Protoplasma DOI 10.1007/s00709-014-0744-7

ORIGINAL ARTICLE

Overexpression of AT14A confers tolerance to drought stress-induced oxidative damage in suspension cultured cells of Arabidopsis thaliana Lin Wang & Jie He & Haidong Ding & Hui Liu & Bing Lü & Jiansheng Liang & L. Wang & J. He & H. D. Ding & H. Liu & B. Lü & J. S. Liang

Received: 26 September 2014 / Accepted: 2 December 2014 # Springer-Verlag Wien 2014

Abstract Drought stress can affect interaction between plant cell plasma membrane and cell wall. Arabidopsis AT14A, an integrin-like protein, mediates the cell wall-plasma membrane-cytoskeleton continuum (WMC continuum). To gain further insight into the function of AT14A, the role of AT14A in response to drought stress simulated by polyethylene glycol (PEG-6000) in Arabidopsis suspension cultures was investigated. The expression of this gene was induced by PEG-6000 resulting from reverse transcription-PCR, which was further confirmed by the expression data from publically available microarray datasets. Compared to the wild-type cells, overexpression of AT14A (AT14A-OE) in Arabidopsis cultures exhibited a greater ability to adapt to water deficit, as evidenced by higher biomass accumulation and cell survival rate. Furthermore, AT14A-OE cells showed a higher tolerance to PEG-induced oxidative damage, as reflected by less H 2 O 2 content, lipid peroxidation (malondialdehyde (MDA) content), and ion leakage, which was further verified by maintaining high levels of activities of antioxidant defense enzymes such as ascorbate peroxidase and guaiacol peroxidase and soluble protein. Taken together, our results suggest that overexpression of AT14A improves Handling Editor: Bhumi Nath Tripathi Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0744-7) contains supplementary material, which is available to authorized users. L. Wang : J. He : H. Ding : H. Liu : B. Lü : J. Liang : L. Wang : J. He : H. D. Ding : H. Liu : B. Lü : J. S. Liang (*) Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China e-mail: [email protected] L. Wang e-mail: [email protected]

drought stress tolerance and that AT14A is involved in suppressing oxidative damage under drought stress in part via regulation of antioxidant enzyme activities Keywords Antioxidant enzymes . Arabidopsis thaliana . AT14A . Drought stress . Integrin-like protein . Oxidative stress

Introduction Drought stress is one of the most adverse environmental factors affecting plant growth and development (Pyngrope et al. 2013). When plants are subjected to water deficit, plant cells must perceive the drought signal to trigger a cellular signal transduction pathway enabling them to survive (Zhu 2002). It is understandable that the cell wall and/or plasma membrane are the most possible candidates for the primary site(s) to sense drought stress (Lü et al. 2007a). The plant cell wall may interact with extrinsic and intrinsic membrane proteins, of which one is the integrin-like proteins similar to integrins in animal cells. Integrins are heterodimeric transmembrane receptors in animal cells. They establish cell adhesion to the extracellular matrix (ECM) and bind inside the cell to cytoskeleton, which in turn interact with different signal transduction components that regulate cell migration, polarity, survival, growth, differentiation, and gene expression (Hynes 2002). In plants, integrins have not been identified so far, but seem to share certain analogies with animal integrins. Integrin-like proteins have been detected in many plant species (Nick 2013). Accumulating evidence has shown that there is a cell wall-plasma membrane-cytoskeleton continuum (WMC continuum) in plant cells, which plays important roles in the

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regulation of plant responses to environmental cues (Baluška et al. 2003; Yu et al. 2009; Nick 2013). But, its molecular basis mediated by the membrane protein is largely unknown. The plant integrin analogues have been proposed to be involved in fungal toxin penetration, mechanoperception, cell wall adhesion to the plasmamembrane, and biotic and abiotic stress responses (Knepper et al. 2011). Arabidopsis thaliana AT14A is a membrane protein and has a small domain that has sequence similarities to integrins from animals. Its complementary (cDNA) was isolated by immuno-screening with an anti-integrin antibody from an expression library and was considered as an integrin-like protein (Nagpal and Quatrano 1999). Our recent studies have revealed that AT14A is an essential core component of the WMC continuum, which serves as a transmembrane linker between the cell wall and the cytoskeleton in plants just as integrin in animals, and plays an important role in controlling polarity and morphogenesis (Lü et al. 2012). However, the physiological function of AT14A is unclear. Knepper et al. (2011) identified an integrin-like protein NDR1, known as disease-resistant protein, with a broad role in mediating cell wall-plasma membrane association and in mediating electrolyte leakage and, in part, regulating processes associated with desiccation and drought tolerance. Our previous results identified the presence of integrin-like proteins in A. thaliana and Zea mays plasma membrane, which mediated the interaction between the cell wall and the plasma membrane, and cell responses to osmotic stress (Lü et al. 2007a, b). Integrin-mediated signal transduction involved in osmosensing and oxidative stress has been found in animal cells (Vom et al. 2003; Vecchione et al. 2009). Therefore, the objective of this work was to explore whether AT14A was involved in drought stress, and if so, what was the possible mechanism of AT14A-mediated response. Accordingly, we investigated the effects of polyethylene glycol (PEG-6000)-induced drought stress on cell growth, cell survival, oxidative damage, and antioxidant enzyme activities in the suspension cultures of A. thaliana wild type and overexpressing AT14A.

Materials and methods Culture of A. thaliana cells The cells of transgenic line overexpressing AT14A (AT14AOE) were subcultured from the stable transgenic cells constructed by Lü et al. (2012). A. thaliana (Columbia-0) ecotype cells were used as wild-type (WT) control. The cell lines were maintained in Murashige and Skoog (MS) liquid medium containing 1 μg mL−1 2, 4-D and subcultured every week with 5 % inoculums. The suspension cultures were cultured under 120-rpm rotation speed and at 24 °C in the dark.

Drought treatment The suspension cultures were treated according to Zhao et al. (2008) with some modifications. After subculture, suspension cells grown for 4 days were used for the experiment. The two genotype cells were placed in 30 mL MS medium containing 20 % (w/v) PEG-6000 for indicated times (12, 24, 48, and 72 h) at 24 °C with shaking at 120 rpm in the dark. At indicated times during the treatment period, the cells were harvested by filtration through a nylon mesh of 400 screen meshes, washed with deionized water to remove medium residues, and then blotted with filter paper to remove excess water. The fresh cells were used for biomass and cell survival assay immediately, and the others were frozen in liquid nitrogen and stored at −70 °C for further analysis. Microarray database analysis The data of AT14A (At3g28300) expression was obtained from Arabidopsis eFP Browser (Winter et al. 2007; http:// b a r. u t o r o n t o . c a / e f p / c g i - b i n / e f p We b . c g i ) a n d GENEVESTIGATOR (Zimmermann et al. 2004; https:// www.genevestigator.com/gv/plant.jsp). Log2 signal values were shown in figures made by Origin 8.0 (OriginLab Corporation). RNA isolation and semi-quantitative RT-PCR Total RNA was extracted from suspension-cultured cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, California, USA). Total RNA (1 μg) was used as a template to generate the cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI). The following genespecific primers were used: AT14A-F (5′-CCAACACGCA GCATACAAGG-3′) and AT14A-R (5′-ACGCACCGATGA AAAGCAC-3′); ACTIN-F (5′-GAAAATGGCTGATGGT GAAG-3′) and ACTIN-R 5′-CATAGATAGGAACAGTGT GG-3′. The semi-quantitative reverse transcription (RT)-PCR was performed in a total volume of 20 μL with the following amplification conditions: 5 min at 94 °C; followed by 28 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C; and, lastly, 5 min at 72 °C. PCR products were confirmed by 1.0 % agarose gel electrophoresis. Characterization of cell growth Cell growth was monitored by the estimation of fresh weight of cell suspension. Fresh weight (3 g) were inoculated into 30 mL media with or without PEG-6000 for 24, 48, and 72 h with shaking at 120 rpm and at 24 °C in the dark. The fresh weight of cells after inoculture were measured as described previously (Islam et al. 2009).

Overexpression of AT14A

Determination of cell survival rate The survival rate of suspension cultured cells was quantitatively evaluated by a spectrophotometric assay of Evans blue stain retained by cells as described by Ikegawa et al. (1998) with minor modifications. As Evans blue can only permeate ruptured plasma membranes, it specifically invades dead cells. Briefly, 40 mg cells were suspended in 0.05 % Evans blue solution, gently shaken for 15 min at room temperature, and then washed to remove unabsorbed dye. Dye that had been absorbed by dead cells was extracted with 1 % SDS plus 50 % methanol for 1 h at 60 °C. The absorbance at 600 nm in the supernatant was measured (A1). To determine the total cell death, cells were deliberately killed by heating at 95 °C for 10 min and stained with Evans blue. The absorbed stain was quantified in parallel with unboiled cells (A2). Cell survival rate was calculated as: Cell survival rate(%)=[(A2−A1)/ A2]×100. Measurement of H2O2 content The content of H2O2 was determined using the method of titanium-peroxide complex as described by Patterson et al. (1984) with some modifications. Suspension-cultured cells (0.5 g) were homogenized with 3 mL of cooled acetone in an ice-bath. Of supernatant, 1.0 mL was mixed with 0.1 mL of 20 % titanium reagent and 0.2 mL ammonium solution. The mixture was incubated at 25 °C for 10 min, precipitated, and dissolved in 3 mL of 2 M H2SO4. The absorbance was read at 415 nm after centrifugation. The H2O2 content was calculated using a standard curve generated from known concentrations of H2O2. Ion leakage measurement Relative ion leakage was determined according to Zhao et al. (2004). The suspension cultures (0.3 g) were washed in deionized water and placed in tubes with 5 mL of deionized water at 25 °C. After 2 h, the conductivity was measured (C1) and the samples were boiled for 20 min to achieve 100 % electrolyte leakage (C2). Relative ion leakage was expressed as: Relative ion leakage(%)=(C1/C2)×100. Analysis of lipid peroxidation Oxidative damage to lipids was determined by measuring the amount of produced malondialdehyde (MDA) by thiobarbituric acid (TBA) reaction as described by Jiang and Zhang (2001). The crude extract was mixed with the same volume of 0.65 % (w/v) TBA solution containing 20 % (w/v) tricholoroacetic acid, incubated at 95 °C for 30 min, and then quickly cooled in an ice-bath. The mixture was centrifuged at 10,000g for 10 min, and the absorbance of the supernatant was

monitored at 532 and 600 nm. After subtracting the nonspecific absorbance (600 nm), the MDA content was calculated from the extinction coefficient (155 mM−1 cm−1). Antioxidant enzyme activity assay The suspension cultures (50 mg) were homogenized in 1.6 mL of 50 mM potassium phosphate buffer, pH 7.5, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 2 % soluble polyvinylpyrrolidone (PVP), with the addition of 1 mM ascorbate in the case of APX assay. The homogenate was centrifuged at 12,000g for 30 min at 4 °C, and the supernatant was immediately frozen under liquid N2 and stored at −70 °C for the following assays. SOD (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) according to the method of Beauchamp and Fridovich (1971). One unit of SOD activity was defined as the amount of enzyme required to cause a 50 % inhibition of the rate of NBT reduction at 560 nm. SOD activity was expressed as units per milligram protein. POD (EC1.11.1.7) activity was estimated using guaiacol as a substrate (Nickel and Cunningham 1969). The increase in absorbance at 470 nm due to the guaiacol oxidation was recorded for 3 min (extinction coefficient 26.6 mM−1 cm−1). The activity was expressed as micromoles per minute per milligram protein. CAT (EC 1.11.1.6) activity was determined by following the reduction of H2O2 (extinction coefficient 39.4 mM−1 cm−1) at 240 nm according to the method of Aebi (1984). The activity was expressed as nanomoles per minute per milligram protein. APX (EC 1.11.1.11) activity was determined according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm (extinction coefficient 2.8 mM−1 cm−1). The activity of CAT was calculated as micromoles per minute per milligram protein. Protein content Protein content was determined according to the method of Bradford (1976) with bovine serum albumin (BSA) as standard. Briefly, 10 μL of crude enzyme solution was added to 1.5 mL G-250 solution, and the absorbance was measured at 595 nm using a spectrophotometer (DU 730, Beckman Coulter, USA). Statistical analysis Each value in this study was presented as the mean±standard error (SE) calculated from at least three independent experiments. All data were subjected to one-way analysis of

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variance (ANOVA), and the differences among treatments were compared by Student’s t test at P