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Immunochemical properties of seed proteins as systematic markers in Cactaceae
Biochemical Systematics and Ecology 44 (2012) 20–26
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Immunochemical properties of seed proteins as systematic markers in Cactaceae Marcelo J. Galvez*, María I. Prat, Carlos B. Villamil Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2012 Accepted 6 April 2012 Available online xxx
The purpose of this study was to compare seed protein patterns of 10 species of Cactaceae, a native New World plant family distributed from Canada to Argentina, in an attempt to assess their usefulness for systematic studies. Particular attention was paid to analysing antigenic patterns derived from western blotting carried out with different antisera. Similarity (SJ) and distance (DBC) indices were further used to carry out Cluster Analysis (UPGMA) and Principal Coordinate Analysis. Antigenic patterns of species of Opuntioideae and Cactoideae were obtained using anti-Cereus aethiops. Between both subfamilies SJ varied from 0.412 to 0.697 while DBC varied from 0.172 to 0.387. On the other hand, between the two species of Cereus SJ was 0.971 and DBC was 0.091. Also, antigenic patterns of species of Cactoideae and Opuntia were obtained using anti-Opuntia elata var. cardiosperma. Between both subfamilies SJ ranged between 0.537 and 0.738 while DBC ranged between 0.128 and 0.247. Among species of Opuntia SJ varied from 0.744 to 0.946 while DBC varied from 0.082 to 0.168. Thus, findings from our study demonstrate that variability is lowest at the intraspecific level while it increases when different species of the same genus, subfamily, family or class are considered. This indicates that differences in antigenic patterns are tightly related to systematic affinities. It can thus be concluded that western blotting could help bring consensus to the current different points of view on systematic relationships at low taxonomic levels. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Cactoideae Opuntioideae Seed antigens Western blotting SDS-PAGE
1. Introduction Cactaceae, a native New World plant family, are typically found in a vast territory which extends from British Columbia and Alberta (56150 N) in Canada to Patagonia (50 S) in Argentina (Anderson, 2001). Four subfamilies of the Cactaceae have been identified by Anderson (2001), namely Cactoideae, Opuntioideae, Pereskioideae and Maihuenioideae. The main characteristic of species belonging to the subfamily Cactoideae, which is, in fact, the largest subfamily of the Cactaceae, is their diversity. Anderson (2001) observed that Cactoideae subfamily include 9 tribes and 108 genera which agrees, though with minor differences, with Buxbaum’s tribal classification of Cactoideae (Buxbaum, 1958; Endler and Buxbaum, 1974). As to the subfamily Opuntioideae, previous research has demonstrated that systematic relationships within this subfamily have not
Abbreviations: Caet, Cereus aethiops; DBC, Bray–Curtis’s Index; MW, Molecular Weight; Oela, Opuntia elata var. cardiosperma; PCO, Principal Coordinate Analysis; SDS-PAGE, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; SJ, Jaccard’s Index; TBS, Tris Buffered Saline; UPGMA, Unweighted PairGroup Method using Arithmetic Averages. * Corresponding author. Tel.: þ54 291 4595129; fax: þ54 291 4595130. E-mail address: [email protected] (M.J. Galvez). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.04.002
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only been but also still are a matter of controversy (Griffith and Porter, 2009). Opuntioideae includes 15 genera, the majority of whose species is included in Opuntia s.s Mill. Cactaceae in South America, in particular, is composed of 328 species and 39 genera of Cactoideae and of 66 species and 11 genera of Opuntioideae (Kiesling et al., 2008). Representative species of these taxa were therefore selected for this study. Since the late twentieth century large plantations of Cactaceae have been established and crops of several of its species have become of importance for economy enterprises (Shedbalkar et al., 2010). Kiesling (2001) recorded 12–15 genera and 50– 80 species with agricultural aptitude, the majority of which belong to the subfamily Cactoideae. Cactus fruits are at present merchandized for human and animal consumption and cactus plants are cultivated as ornamentals (Shedbalkar et al., 2010). Furthermore, as some species have either antioxidative effects (Huang et al., 2009) or anti-hyperglycemic properties (Andrade-Cetto and Wiedenfeld, 2011), they have also become of relevance for the pharmaceutical industry. Improvements in protein extraction and multiseparative methods (1D and 2D electrophoresis and capillary electrophoresis) have facilitated the exploitation of seed proteins as molecular markers of genetic variability (Cooke, 1995; Reynolds, 2007). Seed protein homology has also been the focus of attention in studies in which cross reactivities are compared using specific antibodies. The usefulness of traditional immunochemical methods has been demonstrated by Petersen and Fairbrothers (1985) and Shneyer et al. (2003) in other plant systematic groups. Also, results from our laboratory demonstrated that in Opuntioideae, western blotting not only broadens the usefulness range of traditional immunological techniques at the specific level but also complements data derived from electrophoretic patterns (Galvez et al., 2009). Compared to traditional techniques, western blotting is also a very useful research tool as it improves both reproducibility and sensitivity. In view of the above, this study is aimed to analyse and compare seed protein patterns of several species of Cactaceae, paying particular attention to the antigenic properties of seed proteins, in an attempt to assess their usefulness for the systematic study of this family. Immunological relationships with other families are also tested. 2. Materials and methods 2.1. Plant material Seeds of mature fruits from Argentinean spontaneous populations of the following species were studied: Harrisia pomanensis (F.A.C. Weber ex K. Schum.) Britton & Rose subsp. pomanensis; Trichocereus candicans (Gillies ex Salm-Dyck) Britton & Rose; Cereus forbesii Otto ex C.F. Först. and Cereus aethiops Haw. –Cactoideae–; Opuntia elata Salm-Dyck var. cardiosperma (K. Schum.) R. Kiesling; Opuntia megapotamica Arechav. (¼Opuntia salagria A. Cast.); Opuntia quimilo K. Schum.; Opuntia ficus-indica (L.) Mill.; Cylindropuntia imbricata (Haw.) Knuth and Tephrocactus articulatus (Pfeiff.) Backeb. var. articulatus –Opuntioideae–; Cucurbita maxima Duchesne –Cucurbitaceae–; Amaranthus hybridus L. subsp. hybridus –Amaranthaceae– and Chenopodium quinoa Willd. var. quinoa –Chenopodiaceae– (Zuloaga et al., 2008). Fruits of O. ficus-indica and seeds of A. hybridus and C. quinoa were purchased. All seeds were stored at 4 C until required. Voucher specimens were deposited at the herbarium of the Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Argentina (BBB). 2.2. Protein extraction Three grams of mature seeds of each sample of Opuntioideae were ground mechanically (Galvez et al., 2009). One gram of mature seeds corresponding to each sample of Cactoideae was ground in a pestle and mortar and the resulting powder was subsequently deoiled, air dried and kept at 4 C until required. Five mature seeds of C. maxima (0.85 g) and 1.5 g of mature seeds of A. hybridus and C. quinoa were processed in the same way. Seed proteins were extracted in 0.025 M Tris–0.192 M glycine buffer, pH 8.3 (10 ml/g of dry weight) at room temperature for 1 h. The suspension was centrifuged at 6000g during 10 min and the supernatant was stored at 20 C. Extract protein content was estimated following Bradford (1976) method and using bovine serum albumin as standard. 2.3. Antisera The seed protein extract obtained in Tris–glycine buffer from a population of Caet (C. aethiops, Salitral de la Vidriera, Villarino, Buenos Aires, Argentina) was used in order to produce antiserum from New Zealand rabbits (Galvez et al., 2009). Previously prepared anti-Oela (anti-O. elata var. cardiosperma) (Galvez et al., 2009) was also employed. 2.4. SDS-PAGE and western blotting Tricine-SDS-PAGE was performed in slab gels following the methodology previously described (Galvez et al., 2009). Prior to electrophoresis extracts were added to a denaturing sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue) and boiled for 3 min. After the run, gels were stained with 0.5% (w/v) Coomassie Brilliant Blue R-250. Alternatively, the proteins separated by electrophoresis were blotted onto a nitrocellulose membrane (Galvez et al., 2009). The membrane was blocked overnight at 4 C in 50 mM Tris buffered saline (TBS) solution (pH 7.4)
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containing 0.5% powdered skimmed milk. It was subsequently incubated at room temperature for 2 h in a 1:50 dilution of polyclonal serum raised against Caet or Oela. After several washings with TBS, the membrane was incubated at room temperature for 2 h in a 1:300 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. Washings with TBS were subsequently repeated. The colour was developed in the dark by treatment with 4-chloro-1-naphthol and hydrogen peroxide. No bands were observed when the membrane was incubated with pre-immune serum instead of with polyclonal antiserum. Phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa) were used as MW standards (Amersham Bioscience, Buckinghamshire, UK). 2.5. Data analysis Gels and membranes were scanned on a G-710 apparatus (Bio-Rad, Hercules, USA) and the corresponding densitometries were traced using Quantity One 4.0.2 software (Bio-Rad). The degree of similarity between the samples was determined by Jaccard’s Index (Krebs, 1999). Each band was scored as a binary character for either absence (0) or presence (1). The degree of distance between the samples was estimated by Bray–Curtis Index (Krebs, 1999). The relative heights of each peak in the densitometries were taken as a measure of relative concentration. Similarity and distance matrices were analysed phenetically with clustering (UPGMA) and ordination (PCO) methods using Multivariate Statistical Package 3.1 software for Windows (Kovach Computing Services, Wales, UK, 2002). Results were compared with the relationships traditionally considered for Cactaceae. 3. Results Our comparative analysis of electrophoretic and antigenic patterns from samples of two populations of Caet (Fig. 1) revealed that the former showed a total of 36 bands, all of which were found to be common to both populations (SJ: 1.000), and non-significant quantitative relative differences were observed (DBC: 0.076). The antigenic patterns, obtained using antiCaet as antiserum, showed 35 bands, all of which were also found to be common to both populations (SJ: 1.000). At least five bands (15, 67, 82.5, 85.5 and 102 kDa) of high intensity were observed in both patterns, yielding a DBC of 0.045. The SDS-PAGE patterns corresponding to 10 species of Cactaceae showed a total of 41 bands with MW between 7 and 103 kDa (Fig. 2A). Thirteen of these bands were common to all these species, i.e. 32% of the total pattern. In particular, a total of 38 bands was found in the species of Opuntioideae, two of which (12 and 15.3 kDa) were unique to this subfamily. On the other hand, a total of 39 bands was observed in the species of Cactoideae, 28 of which were common to the four species compared and three (31.5, 45 and 82.5 kDa) were found only in Cactoideae. In the case of the species of Cereus, qualitative differences were particularly observed only in two bands (24.3 and 82.5 kDa). SJ was 0.946 and DBC was 0.117. Among the species of Opuntioideae, SJ varied from 0.444 to 0.971 and among the species of Cactoideae, SJ was found to vary from 0.789 to 0.946 whereas DBC was observed to vary from 0.107 to 0.580 and between 0.117 and 0.243, respectively. The patterns corresponding to both subfamilies showed that SJ ranged from 0.421 to 0.821 while DBC ranged from 0.245 to 0.649. In addition, the dendrogram based on the similarity matrix showed two groups, one of which included C. imbricata and species of Opuntia while the other included species of Cactoideae, T. articulatus being distant from them all. Furthermore, the PCO graph
Fig. 1. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to Cereus. Anti-C. aethiops was used as antiserum in the western blotting. Ca1: Cereus aethiops, Salitral de la Vidriera, Buenos Aires (reference pattern in B); Ca2: C. aethiops, Salina Chica, Buenos Aires; Cf: C. forbesii, Serrezuela, Córdoba. On the left molecular mass standards (kDa).
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Fig. 2. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to species of Cactaceae. Anti-C. aethiops was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops (reference pattern in B); 5: Cylindropuntia imbricata; 6: Opuntia elata var. cardiosperma; 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica; 10: Tephrocactus articulatus. On the left molecular mass standards (kDa).
obtained from the distance matrix showed species of Cactoideae on the upper plane and species of Opuntioideae on the lower plane. T. articulatus was observed to remain distant from the other species (see Supplementary Material, SM1). The antigenic patterns corresponding to the 10 species of Cactaceae detected using anti-Caet as antiserum showed a total of 40 components between 12.5 and 113 kDa (Fig. 2B). All the species shared 11 bands, i.e. 28% of the total antigenic pattern. In Opuntioideae four (12.7, 23, 59.5 and 77.8 kDa) out of the 34 bands were unique to this subfamily whereas in Cactoideae six (17, 19, 23.5, 34, 36.5 and 72.5 kDa) out of the 36 bands were only found in this subfamily. The species of Cactoideae shared 25 bands, i.e. 69% of the subfamily antigenic pattern. A 96 kDa band was the only qualitative difference observed between the species of Cereus in which SJ was 0.971 and DBC was 0.091. For the six species of Opuntioideae analysed, SJ varied between 0.484 and 0.857 whereas DBC varied from 0.060 to 0.260. Our comparative analysis of the four species of Cactoideae showed that SJ varied between 0.722 and 0.971 whereas DBC ranged from 0.091 to 0.201. In addition, the comparison of the patterns corresponding to both subfamilies revealed that SJ varied between 0.412 and 0.697 whereas DBC ranged from 0.172 to 0.387. The dendrogram obtained from the similarity matrix (Fig. 3A) showed higher affinity between the species of Cereus than among the other eight species analysed. In addition, the species considered evolutionarily closer to the reference sample (Caet) showed higher affinity than the most distant ones. Fig. 3B shows the PCO graph made with the distance matrix. The antigenic pattern corresponding to Cactoideae and Opuntia obtained using anti-Oela as antiserum showed a total of 44 bands (13.5–112 kDa) (Fig. 4). A band (16 kDa) of relative high intensity was observed to be quantitatively characteristic of Opuntia. Among the species of Cactoideae SJ varied from 0.730 to 0.875 and among the species of Opuntia it ranged between 0.744 and 0.946. DBC varied from 0.094 to 0.171 and it ranged between 0.082 and 0.168. The antigenic patterns of Cereus showed qualitative (SJ: 0.875) and quantitative (DBC: 0.101) differences. The SJ estimated comparing patterns of species of Cactoideae and Opuntia varied between 0.537 and 0.738 whereas DBC ranged from 0.128 to 0.247. Qualitative affinity was highest within Cereus and quantitative distance was lowest between T. candicans and C. forbesii (see Supplementary Material, SM2). Comparison was also made of SDS-PAGE and antigenic patterns (Fig. 5) corresponding to species of Cactaceae (Oela and Caet), Chenopodiaceae (C. quinoa), Amaranthaceae (A. hybridus) and Cucurbitaceae (C. maxima). A total of 51 bands was observed in the electrophoretic pattern. Twenty of these bands were common to these five species, 29 bands revealed variable presence, one band (59.5 kDa) was observed only in the A. hybridus pattern and another one (44 kDa) was observed only in the Caet pattern. SJ varied from 0.521 to 0.750. The antigenic patterns obtained using anti-Caet as antiserum showed a total of 39 bands, of which five (48.5, 62.7, 82.7, 85.7 and 90.7 kDa) were shared by all of the species, 12 evidenced variable presence and one (89 kDa) was only observed in A. hybridus. Both species of Cactaceae exhibited 21 unique bands and SJ varied from 0.286 to 0.714. The dendrogram derived from SJ showed the Cactaceae grouped and separated from the other species analysed.
4. Discussion The introduction of new chemical tools, such as 2D electrophoresis and DNA-base analysis, and statistical procedures to study the macromolecular components of plants has paved the way to proteomics and genomics (Reynolds, 2007). When findings from these disciplines are complementarily used with traditional characters derived from phenotypic expression, they not only facilitate phylogeny reconstruction but also clarify the interpretation of biodiversity. In addition, modern immunochemical methods are potentially useful for the identification and study of interrelations among plants. The presence of an epitope in a protein is determined by its ability to specifically bind to an antibody. As a rule, these epitopes are located on hydrophilic, flexible and superficial regions of a native protein, where a few aminoacidic, polar and charged residues are involved in the interaction with the antibody. These regions, in turn, can be found in different
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Fig. 3. A. Neighbour joining tree (UPGMA) obtained from analysis of antigenic patterns of Cactaceae using Jaccard’s Index matrix. B. Graph of distribution following PCO (firsts three factors, 89.00% of the variance). Anti-C. aethiops was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops (Cactoideae, -); 5: Cylindropuntia imbricata; 6: Opuntia elata var. cardiosperma; 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica; 10: Tephrocactus articulatus (Opuntioideae, :).
polypeptides that explain cross-reaction (Harlow and Lane, 1988). The level at which diverse epitopes are shared by different taxa can help to infer possible taxonomic affinities as it is related to epitope structural homology. The electrophoretic and antigenic patterns shown using anti-Caet in Caet populations, were found to be qualitatively identical and only minor quantitative differences could be observed (Fig. 1). Similar results were collected in a recent study in which intraspecific variability of Oela was analysed (Galvez et al., 2009). The electrophoretic and antigenic patterns of species of Cereus obtained with anti-Caet evidenced high uniformity although further qualitative and quantitative analyses revealed more differences than at the intraspecific level (Figs. 1–3). Similarly, the species of Cereus evidenced higher affinity with respect to species of other genera (Fig. 3). Nevertheless, this was not the case when Cereus patterns were obtained using antiOela (see Supplementary Material, SM2) as it is discussed below. The four species of Cactoideae showed patterns sharing 72% of the bands separated by SDS-PAGE whereas 69% of such bands were shared when anti-Caet was used. In both cases the degree of homology decreased to near 30% when species of the
Fig. 4. Antigenic pattern of seed proteins corresponding to species of Cactaceae. Anti-Opuntia elata var. cardiosperma was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops; 6: O. elata var. cardiosperma (reference pattern); 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica. On the left molecular mass standards (kDa).
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Fig. 5. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to species of Magnoliopsida. Anti-C. aethiops was used as antiserum in the western blotting. 1: Cucurbita maxima; 2: Amaranthus hybridus; 3: Chenopodium quinoa; 4: Cereus aethiops (reference pattern in B); 5: Opuntia elata var. cardiosperma. On the left molecular mass standards (kDa).
subfamily Opuntioideae were included in the comparative analysis. This suggests that differences increases when distantly related taxa within the family are included. On the other hand, when anti-Caet was used, numerous bands of the antigenic pattern, such as those corresponding to 17– 43 kDa (Fig. 2B), could be detected in the Cactoideae while they were very weak in the Opuntioideae. Variability in cross reactivity could be attributed to the fact that the species belong to two different subfamilies. Likewise, the high similarity in the patterns corresponding to species of Opuntioideae was found to be noteworthy (Fig. 3). Nonetheless, it must be taken into account that comparisons must be made using the reference reaction, in this particular case, Caet pattern. It could thus be inferred that all the species of Opuntioideae share the same group of structural components as those of Caet. Likewise, high uniformity was observed among the Cactoideae when anti-Oela was used. Based on the reference pattern, the quantitatively most important bands in Oela were found to be 16, 30 and 61 kDa polypeptides (Fig. 4). It was also observed that although antigenicity of 61 kDa polypeptides decreased, it remained important among the Cactoideae (except in H. pomanensis). On the other hand, 16 and 30 kDa polypeptides were either not observed at all or they were weakly shown among the Cactoideae. Only among Opuntia species were these bands found to be equally relevant in terms of relative intensity (Galvez et al., 2009), thus confirming them as important molecular markers for the genus. In order to compare the protein patterns of Cactaceae with more distant taxonomic groups (Takhtajan, 2009), samples of Oela and Caet were compared with members of other subclasses, namely C. quinoa and A. hybridus (Caryophyllidae), and C. maxima (Dilleniidae) (Fig. 5). The numerous bands with matching molecular weights are not necessarily indicative of common molecular components, particularly when remote evolutionary histories are accepted. The patterns obtained using anti-Caet were found to clearly show closer affinities among Cactaceae as compared with other taxonomic groups. Relatively few epitopes of the Cactaceae were shared with species of the other families considered in this study, including those belonging to the same subclass. These common epitopes can be interpreted as an evidence of evolutionary conserved aminoacidic sequences. Furthermore, complexity in the electrophoretic pattern in contrast to the relative simplicity observed in the antigenic pattern demonstrates the high degree of seed protein polymorphism. Electrophoresis of seed proteins and enzymes has been successfully used for the identification of other plant taxa. In this respect, either direct comparison of band patterns of different samples or the evaluation of the frequency with which bands are detected are usual procedures in this type of studies (Cooke, 1995) and they can both be applied as well to western blotting. On the other hand, immunochemical analysis is a high-specificity procedure both for the detection of homologies and the mapping of conserved regions shared among samples. Taken together, findings from our study demonstrate that differences in antigenic patterns are tightly related to systematic affinity. They also show that it decreases at the intraspecific level while it increases when different species of the same genus, subfamily, family or class are considered. The notorious morphological features of particular species of either Cactoideae or Opuntioideae facilitate characterization at this taxonomic level, our results therefore suggest that western blotting could help bring consensus to the different points of view on systematic relationships at low taxonomic levels. In this respect, only O. elata var. cardiosperma (and other species of the genus) was found to show bands that were not observed in other species of Cactaceae. Also unique bands were identified only in C. aethiops and C. forbesii when the reference pattern was used.
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It can therefore be concluded that western blotting, particularly the use of antigenic patterns, serves as a useful tool to study Cactoideae, the most numerous subfamily which, on account of this, has led to conflicting disagreement on its relationships. Furthermore, although genome analysis has contributed to clarifying such dissent, it has also given rise to new queries (Nyffeler, 2002). Among the Opuntioideae, a pending challenge not yet fully elucidated is to interpret the internal systematics of the genus Opuntia and its relatives (Griffith and Porter, 2009). Future research about specific cases of dissent on systematic relationships will thus help precise the role of immunochemical methods in their clarification. Appendix A. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.bse.2012.04.002. References Anderson, E.F., 2001. The Cactus Family. Timber Press, Portland, Oregon. Andrade-Cetto, A., Wiedenfeld, H., 2011. Anti-hyperglycemic effect of Opuntia streptacantha Lem. J. Ethnopharmacol. 133, 940–943. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Buxbaum, F., 1958. The phylogenetic division of the subfamily Cereoideae, Cactaceae. Madroño 14, 177–206. Cooke, R.J., 1995. Gel electrophoresis for the identification of plant varieties. J. Chromatogr. A 698, 281–299. Endler, J., Buxbaum, F., 1974. Die Pflazenfamilie der Kakteen. Albert Philler Verlag, Minden. Galvez, M.J., Castro, H.A., Villamil, C.B., 2009. Antigenic patterns of seed proteins in Opuntioideae (Cactaceae). Biochem. Syst. Ecol. 37, 91–97. Griffith, M.P., Porter, J.M., 2009. Phylogeny of Opuntioideae (Cactaceae). Int. J. Plant Sci. 170, 107–116. Harlow, E., Lane, D., 1988. Antibody-antigen interaction. In: Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Plainview, New York, pp. 23–35. Huang, X., Li, Q., Li, H., Guo, L., 2009. Neuroprotective and antioxidative effect of cactus polysaccharides in vivo and in vitro. Cell. Mol. Neurobiol. 29, 1211–1221. Kiesling, R., 2001. Cactáceas de la Argentina promisorias agronómicamente. J. Prof. Assoc. Cactus Dev. 6, 11–14. Kiesling, R., Larocca, J., Faúndez, L., Metzing, D., Albesiano, S., 2008. Cactaceae. In: Zuloaga, F.O., Morrone, O., Belgrano, M.J. (Eds.), Catálogo de las Plantas Vasculares del Cono Sur (Argentina, Sur de Brasil, Chile, Paraguay y Uruguay). Dicotyledoneae: Acanthaceae-Fabaceae (Abarema-Schizolobium). Monographs in Systematic Botany 107, vol. 2. Missouri Botanical Garden Press, Saint Louis, pp. 1715–1830. Krebs, C.J., 1999. Ecological Methodology. A. Wesley Longman, New York, pp. 375–406. Nyffeler, R., 2002. Phylogenetic relationships in the cactus family (Cactaceae) based on evidence from trnK/matK and trnL/trnF sequences. Am. J. Bot. 89, 312–326. Petersen, F.P., Fairbrothers, D.E., 1985. A serotaxonomic appraisal of Amphipterygium and Leitneria – two amentiferous taxa of Rutiflorae (Rosidae). Syst. Bot. 8, 134–148. Reynolds, T., 2007. The evolution of chemosystematics. Phytochemistry 68, 2887–2895. Shedbalkar, U.U., Adki, V.S., Jadhav, J.P., Bapat, V.A., 2010. Opuntia and other cacti: applications and biotechnological insights. Trop. Plant Biol. 3, 136–150. Shneyer, V.S., Kutyavina, N.G., Pimenov, M.G., 2003. Systematic relationships within and between Peucedanum and Angelica (Umbelliferae-Peucedaneae) inferred from immunological studies of seed proteins. Plant Syst. Evol. 236, 175–194. Takhtajan, A., 2009. Flowering Plants. Springer, Berlin. Zuloaga, F.O., Morrone, O., Belgrano, M.J. (Eds.), 2008. Catálogo de las Plantas Vasculares del Cono Sur (Argentina, Sur de Brasil, Chile, Paraguay y Uruguay). Dicotyledoneae: Acanthaceae-Fabaceae (Abarema-Schizolobium), Monographs in Systematic Botany 107, vol. 2. Missouri Botanical Garden Press, Saint Louis.
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Immunochemical properties of seed proteins as systematic markers in Cactaceae Marcelo J. Galvez*, María I. Prat, Carlos B. Villamil Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2012 Accepted 6 April 2012 Available online xxx
The purpose of this study was to compare seed protein patterns of 10 species of Cactaceae, a native New World plant family distributed from Canada to Argentina, in an attempt to assess their usefulness for systematic studies. Particular attention was paid to analysing antigenic patterns derived from western blotting carried out with different antisera. Similarity (SJ) and distance (DBC) indices were further used to carry out Cluster Analysis (UPGMA) and Principal Coordinate Analysis. Antigenic patterns of species of Opuntioideae and Cactoideae were obtained using anti-Cereus aethiops. Between both subfamilies SJ varied from 0.412 to 0.697 while DBC varied from 0.172 to 0.387. On the other hand, between the two species of Cereus SJ was 0.971 and DBC was 0.091. Also, antigenic patterns of species of Cactoideae and Opuntia were obtained using anti-Opuntia elata var. cardiosperma. Between both subfamilies SJ ranged between 0.537 and 0.738 while DBC ranged between 0.128 and 0.247. Among species of Opuntia SJ varied from 0.744 to 0.946 while DBC varied from 0.082 to 0.168. Thus, findings from our study demonstrate that variability is lowest at the intraspecific level while it increases when different species of the same genus, subfamily, family or class are considered. This indicates that differences in antigenic patterns are tightly related to systematic affinities. It can thus be concluded that western blotting could help bring consensus to the current different points of view on systematic relationships at low taxonomic levels. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Cactoideae Opuntioideae Seed antigens Western blotting SDS-PAGE
1. Introduction Cactaceae, a native New World plant family, are typically found in a vast territory which extends from British Columbia and Alberta (56150 N) in Canada to Patagonia (50 S) in Argentina (Anderson, 2001). Four subfamilies of the Cactaceae have been identified by Anderson (2001), namely Cactoideae, Opuntioideae, Pereskioideae and Maihuenioideae. The main characteristic of species belonging to the subfamily Cactoideae, which is, in fact, the largest subfamily of the Cactaceae, is their diversity. Anderson (2001) observed that Cactoideae subfamily include 9 tribes and 108 genera which agrees, though with minor differences, with Buxbaum’s tribal classification of Cactoideae (Buxbaum, 1958; Endler and Buxbaum, 1974). As to the subfamily Opuntioideae, previous research has demonstrated that systematic relationships within this subfamily have not
Abbreviations: Caet, Cereus aethiops; DBC, Bray–Curtis’s Index; MW, Molecular Weight; Oela, Opuntia elata var. cardiosperma; PCO, Principal Coordinate Analysis; SDS-PAGE, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; SJ, Jaccard’s Index; TBS, Tris Buffered Saline; UPGMA, Unweighted PairGroup Method using Arithmetic Averages. * Corresponding author. Tel.: þ54 291 4595129; fax: þ54 291 4595130. E-mail address: [email protected] (M.J. Galvez). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.04.002
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only been but also still are a matter of controversy (Griffith and Porter, 2009). Opuntioideae includes 15 genera, the majority of whose species is included in Opuntia s.s Mill. Cactaceae in South America, in particular, is composed of 328 species and 39 genera of Cactoideae and of 66 species and 11 genera of Opuntioideae (Kiesling et al., 2008). Representative species of these taxa were therefore selected for this study. Since the late twentieth century large plantations of Cactaceae have been established and crops of several of its species have become of importance for economy enterprises (Shedbalkar et al., 2010). Kiesling (2001) recorded 12–15 genera and 50– 80 species with agricultural aptitude, the majority of which belong to the subfamily Cactoideae. Cactus fruits are at present merchandized for human and animal consumption and cactus plants are cultivated as ornamentals (Shedbalkar et al., 2010). Furthermore, as some species have either antioxidative effects (Huang et al., 2009) or anti-hyperglycemic properties (Andrade-Cetto and Wiedenfeld, 2011), they have also become of relevance for the pharmaceutical industry. Improvements in protein extraction and multiseparative methods (1D and 2D electrophoresis and capillary electrophoresis) have facilitated the exploitation of seed proteins as molecular markers of genetic variability (Cooke, 1995; Reynolds, 2007). Seed protein homology has also been the focus of attention in studies in which cross reactivities are compared using specific antibodies. The usefulness of traditional immunochemical methods has been demonstrated by Petersen and Fairbrothers (1985) and Shneyer et al. (2003) in other plant systematic groups. Also, results from our laboratory demonstrated that in Opuntioideae, western blotting not only broadens the usefulness range of traditional immunological techniques at the specific level but also complements data derived from electrophoretic patterns (Galvez et al., 2009). Compared to traditional techniques, western blotting is also a very useful research tool as it improves both reproducibility and sensitivity. In view of the above, this study is aimed to analyse and compare seed protein patterns of several species of Cactaceae, paying particular attention to the antigenic properties of seed proteins, in an attempt to assess their usefulness for the systematic study of this family. Immunological relationships with other families are also tested. 2. Materials and methods 2.1. Plant material Seeds of mature fruits from Argentinean spontaneous populations of the following species were studied: Harrisia pomanensis (F.A.C. Weber ex K. Schum.) Britton & Rose subsp. pomanensis; Trichocereus candicans (Gillies ex Salm-Dyck) Britton & Rose; Cereus forbesii Otto ex C.F. Först. and Cereus aethiops Haw. –Cactoideae–; Opuntia elata Salm-Dyck var. cardiosperma (K. Schum.) R. Kiesling; Opuntia megapotamica Arechav. (¼Opuntia salagria A. Cast.); Opuntia quimilo K. Schum.; Opuntia ficus-indica (L.) Mill.; Cylindropuntia imbricata (Haw.) Knuth and Tephrocactus articulatus (Pfeiff.) Backeb. var. articulatus –Opuntioideae–; Cucurbita maxima Duchesne –Cucurbitaceae–; Amaranthus hybridus L. subsp. hybridus –Amaranthaceae– and Chenopodium quinoa Willd. var. quinoa –Chenopodiaceae– (Zuloaga et al., 2008). Fruits of O. ficus-indica and seeds of A. hybridus and C. quinoa were purchased. All seeds were stored at 4 C until required. Voucher specimens were deposited at the herbarium of the Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Argentina (BBB). 2.2. Protein extraction Three grams of mature seeds of each sample of Opuntioideae were ground mechanically (Galvez et al., 2009). One gram of mature seeds corresponding to each sample of Cactoideae was ground in a pestle and mortar and the resulting powder was subsequently deoiled, air dried and kept at 4 C until required. Five mature seeds of C. maxima (0.85 g) and 1.5 g of mature seeds of A. hybridus and C. quinoa were processed in the same way. Seed proteins were extracted in 0.025 M Tris–0.192 M glycine buffer, pH 8.3 (10 ml/g of dry weight) at room temperature for 1 h. The suspension was centrifuged at 6000g during 10 min and the supernatant was stored at 20 C. Extract protein content was estimated following Bradford (1976) method and using bovine serum albumin as standard. 2.3. Antisera The seed protein extract obtained in Tris–glycine buffer from a population of Caet (C. aethiops, Salitral de la Vidriera, Villarino, Buenos Aires, Argentina) was used in order to produce antiserum from New Zealand rabbits (Galvez et al., 2009). Previously prepared anti-Oela (anti-O. elata var. cardiosperma) (Galvez et al., 2009) was also employed. 2.4. SDS-PAGE and western blotting Tricine-SDS-PAGE was performed in slab gels following the methodology previously described (Galvez et al., 2009). Prior to electrophoresis extracts were added to a denaturing sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue) and boiled for 3 min. After the run, gels were stained with 0.5% (w/v) Coomassie Brilliant Blue R-250. Alternatively, the proteins separated by electrophoresis were blotted onto a nitrocellulose membrane (Galvez et al., 2009). The membrane was blocked overnight at 4 C in 50 mM Tris buffered saline (TBS) solution (pH 7.4)
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containing 0.5% powdered skimmed milk. It was subsequently incubated at room temperature for 2 h in a 1:50 dilution of polyclonal serum raised against Caet or Oela. After several washings with TBS, the membrane was incubated at room temperature for 2 h in a 1:300 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. Washings with TBS were subsequently repeated. The colour was developed in the dark by treatment with 4-chloro-1-naphthol and hydrogen peroxide. No bands were observed when the membrane was incubated with pre-immune serum instead of with polyclonal antiserum. Phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa) were used as MW standards (Amersham Bioscience, Buckinghamshire, UK). 2.5. Data analysis Gels and membranes were scanned on a G-710 apparatus (Bio-Rad, Hercules, USA) and the corresponding densitometries were traced using Quantity One 4.0.2 software (Bio-Rad). The degree of similarity between the samples was determined by Jaccard’s Index (Krebs, 1999). Each band was scored as a binary character for either absence (0) or presence (1). The degree of distance between the samples was estimated by Bray–Curtis Index (Krebs, 1999). The relative heights of each peak in the densitometries were taken as a measure of relative concentration. Similarity and distance matrices were analysed phenetically with clustering (UPGMA) and ordination (PCO) methods using Multivariate Statistical Package 3.1 software for Windows (Kovach Computing Services, Wales, UK, 2002). Results were compared with the relationships traditionally considered for Cactaceae. 3. Results Our comparative analysis of electrophoretic and antigenic patterns from samples of two populations of Caet (Fig. 1) revealed that the former showed a total of 36 bands, all of which were found to be common to both populations (SJ: 1.000), and non-significant quantitative relative differences were observed (DBC: 0.076). The antigenic patterns, obtained using antiCaet as antiserum, showed 35 bands, all of which were also found to be common to both populations (SJ: 1.000). At least five bands (15, 67, 82.5, 85.5 and 102 kDa) of high intensity were observed in both patterns, yielding a DBC of 0.045. The SDS-PAGE patterns corresponding to 10 species of Cactaceae showed a total of 41 bands with MW between 7 and 103 kDa (Fig. 2A). Thirteen of these bands were common to all these species, i.e. 32% of the total pattern. In particular, a total of 38 bands was found in the species of Opuntioideae, two of which (12 and 15.3 kDa) were unique to this subfamily. On the other hand, a total of 39 bands was observed in the species of Cactoideae, 28 of which were common to the four species compared and three (31.5, 45 and 82.5 kDa) were found only in Cactoideae. In the case of the species of Cereus, qualitative differences were particularly observed only in two bands (24.3 and 82.5 kDa). SJ was 0.946 and DBC was 0.117. Among the species of Opuntioideae, SJ varied from 0.444 to 0.971 and among the species of Cactoideae, SJ was found to vary from 0.789 to 0.946 whereas DBC was observed to vary from 0.107 to 0.580 and between 0.117 and 0.243, respectively. The patterns corresponding to both subfamilies showed that SJ ranged from 0.421 to 0.821 while DBC ranged from 0.245 to 0.649. In addition, the dendrogram based on the similarity matrix showed two groups, one of which included C. imbricata and species of Opuntia while the other included species of Cactoideae, T. articulatus being distant from them all. Furthermore, the PCO graph
Fig. 1. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to Cereus. Anti-C. aethiops was used as antiserum in the western blotting. Ca1: Cereus aethiops, Salitral de la Vidriera, Buenos Aires (reference pattern in B); Ca2: C. aethiops, Salina Chica, Buenos Aires; Cf: C. forbesii, Serrezuela, Córdoba. On the left molecular mass standards (kDa).
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Fig. 2. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to species of Cactaceae. Anti-C. aethiops was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops (reference pattern in B); 5: Cylindropuntia imbricata; 6: Opuntia elata var. cardiosperma; 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica; 10: Tephrocactus articulatus. On the left molecular mass standards (kDa).
obtained from the distance matrix showed species of Cactoideae on the upper plane and species of Opuntioideae on the lower plane. T. articulatus was observed to remain distant from the other species (see Supplementary Material, SM1). The antigenic patterns corresponding to the 10 species of Cactaceae detected using anti-Caet as antiserum showed a total of 40 components between 12.5 and 113 kDa (Fig. 2B). All the species shared 11 bands, i.e. 28% of the total antigenic pattern. In Opuntioideae four (12.7, 23, 59.5 and 77.8 kDa) out of the 34 bands were unique to this subfamily whereas in Cactoideae six (17, 19, 23.5, 34, 36.5 and 72.5 kDa) out of the 36 bands were only found in this subfamily. The species of Cactoideae shared 25 bands, i.e. 69% of the subfamily antigenic pattern. A 96 kDa band was the only qualitative difference observed between the species of Cereus in which SJ was 0.971 and DBC was 0.091. For the six species of Opuntioideae analysed, SJ varied between 0.484 and 0.857 whereas DBC varied from 0.060 to 0.260. Our comparative analysis of the four species of Cactoideae showed that SJ varied between 0.722 and 0.971 whereas DBC ranged from 0.091 to 0.201. In addition, the comparison of the patterns corresponding to both subfamilies revealed that SJ varied between 0.412 and 0.697 whereas DBC ranged from 0.172 to 0.387. The dendrogram obtained from the similarity matrix (Fig. 3A) showed higher affinity between the species of Cereus than among the other eight species analysed. In addition, the species considered evolutionarily closer to the reference sample (Caet) showed higher affinity than the most distant ones. Fig. 3B shows the PCO graph made with the distance matrix. The antigenic pattern corresponding to Cactoideae and Opuntia obtained using anti-Oela as antiserum showed a total of 44 bands (13.5–112 kDa) (Fig. 4). A band (16 kDa) of relative high intensity was observed to be quantitatively characteristic of Opuntia. Among the species of Cactoideae SJ varied from 0.730 to 0.875 and among the species of Opuntia it ranged between 0.744 and 0.946. DBC varied from 0.094 to 0.171 and it ranged between 0.082 and 0.168. The antigenic patterns of Cereus showed qualitative (SJ: 0.875) and quantitative (DBC: 0.101) differences. The SJ estimated comparing patterns of species of Cactoideae and Opuntia varied between 0.537 and 0.738 whereas DBC ranged from 0.128 to 0.247. Qualitative affinity was highest within Cereus and quantitative distance was lowest between T. candicans and C. forbesii (see Supplementary Material, SM2). Comparison was also made of SDS-PAGE and antigenic patterns (Fig. 5) corresponding to species of Cactaceae (Oela and Caet), Chenopodiaceae (C. quinoa), Amaranthaceae (A. hybridus) and Cucurbitaceae (C. maxima). A total of 51 bands was observed in the electrophoretic pattern. Twenty of these bands were common to these five species, 29 bands revealed variable presence, one band (59.5 kDa) was observed only in the A. hybridus pattern and another one (44 kDa) was observed only in the Caet pattern. SJ varied from 0.521 to 0.750. The antigenic patterns obtained using anti-Caet as antiserum showed a total of 39 bands, of which five (48.5, 62.7, 82.7, 85.7 and 90.7 kDa) were shared by all of the species, 12 evidenced variable presence and one (89 kDa) was only observed in A. hybridus. Both species of Cactaceae exhibited 21 unique bands and SJ varied from 0.286 to 0.714. The dendrogram derived from SJ showed the Cactaceae grouped and separated from the other species analysed.
4. Discussion The introduction of new chemical tools, such as 2D electrophoresis and DNA-base analysis, and statistical procedures to study the macromolecular components of plants has paved the way to proteomics and genomics (Reynolds, 2007). When findings from these disciplines are complementarily used with traditional characters derived from phenotypic expression, they not only facilitate phylogeny reconstruction but also clarify the interpretation of biodiversity. In addition, modern immunochemical methods are potentially useful for the identification and study of interrelations among plants. The presence of an epitope in a protein is determined by its ability to specifically bind to an antibody. As a rule, these epitopes are located on hydrophilic, flexible and superficial regions of a native protein, where a few aminoacidic, polar and charged residues are involved in the interaction with the antibody. These regions, in turn, can be found in different
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Fig. 3. A. Neighbour joining tree (UPGMA) obtained from analysis of antigenic patterns of Cactaceae using Jaccard’s Index matrix. B. Graph of distribution following PCO (firsts three factors, 89.00% of the variance). Anti-C. aethiops was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops (Cactoideae, -); 5: Cylindropuntia imbricata; 6: Opuntia elata var. cardiosperma; 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica; 10: Tephrocactus articulatus (Opuntioideae, :).
polypeptides that explain cross-reaction (Harlow and Lane, 1988). The level at which diverse epitopes are shared by different taxa can help to infer possible taxonomic affinities as it is related to epitope structural homology. The electrophoretic and antigenic patterns shown using anti-Caet in Caet populations, were found to be qualitatively identical and only minor quantitative differences could be observed (Fig. 1). Similar results were collected in a recent study in which intraspecific variability of Oela was analysed (Galvez et al., 2009). The electrophoretic and antigenic patterns of species of Cereus obtained with anti-Caet evidenced high uniformity although further qualitative and quantitative analyses revealed more differences than at the intraspecific level (Figs. 1–3). Similarly, the species of Cereus evidenced higher affinity with respect to species of other genera (Fig. 3). Nevertheless, this was not the case when Cereus patterns were obtained using antiOela (see Supplementary Material, SM2) as it is discussed below. The four species of Cactoideae showed patterns sharing 72% of the bands separated by SDS-PAGE whereas 69% of such bands were shared when anti-Caet was used. In both cases the degree of homology decreased to near 30% when species of the
Fig. 4. Antigenic pattern of seed proteins corresponding to species of Cactaceae. Anti-Opuntia elata var. cardiosperma was used as antiserum in the western blotting. 1: Harrisia pomanensis; 2: Trichocereus candicans; 3: Cereus forbesii; 4: C. aethiops; 6: O. elata var. cardiosperma (reference pattern); 7: O. quimilo; 8: O. megapotamica; 9: O. ficus-indica. On the left molecular mass standards (kDa).
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Fig. 5. Electrophoretic (A) and antigenic (B) patterns of seed proteins corresponding to species of Magnoliopsida. Anti-C. aethiops was used as antiserum in the western blotting. 1: Cucurbita maxima; 2: Amaranthus hybridus; 3: Chenopodium quinoa; 4: Cereus aethiops (reference pattern in B); 5: Opuntia elata var. cardiosperma. On the left molecular mass standards (kDa).
subfamily Opuntioideae were included in the comparative analysis. This suggests that differences increases when distantly related taxa within the family are included. On the other hand, when anti-Caet was used, numerous bands of the antigenic pattern, such as those corresponding to 17– 43 kDa (Fig. 2B), could be detected in the Cactoideae while they were very weak in the Opuntioideae. Variability in cross reactivity could be attributed to the fact that the species belong to two different subfamilies. Likewise, the high similarity in the patterns corresponding to species of Opuntioideae was found to be noteworthy (Fig. 3). Nonetheless, it must be taken into account that comparisons must be made using the reference reaction, in this particular case, Caet pattern. It could thus be inferred that all the species of Opuntioideae share the same group of structural components as those of Caet. Likewise, high uniformity was observed among the Cactoideae when anti-Oela was used. Based on the reference pattern, the quantitatively most important bands in Oela were found to be 16, 30 and 61 kDa polypeptides (Fig. 4). It was also observed that although antigenicity of 61 kDa polypeptides decreased, it remained important among the Cactoideae (except in H. pomanensis). On the other hand, 16 and 30 kDa polypeptides were either not observed at all or they were weakly shown among the Cactoideae. Only among Opuntia species were these bands found to be equally relevant in terms of relative intensity (Galvez et al., 2009), thus confirming them as important molecular markers for the genus. In order to compare the protein patterns of Cactaceae with more distant taxonomic groups (Takhtajan, 2009), samples of Oela and Caet were compared with members of other subclasses, namely C. quinoa and A. hybridus (Caryophyllidae), and C. maxima (Dilleniidae) (Fig. 5). The numerous bands with matching molecular weights are not necessarily indicative of common molecular components, particularly when remote evolutionary histories are accepted. The patterns obtained using anti-Caet were found to clearly show closer affinities among Cactaceae as compared with other taxonomic groups. Relatively few epitopes of the Cactaceae were shared with species of the other families considered in this study, including those belonging to the same subclass. These common epitopes can be interpreted as an evidence of evolutionary conserved aminoacidic sequences. Furthermore, complexity in the electrophoretic pattern in contrast to the relative simplicity observed in the antigenic pattern demonstrates the high degree of seed protein polymorphism. Electrophoresis of seed proteins and enzymes has been successfully used for the identification of other plant taxa. In this respect, either direct comparison of band patterns of different samples or the evaluation of the frequency with which bands are detected are usual procedures in this type of studies (Cooke, 1995) and they can both be applied as well to western blotting. On the other hand, immunochemical analysis is a high-specificity procedure both for the detection of homologies and the mapping of conserved regions shared among samples. Taken together, findings from our study demonstrate that differences in antigenic patterns are tightly related to systematic affinity. They also show that it decreases at the intraspecific level while it increases when different species of the same genus, subfamily, family or class are considered. The notorious morphological features of particular species of either Cactoideae or Opuntioideae facilitate characterization at this taxonomic level, our results therefore suggest that western blotting could help bring consensus to the different points of view on systematic relationships at low taxonomic levels. In this respect, only O. elata var. cardiosperma (and other species of the genus) was found to show bands that were not observed in other species of Cactaceae. Also unique bands were identified only in C. aethiops and C. forbesii when the reference pattern was used.
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It can therefore be concluded that western blotting, particularly the use of antigenic patterns, serves as a useful tool to study Cactoideae, the most numerous subfamily which, on account of this, has led to conflicting disagreement on its relationships. Furthermore, although genome analysis has contributed to clarifying such dissent, it has also given rise to new queries (Nyffeler, 2002). Among the Opuntioideae, a pending challenge not yet fully elucidated is to interpret the internal systematics of the genus Opuntia and its relatives (Griffith and Porter, 2009). Future research about specific cases of dissent on systematic relationships will thus help precise the role of immunochemical methods in their clarification. Appendix A. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.bse.2012.04.002. References Anderson, E.F., 2001. The Cactus Family. Timber Press, Portland, Oregon. Andrade-Cetto, A., Wiedenfeld, H., 2011. Anti-hyperglycemic effect of Opuntia streptacantha Lem. J. Ethnopharmacol. 133, 940–943. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Buxbaum, F., 1958. The phylogenetic division of the subfamily Cereoideae, Cactaceae. Madroño 14, 177–206. Cooke, R.J., 1995. Gel electrophoresis for the identification of plant varieties. J. Chromatogr. A 698, 281–299. Endler, J., Buxbaum, F., 1974. Die Pflazenfamilie der Kakteen. Albert Philler Verlag, Minden. Galvez, M.J., Castro, H.A., Villamil, C.B., 2009. Antigenic patterns of seed proteins in Opuntioideae (Cactaceae). Biochem. Syst. Ecol. 37, 91–97. Griffith, M.P., Porter, J.M., 2009. Phylogeny of Opuntioideae (Cactaceae). Int. J. Plant Sci. 170, 107–116. Harlow, E., Lane, D., 1988. Antibody-antigen interaction. In: Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Plainview, New York, pp. 23–35. Huang, X., Li, Q., Li, H., Guo, L., 2009. Neuroprotective and antioxidative effect of cactus polysaccharides in vivo and in vitro. Cell. Mol. Neurobiol. 29, 1211–1221. Kiesling, R., 2001. Cactáceas de la Argentina promisorias agronómicamente. J. Prof. Assoc. Cactus Dev. 6, 11–14. Kiesling, R., Larocca, J., Faúndez, L., Metzing, D., Albesiano, S., 2008. Cactaceae. In: Zuloaga, F.O., Morrone, O., Belgrano, M.J. (Eds.), Catálogo de las Plantas Vasculares del Cono Sur (Argentina, Sur de Brasil, Chile, Paraguay y Uruguay). Dicotyledoneae: Acanthaceae-Fabaceae (Abarema-Schizolobium). Monographs in Systematic Botany 107, vol. 2. Missouri Botanical Garden Press, Saint Louis, pp. 1715–1830. Krebs, C.J., 1999. Ecological Methodology. A. Wesley Longman, New York, pp. 375–406. Nyffeler, R., 2002. Phylogenetic relationships in the cactus family (Cactaceae) based on evidence from trnK/matK and trnL/trnF sequences. Am. J. Bot. 89, 312–326. Petersen, F.P., Fairbrothers, D.E., 1985. A serotaxonomic appraisal of Amphipterygium and Leitneria – two amentiferous taxa of Rutiflorae (Rosidae). Syst. Bot. 8, 134–148. Reynolds, T., 2007. The evolution of chemosystematics. Phytochemistry 68, 2887–2895. Shedbalkar, U.U., Adki, V.S., Jadhav, J.P., Bapat, V.A., 2010. Opuntia and other cacti: applications and biotechnological insights. Trop. Plant Biol. 3, 136–150. Shneyer, V.S., Kutyavina, N.G., Pimenov, M.G., 2003. Systematic relationships within and between Peucedanum and Angelica (Umbelliferae-Peucedaneae) inferred from immunological studies of seed proteins. Plant Syst. Evol. 236, 175–194. Takhtajan, A., 2009. Flowering Plants. Springer, Berlin. Zuloaga, F.O., Morrone, O., Belgrano, M.J. (Eds.), 2008. Catálogo de las Plantas Vasculares del Cono Sur (Argentina, Sur de Brasil, Chile, Paraguay y Uruguay). Dicotyledoneae: Acanthaceae-Fabaceae (Abarema-Schizolobium), Monographs in Systematic Botany 107, vol. 2. Missouri Botanical Garden Press, Saint Louis.