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Correspondence:
Dr. Oscar Adolfo Ruiz. Cátedra de Química Analítica Instrumental. Facultad de Farmacia y Bioquímica.
Universidad de Buenos Aires (UBA). Junín 956 3er Piso. (1113) Capital Federal. Argentina .(Tel-fax: +54 1 964 8254)

E-mail: oruiz@huemul.ffyb.uba.ar

Running title
Analysis of storage proteins of Lotus spp. seeds by CGE

Keywords
Capillary Electrophoresis / Lotus / seeds / proteins / SDS-PAGE

ABSTRACT
The Lotus forage species of major diffusion in Argentine are Lotus tenuis and Lotus corniculatus; and any future intensification in animal production needs an increase in the amount and the quality of forage supply. At this moment, the L. tenuis has adapted to the unfavourable environmental conditions of Flooding Pampa area (the most extensive region for calf production in Argentina), to such an extent that, during spring and summer it becomes the dominant species in the natural grasslands. L. tenuis and corniculatus seeds are morphologically very similar but their prices are quite different. Chromosome number counting is the only test used so far, in the laboratories for the identification of those seeds.

In the present work, we checked the storage protein patterns of different Lotus seed samples by capillary gel electrophoresis (CGE), as an alternative method, comparing it with sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE). The former was faster and displayed a good resolution. CGE becomes thus, a powerful tool in taxonomic investigations.

INTRODUCTION

The "Instituto Tecnológico de Chascomús" is situated near the town of Chascomús in a vast area of about 50.000 Km2 called "Depresión del Río Salado", where cattle and dairy farms are mainly based on the native grassland. In this region, native Lotus is the accessions tenuis. However, Lotus corniculatus is most recently gaining popularity. These species are important pasture legumes adapted to different climatic and soil conditions (Remis et al., 1995). The soils in the area have an alkaline pH value and high salt concentration, significantly decreasing the persistence and yield of other common legume species in the region.

The use of seed protein patterns obtained by SDS-PAGE and reversed-phase liquid chromatography (RP-HPLC) in solving taxonomic and evolutionary problems has been greatly expanded in the last two decades. Recent reports have indicated the CE potential to distinguish among cultivars of wheat (Wrigley, 1995). This is a powerful analytical technique for the separation and quantitation of proteins and peptides especially, based not only on their charge and size but also on their hydrophobicity and stereospecifity. In this respect, the present technique combines the advantages of both SDS-PAGE and HPLC. Generally, CE appeared to be very useful in the separation and characterisation of model proteins, complex protein mixtures and glycoconjugates. The basis of separation in CE is essentially identical to other electrophoretic techniques and its major potential is the versatility in the separations due to its various modes of operation. CGE separations have high efficiency with values around 100,000 theoretical plates and sometimes higher. The electrophoretic separation of proteins on uncoated fused silica capillaries has been more problematic mainly due to the hydrophobic interactions with the inner capillary wall. This causes many problems: poor peak shapes, low efficiency, drifting electroosmotic flow and errors in quantitation. The introduction of capillary gel electrophoresis (CGE) technique overcomes these problems.

The identification of L. corniculatus and L. tenuis seeds is difficult because they are morphologically similar in size and colour, although the price of the latter is higher. For agronomic purposes, up to 10 % of contamination is acceptable. However, only the genetic analysis by chromosome number counting is employed as a confirmatory technique for its identification. Recently, the differentiation of L. tenuis and L. corniculatus seeds through the quantificaation of the flavonol content has been reported. In this method the presence of quercetin-3-O-xyloside only in seeds of L. corniculatus was taken as the base for differentiation (Kade et al., 1997). However this method is time-consuming, so alternative methods for rapid screening and identification in a quality analysis of seed lots are necessary. In this way, CE is rapidly becoming the major technique for the analysis of proteins, nucleic acid, glycoconjugates (Novotny, 1996) and plant secondary metabolites (Tomas-Barberán, 1995). Recent reports have described the species and varietal differentiation in Vicia sativa and Vicia villosa (Lucchese et al, 1997), the differentiation between genotypes of Lupinus species (Pollar et al., 1996) and the identification of bean cultivars (Salmanowicz,1995).

The purpose of this study was to develop a CGE technique that could be useful for Lotus spp differentiation, looking for an improvement in reproducibility through the electrophoretic separation of storage proteins, departing from a previous report based on SDS-PAGE analysis.(Ferrari and Pallares, 1996)

MATERIALS AND METHODS

SAMPLES

Lotus spp seeds were obtained from USDA (United States Department Agriculture) and reproduced in the "introduction garden of INTECh" (Remis et al, 1995). Typically, the seeds were molted in the presence of liquid nitrogen until they became a fine powder, and dissolved finally in a solution prepared by mixing 2-mercaptoetanol, formamide, distilled water and a stock solution in the ratio 1.06:1.76:3:3, respectively. The stock solution was prepared adding 12.5 ml of 1 M Tris.HCl pH 6.80; 20 ml of glycerol, 4 g of SDS and 24 ml of distilled water.

According to the International Seed Testing Association (ISTA, 1992) recommendations, 250 (l of the final solution were added to 20 mg of the seed powder. The samples were extracted overnight at 4º C and centrifuged at 14000 rpm for 1 hour. 10 (l of the supernatant were diluted adding 10 (l of "sample buffer". The latter was prepared mixing 20 ml of glycerol, 10 ml of (-mercaptoethanol, 46 ml of SDS 10 % solution (2,3 % final concentration), 12,5 ml of Tris-HCl pH 6.80 (0.0625 M final concentration) and distillate water up to 200 ml.

We omitted the addition of Bromophenol Blue in the samples employed in CGE. The extracted samples were then, boiled for 5 minutes.

SDS-PAGE

The samples were loaded in a 15 % SDS gel with stacking of 4 %. A Mini-Protean II electrophoresis cell and a LKB 2197 Power Supply were used. The voltage applied was 200 V for 1 h; other conditions as previously described (Ferrari and Pallares, 1996). Protein standards purchased from Sigma were employed.

CGE

Routine CE separations were achieved on a BioFocus 3000 Capillary Electrophoresis System, using a Biofocus Capillary Cartridge with and uncoated capillary of 24 cm total length (18 cm effective length) and 50 (m I.D. We employed the CE-SDS Protein Kit purchased from BioRad, according to the manufacturer's instructions. The CGE separations were performed at 25 ºC and on line UV detection was carried out at 220 nm.

RESULTS AND DISCUSSION

Under the conditions selected, we resolved the protein standard in 12 min. We worked in the range between 14 kDa and 200 kDa (Fig. 1).

Figure 1.

Electropherogram of the protein standard. Benzoic acid (Internal Standard), Lysozyme (14 kDa), Trypsin inhibitor (21.5 kDa), Carbonic Anhidrase (31 kDa), Ovalbumin (45 kDa), Serum Albumin (66.2 kDa), Phosphorylase B (97 kDa), ( Galactosidase (116 kDa) and Myosin (200 kDa), respectively.

Under the same experimental conditions, we performed the CE analysis of L. corniculatus, L. uliginosus (Fig 2); and four L. tenuis varieties (Fig. 3) seed samples. The electropherograms displayed similar patterns for all the seeds tested.

Figure 2.

Storage protein pattern of Lotus seed samples by CGE. a- Lotus corniculatus, b- Lotus uliginosus.

Figure 3.

Storage protein pattern of Lotus seed samples by CGE. Overlaid electropherograms of four seed samples of different varietes of Lotus tenuis: var. "La Plata", var. "El Chajá", var. "Jardín de Introducción INTECh", var. "Agro Verónica 95".

On the other hand, the same seed samples used for CE analysis, showed identical patterns when analysed in SDS-PAGE as can be seen in Fig 4.

Figure 4. Table of contents