In this study, total 8 species (Section Cicercula) belong to 14 different geographic distributions collected from Turkey have been studied for the analysis of seed storage protein profiles to examine their relationship by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE) technique. Hierarchical cluster analysis and Euclidean distance were used for comparison between species and calculating the genetic resemblance respectively. Dendogram was formed using average linkage. Electrophoretic protein profiles of seed cotyledons were showed that all species formed two clusters. The first one consisted of the L. stenophyllus, L. hirsutus, L. chloranthus, L. cicera and L. sativus second one by three species (L. annuus, L. cassius and L. phaselitanus). In addition, very little differences were observed in total protein profiles of the examined sample species (L. cicera, L. hirsutus and L. chloranthus) from nine geographical regions belong to section Cicercula. Protein amount was found to be highest in L. hirsutus and lowest in L. cicera.
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Fabaceae is third largest family of flowering plants comprises about 269 genera and 5100 species in the world (Mabberly, 1997; Ellison et al., 2006). The genus Lathyrus is placed in the tribes of Vicieae of Fabaceae family and divided into 13 sections (Kupicha, 1983). It has 152 species and shows a broad diffusion throughout the world (Alkin et al., 1986). This genus is represented with 70 taxa in the level of species, subspecies and variety and is divided into 10 sections in Turkey (Davis, 1970). Additionally, six taxa have been described from Turkey in recently years (Ertekin and Saya, 1990; Ertekin, 1994; Davis et al., 1988; Maxted and Goyder, 1988; Guner and Ozhatay, 2000).
Legume seeds have a high level of protein content which contain average of 20-25% protein, 2-3 times more than cereals (Cai et al., 2001; Monsoor and Yusuf, 2002; Gepts et al., 2005; Duranti, 2006). Therefore, grain legumes have played a primary role in the search for vegetable sources of proteins and investigations on economically viable legumes as alternative foods broaden the protein sources for human nutrition (Valizadeh, 2001; Sena et al., 2005). Seed proteins are highly stable, being unaffected by environmental conditions and easily to handle. Thus, electrophoretic banding patterns of total seed proteins and seed storage protein subfractions (albumins, globulins, prolamins, glutelins) as revealed by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis) have provided valid evidence for addressing taxonomic and evolutionary problems (Ladizinsky and Hymowitz, 1979; Kamel et al., 2003; Freitas et al., 2004; Ribeiro et al., 2004; Fukuda et al., 2005).
The aim of the present study was to investigate both interspecific and intraspecific variations in Section Cicercula by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis) technique. In this study, total 8 species belong to 14 different geographic distributions were collected and studied for the analysis of seed storage protein profiles to examine their relationship. Protein amounts of samples were also determined.
MATERIALS AND METHODS
Dry seeds of Lathyrus species were collected from various areas of Turkey. Outcross was not observed among studied species. Details about the seed materials are given in Table 1. The laboratory study was carried out in the Molecular Identification Laboratory, Department of Biology, Faculty of Sciences and Art, Firat University, Elazig, Turkey in 2006.
|Table 1:||Localities of investigated Lathyrus species|
Seed proteins were extracted as described by Jha and Ohri (2002). Seed coats were removed prior to extraction and cotyledons were obtained. These were homogenized in the ratio 1:10 (w/v), using 0.1 M Tris-HCl buffer (pH: 7.5). Total protein was extracted after centrifugation at 17.600 g for 20 min at 4° C and supernatants were used for analysis. Proteins in the supernatants were quantified using Bio-Rad DC protein assay Kit (Bio-Rad Laboratories, UK). The samples were boiled for 5 min prior to loading, then average equal protein of each sample was loaded on to the 12% SDS-PAGE (Laemmli, 1970). Electrophoresis was performed in the Protean II electrophoresis cell (Bio-Rad Laboratories, UK) at 20 mA until the bromophenol dye (BDH Laboratory Supplies Poole, England) front had reached the bottom of the gel. The gels were stained in Coomassie Brilliant Blue (Sigma Aldrich Chemie, Germany) solution for 30 min at 67° C and destained in destaining solution for 3-4 h at 67° C to visualise the proteins.
Statistical analysis: Evaluation of protein profiles was done visually with an emphasis on qualitative banding differences. The bands were divided into four distinct groups of strong dark, dark, faint and no band and four groups represented by 4, 3, 2, 1, respectively, in statistical analysis. On the gel, five distinct regions were determined and called as A, B, C, D and E being equivalent to decreasing MWs of 97, 66, 45, 30 and 20.1 kDa (kilodalton), respectively; (Amersham Biosciences, UK) (Fig. 1). We analyzed the gels in a numerical classification for all protein bands. 3 bands in region A (a1 to a3), 3 bands in region B (b1 to b3), 6 bands in region C (c1 to c6), 4 bands in region D (d1 to d4) and, 2 bands in region E (e1 to e2) were distinguished.
Data obtained from electrophoresis of seed proteins were analysed using the SPSS (version 10.0) program (SPSS, Chicago, IL, USA). Hierarchical cluster analysis was used for comparison between species and Euclidean distance was used for calculating the genetic resemblance. Dendogram was formed using average linkage.
RESULTS AND DISCUSSION
The analysis of seed total proteins by SDS-PAGE revealed that seeds of Lathyrus are very rich in storage proteins with a large number of stable bands on the gel (Fig. 1). Many studies based on the electrophoretic analysis of seed proteins have been used to examine genetic variability and systematic problems in several legumes such as genus Astragalus (Ac1k et al., 2004), genus Lupin (Vaz et al., 2004), genus Pisum (Jha and Ohri, 2002), genus Phaseolus (Przybylska and Przybylska, 1993), genus Lathyrus (Przybylska et al., 1999, 2000; Emre et al., 2006), genus Onobrychis (Emre et al., 2007).
The electrophoregrams were evaluated on the basis of band mobility and relative intensity. A dendogram of studied species based on the total seed protein profile is given in Fig. 2 regions of electropherogram was the most informative part of the gel that revealed the largest number of different protein banding profile in the electropherogram of species, belong to Section Cicercula in genus Lathyrus (Fig. 1). Protein profiles of each species showed its own electrophoresis pattern with subunits of varied molecular weights. Differences were observed in both presence and absence of a particular band. A strong dark band was present in d3 band (between 30 and 20.1 kDa major protein band) in all of the species studied except for L. cicera which has dark band. Protein amounts in all species were also quite similar as shown in Table 2. Protein amount was found to be highest in L. hirsutus (225.393 μg mL‾1) from Isparta (Egirdir-Aksu road) and lowest in L.cicera (206.677 μg mL‾1) from Burdur (Burdur Education Faculty campus).
Electrophoretic protein banding profiles of seed cotyledons were showed that all species formed two clusters. The first one consisted of the L. stenophyllus, L. hirsutus, L. chloranthus, L. cicera and L. sativus second one by three species (L. annuus, L. cassius and L. phaselitanus).
|Fig. 1:||SDS-PAGE of total seed proteins in eight species. M: Marker; 1: L. annuus (Mugla); 2: L. cassius (Sanlyurfa); 3: L. cicera (Burdur); 4: L. sativus (Antalya); 5: L. stenophyllus (Antalya); 6: L. phaselitanus (Antalya); 7: L. hirsutus (Konya) 8: L. chloranthus (Isparta)|
|Table 2:||Protein amounts of investigated Lathyrus species|
Protein profiles of L. cicera and L. sativus show a high level of similarity to each other. Both species had almost the same protein bands but some differences were observed in protein profiles. For example, while L. cicera has strong dark band profile, L. sativus has faint band in b2 band. Also in c3 band, L. sativus has strong dark band but L. cicera has dark band. Furthermore, in cluster I, L. stenophyllus also shows similar band patterns with L. hirsutus. In addition L. chloranthus is similar with these two species (L. stenophyllus and L. hirsutus). On the other hand according to band profiles of L. annuus, L. cassius and L. phaselitanus all three species formed Cluster II. L. annuus and L. cassius higher degree of similarity than L. phaselitanus which endemic for Turkey.
These results are agreement with an earlier study done by El-Shanshoury (1997) who found that L. sativus, L. chloranthus, L. cicera and L. hirsutus were in the same cluster but L. annuus was member of the other cluster.
|Fig. 2:||Dendogram of Lathyrus species based on total seed protein profiles|
|Fig. 3:||SDS-PAGE of total seed protein bands among species according to the geographic distribution. M: Marker; 1, 2, 3: L. cicera (Burdur, Elazig, Burdur-Denizli road; respectively); 4, 5, 6, 7: L. hirsutus (Konya, Isparta (Egirdir-Aksu road), Isparta (Egirdir Balkyry village), Elazig); 8, 9: L. chloranthus (Isparta, Mus)|
In another study, isozymic results are partly consistent with our results (Brahim et al., 2002). In this study (done by Brahim et al., 2002), L. sativus and L. cicera formed the same cluster and had great similarity but L. hirsutus was in another cluster. Also in another study (results from RAPD analyses) done by Chtourou-Ghorbel et al. (2002) showed that L. sativus more closely related to L. cicera. Similarly, Present results show that these two species (L. sativus and L. cicera) have great homology each other and also L. hirsutus was in different position in same cluster. In addition, L. annuus has different electrophoretic band patterns and was another cluster. Furthermore L. sativus and L. cicera were reported to be similar with respect to some properties, such as morphological traits (Jackson and Yunus, 1984), karyotype (Yamamoto et al., 1984). On the contrary, Przybylska et al. (2000) indicated that L. sativus and L. cicera are definitely different in the composition of seed albumins and globulins.
Band profiles of the examined taxa from nine geographical regions belong to section Cicercula are shown in Fig. 3. Very little differences were observed in the examples of total seed protein bands among taxa according to the geographic distribution.
According to present results, it seems justified to recommend a wider use of electrophoretic analysis of seed storage proteins in taxonomic investigations of leguminous plants. The other prominent result of our study is similarity of electrophoretic band patterns of L. sativus to band patterns of L. cicera. As a result, further investigation of phylogenetic relationships between these species needed by analysis of genetic variations between the species, such as analysis of subprotein fractions (albumins, globulins etc.) and isozymic studies.
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