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Antigenic patterns of seed proteins in Opuntioideae (Cactaceae)
Biochemical Systematics and Ecology 37 (2009) 91–97
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Antigenic patterns of seed proteins in Opuntioideae (Cactaceae) Marcelo J. Galvez*, Hugo A. Castro, Carlos B. Villamil Departamento de Biologı´a, Bioquı´mica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahı´a Blanca, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2008 Accepted 23 December 2008
The antigenic patterns recognised by Western blotting in seed proteins of species of Opuntioideae (Cactaceae) were analysed in an attempt to evaluate their usefulness in systematics. Total protein profiles were also analysed by SDS–PAGE. The resulting similarity and distance matrices were further used to carry out Cluster Analysis (UPGMA) and Principal Coordinates Analysis. Populations of Opuntia cardiosperma were found to exhibit a prominent morphological uniformity, a unique electrophoretic pattern and a uniform antigenic pattern. The latter was obtained using anti-O. cardiosperma as antiserum. Results from the qualitative and quantitative interspecific analyses of antigenic profiles helped to characterise all the species studied. Tephrocactus articulatus and Cylindropuntia imbricata evidenced lower affinity with O. cardiosperma than the species of Opuntia s.s. Our results demonstrate that in Cactaceae, Western blotting analysis broadens the usefulness range of immunological techniques at the specific level and complements the information collected from electrophoretic profiles. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Opuntia Seed antigen Molecular marker Western blotting SDS–PAGE
1. Introduction The Cactaceae, one of the most typical families in arid and semiarid American regions, include approximately 1500–1800 species, all of which are grouped into more than 100 genera (Barthlott and Hunt, 1993). The following subfamilies are recognised to date: Pereskioideae, Opuntioideae, Cactoideae, and Maihuenioideae (Anderson, 2001). The Argentine flora, in particular, includes 225 species and 36 genera of this family (Kiesling, 1999). Several studies have been conducted to date in order to elucidate the systematic relationships within the Cactaceae as well as to clarify the nomenclatural confusion existing about its genera (Carreras et al., 1997; Leuenberger, 2002; Nyffeler, 2002; Labra et al., 2003). Approximately 11,000 binomials have been published to date, most of which are considered incorrect (Gibson and Nobel, 1986). Opuntia s.s. Mill., the most speciose genus of the subfamily Opuntioideae, includes 181 species as well as 10 natural hybrids (Anderson, 2001). Britton and Rose (1919) proposed the first global classification of the genus Opuntia. Since then, the systematics of this genus has undergone several modifications and it is still at present a point of controversy among researchers (Hunt and Taylor, 1986, 1990; Leuenberger, 2002). Due to their economical importance, several species of this genus are merchandised as either exotic fruit, vegetables or forage in several countries, namely Mexico, the United States, ˜ oz-Urias, 1995). Due to their pharmacological Chile, Argentina, Israel, Italy, Spain and South Africa (Pimienta-Barrios and Mun properties, such as the anti-inflammatory action of O. humifusa, some species have been thoroughly studied (Cho et al., 2006). The systematic complexity of the Cactaceae has generated several studies aiming at designing new methodologies to complement the classical phenotypical characterisation. In this respect, the usefulness of seed protein profiles obtained by electrophoresis has been demonstrated by Wallace and Fairbrothers (1986) in comparative intra- and interpopulation studies
* Corresponding author. Tel.: þ54 (0)291 459 5129; fax: þ54 (0)291 459 5130. (M.J. Galvez). E-mail addresses: [email protected] (M.J. Galvez), [email protected] (C.B. Villamil). 0305-1978/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2008.12.004
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of O. humifusa and by Carreras et al. (1997) in their study on nine Argentine species of this family. Furthermore, different intraand interspecific studies have been carried out in order to analyse specific genomic sequences (Wang et al., 1998; Labra et al., 2003; Griffith, 2004). A few works have been focused in the study of seed proteins of Opuntioideae. Uchoˆa et al. (1998) have isolated and characterised a major albumin 2S seed storage protein (6.5 kDa molecular weight) from O. ficus-indica. Components of albumin (storage and metabolic proteins), glutelin (structural and metabolic proteins) and mainly globulin (storage proteins) fraction constitute the seed proteins herein compared. In addition, immunochemical methods, such as Western blotting, have been used in order to analyse populations of Eragrostis (Castro et al., 1999) and Cucurbitaceae (Castro et al., 2006) although Argentine species of the family Cactaceae have not been included in these studies. In general, the major benefit of immunological methods lies in the specificity of the reaction between an antibody molecule and the corresponding antigenic determinant (epitope), thus allowing to detect molecular structures that are common to two different samples. The detection of epitopes by means of an appropriate antiserum and the further analysis of either their similarities or differences among related samples therefore constitute a highly useful tool for comparative studies. In view of the above, the aim of this study was to analyse and compare electrophoretic and antigenic patterns of several species of Opuntioideae in an attempt to assess their usefulness for taxonomic identifications as well as for studies on the internal relationships of this subfamily. Our results are discussed based on the affinities which have been determined for this subfamily by means of more traditional methods (morphology, pigments, palinology). 2. Materials and methods 2.1. Plant material The following species were analysed in the present study: Opuntia cardiosperma K. Schum. (O. chakensis Speg.; 9 populations); O. elata Hort. Berol. ex Salm-Dyck (O. bonaerensis Speg.; 2 populations); O. salagria A. Cast. (O. megapotamica Arechav.; 4 populations); O. quimilo K. Schum. (O. distans Britton & Rose; 3 populations); O. sulphurea Gillies ex Salm-Dyck var. pampeana (Speg.) Backeb. (O. pampeana Speg.; 1 population); O. ficus-indica (L.) Mill. (1 population); Cylindropuntia imbricata (Haw.) Knuth (1 population); Tephrocactus articulatus (Pfeiff.) Backeb. var. articulatus (1 population). Seeds of mature fruits from Argentine spontaneous populations were used. Fruits of O. ficus-indica were obtained commercially. All seeds were stored at 4 C until required. At least two samples collected in different years (1998–2002) were used for seed ageing analysis of three populations of O. cardiosperma. Voucher specimens were deposited at the herbarium of the Departamento de Biologı´a, Bioquı´mica y Farmacia of the Universidad Nacional del Sur (BBB).
2.2. Protein extraction Three grams of mature seeds corresponding to each sample were ground mechanically and the powder thus obtained was de-oiled with petroleum ether at 4 C. The powder was subsequently air dried and kept at 4 C until required. Seed proteins were extracted in 0.025 M Tris–0.192 M glycine buffer, pH 8.3 (10 ml per g of dry weight) at room temperature for 1 h. The suspension was centrifuged at 6000 g for 10 min and the supernatant was stored at 20 C. The protein content of the extracts was determined by the Lowry method slightly modified (Bensadoun and Weinstein, 1976) using bovine serum albumin as standard.
2.3. Antiserum Seed protein extract from one of the populations of O. cardiosperma (El Cardo´n, Bahı´a Blanca, Buenos Aires) was used in order to obtain antiserum according to Villamil and Gonza´lez (1993). Equal parts of the protein extract (2.8 g/l) and complete Freund’s adjuvant were emulsified and 1 ml of this mixture was inoculated subcutaneously into New Zealand female rabbits every week. From the second injection on, incomplete Freund’s adjuvant was used until an appropriate amount of antibodies was obtained. The antiserum was collected and stored at 20 C until required.
2.4. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) Slab gels, 9 cm 10 cm 1.5 mm, were prepared using a Hoefer Mighty Small apparatus (Amersham Pharmacia Biotech, San Francisco, USA) according to Scha¨ger and von Jagow (1987). Acrylamide was used at a concentration of 13% and 8% for the separating and stacking gels, respectively. Prior to electrophoresis, equal parts of protein extract and Laemmli buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue) were mixed and boiled for 3 min. Then, 100 mg of each protein sample were loaded in each well. The run was performed (44 mA, 90 V) during 6 h. Gels were stained with 0.5% (w/v) Coomassie Brilliant Blue R-250.
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2.5. Western blotting The proteins separated by SDS–PAGE were transferred onto a 0.45 mm pore size nitrocellulose membrane (Sigma, St. Louis, USA) in a Hoefer Te SERIES chamber (350 mA, 75 V) for 90 min, following the procedure described by Towbin et al. (1979). The membrane was blocked overnight at 4 C with 50 mM Tris buffered saline solution (TBS), pH 7.4 containing 0.5% powdered skimmed milk. It was then incubated at room temperature for 2 h in a 1:50 dilution of polyclonal serum raised against O. cardiosperma. 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, followed by washings with TBS. The colour was developed in the dark by treatment with 4-chloro-1-naphthol and hydrogen peroxide. Bands were not observed when the membrane was incubated with pre-immune serum instead of polyclonal antiserum. For SDS–PAGE and Western blotting molecular mass standards, 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 (Amersham Bioscience, Buckinghamshire, UK). 2.6. Data analysis The obtained gels and membranes were scanned on a G-710 equipment (Bio-Rad, Hercules, USA) and the corresponding densitometries were traced using Quantity One 4.0.2 software (Bio-Rad). The degree of similarity among the samples was determined by Jaccard’s Index (SJ). To this end, each band was scored as a binary character for either absence (0) or presence (1). The degree of distances between samples was determined by Bray–Curtis Index (DBC). To this end, the relative heights of each peak in densitometries were taken as a measure of relative concentration. Similarity and distance matrices were analysed phenetically through clustering (Unweighted Pair-Group Method using Arithmetic Averages, UPGMA) and ordination (Principal Coordinates Analysis, 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 3.1. Intraspecific variability The SDS–PAGE patterns showed polypeptides with a molecular weight (MW) ranging between 7 and 103 kDa (Fig. 1A). The electrophoretic patterns corresponding to the samples collected within a population of Opuntia cardiosperma during different years revealed the same band number (33) and position. The electrophoretic patterns corresponding to nine populations of O. cardiosperma (Fig. 1A) also showed 33 components which were common to all these populations (SJ: 1.000). However, nonsignificant quantitative relative differences were observed (DBC: 0.036–0.196). The electrophoretic profiles of different populations of either O. quimilo or O. salagria showed no qualitative differences (SJ: 1.000). As to O. elata, it evidenced qualitative differences only in a low intensity band (SJ: 0.971). Minor quantitative differences were observed in O. elata (DBC: 0.127), being more evident in O. salagria (DBC: 0.043–0.361). A total of 33 components in a range between 13.5 and 112 kDa was found in the antigenic patterns corresponding to nine populations of O. cardiosperma (Fig. 1B). These patterns had been obtained using anti-O. cardiosperma as antiserum. Qualitative differences could be observed in three bands (between 38 and 45 kDa) of low intensity although they were absent in four of these populations. The remaining five populations, including the reference sample (O. cardiosperma, BB, Fig. 1B),
Fig. 1. Seed proteins electrophoretic (A) and antigenic (B) patterns corresponding to nine population of Opuntia cardiosperma. BB: El Cardo´n, Bahı´a Blanca, Buenos Aires (reference pattern in B); MA: Macachı´n, La Pampa; AR: Ataliva Roca, La Pampa; AN: Anguil, La Pampa; HR: Huinca Renanco´, Co´rdoba; Bb: Sarmiento, Bahı´a Blanca, Buenos Aires; VV: Villa Ventana, Buenos Aires; AL: Algarrobo, Buenos Aires; SR: Santa Rosa, La Pampa. On the left, molecular mass standards (kDa).
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shared the same pattern consisting of 33 bands. SJ varied between 0.909 and 1.000 whereas DBC ranged between 0.033 and 0.197. Fig. 1B also shows duplicates of the reference sample (SJ: 1.000 and DBC: 0.030).
3.2. Interspecific variability In addition to O. cardiosperma other five species of Opuntia were analysed (Fig. 2A). Of the 38 SDS–PAGE-separated components 29 were observed in the six species analysed whereas the other nine were absent in at least one of them. The number of bands observed varied from 33 to 35 depending on the species. SJ varied between 0.811 and 0.971. The profiles corresponding to O. quimilo and O. sulphurea shared the same pattern except for one band corresponding to 56 kDa (SJ: 0.970). The SJ estimated by comparing the profiles of population of O. cardiosperma and those of O. elata was 0.811 whereas DBC varied from 0.134 to 0.395. Samples from one population of Cylindropuntia imbricata and one of Tephrocactus articulatus were also analysed (Fig. 2A). An additional component (35 kDa) to the 38 ones found among the species of Opuntia was observed in C. imbricata (34 bands) whereas the 18 components of T. articulatus coincided with those observed in the other species. The species of Opuntia exhibited three bands present only in them. All the species of Opuntioideae shared 15 bands, i.e. a 38% of the total electrophoretic profile. Among the eight species, SJ varied between 0.457 and 0.971 whereas DBC ranged from 0.101 to 0.575. Fig. 3A shows the dendrogram resulting from the cluster analysis (UPGMA) based on the similarity matrix in which the group of species of Opuntia can be distinguished from the species of C. imbricata and T. articulatus. Fig. 3B shows the PCO graph obtained from the distance matrix in which C. imbricata does not separate from the group of species of Opuntia. In both cases the data corresponding to the analysis of the nine populations of O. cardiosperma were included and it was possible to separate this group from the other species. In the antigenic patterns corresponding to the species of Opuntia, a total of 41 components could be observed using anti-O. cardiosperma as antiserum (Fig. 2B). Twenty-five of these components were common to the six species whereas other 16 components were absent in at least one of them, thus indicating a more heterogeneous pattern than the electrophoretic one. The number of bands recorded varied from 32 to 37 depending on the species. SJ varied between 0.737 and 0.946 whereas DBC ranged from 0.074 to 0.166. C. imbricata and T. articulatus exhibited profiles of 30 and 22 bands, respectively (Fig. 2B). T. articulatus was found to have an additional characteristic component (24 kDa) to the 41 recorded among the species of Opuntia. The latter exhibited eight bands which were recorded only in them. On the other hand, the species of Opuntioideae shared 12 bands, i.e. a 29% of the antigenic profile. In the eight species analysed, SJ varied between 0.474 and 0.946 whereas DBC varied between 0.074 and 0.274. Fig. 4A shows the dendrogram resulting from the cluster analysis (UPGMA) based on the similarity matrix. Fig. 4B shows the PCO graph obtained from the distance matrix. In both cases, the species considered evolutionarily closest to the reference sample (O. cardiosperma) evidenced higher affinity than T. articulatus and C. imbricata. 4. Discussion The classical concept of the genus Opuntia s.l. (Castellanos and Lelong, 1943) includeddas subgenerada series of taxa, Cylindropuntia and Tephrocactus, among others, considered now as independent genera (Kiesling, 1984).
Fig. 2. Seed proteins electrophoretic (A) and antigenic (B) patterns corresponding to species of Opuntioideae. C: Cylindropuntia imbricata; 1: O. cardiosperma (reference pattern in B); 2: O. sulphurea; 3: O. quimilo; 4: O. salagria; 5: O. elata; 6: O. ficus-indica; T: Tephrocactus articulatus. On the left, molecular mass standards (kDa).
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Fig. 3. (A) Neighbour joining tree (UPGMA) obtained from analysis of protein electrophoretic patterns of Opuntioideae using Jaccard’s Index matrix. (B) Graph of distribution following PCO (first three factors, 93.32% of the variance). :: 1, Opuntia cardiosperma populations; 2, O. sulphurea; 3, O. quimilo; 4, O. salagria; 5, O. elata; 6, O. ficus-indica; -: Cylindropuntia imbricata; r: Tephrocactus articulatus.
The genus Opuntia includes more than 200 species, a higher number than the average 20 species per genus in Cactaceae (Reyes-Agu¨ero et al., 2006), and exhibits a high degree of morphological variability. Reyes-Agu¨ero et al. (2006) argue on the evolutionary and ecological success of Opuntia, which could be explained in terms of the diversity of reproductive modes. Sexual reproduction and multiplication through fragmentation are common mechanisms. Multiplication via fragmentation produces organisms that are genetically identical in neighbouring populations and allows that unusual morphological characteristics and/or peculiar protein profiles become fixed. The present research demonstrates that seed ageing produces no detectable changes in the electrophoretic patterns. This facilitates further comparisons with samples collected during different years in a period of time of at least 5 years. This agrees with results from Carreras et al. (1997) and permits the use of seeds collected during different reproductive seasons. In agreement with morphological observations, results from the qualitative and quantitative analyses of different populations of O. elata and O. salagria suggest a certain degree of genetic variability in populations geographically distant from each other. In contrast, the populations of O. cardiosperma herein analysed not only exhibited high morphological uniformity but also showed the same electrophoretic pattern (Fig. 1A) as well as a highly uniform antigenic pattern (Fig. 1B) although they grew in localities distant from each other. The differences observed in the degree of cross reactivity determined by Western blotting among populations of O. cardiosperma were not enough to fully characterise each of them. In some cases, the relative composition of the antigenic pattern (e.g. the components of 82.5 kDa in the population of Villa Ventana, Fig. 1B) revealed a characteristic profile. Nevertheless, Bray–Curtis indices for the majority of the populations of O. cardiosperma fell within the range of variability of those calculated for the duplicates of the same sample. The low intraspecific antigenic variability was correlated with that observed in the electrophoretic profiles. This finding coincides with results obtained for cultivars of species of Cucurbitaceae studied by Western blotting (Castro et al., 2006).
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Fig. 4. (A) Neighbour joining tree (UPGMA) obtained from analysis of protein antigenic patterns of Opuntioideae using Jaccard’s Index matrix. (B) Graph of distribution of Opuntioideae following PCO (first three factors, 87.60% of the variance). : 1, Opuntia cardiosperma; 2, O. sulphurea; 3, O. quimilo; 4, O. salagria; 5, O. elata; 6, O. ficus-indica; -: Cylindropuntia imbricata; r: Tephrocactus articulatus.
The uniformity found among different populations of O. cardiosperma as well as the consistent differences among them and those of O. elata support the hypothesis that both taxa are different species, in agreement with Leuenberger’s opinion (Leuenberger, 2001, 2002). On the other hand, the qualitative analysis of the electrophoretic patterns of different species of Opuntia revealed more differences than at the intraspecific level on account of the fact that only 76% of the bands were common to all of them (Fig. 2A). Still, the characterisation of the species analysed was rather difficult because at least two of them differed in only one band. The great similarity of the electrophoretic profiles of O. quimilo and O. sulphurea contrasts with the clear morphological differences between both taxa. In general, the nine variable components in the patterns of the six species of Opuntia were found in low intensity bands. In these cases, complementation with a quantitative analysis of the patterns minimised the possible errors that could be made when considering only the presence/absence of a band. However, this analysis demonstrated a quantitatively comparable variability among species of Opuntia and between the latter and C. imbricata and the phenetic analyses (Fig. 3) showed them grouped. Thus, in order to clearly interpret these results, it should be taken into account that the fact that two bands are located at the same relative position or that two profiles are similar does not necessarily indicate that their components are structurally identical. The use of antibodies detects the sequential epitopesdcontiguous amino acids in their primary protein sequencedthat are present in a sample as well as in the reference sample. The qualitative analysis of the antigenic profiles within the same species showed a lower variability (more than 90% of the bands were common to all the populations of O. cardiosperma, Fig. 1B) than that among the different species of Opuntia (only 61% of the bands were common to all of them, Fig. 2B). The 16 variable components showed characteristic profiles which permitted the qualitative identification of each species (Fig. 4A). In addition, results from the analysis based on Bray–Curtis indices allowed us to clearly differentiate C. imbricata from the
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remaining species (Fig. 4B). It is thus demonstrated that those taxa considered to be of lowest affinity, on the basis of the traditional characteristics, also share a lower number of molecular components. No relatively intense bands were observed in the range of MW between 13.5 and 16 kDa in the electrophoretic profiles. In contrast, one of the most intense bands of the antigenic profile was detected in this zone. Particularly, the band corresponding to a MW of 16 kDa was found in a higher relative concentration in the antigenic profiles of the different species of Opuntia with respect to C. imbricata. This was not observed in T. articulatus (Fig. 2B).The intensity and uniformity of cross-reactions between O. cardiosperma and the remaining species of Opuntia were therefore higher than those observed for O. cardiosperma and C. imbricata, making it possible to characterise the genus Opuntia s.s. A similar situation was observed for the bands close to 61 kDa (Fig. 2B). With traditional immunochemical methods (e.g. bidimensional immunodiffusion) it is possible to make differentiations only at the level of ‘‘groups of species’’ (Jensen and Penner, 1980; Villamil and Gonza´lez, 1993) probably because of the low number of bands that can be separated and which are generally common at low hierarchy systematic units. Conversely, Western blotting contributes to comparing a higher number of components at low taxonomic levels and to better understanding the results yielded by such comparative studies. The present study demonstrates that the application of Western blotting to study the family Cactaceae extends the usefulness of immunological techniques at the specific level. It also complements the information provided by electrophoretic profiles in adding the antigenic properties of the compared proteins. Further studies will allow us to know with greater detail the composition of seed proteins of Cactaceae. Also, other studies including a higher number of species of the subfamily Opuntioideae and the remaining subfamilies of the Cactaceae will be necessary to demonstrate the usefulness of antigenic patterns as a means to resolve specific systematic problems within this family. Acknowledgements Financial support from the Secretarı´a de Ciencia y Tecnologı´a of the Universidad Nacional del Sur to C.B.V. is greatly acknowledged. References Anderson, E.F., 2001. The Cactus Family. Timber Press, Portland, OR. Barthlott, W., Hunt, D.R., 1993. Cactaceae. In: Kubitzki, K., Rohwer, J.G., Bittrich, V. (Eds.), The Families and Genera of Vascular Plants 2. Springer, Berlin, pp. 161–197. Bensadoun, A., Weinstein, D., 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem 70, 241–250. Britton, N.L., Rose, J.N., 1919. The Cactaceae. Carnegie Institute, Washington. Carreras, M.E., Fuentes, E., Merino, E.F., 1997. Seed protein patterns of nine species of Cactaceae. Biochem. Syst. Ecol 25, 43–49. Castellanos, A., Lelong, H.V., 1943. Opuntiales vel Cactales. In: Descole, H. (Ed.), Genera et Species Plantarum Argentinarum 1, pp. 47–142. Castro, H.A., Gonza´lez, S.R., Prat, M.I., Villamil, C.B., 1999. Utilizacio´n del me´todo de inmunotransferencia (Western blotting) en taxonomı´a vegetal. Phyton 65, 185–196. Castro, H.A., Galvez, M.J., Gonza´lez, S.R., Villamil, C.B., 2006. Protein composition of Cucurbita maxima and C. moschata seeds. Biol. Plant 50, 251–256. Cho, J.Y., Park, S.C., Kim, T.W., Kim, K.S., Song, J.C., Kim, S.K., Lee, H.M., Sung, H.J., Park, H.J., Song, Y.B., Yoo, E.S., Lee, C.H., Rhee, M.H., 2006. Radical scavenging and anti-inflammatory activity of extracts from Opuntia humifusa Raf. J. Pharm. Pharmacol 58, 113–119. Gibson, A.C., Nobel, P.S., 1986. The Cactus Primer. Harvard University Press, Cambridge, MA. Griffith, M.P., 2004. The origins of an important cactus crop, Opuntia ficus-indica (Cactaceae): new molecular evidence. Am. J. Bot 91, 1915–1921. Hunt, D.R., Taylor, N.P., 1986. The genera of the Cactaceae: towards a new consensus. Bradleya 4, 65–78. Hunt, D.R., Taylor, N.P., 1990. The genera of the Cactaceae: progress towards consensus. Bradleya 8, 85–107. Jensen, U., Penner, R., 1980. Investigation of serological determinants from single storage plant proteins. Biochem. Syst. Ecol 8, 161–170. Kiesling, R., 1984. Estudios en Cactaceae de Argentina: Maihueniopsis, Tephrocactus y ge´neros afines (Opuntioideae). Darwiniana 25, 171–215. Kiesling, R., 1999. Cactaceae. In: Zuloaga, F.O., Morrone, O. (Eds.), Cata´logo de Plantas Vasculares de la Repu´blica Argentina II, Acanthaceae-Euphorbiaceae (Dicotyledoneae). Monographs in Systematic Botany 74, St Louis, pp. 423–489. Labra, M., Grassi, F., Bardini, M., Imazio, S., Guiggi, A., Citterio, S., Banfi, E., Sgorbati, S., 2003. Genetic relationships in Opuntia Mill. genus (Cactaceae) detected by molecular marker. Plant Sci. 165, 1129–1136. Leuenberger, B.E., 2001. The type specimen of Opuntia cardiosperma (Cactaceae), new synonyms and new records from Argentina and Paraguay. Willdenowia 31, 171–179. Leuenberger, B.E., 2002. The South American Opuntia ser. Armatae (¼O. ser Elatae) (Cactaceae). Bot. Jahrb. Syst 123, 413–439. 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. ˜ oz-Urias, A., 1995. Domestication of Opuntia and cultivated varieties. In: Barbera, G., Inglese, P., Pimienta-Barrios, E. (Eds.), AgroPimienta-Barrios, E., Mun Ecology, Cultivation and Uses of Cactus Pear. FAO International Technical Cooperation Network on Cactus Pear, Rome Italy, pp. 58–63. Reyes-Agu¨ero, J.A., Aguirre, J.R., Valiente-Banuet, A., 2006. Reproductive biology of Opuntia: A review. J. Arid Environ 64, 549–585. Scha¨gger, H., von Jagow, G., 1987. 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Antigenic patterns of seed proteins in Opuntioideae (Cactaceae) Marcelo J. Galvez*, Hugo A. Castro, Carlos B. Villamil Departamento de Biologı´a, Bioquı´mica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahı´a Blanca, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2008 Accepted 23 December 2008
The antigenic patterns recognised by Western blotting in seed proteins of species of Opuntioideae (Cactaceae) were analysed in an attempt to evaluate their usefulness in systematics. Total protein profiles were also analysed by SDS–PAGE. The resulting similarity and distance matrices were further used to carry out Cluster Analysis (UPGMA) and Principal Coordinates Analysis. Populations of Opuntia cardiosperma were found to exhibit a prominent morphological uniformity, a unique electrophoretic pattern and a uniform antigenic pattern. The latter was obtained using anti-O. cardiosperma as antiserum. Results from the qualitative and quantitative interspecific analyses of antigenic profiles helped to characterise all the species studied. Tephrocactus articulatus and Cylindropuntia imbricata evidenced lower affinity with O. cardiosperma than the species of Opuntia s.s. Our results demonstrate that in Cactaceae, Western blotting analysis broadens the usefulness range of immunological techniques at the specific level and complements the information collected from electrophoretic profiles. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Opuntia Seed antigen Molecular marker Western blotting SDS–PAGE
1. Introduction The Cactaceae, one of the most typical families in arid and semiarid American regions, include approximately 1500–1800 species, all of which are grouped into more than 100 genera (Barthlott and Hunt, 1993). The following subfamilies are recognised to date: Pereskioideae, Opuntioideae, Cactoideae, and Maihuenioideae (Anderson, 2001). The Argentine flora, in particular, includes 225 species and 36 genera of this family (Kiesling, 1999). Several studies have been conducted to date in order to elucidate the systematic relationships within the Cactaceae as well as to clarify the nomenclatural confusion existing about its genera (Carreras et al., 1997; Leuenberger, 2002; Nyffeler, 2002; Labra et al., 2003). Approximately 11,000 binomials have been published to date, most of which are considered incorrect (Gibson and Nobel, 1986). Opuntia s.s. Mill., the most speciose genus of the subfamily Opuntioideae, includes 181 species as well as 10 natural hybrids (Anderson, 2001). Britton and Rose (1919) proposed the first global classification of the genus Opuntia. Since then, the systematics of this genus has undergone several modifications and it is still at present a point of controversy among researchers (Hunt and Taylor, 1986, 1990; Leuenberger, 2002). Due to their economical importance, several species of this genus are merchandised as either exotic fruit, vegetables or forage in several countries, namely Mexico, the United States, ˜ oz-Urias, 1995). Due to their pharmacological Chile, Argentina, Israel, Italy, Spain and South Africa (Pimienta-Barrios and Mun properties, such as the anti-inflammatory action of O. humifusa, some species have been thoroughly studied (Cho et al., 2006). The systematic complexity of the Cactaceae has generated several studies aiming at designing new methodologies to complement the classical phenotypical characterisation. In this respect, the usefulness of seed protein profiles obtained by electrophoresis has been demonstrated by Wallace and Fairbrothers (1986) in comparative intra- and interpopulation studies
* Corresponding author. Tel.: þ54 (0)291 459 5129; fax: þ54 (0)291 459 5130. (M.J. Galvez). E-mail addresses: [email protected] (M.J. Galvez), [email protected] (C.B. Villamil). 0305-1978/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2008.12.004
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of O. humifusa and by Carreras et al. (1997) in their study on nine Argentine species of this family. Furthermore, different intraand interspecific studies have been carried out in order to analyse specific genomic sequences (Wang et al., 1998; Labra et al., 2003; Griffith, 2004). A few works have been focused in the study of seed proteins of Opuntioideae. Uchoˆa et al. (1998) have isolated and characterised a major albumin 2S seed storage protein (6.5 kDa molecular weight) from O. ficus-indica. Components of albumin (storage and metabolic proteins), glutelin (structural and metabolic proteins) and mainly globulin (storage proteins) fraction constitute the seed proteins herein compared. In addition, immunochemical methods, such as Western blotting, have been used in order to analyse populations of Eragrostis (Castro et al., 1999) and Cucurbitaceae (Castro et al., 2006) although Argentine species of the family Cactaceae have not been included in these studies. In general, the major benefit of immunological methods lies in the specificity of the reaction between an antibody molecule and the corresponding antigenic determinant (epitope), thus allowing to detect molecular structures that are common to two different samples. The detection of epitopes by means of an appropriate antiserum and the further analysis of either their similarities or differences among related samples therefore constitute a highly useful tool for comparative studies. In view of the above, the aim of this study was to analyse and compare electrophoretic and antigenic patterns of several species of Opuntioideae in an attempt to assess their usefulness for taxonomic identifications as well as for studies on the internal relationships of this subfamily. Our results are discussed based on the affinities which have been determined for this subfamily by means of more traditional methods (morphology, pigments, palinology). 2. Materials and methods 2.1. Plant material The following species were analysed in the present study: Opuntia cardiosperma K. Schum. (O. chakensis Speg.; 9 populations); O. elata Hort. Berol. ex Salm-Dyck (O. bonaerensis Speg.; 2 populations); O. salagria A. Cast. (O. megapotamica Arechav.; 4 populations); O. quimilo K. Schum. (O. distans Britton & Rose; 3 populations); O. sulphurea Gillies ex Salm-Dyck var. pampeana (Speg.) Backeb. (O. pampeana Speg.; 1 population); O. ficus-indica (L.) Mill. (1 population); Cylindropuntia imbricata (Haw.) Knuth (1 population); Tephrocactus articulatus (Pfeiff.) Backeb. var. articulatus (1 population). Seeds of mature fruits from Argentine spontaneous populations were used. Fruits of O. ficus-indica were obtained commercially. All seeds were stored at 4 C until required. At least two samples collected in different years (1998–2002) were used for seed ageing analysis of three populations of O. cardiosperma. Voucher specimens were deposited at the herbarium of the Departamento de Biologı´a, Bioquı´mica y Farmacia of the Universidad Nacional del Sur (BBB).
2.2. Protein extraction Three grams of mature seeds corresponding to each sample were ground mechanically and the powder thus obtained was de-oiled with petroleum ether at 4 C. The powder was subsequently air dried and kept at 4 C until required. Seed proteins were extracted in 0.025 M Tris–0.192 M glycine buffer, pH 8.3 (10 ml per g of dry weight) at room temperature for 1 h. The suspension was centrifuged at 6000 g for 10 min and the supernatant was stored at 20 C. The protein content of the extracts was determined by the Lowry method slightly modified (Bensadoun and Weinstein, 1976) using bovine serum albumin as standard.
2.3. Antiserum Seed protein extract from one of the populations of O. cardiosperma (El Cardo´n, Bahı´a Blanca, Buenos Aires) was used in order to obtain antiserum according to Villamil and Gonza´lez (1993). Equal parts of the protein extract (2.8 g/l) and complete Freund’s adjuvant were emulsified and 1 ml of this mixture was inoculated subcutaneously into New Zealand female rabbits every week. From the second injection on, incomplete Freund’s adjuvant was used until an appropriate amount of antibodies was obtained. The antiserum was collected and stored at 20 C until required.
2.4. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) Slab gels, 9 cm 10 cm 1.5 mm, were prepared using a Hoefer Mighty Small apparatus (Amersham Pharmacia Biotech, San Francisco, USA) according to Scha¨ger and von Jagow (1987). Acrylamide was used at a concentration of 13% and 8% for the separating and stacking gels, respectively. Prior to electrophoresis, equal parts of protein extract and Laemmli buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue) were mixed and boiled for 3 min. Then, 100 mg of each protein sample were loaded in each well. The run was performed (44 mA, 90 V) during 6 h. Gels were stained with 0.5% (w/v) Coomassie Brilliant Blue R-250.
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2.5. Western blotting The proteins separated by SDS–PAGE were transferred onto a 0.45 mm pore size nitrocellulose membrane (Sigma, St. Louis, USA) in a Hoefer Te SERIES chamber (350 mA, 75 V) for 90 min, following the procedure described by Towbin et al. (1979). The membrane was blocked overnight at 4 C with 50 mM Tris buffered saline solution (TBS), pH 7.4 containing 0.5% powdered skimmed milk. It was then incubated at room temperature for 2 h in a 1:50 dilution of polyclonal serum raised against O. cardiosperma. 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, followed by washings with TBS. The colour was developed in the dark by treatment with 4-chloro-1-naphthol and hydrogen peroxide. Bands were not observed when the membrane was incubated with pre-immune serum instead of polyclonal antiserum. For SDS–PAGE and Western blotting molecular mass standards, 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 (Amersham Bioscience, Buckinghamshire, UK). 2.6. Data analysis The obtained gels and membranes were scanned on a G-710 equipment (Bio-Rad, Hercules, USA) and the corresponding densitometries were traced using Quantity One 4.0.2 software (Bio-Rad). The degree of similarity among the samples was determined by Jaccard’s Index (SJ). To this end, each band was scored as a binary character for either absence (0) or presence (1). The degree of distances between samples was determined by Bray–Curtis Index (DBC). To this end, the relative heights of each peak in densitometries were taken as a measure of relative concentration. Similarity and distance matrices were analysed phenetically through clustering (Unweighted Pair-Group Method using Arithmetic Averages, UPGMA) and ordination (Principal Coordinates Analysis, 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 3.1. Intraspecific variability The SDS–PAGE patterns showed polypeptides with a molecular weight (MW) ranging between 7 and 103 kDa (Fig. 1A). The electrophoretic patterns corresponding to the samples collected within a population of Opuntia cardiosperma during different years revealed the same band number (33) and position. The electrophoretic patterns corresponding to nine populations of O. cardiosperma (Fig. 1A) also showed 33 components which were common to all these populations (SJ: 1.000). However, nonsignificant quantitative relative differences were observed (DBC: 0.036–0.196). The electrophoretic profiles of different populations of either O. quimilo or O. salagria showed no qualitative differences (SJ: 1.000). As to O. elata, it evidenced qualitative differences only in a low intensity band (SJ: 0.971). Minor quantitative differences were observed in O. elata (DBC: 0.127), being more evident in O. salagria (DBC: 0.043–0.361). A total of 33 components in a range between 13.5 and 112 kDa was found in the antigenic patterns corresponding to nine populations of O. cardiosperma (Fig. 1B). These patterns had been obtained using anti-O. cardiosperma as antiserum. Qualitative differences could be observed in three bands (between 38 and 45 kDa) of low intensity although they were absent in four of these populations. The remaining five populations, including the reference sample (O. cardiosperma, BB, Fig. 1B),
Fig. 1. Seed proteins electrophoretic (A) and antigenic (B) patterns corresponding to nine population of Opuntia cardiosperma. BB: El Cardo´n, Bahı´a Blanca, Buenos Aires (reference pattern in B); MA: Macachı´n, La Pampa; AR: Ataliva Roca, La Pampa; AN: Anguil, La Pampa; HR: Huinca Renanco´, Co´rdoba; Bb: Sarmiento, Bahı´a Blanca, Buenos Aires; VV: Villa Ventana, Buenos Aires; AL: Algarrobo, Buenos Aires; SR: Santa Rosa, La Pampa. On the left, molecular mass standards (kDa).
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shared the same pattern consisting of 33 bands. SJ varied between 0.909 and 1.000 whereas DBC ranged between 0.033 and 0.197. Fig. 1B also shows duplicates of the reference sample (SJ: 1.000 and DBC: 0.030).
3.2. Interspecific variability In addition to O. cardiosperma other five species of Opuntia were analysed (Fig. 2A). Of the 38 SDS–PAGE-separated components 29 were observed in the six species analysed whereas the other nine were absent in at least one of them. The number of bands observed varied from 33 to 35 depending on the species. SJ varied between 0.811 and 0.971. The profiles corresponding to O. quimilo and O. sulphurea shared the same pattern except for one band corresponding to 56 kDa (SJ: 0.970). The SJ estimated by comparing the profiles of population of O. cardiosperma and those of O. elata was 0.811 whereas DBC varied from 0.134 to 0.395. Samples from one population of Cylindropuntia imbricata and one of Tephrocactus articulatus were also analysed (Fig. 2A). An additional component (35 kDa) to the 38 ones found among the species of Opuntia was observed in C. imbricata (34 bands) whereas the 18 components of T. articulatus coincided with those observed in the other species. The species of Opuntia exhibited three bands present only in them. All the species of Opuntioideae shared 15 bands, i.e. a 38% of the total electrophoretic profile. Among the eight species, SJ varied between 0.457 and 0.971 whereas DBC ranged from 0.101 to 0.575. Fig. 3A shows the dendrogram resulting from the cluster analysis (UPGMA) based on the similarity matrix in which the group of species of Opuntia can be distinguished from the species of C. imbricata and T. articulatus. Fig. 3B shows the PCO graph obtained from the distance matrix in which C. imbricata does not separate from the group of species of Opuntia. In both cases the data corresponding to the analysis of the nine populations of O. cardiosperma were included and it was possible to separate this group from the other species. In the antigenic patterns corresponding to the species of Opuntia, a total of 41 components could be observed using anti-O. cardiosperma as antiserum (Fig. 2B). Twenty-five of these components were common to the six species whereas other 16 components were absent in at least one of them, thus indicating a more heterogeneous pattern than the electrophoretic one. The number of bands recorded varied from 32 to 37 depending on the species. SJ varied between 0.737 and 0.946 whereas DBC ranged from 0.074 to 0.166. C. imbricata and T. articulatus exhibited profiles of 30 and 22 bands, respectively (Fig. 2B). T. articulatus was found to have an additional characteristic component (24 kDa) to the 41 recorded among the species of Opuntia. The latter exhibited eight bands which were recorded only in them. On the other hand, the species of Opuntioideae shared 12 bands, i.e. a 29% of the antigenic profile. In the eight species analysed, SJ varied between 0.474 and 0.946 whereas DBC varied between 0.074 and 0.274. Fig. 4A shows the dendrogram resulting from the cluster analysis (UPGMA) based on the similarity matrix. Fig. 4B shows the PCO graph obtained from the distance matrix. In both cases, the species considered evolutionarily closest to the reference sample (O. cardiosperma) evidenced higher affinity than T. articulatus and C. imbricata. 4. Discussion The classical concept of the genus Opuntia s.l. (Castellanos and Lelong, 1943) includeddas subgenerada series of taxa, Cylindropuntia and Tephrocactus, among others, considered now as independent genera (Kiesling, 1984).
Fig. 2. Seed proteins electrophoretic (A) and antigenic (B) patterns corresponding to species of Opuntioideae. C: Cylindropuntia imbricata; 1: O. cardiosperma (reference pattern in B); 2: O. sulphurea; 3: O. quimilo; 4: O. salagria; 5: O. elata; 6: O. ficus-indica; T: Tephrocactus articulatus. On the left, molecular mass standards (kDa).
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Fig. 3. (A) Neighbour joining tree (UPGMA) obtained from analysis of protein electrophoretic patterns of Opuntioideae using Jaccard’s Index matrix. (B) Graph of distribution following PCO (first three factors, 93.32% of the variance). :: 1, Opuntia cardiosperma populations; 2, O. sulphurea; 3, O. quimilo; 4, O. salagria; 5, O. elata; 6, O. ficus-indica; -: Cylindropuntia imbricata; r: Tephrocactus articulatus.
The genus Opuntia includes more than 200 species, a higher number than the average 20 species per genus in Cactaceae (Reyes-Agu¨ero et al., 2006), and exhibits a high degree of morphological variability. Reyes-Agu¨ero et al. (2006) argue on the evolutionary and ecological success of Opuntia, which could be explained in terms of the diversity of reproductive modes. Sexual reproduction and multiplication through fragmentation are common mechanisms. Multiplication via fragmentation produces organisms that are genetically identical in neighbouring populations and allows that unusual morphological characteristics and/or peculiar protein profiles become fixed. The present research demonstrates that seed ageing produces no detectable changes in the electrophoretic patterns. This facilitates further comparisons with samples collected during different years in a period of time of at least 5 years. This agrees with results from Carreras et al. (1997) and permits the use of seeds collected during different reproductive seasons. In agreement with morphological observations, results from the qualitative and quantitative analyses of different populations of O. elata and O. salagria suggest a certain degree of genetic variability in populations geographically distant from each other. In contrast, the populations of O. cardiosperma herein analysed not only exhibited high morphological uniformity but also showed the same electrophoretic pattern (Fig. 1A) as well as a highly uniform antigenic pattern (Fig. 1B) although they grew in localities distant from each other. The differences observed in the degree of cross reactivity determined by Western blotting among populations of O. cardiosperma were not enough to fully characterise each of them. In some cases, the relative composition of the antigenic pattern (e.g. the components of 82.5 kDa in the population of Villa Ventana, Fig. 1B) revealed a characteristic profile. Nevertheless, Bray–Curtis indices for the majority of the populations of O. cardiosperma fell within the range of variability of those calculated for the duplicates of the same sample. The low intraspecific antigenic variability was correlated with that observed in the electrophoretic profiles. This finding coincides with results obtained for cultivars of species of Cucurbitaceae studied by Western blotting (Castro et al., 2006).
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Fig. 4. (A) Neighbour joining tree (UPGMA) obtained from analysis of protein antigenic patterns of Opuntioideae using Jaccard’s Index matrix. (B) Graph of distribution of Opuntioideae following PCO (first three factors, 87.60% of the variance). : 1, Opuntia cardiosperma; 2, O. sulphurea; 3, O. quimilo; 4, O. salagria; 5, O. elata; 6, O. ficus-indica; -: Cylindropuntia imbricata; r: Tephrocactus articulatus.
The uniformity found among different populations of O. cardiosperma as well as the consistent differences among them and those of O. elata support the hypothesis that both taxa are different species, in agreement with Leuenberger’s opinion (Leuenberger, 2001, 2002). On the other hand, the qualitative analysis of the electrophoretic patterns of different species of Opuntia revealed more differences than at the intraspecific level on account of the fact that only 76% of the bands were common to all of them (Fig. 2A). Still, the characterisation of the species analysed was rather difficult because at least two of them differed in only one band. The great similarity of the electrophoretic profiles of O. quimilo and O. sulphurea contrasts with the clear morphological differences between both taxa. In general, the nine variable components in the patterns of the six species of Opuntia were found in low intensity bands. In these cases, complementation with a quantitative analysis of the patterns minimised the possible errors that could be made when considering only the presence/absence of a band. However, this analysis demonstrated a quantitatively comparable variability among species of Opuntia and between the latter and C. imbricata and the phenetic analyses (Fig. 3) showed them grouped. Thus, in order to clearly interpret these results, it should be taken into account that the fact that two bands are located at the same relative position or that two profiles are similar does not necessarily indicate that their components are structurally identical. The use of antibodies detects the sequential epitopesdcontiguous amino acids in their primary protein sequencedthat are present in a sample as well as in the reference sample. The qualitative analysis of the antigenic profiles within the same species showed a lower variability (more than 90% of the bands were common to all the populations of O. cardiosperma, Fig. 1B) than that among the different species of Opuntia (only 61% of the bands were common to all of them, Fig. 2B). The 16 variable components showed characteristic profiles which permitted the qualitative identification of each species (Fig. 4A). In addition, results from the analysis based on Bray–Curtis indices allowed us to clearly differentiate C. imbricata from the
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remaining species (Fig. 4B). It is thus demonstrated that those taxa considered to be of lowest affinity, on the basis of the traditional characteristics, also share a lower number of molecular components. No relatively intense bands were observed in the range of MW between 13.5 and 16 kDa in the electrophoretic profiles. In contrast, one of the most intense bands of the antigenic profile was detected in this zone. Particularly, the band corresponding to a MW of 16 kDa was found in a higher relative concentration in the antigenic profiles of the different species of Opuntia with respect to C. imbricata. This was not observed in T. articulatus (Fig. 2B).The intensity and uniformity of cross-reactions between O. cardiosperma and the remaining species of Opuntia were therefore higher than those observed for O. cardiosperma and C. imbricata, making it possible to characterise the genus Opuntia s.s. A similar situation was observed for the bands close to 61 kDa (Fig. 2B). With traditional immunochemical methods (e.g. bidimensional immunodiffusion) it is possible to make differentiations only at the level of ‘‘groups of species’’ (Jensen and Penner, 1980; Villamil and Gonza´lez, 1993) probably because of the low number of bands that can be separated and which are generally common at low hierarchy systematic units. Conversely, Western blotting contributes to comparing a higher number of components at low taxonomic levels and to better understanding the results yielded by such comparative studies. The present study demonstrates that the application of Western blotting to study the family Cactaceae extends the usefulness of immunological techniques at the specific level. It also complements the information provided by electrophoretic profiles in adding the antigenic properties of the compared proteins. Further studies will allow us to know with greater detail the composition of seed proteins of Cactaceae. 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