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Changes in Peroxidase Isoenzyme and Protein Patterns in Oat Cultivars under Cold Stress İmren KUTLU1*, Ece TURHAN2 Eskisehir Osmangazi University, Faculty of Agriculture, Department of Biosystem Engineering, 26160 Eskisehir/TURKEY 2 Eskisehir Osmangazi University, Faculty of Agriculture, Department of Agricultural Biotechnology, 26160 Eskisehir/TURKEY * Corresponding author: [email protected] 1

ABSTRACT The possible role of peroxidase isoenzymes and protein patterns induced by cold stress were investigated in 'Faikbey' (relatively sensitive) and 'Yeniçeri' (relatively tolerant) oat (Avena sativa L.) cultivars. The seeds of the oat cultivars were sown in perlite:torf:sand (1:1:1) media and germinated at 25/15 °C (day/night) in a controlled greenhouse. Plants were harvested after 21 days and half of the plants for 12 hours while the other half was acclimated for 24 hours at 4 °C to low temperatures. After that, the leaves were exposed to controlled freezing test at 0, -6, -12 and -18 ºC for 12 hours in manually-controlled low temperature freezer. Samples were then removed from the freezer at each temperature and placed at 4 °C overnight for slow thawing. Peroxidase (POX) profiles and protein patterns of the leaf tissues were performed by native polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, respectively. In native PAGE, different acidic and basic isoperoxidase bands (between Rf=0.04 and Rf=0.88) were observed commonly with different band intensities in both cultivars with respect to the cold treatments and duration of acclimation. In addition, according to the SDS-PAGE analysis different protein bands, in mass range 9-94 kDa, were detected in the oat cultivars. The intensities of some proteins generally decreased and/or disappeared and a few new proteins appeared in response to cold stress and duration of acclimation. In conclusion, we supposed that isoperoxidases and proteins may responsible to cold tolerance in oat cultivars. Keywords: Avena sativa L., cold acclimation, low temperature stress, peroxidase, soluble protein INTRODUCTION Low temperatures (cold and frost) represent a severe threat to plant survival and economic crop productivity in many regions. The process whereby certain plants increase in freezing tolerance in response to low non-freezing temperatures are known as cold-acclimation (or cold-hardiness) (Thomashow 2010). In temperate latitudes, coldacclimation is established in the autumn, when the temperatures are low but positive and the photoperiod decreases, and it reaches a maximum in winter (Reulland et al. 2009). The ability of plants to acclimate to low temperatures is crucial for their survival. Plant responses to low temperature stress have been associated with activated forms of oxygen, including superoxide (O2.-), hydrogen peroxide (H2O2), hydroxyl (-OH) radicals and singlet oxygen (O21) (Foyer and Noctor 2005, Suzuki and Mittler 2006). Cold stress induces or enhances the active oxygen speciesscavenging enzymes like, catalase (CAT: EC, peroxidase (POX:EC, ascorbate peroxidase (APX: EC, and glutathione reductase (GR: EC (Turhan et al. 2012a, 2012b). Peroxidases are a large group of isoenzymes with an extreme range of isoelectric points, serving a multitude of functions (Huystee 1987). Peroxidases a class of enzymes in plant, catalyze oxido reduction between H2O2 and various reductans (Hiraga et al. 2001). Therefore, peroxidases are known one of the important parts of the enzymatic defence system of plant cells under stress conditions (Gaspar et al. 1982). Biochemical changes that have been associated with cold-acclimation include increased sugar and soluble protein content, expression of specific proteins and the appearance of new isozymes (Eris et al. 2007, Gulen et al. 2008, Turhan 2012, Liu et al. 2013). Protein folding is influenced by temperature changes. Temperature-induced changes in the physical state of membranes and proteins are expected to change the metabolic reactions and thus the metabolite concentrations. Therefore, plant cells can sense cold stress through membrane rigidification, protein/nucleic acid conformation, and/or metabolite concentration (Chinnusamy et al. 2010). The commercial value of oat is derived both from its high-energy grain and from superior break-crop benefits. Today oat is mainly used as animal feed, but it is one of the most promising future cereals in the functional food area (Bräutigam et al. 2005). Oats are grown in Turkey where occasional frost may occur. In

Turkey, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop cold hardy oat cultivars, more knowledge about mechanisms of cold tolerance in oat is required (Bräutigam et al. 2005). Livingston et al. (2005) reported that under freezing temperatures, most of the aerial plant tissue in oat is destroyed and recovery from freezing is mainly dependent on surviving cells from the crown. In addition, the close involvement of soluble carbohydrates in cold tolerance is well established (Livingston and Henson 1998) and cold tolerance has been found to be correlated to the activity of antioxidative enzymes in oat plant (Liu et al. 2013, Kutlu and Turhan 2015). However, more detailed data are needed on isozymes and proteins to understand and explain the mechanisms of cold-hardiness in oat. Changes in the qualitative activities of POX and proteins at two durations of acclimation (12 and 24 hours) and non-stressed plants (control) in two oat cultivars were under cold stress assessed in the present study. Changes associated with cold stress were analysed for a better understanding of the general cultivar differences and for providing preliminary information for later, more detailed, studies on oat cultivars. MATERIALS AND METHODS The seeds of the oat (Avena sativa L.) cultivars ('Faikbey'; relatively cold sensitive and 'Yeniçeri; relatively cold tolerant') (Kutlu and Turhan 2015) were sown in perlite:torf:sand (1:1:1) media and germinated at 25/15 °C in a controlled greenhouse. Plants were harvested after 21 days and half of the plants for 12 hours while the other half was acclimated for 24 hours at 4°C to low temperatures. Samples were exposed to low temperatures according to the method of Arora et al. (1992) with some modifications. Briefly, leaves from each cultivar were wrapped (10 leaves per temperature in each replication) in aluminium foil along with moistened paper and placed in a manually controlled low-temperature freezer. The plant tissue temperature was monitored with a copper-constant thermocouple (Testo 925, Omni Inst., Scotland, UK) inserted in the foil pouch. The temperature was decreased stepwise at approximately 1.5ºC/h to 0ºC and 6ºC/h thereafter to a final value of -18ºC. The samples were exposed to low temperatures of 0, -6, -12 and -18 ºC for 12 h. The samples were then removed from the freezer at each temperature and placed at 4ºC overnight for slow thawing. The leaf material was collected from each plant group at each temperature application step. Triplicate samples of leaf tissues were frozen immediately put in liquid N2 and stored at −80 °C until used for POX isozymes and protein analysis. In addition, plants that are unstressed used as control plants. POX was extracted from leaf tissues using the extraction methods described by Gulen et al. (2002), with modifications as follows. Ground leaf tissues (0.1 g) were homogenized at 4 °C in 0.6 mL extraction buffer (0.1M potassium phosphate pH 7.5, 30mM boric acid, 50mM L-ascorbic acid, 17mM sodium metabisulfite, 16mM diethyldithiocarbamic Acid (DIECA), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% (w/v) polyvinylpolypyrrolidone (PVPP); 4% (w/v) PVP-40 was omitted and the final pH was readjusted to 7.5 with sodium hydroxide). Homogenates were centrifuged at 21,000×g for 20 min and the supernatant was used for electrophoresis. Discontinuous polyacrylamide gel electrophoresis (PAGE) was performed with a PROTEAN tetra vertical electrophoresis unit (Bio-Rad, Hercules, CA) for acidic and basic POX, respectively, according to Davis (1964) and Reisfeld et al. (1962). Five percent stacking gels and 10% separating gels were prepared for both systems. Equal volumes of the protein (30 µg) were loaded for each sample. Electrophoresis was performed at 20 mA for 30 min, followed by 40 mA for 3 h. Gels were stained for POX using the method of Wendel and Weeden (1989). The relative distance (Rf-value) of the bands on the gel was calculated as described by Manganaris and Alston (1992) using Rf =1.0, distance to the fastest band and Rf = 0.0, the starting point. Scanned isozymes profiles gels were analyzed by using the Public Domain NIH Image program (available on the internet at (verified 28 July 2008) ImageJ Software. Total soluble proteins (TPS) were extracted in the light of Shen et al. (2003) method with some modifications. Exactly 0.25 g of each sample was homogenized in 1 mL homogenate buffer containing 25 mM Tris-base (pH 7.8), 275 mM sucrose, 2 mM EDTA, 10 mM Dithiothreitol (DTT), 0.5 mM Phenylmethylsulfonyl fluoride (PMSF), and 1% PVPP. The homogenate was transferred into an eppendorf tube and then centrifuged at 10.000 rpm for 10 min at 4°C. Protein content was measured using the Bradford assay method (Bradford 1976). Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) was performed with a PROTEAN tetra vertical electrophoresis unit (Bio-Rad, Hercules, CA, USA) using 0.04 stacking gel and 0.125 separating gel. An equal amount of total protein (10 mg) was loaded for each sample and gels were stained with Coomassie Brilliant Blue G-250. RESULTS Native PAGE analysis of the samples was repeated at least three times with similar results and data from a single, representative analysis are presented herein. The results showed that there were changes in intensities and

number of isozyme bands of leaf tissues subjected to low temperatures for two durations of acclimation. Analysis of acidic POX zymograms patterns, showed varied expression of isoforms in two oat cultivars studied, the number of these isozymes varies from 1 to 4 depending on each cultivar studied. These isoforms, named POX1-POX4 were expressed in both tolerant and sensitive cultivars but variations in the intensity of the bands were observed among the cold treatments in comparison with the control (Figure 1 to Figure 2). We noticed, in cv.'Faikbey', three isozymes of POX (Rf 0.14, POX1; Rf 0.27, POX2; Rf 0.88, POX4) in cv.'Yeniçeri' four isozymes of POX (Rf 0.14, POX1; Rf 0.27, POX2; Rf 0.83, POX3; Rf 0.88, POX4) in both durations of acclimation (Figure 1A, B).

Figure 1. Native PAGE of acidic POX activities of the leaf tissues of oat cultivars under low temperature treatments at two durations of acclimation (12h:Panel A and 24 h: Panel B) and unstressed (Control:C) plants and their band intensities based on the quantitative measurements. Equal volumes of the crude extracts, 20 µL, were loaded in each lane. POX1 (Rf 0.14), POX2 (Rf 0.27), POX3 (Rf 0.83), POX4 (Rf 0.88). In general POX activities were the highest level in control plants, whereas low temperatures (particularly 4ºC) inhibited the POX activity in cv.'Faikbey' at both durations of acclimation. However the band intensity generally increased with low temperature treatments in cv.'Yeniçeri' especially at 12h duration of acclimation. Indeed POX band intensities of cv.'Yeniçeri' were higher than cv. 'Faikbey' in both durations of acclimation (Fig.1A, B). Moreover, only relatively cold tolerant cultivar cv.'Yeniçeri' has a capacity to activate POX3 under cold stress.

Figure 2. Native PAGE of basic POX activities of the leaf tissues of oat cultivars under low temperature treatments at two durations of acclimation (12h: Panel A and 24 h: Panel B) and unstressed (Control:C) plants and their band intensities based on the quantitative measurements. Equal volumes of the crude extracts, 20 µL, were loaded in each lane. POX1 (Rf 0.04), POX2 (Rf 0.36), POX3 (Rf 0.48), POX4 (Rf 0.63). Four basic POX isozymes were detected at two durations of acclimation in both cultivars during cold stress at (Figure 2A, B). The intensity of basic POX isoenzymes in cv.‘Faikbey’ generally increased, while the intensity of four basic isozymes, POX1 (Rf 0.04), POX2 (Rf 0.36), POX3 (Rf 0.48) and POX4 (Rf 0.63) decreased at 12h duration of acclimation in cv.'Yeniçeri' and was stronger in cv.‘Faikbey’ than in cv.‘Yeniçeri’. The bands were observed commonly with different band intensities in all over the low temperature treatments at two durations of acclimation. The intensities of the bands generally significantly increased especially at 0°C and -6 °C treatments at 12h acclimation and at 0 °C at 24 h acclimation in both cultivars. Band intensities of basic isoperoxidases of both cultivars showed some fluctuations (up and down) at two durations of acclimation under cold stress (Figure 2A, B). SDS-PAGE analyses of samples of leaf proteins were repeated at least three-times with similar results. Data from a single, representative SDS-PAGE profile are presented (Figure 3A, B). According to the SDSPAGE profiles, at 12 h acclimation stage, four protein bands estimated at 9, 15, 18 and 26 kDa were considered in cv. 'Faikbey'. 13, 15, 34, 72 and 88 kDa size of bands have been observed in all cold treatments in cv.'Yeniçeri' (Figure 3A). While, the intensities of 9, 15 and 26 kDa size of bands decreased at -18 °C, 18 kDa protein band disappeared at 0 C° in cv.'Faikbey' at 12 h acclimation stage. The 94 kDa protein band was observed from the control to -18 ºC in cv.'Faikbey at 24 h acclimation stage'. However, the 94 kDa protein band disappeared after 4 °C treatment in cv.'Yeniçeri'. New protein bands with 13, 15, 18, 20, 26, 28, 36, 40, 94 kDa appeared and with 9 kDa band disappear in cv. 'Faikbey' at 24 h acclimated stage. However generally all protein bands disappeared except 13 kDa detected at 12 h acclimated stage and new protein bands with 84 and 94 kDa protein bands in cv.'Yeniçeri' (Figure 3B).

Figure 3. Total soluble protein (TSP) profiles (SDS-PAGE) of the leaf tissues of oat cultivars under low temperature treatment at 12h (Panel A) and 24 h (Panel B) durations of acclimation and unstressed (control) plants. 10 µg of protein was loaded on each lane. Molecular weight markers (M) and their molecular masses (kDa) are shown on the left side of each Panel. DISCUSSION Plant peroxidases are important in diverse cellular functions such as lignin biosynthesis, hormone generation, and detoxification of hydrogen peroxide (Conroy et al. 1982). Plant peroxidases are usually classified as acidic, neutral, or basic, according to their isoelectric points (Yoshida et al. 2003). Each group is thought to have a different function in the cell. Acidic peroxidases are the isoenzymes most likely involved in lignin formation and wall-associated, whereas function of basic isoenzymes has been suggested that they might provide H2O2 for other peroxidases (Walter 1992). In the present study, data from native PAGE indicated three and four asidic isoperoxidases with different intensities in cv. 'Faikbey' and in cv.'Yeniçeri', respectively at both durations of acclimation under cold stress (Figure 1, 2). The increased POX activities under low temperature in oat were linked with expression of isoform POX3 only in relatively cold tolerant cultivar. It seems that the cold tolerant cultivar display a better overall POX activity. In addition, four basic isoperoxidases stated at two durations of acclimation in both cultivars with different intensities. Since data indicated a linear relationship between band intensities and the low temperature treatment, it is correlated to cold-acclimation of oat leaf tissues. On the other hand, the data indicated that expression of isozymes in response to cold stress depends on the temperatures and is higher in tolerant cultivar than in sensible one. Similar results also obtained in Medicago under cold stress (Nourredine et al. 2015). In addition Liu et al. (2013) reported that POX activities in naked oats (Avena nuda L.) were higher under low temperature than normal temperature. Kuk et al. (2003) showed, in rice plants, that isozyme profile and activity of peroxidase were significantly expressed under cold stress and deduced that peroxidase were most important for cold acclimation and chilling tolerance. Cansev et al. (2005) reported expression of acidic peroxidase bands with different band intensities which are responsible for tolerance to cold stress of 15 olive cultivars. Gulen et al. (2008) also pointed out that one acidic (Rf=0.23) and one basic (Rf=0.17) POX bands were observed commonly in cold subjected strawberry plants with different band intensities, and, they suggested that it may be associated with cold-acclimation in strawberry plants under low temperature stress. The effects of low temperature stress on protein profile on both cultivars were different. Protein banding patterns of cv.'Faikbey' were between 9 kDa and 26 kDa at 12 h duration of acclimation and between 15 kDa and

94 kDa at 24 h duration of acclimation with reference to the marker (Figure 3). As shown in Figure 3, protein banding patterns of cv. 'Yeniçeri' were between13 kDa and 88 kDa at 12 h duration of acclimation and between 13 kDa and 94 kDa at 24 h duration of acclimation with reference to the marker under low temperature stress. Different band positions between cv.'Faikbey' and cv.'Yeniçeri' indicated their specific response towards the given low temperature stress and also it reflects their different genetic variation. The banding intensity of different polypeptide bands (15kDa) of cv.'Yeniçeri' was higher than cv.'Faikbey' under low temperature treatment. It was founded that protein banding patterns of rice plant between 30 kDa and 15 kDa with reference to the marker under low temperature stress (Perveen et al. 2013). In conclusion, our data indicated that acidic POX3 isoform (Rf 0.83) can be associated with coldacclimation in oat plants under cold stress. In addition we supposed TSP may responsible to cold tolerance in oat cultivars. Differences in the TSP contents of leaf tissues occurred between the duration of acclimation. The 84 kDa leaf proteins appeared that to be related to cold-acclimation in oat. Further research is in progress to characterise these proteins and to immunolocalise them. REFERENCES Arora R, Wisniewski ME, Scorza R (1992). Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch) I: Seasonal changes in coldhardiness and polypeptides of bark and xylem tissues. Plant Physiol 99:1562-1568. Bradford MM (1976). A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254. Bräutigam M, Lindlöf A, Zakhrabekova S, Gharti-Chhetri G, Olsson B, Olsson O (2005). Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa. BMC Plant Biology 5:18. Cansev A, Köksal N, Gülen H, İpek A, Eriş A (2005). Düşük sıcaklık stresi altındaki bazı zeytin çeşitlerinin peroksidaz aktivitesine göre gruplanması. XIV. Biyoteknoloji Kongresi, Bildiriler Kitabı, 313-317. Chinnusamy V, Zhu JK, Sunkar R (2010). Gene regulation during cold stress acclimation in plants. Methods Mol Biol 639: 39–55. Conroy JM, Borzelleca DC, Mcdonell LA (1982). Homology of plant peroxidases. Plant Physiol 69: 28-31. Davis BJ (1964). Disc electrophoresis, method and application to human serum proteins. Ann NY Acad Sci 121: 404-427. Eris A, Gulen H, Barut E, Cansev A (2007). Annual patterns of total soluble sugars and proteins related to coldhardiness in olive (Olea europaea L.‘Gemlik’). Journal of Horticultural Science and Biotechnology 82: 597–604. Foyer CH, Noctor G (2005). Oxidant and antioxidant signaling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056-1071. Gaspar T, Penel CL, Thorpe T, Greppin H (1982). Peroxidases. A survey of their biochemical and physiological roles in higher plants. Universite de Geneve Press, Geneve. Gulen H, Çetinkaya C, Kadıoğlu M, Kesici M, Cansev A, Eriş A (2008). Peroxidase activity and lipid peroxidation in strawberry (Fragaria X ananassa) plants under low temperature. J Biol Environ Sci 2(6): 95-100. Gulen H, Arora R, Küden A, Krebs SL, Postman J (2002). Peroxidase isozyme profiles in compatible and incompatible pear/quince graft combinations. J Amer Soc Hort Sci 127(2): 152-157. Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H (2001). A large family of class III plant peroxidases. Plant Cell Physiol 42(5):462-468. Huystee RBV (1987). Some molecular aspects of plant peroxidase biosynthetic studies. Annu Review Plant Physiol 38: 205219. Kuk Y, In, SJS, Burgos NR, Hwang T, Eak HO, Cho BH, Jung S, Guh JO (2003). Antioxidative enzymes offer protection from chilling damage in rice plants. Crop Sci 43: 2109-2117. Kutlu I, Turhan E (2015). Yulaf (Avena sativa L.) bitkisinde düşük sıcaklık uygulamalarının teşvik ettiği bazı fizyolojik değişiklikler ve antioksidan enzim aktivitelerinde değişim. 11. Tarla Bitkileri Kongresi, Poster Bildirileri Cilt I, Tahıllar, Yemeklik Dane Baklagiller, Bitki Biyoteknolojisi, 7-10 Eylül 2015 Çanakkale, s:459-462. Liu W, Yu K, He T, Li F, Zhang D, Liu J (2013). The low temperature induced physiological responses of Avena nuda L., a cold-tolerant plant species. The Scientific World Journal. Livingston III DP, Tallury SP, Premkumar R, Owens SA, Olien CR (2005) Changes in the histology of coldhardened oat crowns during recovery from freezing. Crop Science 45: 1545–1558.

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