Introduction

A primary goal of molecular biology is to understand how the genetic information is organized and how the genetic expression is regulated. Thus, extracting DNA from any tissue of an organism at any stage of development does not change their DNA sequence; meaning it is stable and identical to the original sequence. Active zones of this macromolecule with coding capacity, are called exons, and they stimulate the synthesis of specific enzymes in the different stages of development of an organism. The catalytic property of these enzymes reveal its presence by histochemical methods. Since 1957 geneticists have used electrophoresis to identify thousands of proteins contained in a raw cellular extract. Biomolecules that have the same catalytic property are called isozymes.

With isozymes or biochemical markers it is possible to perform genetic variability studies, where tissue variability (TV) means that various tissues or organs from the same individual show different molecular forms or different relative amounts of the same isozyme. In addition, the ontogenetic variability (OV) refers that in the same organ at certain stages of development (phenological stages) different molecular forms or different relative amounts of the same isozyme are shown. Thus, we can infer that both the TV and the OV are the result of differential regulation. So, every tissue of an organism has different genes expressions and in the same tissue at different points of the phenological cycle diverse groups of genes are expressed. In an electrophoresis gel each band that appears on the support matrix corresponds to a different protein whose synthesis may be controlled by one or more genes. Due to the fact that the order and nature of the amino acids determine the primary structure of the protein, this sequence depends only on the information contained in the DNA, which encodes a particular polypeptide. Thus, the phenotypes observed on the support matrix can be interpreted in terms of genotypes, in other words, in terms of genes and their alleles.

For geneticists, some of the most important isozyme’s characteristics are: 1) A proportion of these genes are polymorphic, this means that they are in the form of one or more alleles; and 2) these alleles are codominant, in other words, both alleles are expressed in heterozygous organisms; this phenomenon allows to relate the observed phenotypes with a given genotype. When interpreting isozymatic phenotypes in a support matrix, it should be considered its quaternary structure, depending on the number of polypeptide subunits (one, two, three or four). This allows us to classify them as monomers, dimers, trimers or tetramers, respectively, and the banding pattern for homozygous is represented, in all cases, by a single allele.

Nevertheless, in heterozygotes the transcripts encode for proteins associated with two alleles (monomers), three alleles (dimers), four alleles (trimers) and five alleles (tetramers). Hence, a study using isozymes can detect the presence of different molecular forms of these proteins at specific stages of development of an organism, from which it is inferred that the presence of a new or different isozyme in a specific state or stage of development might be controlling a process by the isozymes themselves. Therefore, to increase the efficiency of the technique, the developmental stages of the plant could be identified when the protein is expressed (^{Kephart, 1990}).

Electrophoresis in polyacrylamide or starch gels is used to calculate and detect gene products. The isozyme analysis by electrophoresis is a strong, reproducible and powerful technique with numerous applications because isozymes present co-dominant expression (^{Kephart, 1990}) and because they do not exhibit ambiental effects (^{Marquard and Chan, 1995}). Besides the method’s reliability, they are relatively cheaper than those of DNA analysis and allows us to obtain enzymatic information from different tissues (Vincent and Fulton, 2003). The isozyme’s analysis have been performed successfully in several plant species: Nicotiana tabacum L. (^{Stafford and Galston, 1970}), Zea mays L. (^{Scandalios, 1964}, ^{Efron, 1970}; ^{Rao et al., 1997}), Hordeum vulgare L. (Simonsen and ^{Pedersen, 1987}), Cereus peruvianus (^{Machado et al., 1993}), Cucurbita pepo L. (^{Carpin et al., 1999}), Brassica oleraceae (^{Bellani et al., 2002}), plants belonging to the family Asteraceae (^{Ros-Barcelo and AznarAsensio, 2002}) Mammillaria gracillis Pfeiff. (^{Balen et al., 2003}), Tigridia pavonia (^{Arzate-Fernández et al., 2005a}) and Lilium spp. (^{Arzate-Fernández et al., 2005b}).

Sprekelia formosissima (L.) Herbert, also known as Aztec lily, is a species native to Mexico that is still considered wild, it belongs to the Amaryllidaceae family and is characterized by being bulbous, annual and of short of cycle. It grows in grasslands and stony fields in the states of Durango, Jalisco, Guanajuato, Hidalgo, Estado de Mexico, Mexico City, Puebla, Veracruz, Guerrero and Oaxaca (^{Lopez-Ferrari and Espejo-Serna, 2002}). The ornamental value of this species is given by its scarlet flowers; therefore, in Mexico it is frequently seen in gardens and used as a medicinal plant (^{Vázquez-García et al., 1998}). Despite the ornamental potential of the species, no studies have been carried out using isozymes for S. formosissima. Therefore, the objective of this study was to evaluate the differential expression (tissue variation and ontogenetic variation) in this wild plant with biochemical markers, to determine the different enzyme forms expressed in the vegetative and reproductive stages.

Materials and methods

In this study we used Aztec lily plants (S. formosissima (L.) Herbert cv. Terciopelo, obtained from cloned bulbs and grown in a substrate composed of: leaf litter, sand and bovine manure in a 1:1:1 ratio for 78 days, in a rustic greenhouse at the Faculty of Agricultural Sciences at the Universidad Autonoma del Estado de Mexico (UAEMex).

The S. formosissima cultivation cycle was divided into two phenological stages to evaluate their differential expression: vegetative stage and reproductive stage (flowering). During the vegetative stage only tissue samples from the leaves (H) were collected. In the reproductive stage, which initiated after the first sprouting of leaves, and continued for approximately 40 d, the tissue samples were obtained from tepals (T), stigma-style (G), stamens (E) and leaves (H).

Once the seedlings emerged, the tissue sampling took place every 8 days, in each sampling all available tissues were collected. During the reproductive stage three samplings were carried out: the first was only H, the second of T, G and E, and the third H, T, G and E. In the vegetative stage seven samples were taken, all of H. Each sample consisted in collecting tissue samples of at least three plants, the samples were placed into Ziploc bags, and placed in a cooler and then kept at -20 °C in a domestic freezer. All samples were processed at the Laboratory of Plant Molecular Biology, in the Faculty of Agricultural Sciences of the UAEMex.

Results and discussion

Bands with enough sharpness and resolution for registration and further analysis were observed in five electrophoretic systems (Table 1). The PGI, PGM and MDH isozymes showed better resolution in the citric acid-histidine system at pH 5.7; whereas the ACP isozyme showed improved resolution in the same buffer, but at pH 6.5. In the POX case, better resolution was provided by the Tris citrate/Trishistidine system at pH 7.8.

Table 1 Isozyme banding patterns (IBP) observed in the different phenological development stages (reproductive and vegetative) in Sprekelia fromosissima (L) Herbert. 

Numbers (1) to (10) means the number of sampling; “o”: Origin of electrophoresis; H: Leaf; T: Tepal; G: Stigma-Style; E: Stamen; m: monomer; d: dimer.

The five isozymes produced 15 bands and nine isozyme banding patterns (IBP) (Figure 1): two for POX, three for ACP, one for PGI, two for PGM and one for MDH. The IBP obtained with each isozyme in both phenological stages is described below.

Figure 1 Schematic representation of isozyme banding patterns (IBP) in Sprekelia formosissima using biochemical markers:(A) POX in Tris-citrate system, pH 7.8; (B) ACP in Histidine-citric acid system, pH 6.5; (C) PGI in Histidine-citric acid system, pH 5.7; (D) PGM in Histidine-citric acid system, pH 5.7; (E) MDH in Histidine-citric acid system, pH 5.7. Numbers figure footer represent the number of isozyme banding pattern (IBP), while “o” represents the origin of the electrophoresis. 

Conclusions

With the horizontal electrophoresis technique on starch gels, differential isozyme banding patterns were identified throughout two phenological stages of Sprekelia formosissima. This technique has advantages over molecular DNA markers, given that several gels can be obtained at the same time and the gels can be dyed with up to seven enzymes, reducing times and costs.

The IBP obtained in our study with the following biochemical markers: peroxidase, acid phosphatase, glucose phosphate isomerase and phosphoglucomutase indicate that this technique is useful in investigations of differential expression in vegetative and reproductive stages of S. formosissima as well as a diagnosis tool in breeding programs or in a genetic variability or diversity studies.