Acta Botanica 1-2017 - za web.indd ACTA BOT. CROAT. 76 (1), 2017 49 Acta Bot. Croat. 76 (1), 49–54, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0038 ISSN 0365-0588 eISSN 1847-8476 Response of dihaploid tobacco roots to salt stress Tihana Marček1*, Željka Vidaković-Cifrek2, Mirta Tkalec2, Marin Ježić2, Mirna Ćurković-Perica2 1 Subdepartment of Biology and Microbiology, Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuhača 20, HR-31000 Osijek, Croatia 2 Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia Abstract – Salinity is a common abiotic factor that affects plant growth and development. Seedlings of to- bacco (Nicotiana tabacum L.) F1 hybrid DH10 and three dihaploid lines (207B, 238C and 239K) obtained by diploidization of anther-derived haploids of hybrid DH10 were subjected to 0, 100 and 200 mM NaCl in in vitro conditions for 33 days and the effect on roots was evaluated. In all lines and in the hybrid DH10 exposed to 200 mM NaCl evident root growth inhibition and increased proline content were noticed. However, in some cases lines differed in the activity of antioxidative enzymes, which could account for differences in their salinity tolerance. Increased activity of catalase and peroxidase in roots of line 239K could contribute to the more pronounced salinity tolerance previously reported for shoots of this line. Keywords: antioxidative enzymes, Nicotiana tabacum L., proline, root, salinity, tobacco dihaploid lines * Corresponding author, e-mail: tihana.marcek@ptfos.hr Introduction Salinity is one of the most adverse environmental fac- tors that reduce the growth and productivity of agricultural crops (Horie et al. 2012). In nature, it often comes together with drought and heat and all these stress factors may cause changes in plant water status, decreased activity and dena- turation of structural proteins and leakiness of membranes caused by phospholipid bilayer disruption (Wang et al. 2003, Mahajan and Tuteja 2005). Generally, the detrimental effects of salinity in plants are characterized by a decrease in growth rate, a change in root/shoot ratio and development of chlorosis (Parida and Das 2005). Reduced growth mainly occurs due to low wa- ter potential in the soil. In such conditions the possibility of cell dehydration is increased due to diffi culty in the acquisi- tion of water by plant roots. In addition, increased concen- tration of Na+ and Cl– in plant organs disturbs the intake of essential microelements such as K+, leading to altered K+/ Na+ ratios and disruption of cellular homeostasis (Apse and Blumwald 2007, Munns and Tester 2008). In order to adjust osmotic potential, plants accumulate compatible osmolytes. The most common among them is the amino acid proline, which is broadly present in many organisms. Besides being an osmolyte, proline acts as a molecular chaperone: it enhances the activities of the en- zymes, controls plant development and acts as a signal mol- ecule. Proline also shows antioxidant property through re- active oxygen species (ROS) scavenging activity (Szèkely et al. 2008, Szábados and Savourè 2009). Under the conditions of increased salinity, the levels of ROS can dramatically increase. In order to deal with these highly reactive species, antioxidative enzymes such as cata- lase (CAT), non-specifi c peroxidase (POD), superoxide dis- mutase (SOD) and ascorbate peroxidase (APX) cooperate together to mitigate cellular damage such as unspecifi c oxi- dation of proteins, membrane lipids and nucleic acids. SOD converts superoxide radical (O2•–) into oxygen and hydro- gen peroxide while POD and CAT are involved in H2O2 de- toxifi cation (Bandeoğlu et al. 2004, Parvanova et al. 2004). Numerous studies emphasize that the antioxidative re- sponse strongly correlates with susceptibility and resistance to increased salinity (Acar et al. 2001, Tűrkan et al. 2005). In previous work we investigated the response of shoots of F1 hybrid DH10 and four dihaploid tobacco lines de- rived from that hybrid (207B, 238C, 239K, 244B) to 100 and 200 mM NaCl. F1 hybrid and dihaploid lines were pre- viously proved to be tolerant to potato virus Y (PVY) (Šmalcelj and Ćurković-Perica 2000). Salt stress caused growth retardation and induced proline and sodium accu- mulation in shoots of all dihaploids and hybrid DH10. In line 239K, salt induced higher activities of SOD, CAT and POD. Our results revealed that lines 238C and 244B were more susceptible than lines 207B and 239K to increased sa- linity, suggesting that tolerance to the virus is not associated with salinity tolerance (Marček et al. 2014). Although the roots are the fi rst to encounter excess salinity and are poten- tially the fi rst sites of damage or “line of defence” (Rewald et al. 2013), most studies of plant tolerance to high salinity MARČEK T., VIDAKOVIĆ-CIFREK Ž., TKALEC M., JEŽIĆ M., ĆURKOVIĆ-PERICA M. 50 ACTA BOT. CROAT. 76 (1), 2017 focus on traits in the aboveground tissues; data on the phe- notypical and physiological plasticity of root systems under salt stress are rare. Except having a role in the uptake of water and nutrients, roots also act as a sensory system, inte- grating changes in nutrient availability, water content and salinity in order to adjust root morphology for better exploi- tation of available resources (Gruber et al. 2013). There- fore, the purpose of this study was to expand our previous research by investigating the effect of NaCl on roots of di- haploid tobacco lines and hybrid DH10 in order to reveal if root responses to salinity may contribute to the salt toler- ance of investigated tobacco lines. Materials and methods Plant material and treatments Tobacco (Nicotiana tabacum L.) F1 hybrid DH10 (hy- brid of cv. Virginia D and line GV3) and four dihaploid lines (207B, 238C, 239K, 244B) obtained by diploidization of anther-derived haploids of the hybrid DH10 (Šmalcelj and Ćurković-Perica 2000) were tested for salinity toler- ance. Seeds, provided by the Duhanski Institut Zagreb (Cro- atia), were grown in pots containing a mixture of peat-sand (2:1). Ten weeks after planting, the seedlings were surface sterilised in 70% ethanol for 10–15 s and then for 15 min in 1% (w/v) sodium hypochlorite (NaOCl) supplemented with 0.05% (v/v) Tween. After extensive rinses in sterile distilled water, seedlings were transferred to solid Murashige and Skoog (MS) medium (Murashige and Skoog 1962) contain- ing 3% (w/v) sucrose and 0.8% agar (w/v) without growth regulators (pH 5.7). Plants were grown in a climate cham- ber at 24±2 °C under 16 h light/8 h dark photoperiod and artifi cial fl uorescent lamp (90 μmol s–1 m–2) and multiplied by subcultivation every 4 to 6 weeks using nodal segments. Four-week-old plants were exposed to salt stress by supplementing MS medium with 100 or 200 mM NaCl. MS medium without added NaCl was used as control. After 33 days of treatment, roots were gently separated from shoots and carefully washed to remove medium residues between root branches. Root tissue was stored at –80 °C until analy- ses. Due to severe growth retardation in plants exposed to the higher concentration of NaCl (200 mM), roots from several plant samples had to be pooled together. Line 244B did not develop a root system at any salt treatment hence this line was omitted from further analyses. Proline content Free proline content was determined according to Bates et al. (1973). 10–25 mg of fresh plant tissue was homoge- nized in 1.5 ml of 3% (w/v) sulphosalicylic acid and the re- sidue was removed by centrifugation at 15 000 g for 15 min. Supernatant, ninhydrin and glacial acetic acid were heated at 100 °C for 1 h, and the reaction was stopped in an ice bath. The absorbance of the free proline fraction with tolu- ene aspirated from the liquid phase was read spectrophoto- metrically at 520 nm. Proline concentration was determined using a calibration curve obtained with L-proline solutions ranging from 10 to 160 μM and expressed as micromols per gram of fresh weight [μmol g–1 FW]. Enzyme extraction Root tissue (50 mg) was homogenized in 1 ml ice cold 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ascorbic acid and polyvinylpolypyrrolidone (PVPP). Ho- mogenate was centrifuged at 25 000 g for 30 min at 4 °C. The content of soluble proteins in the supernatant was de- termined according to Bradford (1976) using bovine serum albumin as a standard. Obtained supernatants were used for assays of antioxidative enzyme activities. Assays of antioxidant enzyme activities POD (EC 1.11.1.7) activity was analysed by measuring the oxidation of guaiacol in the presence of hydrogen per- oxide at 470 nm according to Chance and Maehly (1955). CAT (EC 1.11.1.6) activity was estimated according to Aebi (1984) by monitoring the decline in absorbance as a consequence of hydrogen peroxide consumption at 240 nm. The activity of SOD (EC 1.15.1.1) was determined by mea- suring the inhibition of the photochemical reduction of ni- tro blue tetrazolium (NBT) at 560 nm as described by Beau- champ and Fridovich (1971). One unit of SOD activity was defi ned as the amount of enzyme that caused 50% inhibi- tion of NBT reduction. The enzyme activities were expressed as units (U) of enzyme activity per milligram of protein [μmol min–1 mg–1proteins]. Statistical analysis All results were expressed as means of three replicates with the corresponding standard errors, except for lines 238C and 239K at the highest salt concentration (200 mM NaCl) where only one replicate was obtained. Data were subjected to the analysis of variance (ANOVA) with a 3×4 factorial approach replicated three times with missing val- ues. Differences between means were compared at P≤0.05 of signifi cance using Fisher’s least signifi cant difference test. Principal component analysis (PCA) was performed to evaluate and discriminate the roots responses of different tobacco lines exposed to NaCl. The data set used for PCA comprised 5 variables (protein concentration, proline con- tent, activity of POD, CAT and SOD). Data were analysed with STATISTICA 10.0 (Stat Soft Inc., USA) software package. Results After 33 days of treatment all control plants of tested lines (207B, 239K and 238C) and hybrid DH10 as well as those treated with 100 mM NaCl developed roots. In all to- bacco lines roots of control plants were fi brous and thick, 3.5–5 cm long, with lateral branches, while plants exposed to 100 mM NaCl had shorter roots (1–2.5 cm). The most pronounced effect of salt stress was noticed at 200 mM NaCl where growth of roots was severely inhibited and roots were only 0.5–1 cm long (Fig. 1) or even not devel- oped, as in line 244B which was therefore omitted from further analysis. DIHAPLOID TOBACCO ROOTS UNDER SALINITY ACTA BOT. CROAT. 76 (1), 2017 51 Considering total protein concentration, results revealed that in line 207B protein concentration was higher in roots of plants exposed to 200 mM NaCl, especially compared to untreated (control) plants (30% above the control value in 207B). Among different tobacco lines control plants of line 239K had the lowest protein content (Fig. 2a). Salt stress caused remarkable proline accumulation in the roots of all tobacco lines treated with 200 mM NaCl in comparison to their respective controls (from 2-fold above the control level in hybrid DH10 to above 8-fold in 207B). The proline accumulation was lowest in roots of 239K line. Treatment with 100 mM NaCl caused signifi cant proline accumulation in all dihaploid lines (5-fold in 207B and 4-fold in 239K and 238C) but not in hybrid DH10 (Fig. 2b). When the proline content in the control plants of all experi- mental groups was compared, a signifi cantly higher level was measured in hybrid DH10. Salt stress mostly did not cause any considerable differ- ences in activities of antioxidative enzymes in the roots of all the tobacco lines tested. However, statistical analysis re- vealed a higher CAT activity in roots of line 239K exposed to 100 mM NaCl than in the roots of line 207B exposed to the same salt concentration (Fig. 3a). In a comparison of POD activity in control plants of different tobacco lines, line 239K had signifi cantly higher POD activity than line Fig. 1. Roots of line 207B after 33 days of treatment with 0, 100 and 200 mM NaCl. bc bc ab d bcd bc bcd cdcd a ab bcd 0 0.04 0.08 0.12 DH10 207B 238C 239K P ro te in c o n te n t (m g g -1 F W ) Tobacco lines 0 mM 100 mM 200 mM a c d d d c bc c c a a ab bc 0 2 4 6 DH10 207B 238C 239K P ro li n e c o n te n t (m m o l g -1 F W ) Tobacco lines 0 mM 100 mM 200 mM b abc c bc abc abc bc abc a abc bc abc ab 0 0.03 0.06 0.09 0.12 0.15 DH10 207B 238C 239K C A T a c ti v it y (U m g -1 p ro te in s ) Tobacco lines 0 mM 100 mM 200 mM bc c bc ab bc abc bc a ab abc bc ab 0 50 100 150 DH10 207B 238C 239K P O D a c ti v it y (U m g -1 p ro te in s ) Tobacco lines 0 mM 100 mM 200 mM b a bcd bc bc cd ab bcd bcd d ab bc bcd 0 1000 2000 3000 4000 DH10 207B 238C 239K S O D a c ti v it y (U m g -1 p ro te in s ) Tobacco lines 0 mM 100 mM 200 mM a c Fig. 2. Protein concentration (a) and proline content (b) in roots of tobacco F1 hybrid DH10 and dihaploid lines 207B, 238C and 239K exposed to 0, 100 and 200 mM NaCl for 33 days. Values are means±SE (n=3). Different letters indicate signifi cantly different values (P<0.05) between means. Fig. 3. Activity of catalase (a), peroxidase (b) and superoxide dismutase (c) in roots of tobacco F1 hybrid DH10 and dihaploid lines 207B, 238C and 239K exposed to 0, 100 and 200 mM NaCl for 33 days. Values are means±SE (n=3). Different letters indicate signifi cantly dif- ferent values (P<0.05) between means. MARČEK T., VIDAKOVIĆ-CIFREK Ž., TKALEC M., JEŽIĆ M., ĆURKOVIĆ-PERICA M. 52 ACTA BOT. CROAT. 76 (1), 2017 207B and increased enzyme activity at 100 mM NaCl in comparison to 238C and DH10 (Fig. 3b). The activity of SOD signifi cantly decreased in hybrid DH10 in NaCl sup- plemented medium as compared to its respective control. Among different tobacco lines the highest SOD activity was observed in control plants of hybrid DH10 while salt- treated roots of line 207B had signifi cantly higher SOD ac- tivity than hybrid DH10 under the same treatment (Fig. 3c). PCA yielded two signifi cant components explaining 70% of data variance. The fi rst component (PC1) was large- ly determined by POD and CAT activity as well as protein content and SOD activity and the second component (PC2) by protein and proline contents (Fig. 4a). The correspond- ing scores plot (Fig. 4b) showed that unstressed plants of lines 207B, 238C and hybrid DH10 grouped together in cluster I while unstressed plants of line 239K separated due to lower protein content. Due to higher CAT activity and higher SOD activity, plants of lines 239K and 207B sepa- rated from other lines treated with 100 mM NaCl (DH10 and 238C, that form cluster II). Plants of all lines exposed to 200 mM NaCl were differentiated from others due to their high proline content but they further separated into two clusters, cluster III with plants from lines 207B and 238C characterized with higher protein content, and cluster IV with plants from lines 239K and hybrid DH10 character- ized with increased POD activity. Discussion Root growth reduction is one of the most common ef- fects of increased salinity (Ceccoli et al. 2011). In our ex- periment, salt stress caused prominent growth inhibition of the roots of all tobacco lines and hybrid DH10. It even completely inhibited root development, especially in line 244B which was therefore omitted from further analysis. Low water potential of nutrient medium, osmotic and toxic effect of NaCl and disturbed uptake of essential minerals can all account for the observed effect (Haq et al. 2009). Previously obtained results revealed that in tobacco shoots salt stress also reduced growth but not very severely, espe- cially in lines 207B and 239K (Marček et al. 2014). This might indicate that shoots at least partially avoided toxic ef- fect of NaCl, retaining it by apoplastic barriers in root or by disposal into parenchyma cells of the root cortex (Munns and Tester 2008). The same effect was observed in wheat (Triticum sp.) (Datta et al. 2009) and Persian clover (Trifo- lium resupinatum L.) (Ates and Tekeli 2007). With the application of higher NaCl concentration (200 mM) a signifi cant increase in root protein content in line 207B was observed. This effect was determined earlier in roots of wheat cultivars (Shaddad et al. 2013). Increased protein content can be an indicator of salinity tolerance if it is caused by synthesis of stress proteins or proteins contrib- uting to antioxidative defence (Mohammadkhani and Hei- dari 2008, Manaa et al. 2011). However, a degradation pro- cess caused by growth retardation can also increase protein level (Parvaiz and Satyavati 2008, Goudarzi and Pakniyat 2009, Keshtehgar et al. 2013, Gupta and Huang 2014). Con trary to the results obtained in this study, protein con- tent in shoots, investigated previously, remained unchanged within lines 207B and 238C exposed to salinity (Marček et al. 2014). It has been reported that salinity can cause differ- ent changes in the protein content in various parts of the plant (Elsamad and Shaddad 1997). In all dihaploid lines and hybrid DH10, salt stress caused signifi cant increase in proline production in roots, implying that proline contributes to osmotic adjustment. Furthermore, remarkable proline production in 207B, 238C and 239K at both salt treatments suggests that in these genotypes proline could be connected with salt resistance (Ahmed et al. 2008). Interestingly, despite higher basal proline content in control plants, roots of hybrid DH10 did not show any more promi- nent tolerance to 100 mM NaCl. According to Tammam (2003), sodium accumulation in root causes increased pro- line production thus protecting root cells against dehydra- tion. Increased proline content was noticed in the root of a CAT POD -1.0 -0.5 0.0 0.5 1.0 Factor 1 : 45.34% -1.0 -0.5 0.0 0.5 1.0 F a c to r 2 : 2 6 .1 5 % SOD PRO PROTEINS DH10-200 207-0 207-100 207-200 239-0 239-100 239-200 238-0 238-200 -4 -3 -2 -1 0 1 2 3 4 Factor 1: 45.34% -4 -3 -2 -1 0 1 2 3 4 F a c to r 2 : 2 6 .1 5 % DH10-0 DH10-100 238-100 a b Fig. 4. Principal component analysis of combined tobacco lines data sets. Loadings (a) and scores (b) of the fi rst two factors. Protein con- centration (PROTEINS), proline content (PRO) and activity of SOD, POD and CAT represent variables. Numbers 0, 100 and 200 follow- ing names of tobacco lines (DH10, 207B, 238C and 239K) represent concentrations of NaCl – 0, 100 and 200 mM, respectively. DIHAPLOID TOBACCO ROOTS UNDER SALINITY ACTA BOT. CROAT. 76 (1), 2017 53 tobacco cultivar (N. tabacum L. Wisconsin) exposed to 300 mM NaCl (Razavizadeh et al. 2009). When proline produc- tion in shoots of tobacco dihaploid lines and hybrid DH10 exposed to NaCl was considered, signifi cant proline pro- duction in plants at 200 mM NaCl was recorded (Marček et al. 2014). Much higher proline content in shoots than in the roots of these lines suggests that tobacco, like most glyco- phytes, could have a poor ability to exclude salt, so Na+ is concentrated in the cell vacuoles of transpiring leaves. In such conditions, proline should accumulate in the cytoplasm and organelles to balance the osmotic pressure of the ions in the vacuole (Munns 2002). Similar results have already been reported for barley (Hordeum vulgare L.) (Ueda et al. 2007) and maize (Zea mays L.) (Versules and Sharp 1999). CAT, POD and SOD are considered major scavenging enzymes involved in removal of ROS produced under vari- ous stress conditions, including salinity (Parvanova et al. 2004). Among investigated tobacco lines, roots of line 239K treated with 100 mM NaCl exhibited higher CAT ac- tivity than line 207B and increased POD activity compared to line 238C and hybrid DH10; those increased activities of both enzymes in line 239K might ensure better scavenging of ROS under 100 mM NaCl. On the other hand, roots of lines 207B and 238C treated with 200 mM NaCl showed remarkable induction of SOD activity compared to hybrid DH10 which implies that in the roots of these tobacco lines SOD might be more involved in defence response. In our previous study (Marček et al. 2014), shoots of line 239K also had pronounced SOD, CAT and POD activity under salt levels so it seems that line 239K generally had more ef- fective antioxidative defence mechanism and is thus less susceptible to salt stress than other dihaploids and hybrid DH10. Untreated roots of hybrid DH10 had higher SOD ac- tivity than other tobacco lines indicating that its expression is constitutive under physiological level (Rangani et al. 2016). Moreover, roots of hybrid DH10 exposed to salt treatments showed the same inhibition of SOD activity as shoots (Marček et al. 2014) which could be connected with the lack of zinc (Tavallali et al. 2010), an enzyme cofactor, since salinity disturbs plant nutrient uptake and ion distri- bution (Fernández-García et al. 2004, Bameri et al. 2012). The decline in SOD activity in the salt-exposed roots was also demonstrated in maize salt-sensitive cultivar (SK) (Keyster et al. 2013). Observed differences in enzyme ac- tivity between lines included in this experiment might be the result of their genetic properties. Compared to shoots (Marček et al. 2014), the roots of all dihaploids and hybrid DH10 exhibited high CAT, POD and SOD activity as a re- sponse to increased salinity suggesting that roots of tobacco dihaploid lines have a stronger antioxidative defence. Simi- larly, more prominent antioxidative response of root than of shoot has been reported for the lentil (Lens culinaris M.) (Bandeoğlu et al. 2004). In conclusion, NaCl stress caused root growth inhibition and remarkable proline accumulation in all tobacco dihap- loid lines 207B, 238C, 239K and hybrid DH10 but certain differences among antioxidative enzyme activities as re- sponse to salinity were noticed. 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