Superoxide dismutase polymorphisms in wild populations of herb Paris (Paris quadrifolia L., Trilliaceae)

Vaida Jogaite, Polymorphism of superoxide dismutase (SOD) was investigated in leaves of Violeta KIeizaite, herb Paris (Paris quadrifolia L., Trilliaceae). The plants were collected dur­ ing the summer and autumn of 2001 from different natural locations in Asta Stapulionyte, Lithuania and Norway. Crude extracts from leaves were analyzed using elec­ Dag K. Bjerketvedt* trophoresis in polyacrylamide gel for SOD polymorphism detection. By means of analysis of plants from different locations, some differences in the elec­ Department of Botany and trophoretic mobility and the phenotypes of SOD bands were detected. Dif­ Genetics, Vilnius University, ferences appeared between the Lithuanian and the Norwegian samples and M. K. Ciurlionio 21, among the Lithuanian samples from different locations as well as inside LT-2009 Vilnius, Lithuania them. These findings indicate a polymorphism in plants from Lithuania and * Telemark University College, Norway. Analysis of the results revealed five types of SOD isozyme spectra Department ofArts and Sciences, in both countries. SOD isozyme spectra also differed in leaves, seeds, roots 3800 Bfj), Norway and rootlets. Key words: enzyme electrophoresis, Paris quadnfolia, polymorphism, super­ oxide dismutase

INTRODUCTION ing from exposure to environmental oxidants, toxi­ cants, radiation, or numerous biostressors perturbs Herb Paris (Paris quadrifolia L., Trilliaceae) is wi­ the cellular redox balance Cto a more oxidized sta­ despread in deciduous and mixed forests of Europe te) and disrupts normal biological functions. This and Central Asia (Lietuvos... 1963; Meusel et aI., condition is referred to as "oxidative stress" and 1965). Its large habitat along the continent of Eu­ may be detrimental to the organism by contributing rasia with various ecological conditions allows to pre­ to the pathogenesis of disease and aging, and nu­ sume a possibility of polymorphisnl. Various enzy­ merous physiologic dysfunctions leading to Gell death me systems have been applied for polymorphism de­ (Kemodle et aI., 2001). tection in different organisms (Asins et aI., 1995; The aim of this work was to investigate whether Kertadikara et aI., 1995). Higher plants possess a polymorphism is present within wild populations of number of superoxide dismutase isozymes that have herb Paris, using superoxide dismutase as a molecu­ been used as a molecular markers in polymorphism lar marker. studies (Kertadikara et aI., 1995; Pszybylska et aI., 1992; Zvingila et aI., 1993). Superoxide dismutases MATERIALS AND METHODS Csuperoxide: superoxide oxidoreductase; SOD; EC 1.15.1.1) are ubiquitous enzymes found in all the Plant material aerobes and involved in protection from oxygen to­ xicity. These metalloproteins catalyze the dismuta­ Leaves of Paris quadrifolia were used in experiments tion of the superoxide radical to molecular oxygen for the detection of SOD polymorphism in different and hydrogen peroxide. The superoxide (Oz" -) and populations. Leaves, rhizome, rootlets and seeds we­ hydroxyl C' OH) radicals together with hydrogen pe­ re used for the detection of SOD polymorphism in roxide (HzOz) are the so-called reactive oxygen spe­ plant tissues of different organs. The samples were cies (ROS) that pose a serious threat to all orga­ collected in different locations of Lithuania (Fig. 1) nisms. ROS are also crucial for many physiologic and NOlWay during the summer and autumn of the processes and usually exist in the cell in a balance year 2001. Five different natural locations of herb with the antioxidants. However, excess ROS result- Paris in Lithuania were chosen: Joniskis district,

ISSN 0235-7224. E k 0 log i j a (Vilnius). 2003. Nr. 1

59 Vilida Jogaitl!, Violeta Kleizaite, Asta Stapulionyte, Dag K. Bjerketvedt

o 11 10 9 8 7

5

2

+ 11 III IV v VI VII

Fig. 2. Electrophoregrams in native - PAGE (9%) of SOD from leaves of Paris herb (Paris quadrifolia L.) collected Fig. 1. Map showing the populations of herb Paris (Paris in different locations: I - Joniskis, II - Trakai, III - Va­ quadrifolia L.) examined in Lithuania: 1 - Joniskis, 2 ­ rena, IV - Labanoras, V- Kairenai, VI - H~rte (Nor­ Labanoras wood, 3 - Kairenai, 4 - Vingis (Vilnius), 5 ­ way), VII - Vingis. Bands are numerated from fastest to Trakai, 6 - Varena slowest

Svencionys district (Labanoras forest), liakai district, fer and for 1 g of seeds 4 ml of the extraction Varena district, and Vilnius district (Botanical Gar­ buffer were taken. Subsequent sample preparations den of Vilnius University in Kairenai and Vingis). for electrophoresis were the same as described In Norway the samples were collected from 16 dif­ above. ferent natural locations. Randomly sampled plants were stored at -18°C until further analysis. From Polyacrylamide gel electrophoresis and enzyme detection each location at least three plants were taken for the preparation of crude extract and tested using SOD was analyzed on 4% concentrating and 9% polyacrylamide gel electrophoresis. discontinuous nondenaturing polyacrylamide gels Only one population from Norway was used for (PAG) by vertical gel electrophoresis (Davis, 1964) comparison with the other populations from Lithu­ (at 200 V, 40 mA) for about 3 h .at 4 °C (Beau­ ania, because the intensity of bands at the catodic champs et al., 1971) using Tris-glycine buffer (pH zone was very low in all the samples from Norway 8.3). About 30 /!l of crude extract from leaves and and it was impossible to make a reasonable compa­ 20 /!l from other organs were loaded in each lane. rison among them. The low quality of electropho­ The zones of SOD activity were detected by stai­ regrams from the Norwegian material may be caused ning the gel in a following mixture: 100 ml 0.1 M by storage conditions as the plants were collected Tris-HO buffer (pH 8.5), 15 mg tetranitro blue tet­ in 2001 and kept at -18°C for almost a whole razolium, 15 mg phenazine methosulphate and 20 mg year. Since the electrophoregrams from H0rte po­ magnesium chloride, incubating for an hour at 37 pulation were most intensive (Fig. 2), they were used °C in the dark. as representative ones for all the Norwegian popu­ SOD densitograms were made using a DM-1 lation in this investigation. densitometer with a white filter No. 5.

Crude extract RESULTS AND DISCUSSION

For crude enzyme extract, 1-2 leaves of each plant Six to nine zones of superoxide dismutase activity were grinded in a mortar with sand in the pre­ were observed in our experiments (Figs. 2, 3). Pre­ cooled extraction buffer. One millilitre of 0.1 M po­ vious papers of other researches also indicated SOD tassium phosphate extraction buffer (pH 8.2) was ta­ polymorphisms in other plant species: three zones ken for 1 g (fresh weight) of leaves (Beauchamp et of SOD activity were identified in pea leaves (Pal­ al., 1971). The homogenate was centrifuged at ma et al., 1998), four zones of SOD were detected 13000x g for 10 min. The supernatant was used for in sunflower leaves (Palomo et al., 1999). In di­ further analysis. All procedures were performed at ploids, an enzyme band is coded by one or two 4 cc. copies of an allele. It is therefore difficult to de­ When homogenizing rhizomes and rootlets, for termine the exact genotype and allele frequency in 1 g (fresh weight) of tissue 3 ml of extraction buf- a polyploid without genetic analysis of crosses (Ny-

60 Superoxide dismutase polymorphisms in wild populations of herb Paris (Paris quadrifolia L., Trilliaceae)

tributed to the first SOD isozyme spectrum. Zy­ mograms of herb Paris from Trakai and Varena po­ pulations were attributed to the second type and from Labanoras to the third type of SOD isozyme spectrum. The fourth type of spectrum included zy­ mograms of herb Paris from Kairenai and H0rte 5 (Norway) populations, while the Vingis population had its own type of SOD isozyme spectrum. The anodic zone showed no polymorphism in all spec­ tra, probably due to a low variation of frequencies at this locus. The medium mobility band of the first spectrum type was faster than in the rest zymograms of the other spectra. A certain difference in rapidi­ ty at the medium mobility zone was observed inside the Labanoras population. This could indicate a polymorphism within the population. Additional ex­ periments are needed to prove it. As mentioned Fig. 3. Densitogram of superoxide dismutase from leaves above, three phenotypes were found at the catodic of herb Paris (Paris quadrifolia L.) (Vingis location). Num­ zone of SOD activity. The first phenotype was three­ bers of peaks correspond with numbering of bands in banded, the second four-banded, while the third had electrophoregram in Fig. 3 five bands. SOD polymorphism in Lithuanian populations of herb Paris was compared with that in the Norwe­ berg Berglund et aI., 2001). Since it is complicated gian populations. Anodic two-banded zones showed to distinguish allozymes (genetically determined no polymorphism. Activity of catodic bands in Nor­ forms of enzymes) from isozymes (any multiple wegian populations was weaker in comparison with forms of enzymes) in polyploids without extensive Lithuanian populations. Therefore a precise estima­ analyses of segregating progeny, we used the term tion of SOD activity and mobility was impossible enzyme bands instead of allozymes and isozymes. because of a poor quality of electrophoregrams from The zones of SOD activity were distributed to the the Norwegian plants. Thus it was difficult to make fastest anodic, the slowest catodic and the medium a comparison both within the Norwegian and Li­ mobility bands. Only one two-banded phenotype was thuanian populations. We have performed 9% PAGE to define the observed in the anodic zone. Three phenotypes we­ SOD banding pattern in tissues of different organs re distinguished in the medium mobility zone. Three from herb Paris. The leaves, rhizome, rootlets and phenotypes were observed in the catodic zone of seeds of two plants from Vingis were analyzed. The SOD activity as well (Fig. 2). rhizome and rootlets showed the same banding pat­ General analysis of the zymograms revealed five tern, however, it differed from the banding pattern types of SOD isozyme spectra (Fig. 4). Zymograms in leaves and seeds (Fig. 5). The main difference of P. quadrifolia from Joniskis population were at- appeared in five additional anodic bands not obser­ ved earlier in zymograms from leaves. A variation was also defined at the anodic zone: the faster band o was less intensive as in leaves. Moreover, SOD ac­ tivity was weaker in the medium mobility and cato­ dic zones in underground organs of the plants. This may depend on a variety of reasons such as dosage effect of gene copies, gene silencing or difference in kinetic activity (Nyberg Berglund et aI., 2001). Besides, SOD activity at the catodic zone was stron­ ger in plants collected during the summer and au­ + tumn of 2001 than in plants collected in April 2002. I II III IV V This difference can be related to the maturity stage Fig. 4. Types of SOD isozyme spectra in leaves of herb of the plants collected at the begining and end of Paris (Paris quadrifolia L.): vegetation. SOD electrophoresis in tissues of diffe­ 1- Joniskis, 11 -Trakai and Varena, III - Labanoras, IV­ rent organs of herb Paris, as in maize (Baum et aI., Kairenai and H0rte (Norway), V- Vingis 1981), indicates a dependency of isozyme activities

61 l/aida Jogaite, Violeta Kleizaite, Asta Stapulionyte, Dag K. Bjerketvedt

6. Kertadikara AW S., Prat D. Isozyme variation among teak (Tectona-grandis L.F.) provenances. Theoretical and Applied Genetics. 1995. Vo!. 90. No. 6. P. 803-810. 7. Lietuvos TSR flora. Vilnius: Valstybine politines ir mokslines literatUros leidykla. 1963. T. n. P. 548-549. 8. Meusel H., Jager E., Weinert E. Vergleichende Chro­ nologie der Zentraleuropiiischen Flora. VEB Gustav Fis­ cher Verlag Jena. 1965. P. 100. 9. Nyberg Berglund A-B., Saura A, Westerbergh A Ge­ netic differentiation of a polyploid plant on ultrama­ fic soils in Fennoscandia. South African Journal of Science. 2001. Vo!. 97. P. 171-183. 10. Palma J. M., Lopez-Huertas E., Corpas F. J. Peroxi­ mal magnesia superoxide dismutase: Purification of the isozyme from pea leaves. PhysiologUz Plantarum. 1998. Vo!. 104. P 720-726. 11. Palomo P. J., Lopez-Valbuena et a1. Sunflower (Heliant­ hus annuus) variability in antioxidant enzyme defences. Free Radical Research. 1999. Vo!. 31. P. 227-233. I 11 III IV 12. Przybylska J., Zimniak-Przybylska Z., Krajewski P. Fig. 5. Electrophoregrams in native PAGE (9%) of SOD Isoenzyme variation in the genetic resources of Vi­ from tissues of different organs of herb Paris (Paris qu­ cia faba L. Genetica Polonica. 1992. Vot. 33. No. 1. adrifolia L.): P. 17-25. I- rootlets, 11 - rhizome, III - leaves, IV - seeds 13. Scandalios J. G. Oxygen stress and superoxide dismu­ tase. Plant Physiology. 1993. Vo!. 101. P. 7-12. 14. Zvingila D., Kleizaite V, Popendikyte V. Investigation relative to the plant tissue and development stage. of molecular polymorphism in barley (Hordeum vul­ Changes in the pattern of SOD isozymes reveal re­ gare L.) cultivars, genetical lines and mutants. Biolo­ gulatory mechanisms controlling the synthesis of gija. 1993. No. 3. P. 111-112. SOD in response to different oxidative stimuli and providing an adequate protection of plants during Vaida Jogaite, Violeta Kleizaite, Asta Stapulionyte, plant growth and development Scandalios, 1993. Dag K. Bjerketvedt Thus, our work revealed SOD polymorphism in SUPEROKSIDDISMUTAZES POLIMORFIZMO the wild populations of herb Paris as well as among 1YRIMAI NATURALIOSE KETURLAPES different organs of this plant. VILKAUOGES (PARIS QUADRIFOLIA L., TRILLIACEAE) POPULIACIJOSE References Santrauka 1. Asins M. J., Herrero R., Navarro L. Factors affecting Superoksiddismutazes (SOD) polimorfizmas buvo tiriamas Citrus tree isozyme-gene expression. Theoretical and keturlapes vilkauoges (Paris quadrifolia L., Trilliaceae) la­ Applied Genetics. 1995. Vo!. 90. No. 6. P 892-898. puose. Tyrimams augalai surinkti Lietuvos bei Norvegijos 2. Baum J. A, Scandalios J. G. Isolation and characte­ gamtinese vilkauoges augimvietese. SOD polimorfizmui nu­ rization of the eytosolic and mitochondrial superoxide statyti buvo ruosiami grubiis augalq ekstraktai, kurie istir­ dismutases of maize. Archives of Biochemistry and Biophysics. 1981. Vo!. 206. No. 2. P. 249-264. ti baltymq elektroforezes poliakrilamidiniame gelyje me­ 3. Beauchamp C. 0., Fridovich I. Superoxide dismutase: todu. Buvo nustatyta, kad elektroforezinis izoformq jud­ Improved assays and applicable to acrylamide gels. rumas skiriasi Lietuvos ir Norvegijos vilkauogese, taip pat Analytical Biochemistry. 1971. Vo!. 44. P. 276. augaluose is skirtingq Lietuvos vilkauogiq populiacijq. Pa­ 4. Davis B. J. Disc electrophoresis. Method and appli­ lyginus Lietuvos ir Norvegijos augal4 lapq elektroforegra­ cation to human serum proteins. Annals of the New mas, nustatyti penki SOD izofermentq spektrai. Atlikus is York Academy of Sciences. 1964. Vo!. 121. P 404-427. lapq, seklq, saknq bei sakniastiebiq iSskirtos SOD analizlt, 5. Kernodle S. P, Scandalios J. G. Structural organization, nustatyta, kad skirtingose augalo dalyse SOD izofermentq regulation, and expression of the chloroplastic super­ spektrai skiriasi. oxide dismutase Sod] gene in maize. Archives of Bio­ Raktazodziai: fermentq elektroforeze, Paris quadrifo­ chemistry and Biophysics. 2001. Vo!. 391. No. 1. P 137. lia, polimorfizmas, superoksido dismutaze

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