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ACTA UNIVERSITATIS PALACKIANAE OLOMUCENSISFACULTAS RERUM NATURALIUM (2000) BIOLOGICA 38

ELECTROPHORETIC ANALYSIS OF PLANT ISOZYMES

Miroslav Zeidler

Dept. of Ecology, Palacký University, Tř. Svobody 26, Olomouc, Czech Republic

Received: July 5, 2000; accepted August 10, 2000

Key words: isozyme, allozyme, electrophoresis, PAGE, marker

Abstract

Isozymes (or isoenzyme) are powerful tool for gene variability within and between populations ofplants and animals, yet nowadays new molecular technics based on DNA are used. There are discussedconditions for performance PAGE vertical electrophoresis of isoenzymes and staining procedures. Isoenzy-me are able to solve other questions of population biology, conservation biology and ecology as well. Mainpros and cons of DNA and isoenzymes analysis are mentioned.

Introduction

The basis of electrophoretic analysis of isozymes (from isoenzymes) was laid downin 1957 (Stebbins 1989, McMillin 1983) when Hunter and Mohler discovered theisozymes. In 1959 Markert and Moler introduced the concept of isozymes, which theydefined as the different molecular forms in which proteins may exist with the sameenzymatic specificity (Buth 1984). This means that different variants on the sameenzymes have identical or similar functions and are present in the same individual. Assuch, their importance for understanding gene action in development and differentia-tion was exploited during the1960s in both animals and plants. Nevertheless, isozymesplayed a minor role in research on plant biochemistry until 1966 when geneticpolymorphism for isozymes within the same population was discovered (Stebbins1989, Wendel 1989). That revealed the possibility for population genetics to makeprecise quantitative estimates of genetic variability based upon one parameter of themolecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleaguesand the investigation of both animals and plants increased explosively. This applica-tion, however, necessitated a partitioning of the isozyme concept, since they only used

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the relevant allozyme subset, defined by Prakash (Buth 1984) as the variant proteinsproduced by allelic forms of the same locus, to avoid the now common confusion withisozymes which are the various polymer produced from monomers specified by diffe-rent loci. The development of genetic distance or similarity coefficients (Lukasová1985) allowed the summarization of allozyme data for intersample comparisons andso quantified allozyme data were soon applied to comparative studies of taxa.

Polymorphism and basic principles of isozymes

Polymorphism may be defined as simultaneous occurrence within or betweenpopulations of multiple phenotypic forms of a trait attributable to the allels of a singlegene or the homologs of a single chromosome (Acquaah 1992). In natural populationsrecurrent mutations of genes produce variability. There are polymorphic loci, variablein the sense described above, and monomorphic or nonvariant. The main points of theconcept of isozymes perceived by researchers are summarized below:1. Multiple molecular forms of enzymes (isozymes) are common in organisms.2. Isozymes share a common catalytic activity. Each isozyme has a specific role in the

metabolic pathway and functions in harmony with other enzymes within the orga-nizational framework of cells.

3. Isozymes often exhibit tissue or cell specificity.4. Molecular heterogenity of enzymes confers flexibility, versatility and precision

upon an organism in terms of metabolic functions.5. Molecular multiplicity is desirable for biological efficiency.

Isozymes arise in nature by two general mechanisms, i.e. genetic and epigenetic.The source of gene multiplicity is duplication through mutation, polyploidization andchromosomal aberrations (Hoelzel 1991). Those events constitute the present epige-netic origin of isozymes. Epigenetically formed enzymes are not considered isozymesby some researchers. Epigenetic mechanisms may be divided intoa) post-translational addition,b) post-translational deletion andc) post-translational conformation.

On the other hand there are four genetic mechanisms (Acquaah 1992):a) Multilocus system I – different genes code for independent proteins with the same

enzymatic activity. The various genes are nuclear in origin but their proteinproducts are located in different parts of the cell.

b) Multilocus system II – is similar to system I except that the enzymes involved arepolymeric and the subunits are encoded by more than one locus.

c) Multilocus-polymeric system – enzymes display a series of polymers that consist ofidentical subunits.

d) Allozyme system – the term allozyme describe isozymes encoded by allelic genes.Alleles at various loci may be modified to produce isozymes that are distributed ina population according to Mendelian laws of inheritance (Weeden 1983).

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Principles of protein electrophoresis

Electrophoresis is a versatile biochemical technique to detect genetic variation.Protein molecules migrate in an electric field because they are charged, for details seeHamrick (Hamrick 1990). When an electrical gradient is applied, the moleculesmigrate toward the electrode with the charge opposite to their own, with the resultthat the initial single boundary formed by the mixture of molecules is broken intoseveral boundaries according to the relative mobilities of the mixture. This techniqueis useful for separating and analyzing complex proteins mixtures. Today active mediaare used. The investigator can alter the porosity of medium for more effective separa-tion of molecules that have identical charge densities but that differ in size. Examplesof media are agarose, starch (May 1994) and the now often used acrylamide gels.Some isozymes resolve better when certain combination of buffers (gel and electrode)are used (Weeden 1983). In polyacrylamide gel electrophoresis (PAGE), multiphasicbuffer systems employ two kinds of gel in one run: lower (analysing) gel and upper(stacking) gel (Hamrick 1990, Wendel 1989). The pH of electrophoretic buffers maybe manipulated within range to optimize the resolution of bands of proteins beingelectrophoresed. Electrophoresis operates on two fundamental and interrelated elec-trical principes: electrical current, which is proportional to voltage, and power, whichis directly proportional to the voltage and current. The heat generated during theelectrophoretic process must be dissipated because excessive heat decreases enzymeactivity (Andrews 1986). The fact mentioned above combined with the duration ofelectrophoresis, protein concentration of samples, quality of samples, sample size,principles of staining gels (Vallejos 1983) and protocols strongly influenced results.For more details see Acquaah (Acquaah 1992), Hamrick (Hamrick 1990), May (May1994), Hoelzel (Hoelzel 1991) and Stebbins (Stebbins 1989).

A good analysis expects interpretation of gel patterns. The resulting bandingpattern is an electrophoretic phenotype (Wendel 1989), which usually consist of oneor more colored bands for each individual analysed. In some cases, it may be simpleand consist of a single invariant band in the whole sample. In contrast, some enzymesmay display complex phenotypes with 15 or more bands per individual. So a correctinterpretation of banding patterns in genetic terms requires the proper determinationof the pertinent factors that influence the electrophoretic phenotype. Moreover, e.g.null allels, intergenic heteromultimers, multiple-banded products (“shadows”) andartifacts may act (Šnábel 1995, Weeden 1983).

An explanation of gel patterns in genetic terms means assessing at least the meannumber of alleles per locus (A), the percentage of polymorphic loci (P), the meannumber of alleles per polymorphic locus and effective number of alleles per locus(Hartl 1988, Mahy 1997, Ollejonsson 1996) from this description proceed otherparameters as heterosigosity (H), genetic diversity (G) or others in accordance withthe design (Hamrick 1983, Hamrick 1989, Godt 1996). There are other possibleexplanation only by bands variability – mixed phenotypes (Karkouri 1996, Lehman1997, Etoh 1981).

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Application allozymes as a tool in research

Genetic markers generally have contributed to the study of plant biology byproviding methods for detecting genetic differences among individuals. There aresome important ecological topics which often use allozymes as powerful markers:

Genetic relatedness within and among populations, often with relations to geogra-phic structure. Patterns across a broad range of taxa are mostly consistent with ourunderstanding of the effects of the breeding system (species with selfing tend to poseslower levels of genetic variation within populations), life history (longer-lived peren-nials tend to be more variable) and the distribution of genetic diversity within andamong populations (Clegg 1990, Hamrick 1990, Kudoh 1997, Mahy 1997, Tarayre1997). There is a narrow linkage between geography and the spatial patterns ofgenetic variation (Epperson 1990, Newton 1999) as well as the genetics of plantmigration and colonization (Barrett 1989,1990; Sun 1997).

Mating system estimation. An alternative approach of the study to plant matinguses classifications of mating events to characterize levels of inbreeding and patternsof gene dispersal in a population and other consequences (Holm 1997, Soltis 1987,Brown 1990, Soltis 1989, Sun 1996, Mitchell 1998, Noyes 1996, Wang 1996).

Genetic diversity in clonal plant species. Asexual reproduction is relatively com-mon in plant species and can occur through a number of modes, e.g. vegetative spread,production of vegetative propagules, apomixis (Cruzan 1998). One difficulty with thestudy of genotypic diversity in clonal species is the inadequacy of allozyme markers toreliably identify all genotypes present (Ellstrand 1987, Etoh 1981, Lehman 1997,Ollejonsson 1996, Pooler1993, Sipes 1997, Zeidler 1999).

Forestry and agriculture (Bliss 1990, Hamrick 1989, 1990, 1997; Čurn 1995; Doeb-ley 1989; Gärtner 1996; Kara 1997; Löchelt 1995, Marschal 1990, Muona 1990, Weber1990). It is often necessary to test paternity (Cruzan 1998) or chromosomal locationsand mapping (Peffley 1988, Satovic 1996, Shigyo 1995a,1995b, 1994a,).

Selection and linkage in plant populations and their evolutional consequences(Cruzan 1998, Ennos 1990, Hartl 1988, Hastings 1990, Sun 1996) and some tasks ofpolyploidy (Chase 1988, Kirschner 1995, Maki 1996, Sun 1996).

Conservation biology is a rapidly rising field because of effective molecular tools(Gärtner 1996, Gemmill 1998, Godt 1996, Haig 1998, Newton 1999).

Other studies: Seed bank populations (Cabin 1998, McCue 1998), environmentalchanges and habitat heterogenity (Lehman 1997), combination with cytological stu-dies and other methods (Anderson 1995, Noyes 1995), plant pathology (Leuchtman1996), germaplasm collections (Lamboy 1996), mycorrhizal genetic variation (Kar-kouri 1996, Martin 1998) and phytopatology (Ylimattila 1997, Forbres 1997).

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Limitations of protein electrophoresis

Current electrophoresis detects only the amino acid sequences that result in thenet charge of proteins. As sensitive as electrophoresis may be, only about a third of allamino acid substitutions can be detected by the technique (May 1994, Acquaah 1992).Then the amount of polymorphisms will probably be underestimated as a result of thiscryptic variation. Electrophoresis is also limited by the number of available stainingprotocols. The protocols available are for water-soluble proteins. Another possiblerestriction of electrophoretic analysis of genetic variation is that only the variability inthe coding portions of the DNA can be sampled (May 1994). Another complication isthat some organisms are polyploid in origin and some specific genes have beenduplicated in otherwise diploid organisms (Hoelzel 1994). Obviously, with more locithere are more gene copies coding for a protein (May 1994, Šnábel 1995). Unfortuna-tely, there are some monomorphic species for most allozymes. On average across taxa,less than half of all loci are polymorphic. Narrow genetic endemic species and othersthat have experienced genetic bottlenecks often lack polymorphic loci (Parker 1998).Many statistical analysis in population genetics assume that phenotypic differencesamong allozymes are minimal and selectively neutral, but exceptions are known. Also,codominant inheritance, although generally true for allozymes, is not always observed(Wendel 1989).

Advantages of allozyme analysis

Comparable data from previous studies and a wealth of standard statistical me-thods make allozymes appealing for studies of genetic variation. Most allozymesrepresent codominant loci distributed according to Mendelian laws of inheritance andmany loci express at all stages of the life cycle (Hamrick 1989). So it is feasible to usevariation in allozymes rather than nucleic acids for a particular question. With RAPDsand sequencing allozymes are considered excellent for population structure analysisfor sufficiently polymorphic taxa (Parker 1998). A comparison of the main featuresbetween allozymes and DNA markers are shown in Table 1.

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Table 1 Comparison of main features of molecular methods based on allozymes and DNA as markers

Summary

It is obvious that in recent years there has been an explosion in the number ofdifferent types of genetic markers available, moreover the number of statistical tech-niques have been developed for analyses of molecular data. In most cases, the newDNA-based markers provide the same type of information as allozymes, but allow forclearer resolution of genetic differences. A lot of conservation and rescue projectshold the role “the cheaper the better” because cost can be the crucial point for therethis projects. Costs can be reduced to the necessary conservation measures and by theimplementation of less expensive research techniques. In this case allozymes seem tobe the more appropriate technique. Many recent investigations combine geneticmarker data with ecological information and so follow a general trend towards morestudies of the mechanisms responsible for the development of patterns in populations.

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