Microsoft Word - 3_Moravčíková et al. Nova Biotechnologica et Chimica 15-2 (2016) 122 DOI 10.1515/nbec-2016-0013 © University of SS. Cyril and Methodius in Trnava BETA-1,3-GLUCANASE ACTIVITIES IN WHEAT AND RELATIVE SPECIES JANA MORAVČÍKOVÁ1, DENISA MARGETÍNYOVÁ2, ZDENKA GÁLOVÁ2, IWONA ŽUR3, ZUZANA GREGOROVÁ1, MÁRIA ZIMOVÁ1,4, EVA BOSZORÁDOVÁ1, ILDIKÓ MATUŠÍKOVÁ5 1 Institute of Plant Genetics and Biotechnology Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, Nitra, SK-950 07, Slovak Republic (jana.moravcikova@savba.sk) 2 Slovak University of Agriculture, Faculty of Biotechnology and Food Science, Tr. A. Hlinku 2, Nitra, SK- 949 76, Slovak Republic 3 Polish Academy of Sciences the Franciszek Gorski Institute of Plant Physiology, Niezapominajek 21, Kraków, PL-30-239, Poland 4 Department of Botany and Genetics, Faculty of Natural Sciences, The Constantine Philosopher University, Nábrežie mládeže 91, Nitra, SK-949 74, Slovak Republic 5 Department of Ecochemistry and Radioecology, University of Cyril and Methodius in Trnava, Nám. J. Herdu 2, Trnava, SK-917 01, Slovak Republic Abstract: The (1,3)-β-D-glucan also referred to as callose is a main component of cell walls of higher plants. Many physiological processes are associated with the changes in callose deposition. Callose is synthesised by the callose synthase complex while its degradation is regulated by the hydrolytic enzymes β-1,3-glucanases. The latter one specifically degrade (1,3)-β-D-glucans. This work is aimed to study β-1,3-glucanase activities in the leaves of plants at two leaf stage in two diploids (Agilops tauschii, Triticum monococcum L.), four tetraploids (Ae. cylindrica, Ae. triuncialis, T. araraticum, T. dicoccum) and two hexaploids (T. aestivum L, T. spelta L.). The leaves were subjected to qualitative and quantitative β-1,3-glucanase activity assays. Our results showed that the total β-1,3-glucanase activities were variable and genotype dependent. No significant correlation between β-1,3-glucanase activities and ploidy level was observed. The gel activity assays revealed a single fraction of ~52 kDa Glu1 that was found in all genotypes. The Glu1 fraction corresponds to a single or two acidic Glu isoforms in dependence on genotype. However, none of the acidic Glu fractions can be assigned as a specific for di-, tetra- or hexaploid genotypes. A single basic GluF isoform was detected and found as present in all genotypes. Key words: (1,3)-β-D-glucans, β-1,3-glucanases, callose, laminarin, ploidy, wheat 1. Introduction β-D-glucans are non-starch polysaccharides that are main component of the cell walls of most plants, fungi or microorganisms. Certain β-D-glucans of different origin were studied mainly for their health-promoting effect. They were demonstrated to have hypocholesterolemic and anticoagulant activities; and chemoprotective effect as well. They can also be a potential source of long chain probiotics. Not surprisingly that organisms rich of β-glucans are of economic interest. The biological activities and application potential of β-D-glucans are mainly determined by their chemical structure. β-D-glucans consist of D-glucose monomers Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC 123 Moravčíková, J. et al. linked by (1,3)-β, (1,4)-β or (1,6)-β glycosidic bonds. In higher plants, β-D-glucans are synthesized as linear (1,3)-β-D-glucans, (1,4)-β-D-glucans and mixed (1,3)(1,4)-β-D- glucans. Cellulose or (1,4)-β-D-glucan is the major component of plant cell walls (MINIC et al., 2008) while the mixed (1,3)(1,4)-β-D-glucan is present mainly in the cell walls of starchy endosperm (HAVRLENTOVA and KRAIC, 2006). The (1,3)-β-D-glucans are commonly referred to as callose. During plant growth development it can be found in the cell plate during cell division. Callose deposition is important during pollen development, microsporogenesis or seed germination (PIRSELOVA and MATUSIKOVA, 2013). Besides, callose is synthesized between the cell wall and the plasma membrane after exposure of plants to various (a)biotic stresses (ZAVALIEV et al., 2011). Callose degradation and thus various plant physiological processes are regulated by the β-glucan endohydrolases namely β-1,3-glucanases (EC 3.2.1.39) (PIRSELOVA and MATUSIKOVA, 2013). The β-1,3-glucanases (GH 17) catalyse the hydrolysis of (1,3)-β-D-glucosidic linkages in (1,3)-β-D-glucans but not in (1,3)(1,4)-β-D-glucans (HØJ and FINCHER, 1995). The β-1,3-glucanases have widely been studied for their potential to inhibit fungal pathogen (MORAVCIKOVA et al., 2004), while recently their importance in plant defence against abiotic stresses has also been proven e.g. low temperature (ROMERO et al., 2008), drought (GREGOROVA et al., 2015) or heavy metals (PIRSELOVA et al., 2011). Plant β-1,3-glucanases are referred to as “Pathogenesis-related proteins” (PR2). They are grouped into the classes I-IV. The class I are vacuolar basic proteins that are accumulated in mature leaves and roots upon pathogen infection. The classes II and III are acidic extracellular proteins. The class IV is similar to the class II; however, β-1,3-glucanases of the class IV are not inducible upon pathogen attack (LEUBNER- METZGNER, 2003; MINIC, 2008). Wheat is a plant with complicated genetic information. Due to the evolutionary hybridisation, domestication and/or selection steps; the wheat genome was evolved to diploid, tetraploid and hexaploid genomes. During polyploidization, many duplicated genes were retained and the redundancy conferred by duplicated genes may lead to their novel or divergent functions (CHEN et al., 2007). In this work, we aimed to study the activities of β-1,3-glucanases in two wheat and six crop relatives species at early growth stages. We studied the plants of two diploids (Aegilops tauschii, Triticum monococcum L.), four tetraploids (Ae. cylindrica, Ae. triuncialis, T. araraticum, T. dicoccum) and two hexaploids (T. aestivum L, T. spelta L.) They were grown to two leaf stage and the leaves were subjected to qualitative and quantitative β-1,3-glucanase activity assays. 2. Material and Methods 2.1. Plant material and growth conditions Seeds of wheat and crop relative species (Table 1) were obtained from the Gene Bank of the Slovak Republic (National Agricultural and Food Centre, Pieštany). The seeds were germinated on the watered sterile filter paper in dark at room Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC Nova Biotechnologica et Chimica 15-2 (2016) 124 temperature for 3 days. Then, germinated seeds were transferred to the pots with the commercial substrate BORA and cultivated at 22 oC and 16 h/8 h light/dark photoperiod under 50 µE/m2.s1 light intensity for 3 weeks. The leaves of the plants (10 plants/genotype) at two leaf stage (Zadoks stage 12) were collected and used for further analyses. Table 1. Wheat and crop relatives species used in experiments. Gene Banka Ploidy Genome Triticum monococcum L. AZESVK2009-84 diploid AA m Aegilops tauschii ARMEN06-40 diploid DD Aegilops cylindrical ARMEN06-02 tetraploid CCDD Aegilops triuncialis ARMEN06-06 tetraploid UUCC Triticum araraticum AZESVK2009-47 tetraploid GGAA u Triticum dicoccum AZESVK2009-78 tetraploid BBAA u Triticum aestivum, L. Astella hexaploid BBAA u DD Triticum spelta, L. Brun 5/9 hexaploid BBAA u DD a Accession number in the Gene bank of the Slovak Republic 2.2. Protein extraction Crude protein extracts were isolated from the leaves using an extraction buffer that contained 0.1 mol/dm3 sodium acetate (pH 5.0) and 0.02 % (v/v) β-mercaptoethanol according to the protocol described previously (ŽUR et al., 2013). Protein concentration was determined spectrophotometrically. 2.3. Detection of β-l,3-glucanase activities in the gel Protein extracts (30 µg) were separated on 12.5 % (w/v) SDS-containing polyacrylamide slab gels (LAEMMLI, 1970) with 2.5 % (w/v) laminarin from Laminaria digitata (Sigma L-9634), as an enzyme substrate. The gels were run at 8 oC at a constant voltage of 120 V for 2 h. After electrophoresis, proteins were re-naturated by shaking the gel in 50 mmol/dm3 sodium acetate buffer (pH 5.0), 1 % (v/v) Triton X-100 for 1 hour. Separation of proteins under native conditions (for acidic/neutral or basic/neutral proteins, separately) was performed according to KONOTOP et al. (2012) using 11 % (w/v) acrylamide gels with 2.5 % (w/v) laminarin. The β-l,3-glucanase activity was visualised by boiling the gel for 10 min in 1 mol/dm3 NaOH containing 0.1 % (w/v) 2,3,5-triphenyltetrazolium chloride according to the method of PAN et al. (1991). After enzyme activity detection, the gels were stained with Coomassie Brilliant Blue R 250. 2.4. β-l,3-glucanase quantitative assays β-l,3-glucanase activities were assayed spectrophotometrically with laminarin as a substrate (Sigma L-9634) using dinitrosalicylic acid (DNS) method (MILLER, 1959). The absorbance was measured at 500 nm (Ultra spect 100, Pharmacia Biotech). The enzyme activity was expressed in nmol as an amount of released Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC 125 Moravčíková, J. et al. reducing sugar (D-glucose) per hour per milligram of soluble proteins. All data are the means of three replications. Statistical significance of the experimental results was evaluated by ANOVA/MANOVA and Duncan’s tests, using STATISTICA® ver. 7.1. 3. Results and Discussion Plant β-l,3-glucanases are abundant and highly regulated enzymes that are commonly found throughout the plant kingdom (LEUBNER-METZGNER, 2003). So far, these enzymes were studied mainly for their anti-pathogenic functions (MINIC, 2008) and for their role in reversible deposition of (1,3) β-D-glucans found in the cell- wall sheath surrounding plasmodesmata orifices during plant response to stress (LEVY et al., 2007). Their role in plant growth and developmental processes has been also proven (MOROHASHI and MATSHUSHIMA, 2000; LEUBNER-METZGNER, 2003; RUAN et al., 2004). However, they were mainly studied in dicot plants such as Arabidopsis (DOXEY et al., 2007), tobacco (LEUBNER-METZGNER, 2003), soybean (JIN et al., 1999) or tomato (MOROHASHI and MATSHUSHIMA, 2000). Relatively little is known about β-1,3-glucanases in wheat. So far, the Uniprot/NCBI databases contain up to 11 characterised sequences concerning β-1,3-glucanases only in the hexaploid T. aestivum (Table 2). Moreover, β-1,3-glucanases were studied mainly for their induction upon fungal infection. In this work we aimed to study the activities of β-1,3-glucanases in wheat and crop relatives species with different genome background (Table 1). The seedlings of two wheat and six crop relatives species were grown under controlled conditions up to two leaf stage. Subsequently, the leaves were collected and subjected to the qualitative and quantitative activity assays of the β-l,3-glucanases. The activities were evaluated based on the ability of plant β-l,3-glucanases to hydrolyse laminarin, a linear (1,3)-β-D-glucan with a low degree of glucosyl substitution at 0-6. Even though, there are available other (1,3)-β-D-glucans such as curdlan or pachyman (HRMOVA and FINCHER, 1993), laminarin was found to be the most suitable substrate for plant β-l,3-glucanases (PAN et al., 1991; HRMOVA and FINCHER, 1993; PIRSELOVA et al., 2011; ŽUR et al., 2013; GREGOROVA et al., 2015). The total β-l,3-glucanase activity detected was variable. Data are summarised in Fig. 1. The highest enzyme activity was detected for genotypes Ae. tauschii (4.47 nmol D-glucose/h/mg) and T. spelta (4.11 nmol D-glucose/h/mg) while the lowest activity was detected for Ae. cylindrica (1.82 nmol D-glucose/h/mg). The β-l,3-glucanase activity was shown to be genotype dependent (at p ≤ 0.001) (Fig. 1a, Table 2). However, no significant differences (at p ≤ 0.05) between di-, tetra- and hexaploid genotypes were observed (Fig. 1b). The enzyme activities may be related to diversification of wheat due to domestication, natural hybridization and allopolyploid speciation. For example, the genome of hexaploid T. aestivum is composed of three closely-related and independently maintained genomes as a result of hybridization of tetraploid T. turgidum dicoccum or emmer wheat (genome AABB) with Ae. tauschii (genome DD) (MATSUOKA, 2011). This supports also the fact that even within the group of tetraploid cultivars significant differences (at p ≤ 0.01) in β-l,3-glucanase activities were detected (Table 2). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC Nova Biotechnologica et Chimica 15-2 (2016) 126 T ab le 2 . β- 1, 3- gl uc an as es in T . a es tiv um L . a nd th ei r c ha ra ct er is at io n ba se d on th e d at a in th e U N IP R O T a N C B I d at ab as es (J ul y 20 16 ). U N IP R O T / N am e A A M W D N A D es cr ip ti on /f un ct io n L it er at ur e N C B I [ kD a] [b p] P5 24 09 /   β -1 ,3 -g lu ca na se (G lc 1) 46 1 48 .9 2 07 2 A bi ot ic s tr es s (A l3 + ) C R U Z -O R T E G A e t a l., 1 99 7 U 30 32 3 Q 1E R F8 / E nd o- β- 1, 3- gl uc an as e 3 32 3 5. 0 1 15 0 A bu nd an t i n he al th y le av es H IG A -N IS H IY A M A e t a l., 2 00 6 A B 24 46 41 (T aG lb 2e ) Q 1E R F9 / E nd o- β- 1, 3- gl uc an as e 3 36 3 5. 4 1 09 3 A bu nd an t i n he al th y sp ik es H IG A -N IS H IY A M A e t a l., 2 00 6 A B 24 46 40 (T aG lb 2d ) Q 1E R G 0/ E nd o- β- 1, 3- gl uc an as e 3 36 3 5. 5 1 08 9 A bu nd an t i n he al th y sp ik es H IG A -N IS H IY A M A e t a l., 2 00 6 A B 24 46 39 (T aG lb 2c ) Q 1E R F7 / E nd o- β- 1, 3- gl uc an as e 3 42 3 6. 1 1 18 1 A bu nd an t i n le av es H IG A -N IS H IY A M A e t a l., 2 00 6 A B 24 46 42 (T aG lb 2f ) Q 1E R G 1/ E nd o- β- 1, 3- gl uc an as e 3 40 3 5. 9 1 43 8 B io tic s tr es s H IG A -N IS H IY A M A e t a l., 2 00 6 A B 24 46 38 (T aG lb 2b ) ( E ry si ph e gr am in is , F us ar iu m g ra m in ea ru m ) B 5A 7B 8/ β- 1, 3- gl uc an as e (G lc 2) 3 99 44 .2 2 15 2 – – E U 81 69 11 Q 9X E N 7/ β -1 ,3 -g lu ca na se (G lb 3) 3 34 3 4. 7 1 43 9 B io tic s tr es s L I e t a l., 2 00 1 A F1 12 96 7 ( F us ar iu m g ra m in ea ru m ) Q 9X E N 5/ β -1 ,3 -g lu ca na se (G lb 3) 3 34 3 4. 9 1 26 9 B io tic s tr es s L I e t a l., 2 00 1 A F1 12 96 5 (F us ar iu m g ra m in ea ru m ) Q 4J H 28 / β -1 ,3 -g lu ca na se 33 4 35 .4 1 33 8 B io tic s tr es s – D Q 09 09 46 (P uc ci ni a st ri ifo rm is ) Q 4J K 90 / β -1 ,3 -g lu ca na se 33 4 3 5. 4 1 33 7 B io tic s tr es s – D Q 07 82 55 ( P uc ci ni a st ri ifo rm is ) A A – a m in o ac id s; M W – m ol ec ul ar w ei gh t Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC 127 Moravčíková, J. et al. Fig. 1. Total β-1,3-glucanase activities in wheat and crop relatives species (a) and in dependence on ploidy level (b). The enzyme activities were assayed spectrophotometrically using laminarin as a substrate. The activity was expressed in nmol of D-glucose released per hour per mg of soluble proteins. Bars represent means ± standard deviations of three replications. Distinct letters denote statistically significant differences with Duncan’s test at p ≤ 0.001, ns – not significant at p ≤ 0.05. The total β-l,3-glucanase activities measured can comprise several glucanase isoforms (Fig. 2, Table 3). We identified a single glucanase fraction of a ~52 kDa in all analysed genotypes (Fig. 2b). In literature, wheat β-l,3-glucanases were studied mainly in the context of a/biotic stresses (LI et al. 2001; HIGA-NISHIYAMA et al., 2006; PIRSELOVA et al., 2011; GREGOROVA et al., 2015). Thus, only data from non-stressed wheat plants might be used for comparison. Besides, these experiments were performed mainly on bread wheat T. aestivum, moreover, at different stage of growth. For example, GREGOROVA et al. (2015) observed up to four glucanase isoforms of sizes ranging from ~30 kDa to ~150 kDa in the breeding line SK-196 at growth stage after tillering. Similarly, Western blot analyses performed on the control plants of the cv. BobWhite (T. aestivum L.) revealed three glucanases Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC Nova Biotechnologica et Chimica 15-2 (2016) 128 of molecular weight ranging from ~30 kDa to ~60 kDa (JAYARAJ et al., 2004). A higher number of the glucanase isoforms in latter stages of wheat plant might coincide with reversible callose deposition and degradation growth (JAYARAJ et al., 2004; GREGOROVA et al., 2015). RUAN et al. (2004) showed the expression of the fibre-specific β-1,3-glucanase gene GhGluc1 was undetectable when callose was deposited but became evident at the time of callose degradation. They suggested that callose deposition and degradation might correlate with timing of plasmodesmata closure and reopening that is important for division, growth and differentiation of plant cells. Table 2. Variance analyses. β-1,3-glucanases vs. F empiricala Genotype 10.51*** Ploidy level 1.01 ns Teptraploid species 14.07** a statistical significance at *** p ≤ 0.001; ** p ≤ 0.01, * p ≤ 0.05 Fig. 2. Detection of β-1,3-glucanase activities after separation of crude protein extracts in SDS-PAGE (b); and under native conditions in PAGE for acidic/neutral (c); and basic/neutral proteins (d). Separated proteins were visualised with Coomassie Brilliant Blue R 250 (a). Numbers on the left refer to the molecular mass marker. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC 129 Moravčíková, J. et al. The glucanase fraction of a ~52 kDa can comprise up to five acidic (GluA-GluE) (Fig. 2c) and a single basic (GluF) isoforms (Fig. 2d) (Table 3). Neither of the analysed genotypes contained the all five acidic isoforms. In most of them, a combination of two acidic isoforms was found out. However, none of the acidic isoforms can be associated with ploidy level. A single basic GluF was detected in the all analysed genotypes (Table 3). Based on the classification by STINTZI et al. (1993) the acidic isoforms are considered as extracellular while basic as vacuolar. However, several authors pointed out that such classification cannot be fully applied for wheat. Analyses performed on apoplastic fluid of wheat leaves revealed accumulation of not only acidic but also basic glucanase fractions (VAN DER WESTHUIZEN et al., 1998). Our results (Fig. 1, Fig. 2) proved that β-1,3-glucanases are accumulated in the leaves of all studied genotypes. The genotype has significant effect on β-1,3-glucanase activities. However, no significant differences between di- tetra- and hexaploid genotypes were observed. Since, the β-1,3-glucanases might be involved in defence against pathogens, the role of individual β-1,3-glucanase isoforms during biotic stress might be in future evaluated for its use in stress marker assistance breeding or biotechnology programmes. Table 3. Overview of the β-1,3-glucanase activities in wheat and crop relatives species. β-1,3-glucanase isoform Totala Acidic/neutralb Basic/neutralc Glu1 [~52kDa] GluA GluB GluC GluD GluE GluF Ae. tauschii + - + - - + + T. monococcum L. + - - + + - + T. araraticum + - - - + - + T. dicoccum + - - + - - + Ae. cylindrica + - + - - - + Ae. triuncialis + + + - - + T. aestivum L. + - + + - - + T. spelta L. + + - + - - + a Size of the fraction detected in the gel after re-naturation of separated proteins in the SDS-PAGE (Fig. 2b) b Fractions detected in the gel after separation of the proteins in the PAGE under conditions for acidic/neutral proteins (Fig. 2c) c Fractions detected in the gel after separation of the proteins in the PAGE under conditions for acidic/neutral proteins (Fig. 2d) +/- presence/absence of fractions with β-1,3-glucanase activities 4. Conclusions The activities of β-1,3-glucanases in the leaves of two diploids (Ae.s tauschii, T. monococcum L.), four tetraploids (Ae. cylindrica, Ae. triuncialis, T. araraticum, T. dicoccum) and two hexaploids (T. aestivum L., T. spelta L.) were studied. The total β-1,3-glucanase activities were variable and genotype dependent. We did not observe significant correlation between β-1,3-glucanase activities and ploidy level. The gel activity assays revealed a single ~52 kDa Glu1 fraction that was found in all Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 14:47 UTC Nova Biotechnologica et Chimica 15-2 (2016) 130 genotypes. The Glu1 isoform comprised a single or a combination of two acidic Glu isoforms in dependence on genotypes. Neither of the acidic Glu isoforms could be assigned as specific for di-, tetra- or hexaploid genotypes. The basic GluF isoform was present in all genotypes. 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