ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 6, с. 816-823



УДК 581.1


DURING COLD STRESS1 © 2014 R. Mohammadi*, R. Maali-Amiri*, N. L. Mantri**

*Department of Agronomy and Plant Breeding, University College of Agriculture and Natural Resources,

University of Tehran, Karaj, Iran **School of Applied Sciences, Biotechnology and Environmental Biology, RMIT University, Bundoora, Victoria, Australia

Received January 31, 2014

The effects of TiO2 nanoparticles (NPs) on physiologo-biochemical responses were studied in two chickpea (Cicer arietimun L.) genotypes differing in cold sensitivity (tolerant Sel11439 and sensitive ILC533) during cold stress (CS). The results showed that hydrogen peroxide and MDA contents and electrolyte leakage index (ELI) increased under CS conditions in both genotypes and that these damage indices were higher in ILC533 than in Sel11439 plants. In plants treated with TiO2 NPs, a decreased H2O2 level was accompanied by a decrease in the MDA content and ELI compared to control plants, and these changes occurred more effectively in Sel11439 than in ILC533 plants. The antioxidant enzymes were more effective in cell protection against CS in Sel11439 plants compared to ILC533 plants, as well as in plants treated with TiO2 NPs compared to control plants. The lipoxygenase activity was induced efficiently only in Sel11439 plants treated with TiO2 NPs during CS, probably indicating its role in stress response (which was confirmed by measuring allen oxide synthase activity). TiO2 NPs caused stability of chlorophyll and carotenoid contents during CS. Results suggest that TiO2 NPs confer an increased tolerance of chickpea plants to CS, decreasing the level of injuries and increasing the capacity of defense systems.

Keywords: Cicer arietinum — cold responses — defense systems — injury indices — TiO2 — nanoparticles

DOI: 10.7868/S0015330314050121


The growth, development, and productivity of plants, including chickpea (Cicer arietinum L.) as a fall- or spring-sown crop in Mediterranean climates, are adversely affected by cold stress (CS), so that in these regions, breeding for tolerance to CS is the most economic and environmentally acceptable option to improve chickpea production [1]. CS, like other types of abiotic and biotic stresses, induces ROS accumulation, which excessive production or inefficient deactivation can cause severe injuries because of their high oxidizing potential, including degradation of polysaccharides, denaturation of enzymes, and the nicking,

1 This text was submitted by the authors in English.

Abbreviations: AOS — allen oxide synthase; APX — ascorbate peroxidase; CAT — catalase; Chl — chlorophyll; CS — cold stress; ELI — electrolyte leakage index; GPX — guaiacol peroxidase; LOX — lipoxygenase; NPs — nanoparticles; PPO — polyphenol oxidase; SOD — superoxide dismutase; TiO2 — titanium dioxide. Corresponding author: Reza Maali-Amiri. Department of Agronomy and Plant Breeding, University College of Agriculture and Natural Resources, University of Tehran, Karaj, 31587-77871 Iran; fax: + 98-26-3222-7605; e-mail: rmamiri@ut.ac.ir

cross linking, and scission of DNA strands [2]. As a result, the cells either adapt their physiology to these changes due to homeostasis processes or perish due to inadequate responses.

An important component of breeding for cold tolerance includes strategies, which improve physiological and biochemical plant responses to CS without any genetic modifications. Recently, TiO2 nanoparticles (NPs) have been found to change the dry weight, chlorophyll (Chl) synthesis, and some characteristics of metabolisms in photosynthetic organisms due to their unique properties. The improvement of plant productivity in these studies often was related to significant reductions in some indices of injuries and induced plant defense systems [3, 4]. Therefore, there is potential for expanding the range of TiO2 NP usage for the improvement of crop physiological and biochemical characteristics [5]. However, NPs can cause also a variety of negative effects on metabolic processes [6]. On one hand, TiO2 NP application improves the activities of defense machinery in some organisms and, on the other hand, shows negative effects like ROS accumulation [7, 8]. This may cause the limitation of TiO2 NP

extensive application in agriculture. Thus, to support applications of TiO2 NPs, their role must be further studied, in particular during CS. In our previous study, we have assessed for the first time the effect TiO2 NP concentrations on indices of injury (MDA content and electrolyte leakage index (ELI)) following CS in chickpea genotypes [5]. Our results showed that low concentrations of TiO2 NPs (especially 5 mg/L) have not been found harmful to chickpea genotypes during CS but instead decreased MDA content and ELI in them. It was supposed that during CS, the presence of TiO2 NPs in the cell probably triggers signal pathways, which result in adjusted metabolic alterations partly against cold-induced oxidative stress.

However, information about these responses involved in plant growth and defense is not available for our genotypes. To accomplish such study, classical strategies can be used. In response to cold-induced ox-idative stress, plants have evolved the antioxidant enzymes, including superoxide dismutase (SOD), cata-lase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and polyphenol oxidase (PPO), to scavenge ROS [9]. Lipoxygenases (LOXs), the enzymes involved in membrane degradation through the deoxygenation of unsaturated fatty acids, and allen oxide synthase (AOS), the enzyme involved in some defense pathways, along with antioxidant enzyme activities and injury indices can be helpful in comparing the degree of cold tolerance induced in the chickpea plants that show a high morphological but narrow genetic variation [10, 11] when TiO2 NPs were used. These responses in a time-dependent manner probably could contribute significantly to the study of stress biology of chickpea plants.

The purpose of this work was to study indices of injury, like hydrogen peroxide and MDA contents and ELI, some enzyme activities involved in the ROS-scavenging system, LOX and AOS activities, and pigments contents during CS in two chickpea genotypes differing in cold sensitivity (tolerant Sel11439 and sensitive ILC533). The investigation of physiological and biochemical characteristics during CS could develop a picture of the molecular mechanisms and their functional products in order to understand how chickpea can response to TiO2 NPs. Such findings may open new TiO2 NP applications in agriculture to provide solutions to survival or recovery of plants during CS.


Plant material and growth conditions. Seeds of two chickpea (Cicer arietinum L.) genotypes, cold-sensitive (ILC533) and cold-tolerant (Sel11439), were provided by Dryland Agriculture Research Institute (DARI) of Iran in Maragheh city of Azerbaijan province. Our previous studies have shown that Sel11439 plants are tolerant to CS while ILC 533 plants are sensitive to CS [1]. In this study we examined the effects

of experimental treatments of these two genotypes. Characterization of TiO2 NPs, plant growth conditions, the accumulation of TiO2 NPs in plant leaves have been reported previously [5]. Also, in our previous work, TiO2 NP treatment (5 mg/L) caused a significant decrease in injury indices like ELI and MDA content compared to other concentrations (0, 2, and 10 mg/L) during thermal treatments. Plants were grown in a growth chamber ("Arvin Tajhiz Espadana", Iran) at 23°C (optimum temperature), under white light (220 ^mol/(m2 s)), a photoperiod of 16 h, and 75% relative humidity for 21 days. These seedlings were approximately 20 cm in height with at least 5 branches of 5—8 cm. Plant leaves were sprayed with NP solution (5 mg/L) twice on the 12th and 16th days. Control plants were not treated with TiO2 NPs. Three-week-old seedlings were exposed to a chilling at 4°C (and identical growth conditions) during 6 days. Samplings were conducted in the first and sixth days (as early and late responses, respectively) after exposing the seedlings to CS. All measurements were made on the middle leaves in each treatment. Collected samples were immediately flash frozen in liquid nitrogen and stored at —80° C for further studies. Physiological experiments, e.g., ELI and lipid peroxidation (MDA) assays, were conducted using fresh leaves.

Cell membrane permeability. Cell membrane permeability of plantlets was assessed by the electrolyte leakage index (ELI) in leaf tissues as described previously [1].

Lipid peroxidation analysis. Lipid peroxidation in leaves was assessed by the determination of MDA content as described previously [12]. The amount of MDA was expressed as ^mol/g fr wt.

Hydrogen peroxide assay. H2O2 content was determined as described previously [12]. The content of H2O2 was expressed in ^mol/g fr wt.

Antioxidant enzymes activity. Samples (0.5 g) were ground in liquid nitrogen and homogenized in extraction buffer (50 mM phosphate buffer, pH 7.0, containing 1% (w/v) polyvinylpolypyrrolidone) at 4°C. The homogenate was centrifuged at 15000 g and 4°C for 30 min. The supernatant was used for enzyme assays. For the APX assay, 2 mM ascorbic acid was present in the extraction buffer. Total soluble protein content was determined by the Bradford method [13]. The activities of superoxide dismutase (SOD; EC in U/(min mg protein) and lipoxygenase (LOX; EC in AOD234/(min mg protein) were determined as described previously [11, 14]. Catalase activity (CAT; EC in nmol of H2O2 decom-posed/(min mg protein), ascorbate peroxidase activity (APX; EC in nmol oxidized ascorbate/(min mg protein), guaiacol peroxidase activity (GPX; EC i

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