Interaction effects of fructan and Salicylic acid on chickpea in both biochemical and traditional agronomic indicators

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1 Legume Research, 41 (3) 2018 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Interaction effects of fructan and Salicylic acid on chickpea in both biochemical and traditional agronomic indicators N. Candan Yücel* Department of Chemistry, Dokuz Eylül University, Faculty of Science, Buca, 35390, Izmir, Turkey. Received: Accepted: DOI: /LR-377 ABSTRACT Starch and fructans are accumulated for carbohydrate storage in legumes, while fructans accumulated large amounts than starch. The uses of this natural and biodegradable material counteract stress as cheaper and safer alternatives. Therefore, fructan (F, 0.5 %) and salicylic acid (SA, 0.5 mm) priming were used as exogenous growth enhancers to stimulate chickpea (Cicer arietinum L.) seed vigor against salt stress. The main aim of this study was to address whether priming chickpea with F, SA and F+SA could bring about supplementary benefits particularly against salt stress. Exogenous application of F- or SA-alone improved chickpea development in the presence of salt stress. Nevertheless, the best results in terms of growth, seed vigor and total phenolic flavonoids, chlorophyll carotenoids contents, phenylalanine ammonia-lyase (PAL), ascorbic acid oxidase (AAO) activities and lipid peroxidation level (LPO) were determined in the combined F+SA treatment against salt stress. Key words: Chickpea, Fish flour, Fructan, Phenolic contents, Salinity stress. INTRODUCTION Global warming and the widespread application of inorganic fertilizers have importantly increased the soil salinity. Nowadays, salt stress is one of the major abiotic stresses affecting germination, legume growth and productivity. At the same time, soil salinty is the major risk factor during chickpea development. Legumes require extra adjustability mechanisms for adaptation to stressful environment (Kumar et al., 2017). One adaptation mechanism includes accumulation of fructans and phenolic compounds. Both these compounds play an important role stress responses. Studies show that when plant increases their fructan contents, they increase their stress tolerance (Ende, 2013). For example, fructan accumulation in plants occurs under low temperature for increasing freezing tolerance. Fructan especially has high water soluble capacity for resistance against membrane damage. Salerno et al. (2004) showed that cyanobacteria Anabaena sp synthesize osmo-protective molecules such as sucrose or oligosaccharides against salt stress. At the same time, in many higher plant species, fructans play an important and physiological role to adjust adverse environmental conditions. Salicylic acid is considered as an important plant growth regulator and phenolic compound, which is increased under especially stress conditions by higher plants. In this study, fructans and salicylic acid will be applied to chickpea seeds prior to stress via priming. Thus, the increases of phenolic compounds and fructan polymers in chickpea are intended *Corresponding author s nilgun.candan@deu.edu.tr to provide stress resistance. In this study, fructan and salicylic acid priming use as a process that prepare chickpea seed for a stronger defense without actually beginning those defense responses prior to stress. The close coordinated connection of hormonal pathways and sugar in plants leads effective immune responses (Shalitin and Wolf, 2000). Plants have ability to modulate their sugar pools as a source of carbon and also source of energy or to play roles as signal molecules (Gomez-Ariza et al., 2007). It probably suggested that the combination of fructan with plant stress tolerance hormone (salicylic acid) could bring about supplementary benefits. The aim was to determine whether F, SA used singly or in combination-induced increases in the resistance of chickpea and antioxidant defence enzyme of peroxidase (POD), phenylalanine ammonia-lyase (PAL), ascorbic acid oxidase (AAO) to salt stress. However, the role of AAO enzyme in plants is not yet fully understood. AAO over expression in higher plants affects the expression of defence and signalling genes (Fotopoulos et al., 2008). This is the first report AAO enzyme activity in chickpea against salt stress in relation to F and SA priming. It is known that fructans are reserve carbohydrates and also protective compounds against drought. There are very little studies related to fructans possible protective roles against salt stress. However, fructan is soluble in water and then fructan may play an osmotic adjustment role under salt

2 400 LEGUME RESEARCH An International Journal stress by varying the polymerization level. Is a fructan a true priming molecule? It remains also unclear. The objectives of this study were to determine exogenous fructan and salicylic acid, alone or in combination to chickpea seed may result in an increase in salt tolerance via increasing seed vigor or/and phenolic biosynthesis (individual phenolic compounds). Additionally, the analysis of malondialdehyde was used as indicator of membrane damage. MATERIALS AND METHODS Ten g of chickpea cultivar (Cicer arietinum L.) seeds were soaked in each treatment fructan (fructooligosaccharides from chicory), salicylic acid (SA) and SA+F combined pretreatments with shaking at 150 rpm overnight. Firstly, salicylic acid was dissolved in 100 µl dimethyl sulfoxide and then 0.5 mm SA (ph 6.5) was dissolved with distilled water plus 0.02 % Tween-20. Also, 0.5% (w/v) fructan was prepared in 0.03 (v/v) Tween-80. To examine the effects of salinity on germination percentage replicates of 15 seeds were sown on noncellulosic paper in 500 ml containers. The seeds were constantly based-watered. The containers were sealed to prevent evaporation and maintained at 20 o C in a growth cabinet illuminated (25 µmol m -2 s -1, nm) on a 14-h day/10-h night regime. Chickpea seeds were admitted germinated with the emergence of the radicle. The salinity concentration is 150 mm sodium chloride. Seeds were considered to be germinated when the radicle emerged through the seed coat and reached more than 2 mm in length. The phenylalanine ammonia-lyase (EC ) activity was measured Hodgins method (Hodgins, 1971). Assay concentrations contained 150 mm Tris-base ph 8.5 buffers, 3 mm L-phenylalanine and enzyme. The guaiacoldependent peroxides (EC ) activity assay was composed of 25 mm phosphate buffer (ph 7.0), 0.05% guaiacol, 10 mm H 2 O 2, and enzyme. Activity was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (E = 26.6 mm 1 cm 1 ) (Nakano and Asada, 1981). Estimation of ascorbic acid oxidase (AAO; EC ) activity was determined by the method of Oberbacher and Vine (1963) the rate of oxygen consumption during ascorbic acid oxidation is proportional to the amount of enzyme present. One unit of enzyme activity was defined as the amount of enzyme that oxidizes 1.0 µmol of L-ascorbic acid per min. Total phenolic content in chickpea samples was taken on first, 6 th, 13 th day of germination and was determined according to McCue method and gallic acid ( µg/ ml) was used as a phenolic standard curve (McCue et al., 2000). The flavonoid content in the crude extract was determined by Du and co-workers method with some modifications (Du et al., 2009). LPO was determined based on thiobarbituric acid (TBA) reactivity (Buege and Aust, 1978). The protein content was determined by Bradford assay (bovine serum albumin as a standard) (Bradford, 1976). RESULTS AND DISCUSION Percentage of seed germination, shoot-root length and also fresh weight were studied (Table 1-2). Fresh weight (FW) and germination percentage of Cicer arietinum seedlings grown from all treatments increased gradually from day 1 to 13 under both non-saline and salt stress. Salt stress decreased the fresh weight and germination rate from 130 ± 3 mg to 65 ± 6 mg and from 100 % to 70 % on the 13 th day (p < 0.01), respectively. However, the levels were able to be restored with F-alone, SA-alone and F+SA-combined pretreatment under non-saline and salinity conditions. Applied pre-treatment in the order of increasing fresh weight and germination percentage of the chickpea seedlings were SA-alone < F-alone < F+SA-combined pretreatment under both non-saline and salinity conditions. It was found that the applicationof salicylic acid, fructan and fructan+salicylic acid led to continues increase in the root and shoot length during the experimental period. Application of salt stress decreased the root and shoot length from 6.0 ± 2.0 cm to 1.4 ± 0.6 cm and from 8.1 ± 2.0 cm to 3.7 ± 0.6 cm on the 13 th day, respectively. Applied pre-treatment in the order of increasing root and shoot lengths of the chickpea seedlings were SA-alone < F-alone < F+SAcombined pre-treatment under salt stress. Among cereals, in chickpea fructan levels are very low ( % DM). From this reason, the aim of this study is to show an economically important of chickpea that is able to accumulate fructans against salt stress by seed priming. It is known that salt and drought stresses are major problems during cereal production (Joudi, 2012). Fructose has an important role against adverse environmental conditions in many higher plant species and it is well documented that its concentrations correlate with drought and freezing stress tolerance (Pontis, 1989; Hendry, 1993). But, there are not enough studies with the fructan and its association with other stress conditions. Indeed, this study shows that fructans may play a multi-functional role when applied with salicylic acid. In this study, fructan, salicylic acid and fructan+salicylic acid-combined pre-treatment would be applied to chickpea seeds prior to the salt stress with priming. The germination percentages and fresh weight were decreased under salt stress. As can be seen in Fig.1, the total amount of phenolic and flavonoid contents of chickpea seedlings organs (seed, shoot and root) increased significantly in the all treatment (p < 0.05). However, the contents increased more under salt stress when compared non-saline conditions.

3 Volume 41 Issue 3 (June 2018) 401 Table 1: Growth parameters: Fresh weight and % Germination of chickpea for different treatments; control, F (0.5%), SA (0.5 mm) and F+SA combined pre-treatment under non-saline and salt stress 100 mm NaCl Growth Plant growth (in days) parameters Salinity Treatments % (% +mm) 0 Control 40 ± ± 2 130± % F 50 ± ± 2 ä 183 ± 8 å 0.5 mm SA 50 ± ± 2 ä 171 ± 9 å 0.5% mm F+SA 60 ± ± 2 ä 212 ± 5 å Fresh weight(mg)% 100 Control 30 ± 2 60 ± ± 6 0.5% F 50 ± ± 08 å 149 ± 5 å 0.5 mm SA 50 ± ± 09 å 130 ± 4 å 0.5%+ 0.5 mm F+SA 50 ± ± 11 ä 189 ± 11 å 0 Control % F mm SA %+ 0.5 mm F+SA Germination 100 Control % F mm SA %+ 0.5 mm F+SA Data are mean±s.d the mean ± SD of three independent experiments. ä p<0.05 (probably significant) å p<0.01 (definitely significant) Table 2: Growth parameters: Shoot and root length of chickpea for different treatments; control, control, F (0.5%), SA (0.5 mm) and F+SA combined pre-treatment under non-saline and salt stress 100 mm NaCl Growth Plant growth (in days) parameters Salinity Treatments % (mm +g/10ml) Control ± ± % F ± ± mm SA ± 0.1 ä 5.9 ± 0.2 ä 0.5%+ 0.5 mm F+SA ± 0.2 ä 7.6 ± 0.1 ä Root length(cm) Control ± ± % F ± 0.2 å 5.9 ± 0.5 å 0.5 mm SA 5.2 ± 0.2 å 4.7 ± 0.2 å 0.5%+ 0.5 mm F+SA ± 0.1 å 7.7 ± 0.4 å Control ± ± % F ± 0.7 å 8.7 ± mm SA ± 0.1 å 8.4 ± 0.2 å 0.5%+ 0.5 mm F+SA ± 0.2 å 9.4 ± 0.1 å Shoot length(cm) 100 Control ± ± % F ± 0.2 å 6.9 ± 0.5 å 0.5 mm SA ± 0.1 ä 6.1 ± 0.4 å 0.5%+ 0.5 mm F+SA ± ± 0.1 å Data are mean±s.d the mean ± SD of three independent experiments. ä p<0.05 (probably significant) å p<0.01 (definitely significant Applied pre-treatment in the order of increasing total phenolic and flavonoid contents of the chickpea seedlings were F-alone < SA-alone < F+SA-combined pre-treatment under salt stress. The maximum increase of the total flavonoid and phenolic contents were determined as 1.9-fold, 3.1-fold and 2.4.-fold and 1.6-fold, 1.4-fold and 1.2-fold increases of F+SA combined pre-treatment under salt stress as compared

4 402 LEGUME RESEARCH An International Journal D a ta a r e m e a n ± S.D th e m e a n ± S D o f th r e e - f o u r in d e p e n d e n t e x p e r im e n ts. δ p < ( p r o b a b ly s i g n if ic a n t) ε p < ( d e f in ite l y s ig n if ic a n t) Fig 1: Total soluble phenolic and flavonoid contents in seedlings of chickpea for different treatments; control, F (0.5%), SA (0.5 mm) and F+SA combined pre-treatment under non-saline and salt stress 100 mm NaCl. to non-saline control for shoot, root and seed, respectively (p < 0.01). Fructan and salicylic acid are as signal molecules, have been showed positive effects of plant adaptation against salt stress especially under F+SA-combined pre-treatment. Also, the order of exogenous stimulator were with respect to the rates of increase in the amounts of phenolic and flavonoid compounds as F SA<F+SA, respectively. Table 3 shows PAL, AAO and POD enzyme activities and also LPO levels of chickpea shoots under both non-saline and salt stress condition on the 13 th day. Salt stress markedly increased PAL, AAO and POD enzyme activities (p < 0.01). It is considered that these enzymes activities increase as a result of exposure to salt stress. F+SA-combined pretreatment appeared to be the most important way in minimizing the negative effects of salt stress on PAL, AAO and POD enzyme activities. F+SA-combined pre-treatment resulted in approximately 5- and 9-fold increases, respectively, in PAL and POD under salt stress. The fructan and salicylic acid applied alone increased PAL and POD activity, but the increases were not significantly from each other. Arun et al. also (2017) showed that chemical priming increased POD activity. PAL is a key enzyme of the phenolic compounds synthesis, phytoalexins and lignins. Other studies also proved that drought tolerant wheat produce and store higher fructan Table 3: Parameters: LPO content, PAL, Gua-dep POD and AAO activities; control (non-saline), control (saline), F (0.5%), SA (0.5 mm) and F+SA combined pre-treatment under non-saline and salt stress 100 mm NaCl on the 13 th day Parameter No salinity Salinity stres, 100 mm NaCl Control Non-treatment 0.5% F 0.5 mm SA F +SA PAL(U.mg -1 protein) 22 ±1 48± 3 å 92 ± 18 å 100 ± 12 å 125 ± 14 å Gua-dep POD (U.mg -1 protein) 0.5 ± ± 0.3 å 2.4 ± 0.3 å 2.9 ± 0.2 å 4.5 ± 0.4 å LPO (nmol MDA.g -1 ) 2.5 ± å ± 0.6 å 5.0 ± 1.0 ä 4.6 ± 1 ä 2.6 ± 1.0 å AAO (U.mg -1 protein) 0.4 ± ± 0.1 å 1.6 ± 0.1 å 1.7 ± 0.1 å 2.2 ± 0.1 ä Data are the mean ± SD of three independent experiments. ä p<0.05 (probably significant) å p<0.01 (definitely significant) content than drought sensitive wheat during stress (Kerepesi et al., 1998). Pourcel et al showed that flavonoids play role as signal molecules to adjust responses induced stress conditions. In this study, PAL activities increased twofold under salt stress, and applied pre-treatment in order of increasing PAL activities of the chickpea seedlings were F- alone SA-alone < F+SA-combined treatment (maximum 5-fold) under salt stress. Fructans are associated with stress responses in plants. After fructans, salicylic acid and F+SA-combined priming, antioxidative enzymes activities were improved and the content of malondialdehyde was decreased. In this study, antioxidant antioxidant enzymes POD, AAO and gave similar response, such as the results of PAL. Further, Zhang et al. (2011) showed that a pre-treatment with fructan significantly increased lignin content and the enzyme activities (POD, SOD, glucanase).taken together, these observations determined that different profiles of the antioxidant defences causing to a reducing of LPO level could be because of higher stress tolerance. Fructan (fructose polymers, short-term storage carbohydrate) can induce defensive mechanism against a number of plant diseases by affecting membrane. There may be a direct interaction between fructan and membranes (Ende, 2013). Fructans could be able to eliminate oxidative stresses, because introduction of plant fructan synthesis genes results in stress tolerance.

5 In this study, the fructan pre-treatment of chickpea in priming processes may be involved by making the plant s defence mechanisms to maximum and counteracting salt stress. Indeed, application of F+SA-combined treatment gave better results than application of F- or SA- alone. This result is proved this information from lipid peroxidation levels of bio-membranes. Salt stress is resulted the harmful free radicals production on the membranes and increased lipid peroxidation levels. The lipid peroxidation level could determine of plant tolerance to stresses such as salt stress. In this study, the F+SA-combined treatment had higher decreasing of LPO levels than SA > F, respectively. Like this study, exogenous SA application decreased LPO in sweet basil under salt stress (Song and Roe, 2008). Generally, the combination F+SA treatment is one the most effective treatment in counteracting the adverse effects of salt stress on AAO enzyme activities (6-fold increase under salt stress). Identification of AAO as a possible player in C. linum adaptation to increased salinity was reported recently and they found that AAO activation is potentially both a first aid response with further implication is signalling, and a way to directly counteract salt stress (Caputo et al., 2010). In our study, it is observed that AAO enzyme counteract the negative effects of salt stress easily when the salt-exposed plants treated with both F and SA. Further research is needed to found on the better understand of this point. The sum up, the enhanced antioxidant enzyme activities of chickpea was approximately on correlated with Volume 41 Issue 3 (June 2018) 403 the enhanced accumulation of various antioxidant metabolites. Application of SA and/or F significantly increased total phenolic contents. Priming with F+SAcombination appeared to be the most important treatment in inhibiting the adverse effects of salt stress on total phenolic and flavonoid contents. It is reported that phenolic compounds take an important role in development of plant metabolism. Seed priming with the combinations of F and SA increased the average plant height and fresh weight. The treatment with combinations of F and SA had affect on chickpea vigour response when compared to other treatments in this study. SA could be involved in the phenylpropanoid pathway when combined with F and SA may have had effect like accumulation. These results showed that the combinations of F and SA treatment increased the production of phenolics, therefore give better seed vigour which was translated into plant height and fresh weight. It is known that phenolics are important keys in plant development. But F+SA-combined treatment give better results (2.2-fold for total phenolic content and 5-fold for PAL activities) than SA+Ca-combined treatment. CONCLUSION Taken together, these results show that fructans and salicylic acid could strongly influence chickpea development and stress responses against salt stress. Further research is necessary to show how these specific signalling pathways exactly along with the recently emerging sweet priming concept under abiotic stress responses. REFERENCES Arun, M.N., Bhanuprakash, K., Hebbar, S.S. and Senthivel, T. (2017). Effect of seed priming on biochemical parameters and seed germination in cowpea (Vigna unguiculata L.) walp. Legume Research 40: Bradford, M.M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: Buege, J. A. and Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology 52: Caputo, E., Ceglie, V., Lippolis, M., Rocca, N. and De Tullio, M. C. (2010). 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