Ascorbic Acid and Salicylic Acid Mitigate NaCl

Appl Biochem Biotechnol DOI 10.1007/s12010-014-0890-6

Ascorbic Acid and Salicylic Acid Mitigate NaCl Stress in 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 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

R. U. Rehman Horticulture and Floriculture Institute, Government of Punjab, Rawalpindi, Pakistan M. Zia (*) : B. H. Abbasi Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 45320 e-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

Appl Biochem Biotechnol

Abbreviations AA Ascorbic acid APX Ascorbate peroxidase CAT Catalase FWGR Fresh weight growth rate GR 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 and severely 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 (O−2), and the hydroxyl radical (OH−1) 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 the dismutation of O−2 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), glutathione 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., dehydration 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 [4–6], swelling of thylakoids at earlier stage [7, 8], and deformation of other organelles. 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 hydrogen 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


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 of C. tuberculata used 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 % HgCl2 solution for 10 min followed by a 5×5 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 μM 6-benzyl amino purine (BAP)+9.04 μM 2,4-dichloro-phenoxy acetic acid (2,4-D) along with 9.08×10−3 μM thidiazuron (TDZ) was used to induce callus from shoot tip explants of C. tuberculata. Sucrose (3 %) was added as a carbon source, and pH was adjusted at 5.7±0.1 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 25±2 °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 different concentrations 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 100 and 200 μM) and salicylic acid (SA 100 and 200 μM) 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 with homogenization 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


phosphate buffer (pH 7.8), 1.0 μM EDTA, 26.0 mM L-methionine, 750.0 μM NBT, and 20.0 μM 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 25±2 °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 μL 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, H2O2 was 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 mM−1 cm−1) 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 dye–albumin complex is the base of this assay. The stable dye–albumin 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 (2–3 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 interval in 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 Spurr’s 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 specimens for screening in the transmission electron microscope (JEOL TEM-1230EX) at an accelerating voltage of 60.0 kV.


Data Analysis Percent variation for growth protein content and antioxidant enzymes was calculated as follows: % variationðfor NaCl stressÞ ¼ ððvalue for treated calli−untreated calliÞ=untreated calliÞ 100 % variationðfor mitigantsÞ ¼ ððvalue of treated calli−calli at 300 mM NaClÞ=calli at 300 mM NaClÞ 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