyme extract. Within 1 min of

mixing the enzyme extract, the reduction in absorbance of H2O2 (E=39.4 mM1 cm1) was

recorded at 240 nm on spectrophotometer.

Assay of glutathione reductase (GR; EC 1.6.4.2) was followed by the method of Foyer and

Halliwell [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 buffer

was formulated at pH 7.8 by the addition of 0.2 mM EDTA. Enzyme aliquot was added and

absorbance was recorded.

The measurement of concentration of soluble protein was done by following the method of

Bradford [18]. In this assay, bovine serum albumin was used as standard. Stable dyealbumin

complex is the base of this assay. The stable dyealbumin could be measured at 590 nm

spectrophotometrically. A dye which is known as Coomassie brilliant blue G-250 was weighed

0.01 % (w/v) and was mixed together with ethanol 4.7 % (w/v) and 8.5 % (w/v) phosphoric

acid 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) for

15 day were selected for fixation. Callus (23 mm2) was fixed in 2.5 % glutaraldehyde (v/v) at

room temperature in 0.1 M sodium phosphate buffer (pH 7.4) and then rinsed three times with

same 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 times

with 0.1 M sodium phosphate buffer. The three rinses were given in a way that there should be

10 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 in

absolute acetone. The samples were then penetrated and implanted in Spurrs resin for whole

night. 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) at

an accelerating voltage of 60.0 kV.



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Data Analysis

Percent variation for growth protein content and antioxidant enzymes was calculated as

follows:

% variation for NaCl stress value for treated calliuntreated calli =untreated calli 100

% variation for mitigants value of treated callicalli at 300 mM NaCl =calli at 300 mM NaCl 100

All the experiments were performed in triplicate, and the results are presented as mean

standard error. The values were analyzed by LSD test withP


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Addition of salicylic acid (SA) and ascorbic acid (AA) antagonized the negative effects of NaCl

stress on callus growth. AA and SA at higher concentration (200 M) also showed a growth

promoting 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 M AA and

SA, respectively, in addition with 300 mM NaCl (Table1). 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 and

abiotic 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, cell

division, 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 as

stressed calli, a significant increase in protein contents was observed with the addition of antiox-

idants. SA proved comparatively better for protein contents ofC. tuberculatacallus showing the

highest value of protein contents (3.7 mg/g FW; 208 % increase as compared with 300 mM NaCl-

stressed calli) at 200 M. The non-enzymatic antioxidants provide shield against oxidative burst

and 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 the

concentration of NaCl in the culture media increased, a boost in peroxidase, ascorbate

peroxidase, 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 these

enzymes decreased when AA and SA were also applied in combination with 300 mM NaCl.The reduction was more pronounced by applying 200 M as compared with 100 M. It was

also observed that ascorbate peroxidase reduced at high rate (7582 %) as compared with

peroxidase (2684 %) and catalase (4361 %). The figure also shows that the reduction in

ascorbate 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 %. To

reduce injurious effect of NaCl, SA functioned better as compared with AA and higher

concentration was optimum. It was observed that application of 200 M SA or AA decreased

the POD activity below the level present in control calli (Fig. 1). In salinized cells of

S. nudiflora and cotton, NaCl-induced enhancement of POD activity to decompose H2O2produced (Cherian and Reddy 2003; Lin and Kao 1999). Increase in POD activity confers salt

tolerance 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 NaCl

concentrations, and at 300 mM NaCl, a fourfold increase in APX activity was observed as compared

with control. However, application of SA and AA decreased APX activity five to six times as

compared with control (300 mM NaCl). It was observed that decrease in APX activity was not

dependent on the type of antioxidant and concentration. Stimulation of APX indicates that the

enzyme has a critical role in plant cells dissimulating H2O2produced during O2 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, 0300 mM) and antioxidants (salicylic acid and ascorbic acid; 100 and 200 M)

on SOD, POD, APX, CAT, and GR activities in Caralluma tubarculata calli. Data are the meanSD of three

replicates.Small letters marked on each bar are not significantly different by the LSD test atP0.05. 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 the

stabilization and protection of organs responsible for photosynthesis from damage due to oxidation

[37]. The results also show that AA and SA reduced the POD level many times and diminished toxic

effects of NaCl. However, type and concentration of mitigant did not affect at higher extinct.Catalase also takes away H2O2into water and oxygen produced inside the cell [38], and this

reaction takes place at higher extent in biotic and abiotic stress conditions [39]. An overall

increase in CAT activity was observed when C. tubarcaulata calli was subcultured in the presence

of NaCl. A threefold increase (153 %) in CAT activity was calculated on 300 mM NaCl

concentration as compared with control. Submission of AA and SA reduced the CAT activity

and trimmed down CAT level approximately equal to control calli (Fig. 1). Statistically, not much

difference 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). An

increase (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 SOD

activity was not observed at 200 mM NaCl stress (16 % increase); however, 26 % decrease in

SOD activity was observed in calli regenerated at 300 mM NaCl. Application of AA and SA (100

Fig. 2 Electron micrographs ofCaralluma 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 M), d exposed to 300 mM NaCl+salicylic acid (200 M). Cell wall (CW), cell membrane (CM),

mitochondria (Mt), plastids (P), vacuole (Vac), plastid (P)



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and 200 M) increased SOD activity in the stressed calli as compared with control (300 mM

NaCl); however, in case of GR increase was observed at 100M AA and 200 M SA. It was

observed 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 parvifloraand

Bruguiera 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 H2O2scavenging in plant cells have been well

established in HalliwellAsada pathway [44]. GR catalyzes the rate limiting the last step of

ascorbate-glutathione pathway. Reduced glutathione is a very efficient scavenger of ROS as it is a

powerful reductant. Non-enzymatic antioxidants like reduced ascorbate and glutathione scav-

enged superoxide radicals generated in plants. However, the APX and glutathione reductase

exhibit enhanced activities [42]. The results also show that in C. tuberculata calli, GR plays a

major role to fight against oxidative molecules as compared with SOD.

Ultra-Structural Modifications of Calli Upon Salt and Antioxidant Treatments

Ultra-structural observations ofCaralluma 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 ofCaralluma tuberculata calli describing variations in nucleus a control, b exposed

to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid (200 M),d exposed to 300 mM NaCl+

salicylic acid (200 M). Cell wall (CW), nucleus (N), nucleolus (Nu)



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of the untreated callus (control), the cytoplasm looked granular and thick with several

subcellular organelles. These cells possessed a thick cell wall, continuous and smooth cell

membranes, a central vacuole, a well-shaped nucleus, and few numbers of mitochondria and

plastids (Figs.2,3, and4).

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 or

insignificant; however, disruption of nuclear membrane and increased numbers of nucleoli

were 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 system

with increased number of plastoglobuli (Fig.4). In case of AA treatment along with NaCl, the

cell wall was relatively better in shape, with slight shrinkage of cytoplasmic and increased

number of mitochondria. Addition of AA improved the shape of plastids and nucleus. The

lamellar 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 of

vacuoles 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, b

exposed to 300 mM NaCl alone,c exposed to 300 mM NaCl+ascorbic acid (200 M),d exposed to 300 mM

NaCl+salicylic acid (200 M). Cell wall (CW), cell membrane (CM), plastids (P), plastoglobuli (PG), lamellar

membrane (LM)



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shape of plastid restored, and it turned into elongated with reduced plastoglobuli, while

lamellar membrane became compacted again. The number of plastids remained constant,

and no change was observed in any treatment. Electron microscopy had been used to assess

damages at the ultra-structural and tissue levels to make the foundation of examination

macroscopically 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 in

the cell wall[47], increased number of micro bodies and mitochondria [48], swelling of

thylakoid [49], etc. It has been postulated that increase in salt concentration induces

enhanced F-ATPase activity by increase in mitochondria number to provide excessive

energy supply for osmotic adjustment [50]. While plastids are considered to be at high

risk by oxidative stress due to electron flux, elevated levels of oxygen might be the

reasons for swelling of plastids [51], break down of thylakoid membrane, and higher

number of plastoglobuli.

Conclusions

In conclusion, the higher activities of SOD, CAT, GR, POD, and APX in response to salinity

stress play an important role in salt tolerance in the calli ofC. tuberculata. The physiological

effects at cellular level include cell membrane damage, disruption of nuclear membrane,

variation in nucleoli number, and deformation of plastids. The antioxidant molecules (SA

and AA) successfully mitigated salt toxicity and improved the growth ofC. tuberculata calli

revealed by normal distribution of antioxidant enzymes and revival of cellular structure.

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34 ascorbic acid and salicylic acid mitigate nacl stress in caralluma tuberculata calli

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