Antioxidative response in two Rubus species exposed to salinity

Transcription

1 IZVORNI ZNANSTVENI RAD Antioxidative response in two Rubus species exposed to salinity Tihana MARČEK 1, Darko VELIĆ 1, Mirjana SABO 1, Krunoslav DUGALIĆ 2, Natalija VELIĆ 1, Daniela AMIDŽIĆ KLARIĆ 3 1 Faculty of Food Technology Osijek, F. Kuhača 20, Osijek, Croatia, ( tihana.marcek@ptfos.hr) 2 Agricultural Institute Osijek, Južno predgrađe 17, Osijek, Croatia 3 Clinical Hospital Dubrava, Avenija Gojka Šuška 6, Zagreb, Croatia Abstract Salinity is considered as one of the serious abiotic factors that inhibits plant growth and development. Excessive salt concentration in the soil has negative impact on germination and yields and causes enormous damage in overall economic output. In nature salinity is often present in soils which are concurrently affected by drought and/or heat stress. The aim of this work was to investigate the influence of the salt stress in two Rubus species (Rubus ideus L. var. Himbo top and Rubus fructicosus L. var. čačanska Bestrna) through proline content, level of lipid peroxidation and activities of antioxidative enzymes. Plants were exposed to NaCl (15 and 35 mm) in in vitro conditions for 21 days. In red raspberry salinity (35 mm NaCl) induced remarkably increased catalase (CAT) and ascorbate peroxidase (APX) activities while blackberry showed high guaiacol peroxidase (POD) activity indicating their important role in elimination of reactive oxygen species (ROS). Malondialdehyde (MDA) content showed an increase at 15 mm NaCl while the highest NaCl concentration (35 mm) caused decline in MDA content, in both Rubus species. Decreased proline synthesis in both Rubus suggested that proline is not involved in antioxidative response induced by NaCl. Key words: salinity, antioxidative enzymes, lipid peroxidation, proline, Rubus species Antioksidacijski odgovor dviju vrsta roda Rubus sp. izloženih salinitetu Sažetak Salinitet je važan abiotički čimbenik koji ograničava rast i razvoj biljaka. Previsoke koncentracije soli u tlu negativno utječu na klijanje i prinos te uzrokuju štete u gospodarstvu. U prirodi se salinitet često javlja u tlima koja su istovremeno pogođena sušom i/ili toplinskim stresom. Cilj ovog istraživanja bio je istražiti učinak solnog stresa na malini (Rubus ideus L. var. Himbo top) i kupini (Rubus fructicosus L. var. čačanska Bestrna) određivanjem sadržaja slobodnog prolina, lipidne peroksidacije i mjerenjem aktivnosti enzima antioksidacijskog sustava. Biljke su bile izložene djelovanju NaCl (15 i 35 mm) u uvjetima in vitro tijekom 21 dana. Salinitet (35 mm NaCl) je u maline inducirao značajno povećanu aktivnost katalaze (CAT) i askorbat peroksidaze (APX) te povećanu aktivnost gvajakol peroksidaze (POD) u kupine iz čega slijedi kako ovi enzimi imaju važnu ulogu u uklanjanju reaktivnih kisikovih čestica (ROS). Obje vrste roda Rubus sp. pokazale su porast sadržaja malondialdehida (MDA) na 15 mm NaCl dok je najveća koncentracija NaCl (35 mm) uzrokovala pad sadržaja MDA. Smanjena sinteza prolina u obje vrste roda Rubus sp. ukazuje da prolin nije uključen u antioksidacijski odgovor uzrokovan povišenom koncentracijom NaCl. Proceedings. 50 th Croatian and 10 th International Symposium on Agriculture. Opatija. Croatia ( ) Section 9. Pomology 595

2 Tihana MARČEK, Darko VELIĆ, Mirjana SABO, Krunoslav DUGALIĆ, Natalija VELIĆ, Daniela AMIDŽIĆ KLARIĆ Ključne riječi: salinitet, enzimi antioksidacijskog sustava, lipidna peroksidacija, prolin, vrste roda Rubus sp. Introduction Under natural conditions, plants are exposed to various abiotic and biotic stress factors. Salinity as abiotic stress factor in plants causes a series morphological, physiological, biochemical and molecular changes, inhibits germination and growth leading to reduced yields and enormous damage in overall economy (Ashraf, 2002). Salinization may be the result of natural processes, but occurs more frequently due to human impact on the environment through a series of agronomic activities, like irrigation (Szabolcs, 1994). In nature salinity is often present in soils which are concurrently affected by drought and/or heat stress. In plants salinity causes osmotic and ionic stress, dehydration, inhibition of photosynthesis and protein synthesis, chlorosis and necrosis, growth reduction and production of highly reactive oxygen species (ROS) (Munns and Tester, 2008). Plants have different cellular mechanisms that ameliorate the effects of the environmental stresses. The most common response is synthesis of compatible osmolytes such as proline. Under stress conditions proline has diverse roles such as protection of the membrane integrity, enhancement of the enzyme activities, control of plant development and signal molecule feature (Szekely et al., 2008). Moreover, proline has antioxidant feature protecting cellular functions by scavenging reactive oxygen species (Szábadós and Savouré, 2009). During salt stress the production of ROS is dramatically elevated. As a response to ROS accumulation plants activate antioxidative enzymes such as catalase (CAT), non-specific peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) that cooperate together to mitigate cellular damages like unspecific oxidation of proteins, membrane lipids and nucleic acids (Miller et al., 2009; Pitzschke et al., 2006). Various studies emphasize that antioxidative response is well correlated with susceptibility and resistance to increased salinity. Red raspberry (Rubus ideus L.) and blackberry (Rubus fructicosus L.) are glycophytes, salt sensitive plants, which can tolerate salt concentration in cytosol in the range mm NaCl (Munns and Tester, 2008). Red raspberry showed growth reduction at 30 mm NaCl (Neocleous and Vasilakakis, 2007) whiles for blackberry no information was found. The objective of this research was to investigate the effects of the salt stress (15 and 35 mm NaCl) on two in vitro Rubus species (Rubus ideus L. var. Himbo top and Rubus fructicosus L. var. čačanska Bestrna) through physiological responses on oxidative stress and proline content. Material and methods In the present study, red raspberry (Rubus ideus L.) and blackberry (Rubus fructicosus L.) were used. Seedlings, kindly provided by the Agricultural Institute Osijek (Croatia), were grown in a growth chamber (24 ± 2 C, light intensity 2,963 µmol s 1 m 2 and photoperiod 16 h light/8 h darkness) and multiplied every 3-4 weeks using nodal segments. Explants were grown on solid Driver and Kuniyuki medium (DKW) (Driver and Kuniyuki, 1984) without growth regulators. Salt stress was induced by addition of 15 and 35 mm NaCl to DKW while medium without addition of salt was used as control. After 21 days of treatment, plants were sampled and stored till analyses. Enzyme extraction. Plant tissue (250 mg) was homogenized in 50 mm potassium phosphate buffer (ph 7.0) containing 0.1 mm ethylenediaminetetraacetic acid, 5 mm ascorbate acid and polyvinylpolypyrrolidone and centrifuged at g for 30 min at 4 C. Total protein content was measured according to Bradford (1976). POD activity was analysed by measuring peroxidation of hydrogen peroxide with guaiacol as an electron donor at 470 nm (Chance and Maehly, 1955). CAT activity was estimated by the decrease in absorbance at 240 nm (Aebi, 1984). APX activity was analysed by monitoring the decline in absorbance of ascorbate at 290 nm (Nakano and Asada, 1981). The enzyme activities were expressed as units (U) of enzyme activity per milligram of protein [U mg 1 proteins ]. Free proline content mg of fresh tissue was extracted in 3% (w/v) sulphosalicylic acid and centrifuged at g for 40 min. Mixture of supernatant, acid ninhydrin and glacial acetic acid was heated at 100 C for 1 h and cooled in an ice bath. Free proline was separated with toluene and absorbance was read spectrophotometrically at 520 nm (Bates et al., 1973). Proline concentration was determined using a calibration curve obtained with L-proline solutions ranging from 50 to 700 µm and expressed as micromols per gram of fresh weight [µmol g -1 FW ] th Croatian and 10 th International Symposium on Agriculture

3 Antioxidative response in two Rubus species exposed to salinity Malondialdehyde (MDA) content. Lipid peroxidation was measured as the amount of MDA (Heath and Packer, 1968). Fresh sample (100 mg) was homogenized using 0.3% (w/v) TBA in 10% (w/v) trichloroacetic acid (TCA), heated at 95 C for 40 min and cooled in an ice bath. After centrifugation ( g for 20 min) the absorbance of the supernatant was read at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. As a blank 0.3% TBA in 10% TCA solution was used. The MDA content was calculated according to the molar extinction coefficient of 155 mm -1 cm -1. All results were expressed as means of three replicates (±SE) and compared by one-way ANOVA followed by Newman-Keul s test at P < Results and discussion Blackberry showed significantly high POD activity at 35 mm NaCl (Fig. 1A) compare to 15 mm NaCl while red raspberry at 35 mm responded with remarkably pronounced CAT and APX activities (Figs. 1B, 1C) compare to control indicating important role of antioxidative enzymes in ROS scavenging (Maia et al., 2010). A positive relationship between salt tolerance and increased activation of POD, CAT and APX has been demonstrated in tolerant mulberry (Morus alba L.) genotype (Sudhakar et al., 2001). MDA content, a product of lipid peroxidation, in blackberry plants treated with 35 mm NaCl showed significant decline compare to 15 mm NaCl (Fig. 1D) which could be connected with increased POD activity (Fig. 1A). On the other hand, the MDA content in red raspberry did not change significantly at 35 mm NaCl suggesting that this salt concentration probably caused the oxidative stress in red raspberry leading to enhanced activation of CAT and APX (Figs. 1B, 1C) thereby reducing the MDA content and maintaining the membrane integrity (Maia et al., 2010). Same was obtained in wheat cultivars (Triticum sp.) (Esfandiari et al., 2007) and rice (Oryza sativa L.) (Khan and Panda, 2008). Proline content declined in both Rubus species at the highest salt concentration (35 mm NaCl) (Fig. 1E). Proline decrease under both salt treatments suggests that in observed Rubus plants proline is not involved in protection mechanisms against salinity induced ROS. Decreased proline accumulation might be due to the greater utilization of proline by the plants toward its production (Alaya-Astorga and Alcaraz-Meléndez, 2010). According to Tester and Davenport (2003) inhibited proline production could be a result of effective compartmentalization of Na + ions into vacuoles in order to avoid its toxic effects in the cytosol. Decreased proline content has already been reported for rice (Lutts et al., 1999) and dragon tree (Paulownia fortunei [Seem.] Hemsl.) (Alaya-Astorga and Alcaraz-Meléndez, 2010). Contrarily, the importance of proline in achieving cell osmotic adjustment during salinity and ROS elimination was described in numerous studies (Chen and Dickman, 2005; Marček et al., 2014). Section 9. Pomology 597

4 Tihana MARČEK, Darko VELIĆ, Mirjana SABO, Krunoslav DUGALIĆ, Natalija VELIĆ, Daniela AMIDŽIĆ KLARIĆ Figure 1. Activity of POD (A), CAT (B), APX (C), content of malondialdehyde (MDA) (D) and proline content (E) in Rubus species treated with 0 (<), 15 (<) and 35 (<) mm NaCl for 21 days. Conclusion Red raspberry responded to NaCl by enhanced CAT and APX activities while blackberry showed high POD activity which could explain why salinity did not cause increased level of lipid peroxidation. Decline in proline content in Rubus species suggests that proline is not specific indicator of the salt response th Croatian and 10 th International Symposium on Agriculture

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