Abstract: In this research seedling samples, taken from seed germination, have been studied. Polyacrylamide gel electrophoresis of some enzyme systems have been done. The resulted bands were analyzed by computer. These analyses have been performed by NTSYS-pc software, using UPGMA (Unweighted Paired Group Method Arithmetic average) method and on the basis of Jaccard correlation coefficient. Results show that in the first step the species H. pusillus and in the second step H. insanus are separated from the other species. The species H. arachnoideus and H. niger are very heterogenous. We observe that species relationships depend on the locality of plant samples. Therefore, the study of interspecific relationships depends on the study of intraspecific variation.
INTRODUCTION
The Solanaceae family consists of some plants such as Atropa, Datura, Duboisia, Scopolia and Hyoscyamus that are important from medicinal point of view. This importance is due to their tropane alkaloid content. Hyoscyamine and its derivatives are the major tropane alkaloids found in the Solanaceae family (Evans, 1979).
As mentioned above Hyoscyamus is one of these important genera which contains hyoscyamine, scopolamine and to a lesser extent atropine.
According to Khatamsaz (1998), there are 13 species and two varieties of this genus in Iran. Therefore, Iran with three endemic species (H. malekianus, H. tenuicaulis and H. bornmulleri) is one of the most important diversification centers of this genus.
Because of the importance of this genus, actually, numerous studies from different aspects have been done and reported on it. The genus has been studied in regard to anatomy and morphology (Ganj-Karimy, 1997). Scanning Electron Microscopy (SEM) of pollen grains (Khatamsaz and Zangirian, 1998) has also been described. Karyological studies have also been made on 11 species of this genus collected from Iran and have shown two different basic chromosome number, that is x = 14 and x = 17 (Sheidai et al., 1999). Interrelationship of these species have been studied through seed storage proteins and numerical taxonomy (Sheidai et al., 2000).
Isozymes are practical and useful genetic and biochemical markers as well as good estimators of genetic variability in plant populations (Hamrick and Godt, 1997). Electrophoretic technology facilitated the recognition that enzymes appear in multiple forms (Buth and Murphy, 1999). However, currently the use of molecular techniques (isoenzymes, PCR) has become widespread in the study of biochemical characters (Crawford, 1990; Weising et al., 1995). Today isozymes have been used to study genetic variation between and within different species of plants like Oryza spp. (Second, 1982), Oryza sativa L. (Glaszmann, 1987), Portuguese Tronchuda cabbage and Galega kale (Dias et al., 1994), Tylosema esculentum (Monaghan, 1995), Stylosanthes (Gillies and Abbott, 1999), Pistacia L. (Fasihi Harandi et al., 2000), Lathyrus sativus L. (Tadesse and Bekele, 2001) and Avena (Benchacho et al., 2002).
In this study, we have tried to describe six isoenzyme systems of seedling samples in some species of Hyoscyamus. The objective is to survey the usefulness of such methods to separate taxa and grouping within this genus.
MATERIALS AND METHODS
Plant materials: Different samples of Hyoscyamus species used in this study are shown in Table 1. Voucher specimens are deposited either in the central herbarium of Iran (TARI) or Tehran University Herbarium (TUH). Seed samples were planted in the soil separately. Germination started in about a week.
Enzyme extraction: The enzymes were extracted from the seedlings. Crude extracts were prepared by macerating 1g of seedlings in 1.5 mL of extraction buffer (Tris-Glycine, 0.05 M, pH:8.3). The extract was centrifuged at 19000 rpm for 30 min at 4°C.
| Table 1: | Plant materials |
The supernatant (enzyme extract) was filtered through cheese– cloth and stored at -20°C for electrophoresis.
Enzyme electrophoresis: Polyacrylamide gel electrophoresis (PAGE) was performed in a vertical, discontinuous system using 12.5% gel. Electrophoresis was done at 4°C and a constant current of 5 mA per gel during 24 h. For all the enzymes, 90 μL extract per well was loaded. Staining procedures followed Desborough and Peloquin (1967), Van Loon (1971) and Wendel and Weeden (1990). The enzyme systems analyzed were: esterase (EST, E.C.3.1.1.1.), peroxidase (PRX, E.C.1.11.1.7.), polyphenoloxidase (PPO, E.C. 1.10.3.2), superoxide dismutase (SOD, E.C.1.15.1.1.), malate dehydrogenase (MDH, E.C.1.1.1.37.) and malic enzyme (ME, E.C.1.1.1.40).
Data analysis: The presence (coded as 1)/absence (coded as 0) data matrix was prepared from the zymograms. The bands which are present in all taxa were removed. Relationships among these taxa were inferred with a UPGMA dendrogram from Jaccard’s similarity matrix of the bands. Data were analysed using the SAHN (Sequential Agglomerative Hierarchical and Nested) option of NTSYS-pc (Version 1.60) system of Rohlf (1990).
This research was being done from June 2000 to July 2001 in Laboratory of Plant Physiology, Department of Botany, School of Biology, University College of Sciences, University of Tehran.
RESULTS
Fifty eight electrophoretic bands were detected from the six seedling isozyme systems analyzed.
| Fig. 1: | Photograph and diagram showing isozyme banding pattern for ME |
Isozyme malic enzyme (ME) exhibited single band on the gel for each taxon of Hyoscyamus which is a typical pattern of a monomeric enzyme. It is coded by one gene at locus ME-1 (Fig. 1). ME-1 has two types of alleles ME-11 and ME-12 corresponding to bands which differs in their relative mobility. Therefore a total of two bands with malic enzyme were observed.
| Fig. 2: | Photograph and diagram showing isozyme banding pattern for PRX |
| Fig. 3: | Photograph and diagram showing isozyme banding pattern for EST |
| Fig. 4: | Schematic illustration of zymogram for PPO |
Isozyme peroxidase (PRX) exhibited the most complicated zymogram as it appeared to be controlled by seven loci in H. insanus (sample 3). Many of loci are represented by one allele except for locus four and locus seven exhibiting two alleles per locus. All the alleles do not express simultaneously in one sample. Locus one and locus two only expressed in H. insanus (sample 3).
| Fig. 5: | Schematic illustration of zymogram for MDH |
Locus 8 is restricted to H. turcomanicus (sample 5) and locus 10 only appeared in H. reticulatus (sample 13). A total of 13 bands that expressed by 11 loci were observed for this isozyme system and diagrammatically represented in Fig. 2.
Isozyme esterase (EST) appeared as a monomeric enzyme coded by seven loci. The banding pattern for most of the samples was different and it was possible to differentiate them from each other (Fig. 3).
| Fig. 6: | Schematic illustration of zymogram for SOD |
Locus four only exists in H. arachnoideus 3 (sample 9).
Isozyme polyphenoloxidase (PPO) exhibited 11 bands on the gel which appeared to be coded by six genes at loci PPO-1 to PPO-6 (Fig. 4). The loci detected in the present study clearly exhibited monomeric pattern.
Isozyme malate dehydrogenase (MDH) showed eight bands coded by four genes at loci one to four. In this study it appeared as a mixture of monomeric and dimeric activity (Fig. 5).
Isozyme superoxide dismutase (SOD) appeared as 12 bands expressed by eight loci (Fig. 6). It seems that it is a mixture of monomer and dimer. Locus one and locus two are monomorphic.
| Fig. 7: | Similarity matrix calculated using ‘Jaccard’ correlation coefficient |
| Fig. 8: | Dendrogram based on UPGMA analysis of genetic similarity estimates (Jaccard’s similarity coefficient) |
| Table 2: | Presence/absence data matrix of the isozyme study |
The presence or absence of each band was treated as a binary character in data matrix (Table 2). Based on the data matrix, Jaccard’s similarity index was calculated (Fig. 7). The lowest similarity value (0.156) was observed between H. pusillus and H. arachnoideus 4, while the highest value (0.731) was between H. arachnoideus 4 and H. arachnoideus 2.
Dendrogram was produced from the results of similarity matrix using the UPGMA method (Fig. 8). Two main branches are observed within different taxa of genus Hyoscyamus. One includes H. pusillus, which presents the lowest similarity with the rest taxa (about 0.25). The second node of the tree separates two clusters: The first one includes H. insanus while the second one divides into two parts, with Hyoscyamus arachnoideus 1, 2, 3, 4, 5 and H. reticulatus in the first branch. The second branch has two clusters, one of them consists of H. squarrosus and H. turcomanicus and the other includes H. niger 1, 2, 3 and H. arachnoideus 6,7.
DISCUSSION
Malic enzyme (ME) has been studied in many plants including Pinus halepensis L. (Loukas et al., 1983), Citrullus (Navot and Zamir 1987), Vigna (Vaillancourt and Weeden 1993; Sonnante et al., 1997; Pasquet and Vanderborght, 1999). In all of these studies it is reported that it is expressed by one locus and it displays one zone of activities on the gel.
Peroxidase has been studied in different plants such as Nicotiana (Bhatia et al., 1967), maize (Brewbaker et al., 1985), Musa (Jarret and Litz, 1986), Citrullus (Navot and Zamir, 1987), Watermelon (Navot et al., 1990), Potato (Nieto et al., 1990), Garlic (Pooler and Simon, 1993), Allium (Abdul Wahab, 1994), Beta (Reamon-Buttner et al., 1996), pyrola (Huh et al., 1998), cotton (Farooq et al., 1999). Various numbers of loci from one to thirteen has been reported for this enzyme. For example, one locus has been reported for genus Citrullus while thirteen loci have been reported for maize (Brewbaker et al., 1985).
Esterase has also been studied in different plants such as Nicotiana (Bhatia et al., 1967), Solanum (Desborough and Peloquin, 1967), Musa (Jarret and Litz, 1986), Watermelon (Navot et al., 1987), Citrullus (Navot and Zamir, 1987), Allium (Abdul Wahab, 1994), Lythrum salicaria (Strefeler et al., 1996), Amaranthus (Chan and Sun, 1997), Pinus (González-Andrés et al., 1999), Vigna (Pasquet and Vanderborght, 1999), Cotton (Farooq et al., 1999). It is reported to be either a monomer such as in Solanum (Desborough and Peloquin, 1967) or a dimer like cotton (Farooq and Sayyed, 1999). In some species like Musa acuminata Colla, EST is useful as a diagnostic tool for cultivar identification in view of the extensive polymorphism for this enzyme (Jarret and Litz, 1986).
Polyphenoloxidase has also been studied in other plant systems to a lesser extent. Allium is one of the examples that have been reported for this enzyme (Abdul Wahab, 1994). Sixteen bands have been reported for Allium and most of the species have the same band pattern.
Malate dehydrogenase has been extensively studied in plants such as Peach (Arulsekar et al., 1986), Musa acuminata Colla (Jarret and Litz, 1986), Citrullus (Navot and Zamir, 1987), Festuca (Aiken et al., 1993), Vigna (Vaillancourt and Weeden 1993), Allium (Abdul Wahab 1994), Beta (Reamon-Buttner et al., 1996), Lythrum salicaria (Strefeler et al., 1996), Vigna luteola and Vigna marina (Sonnante et al., 1997), Amaranthus (Chan and Sun, 1997), Pyrola (Huh et al., 1998), Vigna (Pasquet and Vanderborght, 1999). It has been reported to be a dimer in Festuca (Aiken et al., 1993), Corn (Yang et al., 1977) and Celery (Orton, 1983), Musa acuminata Colla (Jarret and Litz, 1986). In the study on Lythrum salicaria (Strefeler et al., 1996) eight bands were observed for MDH.
Superoxide dismutase has been studied in different plants such as Citrullus (Navot and Zamir, 1987), Watermelon (Navot et al., 1990), Beta (Reamon- Buttner et al., 1996), Cotton (Farooq et al., 1999), Pinus (González-Andrés et al., 1999) and Vigna (Pasquet and Vanderborght, 1999). In cotton it has been reported to be monomer (Farooq et al., 1999). In most of the other plant species like maize (Baum and Scandalios, 1981), barely (Jaaska and Jaaska, 1982), Beta (Reamon-Buttner et al., 1996), it has been reported to have dimeric or tetrameric structure.
Separation of the species H. pusillus is confirmed by numerical taxonomy (Based on Single- Linkage method) (Sheidai et al., 2000). On the other hand chromosome number of this species is 2n = 68 which differs from the others (Sheidai et al., 1999). Vegetative form of this species is restricted to annual form (Khatamsaz 1998)). In the second branch the species H. insanus separates from the other ones. According to morphological studies (Khatamsaz 1998), three subgenera for this genus have been reported:
| • | Subgenus Para hyoscyamus includes H. malekianus and H. leptocalyx. |
| • | Subgenus Hyoscyamus includes H. reticulatus, H. kurdicus, H. turcomanicus, H. senecionis, H. arachnoideus, H.squarrosus, H.pusillus and H. niger. |
| • | Subgenus Dendrotrichon includes H. insanus, H. tenuicaulis and H. bornmulleri. |
The members of subgenus Dendrotrichon have chromosome number that is 2n = 28 (Sheidai et al., 1999). The studies mentioned above as well as numerical taxonomy studies (Based on Single-Linkage method) (Sheidai et al., 2000) confirm the separation of this species from the others.
Among the other species H. arachnoideus is very heterogenous and we observe that seven samples of this species that have been collected from different localities don’t have similar isozyme banding patterns. We have a similar manner for the species H. niger with three different samples. This heterogeneity shows the high variation of isozyme banding pattern within species, thus for the study of interspecific relationship, we need to survey intraspecific variation.
The close relationship between the species H. turcomanicus and H. squarrosus and also between H. reticulatus and H. arachnoideus is in accordance with numerical taxonomy studies (Based on UPGMA method) (Mosallanejad, 1996).
ACKNOWLEDGMENTS
The financial support of this research was provided for M. Sc. research projects from Research Council of the University College of Science, University of Tehran. Thanks are due to Dr. Sh. Zarre for his kind assistance in data analysis. We are also grateful to Dr. F. Attar and Mr. M. Mirmasoumi for providing some of the seed samples used in this research.