Lichen Proteins, Secondary Products and Morphology: a Review of Protein Studies in Lichens with Special Emphasis on Taxonomy
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J. Hattori Bot. Lab. No. 76: 235- 248 (Oct. 1994) LICHEN PROTEINS, SECONDARY PRODUCTS AND MORPHOLOGY: A REVIEW OF PROTEIN STUDIES IN LICHENS WITH SPECIAL EMPHASIS ON TAXONOMY JAN-ERIC MATTSSON1 ABSTRACT. Almost 30 protein studies with different objectives for c. 50 lichens species are compared and the taxonomic relevance, in particular, and other aspects of the use of elec trophoretic techniques discussed. The results indicate large enzymatic variation both in vegetat ively and sexually propagating lichens, differences in enzyme patterns between lichenized and non-lichenized bionts but also, correlation between protein banding patterns and secondary product chemistry or spatial separation. INTRODUCTION Electrophoretic studies of lichen proteins and enzymes are still far behind those of other organisms and for taxonomic purposes very few investigations have been made. Reasons for this have been the technical problems with the methods and the uncertainty of how to interpret the electrophoretic data taxonomically. The reliability of earlier techniques such as starch gel or polyacrylamide gel electrophoresis have been questioned, especially when combined with simple methods of plant protein separation (Kershaw et al. 1983). Isoelectric focusing has been shown to be more reliable, and the development of a standardized method of isoelectric focusing for routine use, which could be used by others than chemists and physiologists, has been necessary to provide lichen taxonomists with access to protein data. Although such a method has been developed by Fahselt and co-workers it has rarely been used for taxonomic purposes. The objective of this paper is, from the viewpoint of the taxonomist, to briefly review most of the studies dealing with electrophoretic investigations of lichen proteins and to discuss some of the general problems when interpreting these results taxonom ically. GENERAL PROBLEMS WITH LICHEN ELECTROPHORESIS One of the main problems for electrophoretical investigations of lichens has been the difficulty of breaking the cell walls in a process to yield a reasonable concentration of active protein. With low protein concentration some isozymes may not be detected 1 Dept. of Systematic Botany, Lund University, Ostra Vallgatan 18- 20, S-223 61 Lund, Sweden. Present address: Botanical Museum, Uppsala University, Villavligen 6, S-752 36 Uppsala, Sweden. 236 J. Hattori Bot . Lab. No. 76 I 9 9 4 and cause erroneous information on the variation between different samples. Thus, apparent homogeneity may actually be heterogeneity when methods are changed (Kershaw et al. 1983). Lack of detectable activity does not necessarily indicate absence of a certain enzyme. Alternative extraction methods would yield different kinds of proteins and no extraction method seems, so far, to be successful in extracting the same enzymes from all lichens (Hageman & Fahselt 1984). Some of the studies discussed here use mainly general protein stains, while others look at specific (particular) enzymes. In the former cases there is more problem comparing bands between thalli. There is a greater chance that bands looking the same are not the same. Another problem with lichen enzymes is to make sure that the appropriate enzyme really is stained. The staining methods are often developed for studies of enzymes of higher plants or animals and not for fungus or lichen enzymes; for example esterase and carbonic anhydrase (CAN) have similar function and for C/adonia cristatella these enzymes have zymograms resembling each other (Fahselt 1985). It is possible that CAN in lichens may also have esterase activity just as some CAN isozymes in mammalian cells have (Tashian 1977). It is necessary therefore to be cautious when interpreting isozyme data for genetic or taxonomic purposes. A major problem is also attempts to characterize a species without investigating variation present in either species or populations before deciding on sampling regimes. For example, Mattsson ( 1991) had difficulties in evaluating enzyme data for taxonomic purposes because of the large intraspecific variation compared to the small sample. The sampling problem is a major one with many of the studies reviewed here. FUNGAL AND ALGAL CONTRIBUTIONS TO THE ZYMOGRAMS The co-occurrence of at least two symbiotic organisms is also a complication. Some taxonomists have been uncertain how protein data from electrophoretic investi gations of such organisms as lichens should be interpreted taxonomically. Some bands of a zymogram could represent enzymes composed of mycobiont and photobiont enzymes or parts of them, although most bands may originate from enzymes produced by one or the other of the symbionts. The algae make up about 10- 15 % of the cellular volume of the lichen thallus (Ahmadjian & Paracer 1986) and will to some extent contribute to the enzyme banding patterns. If for taxonomic reasons one prefer to use mycobiont characters it is interesting to know to what extent the photobiont is responsible for the bands of a zymogram of a thallus preparation. For Cladonia cristate/la Fahselt (1985) showed a large similarity between isolated mycob iont and lichen zymograms and concluded that most of the detectable lichen protein could well have been produced by the mycobiont, in some cases under influence of the photobiont. ELECTROPHORETIC STUDIES OF LICHENS Electrophoretic protein studies on lichens have mainly dealt with populations of distinct species (e.g. Hageman & Fahselt 1984, Fahselt 1991, 1992) or more or less J.-E. MATTSSON: Lichen proteins, secondary products and morphology 237 Table 1. Lichen species studied with proteins studied by electrophoretic methods. Nomenclature according to original publication. Species Topic Reference Cetraria alvarensis Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria a/varensis Taxonomy: pop. sp. Mattsson 1991, 1993 Cetraria arenaria lsozyme var. : p., pop. Fahselt & Hageman 1983 Cetraria cucullata Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria ericetorum Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria halei Isozyme var. : pop. Fahselt 1988 Cetraria islandica Taxonomy: g., sp., ssp. Klirnefelt & Mattsson 1987 Cetraria juniperina Taxonomy: g., sp. Klirnefelt & Mattsson 1987 Cetraria juniperina Taxonomy: g., sp. Mattsson 1991, 1993 Cetraria pinastri Taxonomy: g., sp. Mattsson 1991, t 993 Cetraria tilesii Taxonomy: g., sp. Mattsson 1991, 1993 Cladonia cristatella Symbiosis. Fahselt 1985 C/adonia cristatella Morphological. var. Fahselt 1985 Cladonia cristatella Genetic var. in spores Fahselt t 987 C/adonia mitis Environmental effects Fahselt 1992 Cladonia rangiferina Methodology Fahselt 1980 Cladonia rangiferina Isozyme var. : sp. MacFarlane et al. 1983 Cladonia stel/aris Methodology Fahselt 1980 Cladonia stellaris lsozyme var. : sp. Kershaw et al. 1983 Cladonia unicialis Methodology Fahselt 1980 Coe/ocaulon acu/eatum Taxonomy: g., sp. Klirnefelt & Mattsson 1987 Coelocaulon muricatum Taxonomy: g., sp. Karnefelt & Mattsson 1987 Evernia mesomorpha Isozume var.: pop. Fahselt 1988 Dermatocarpon miniatum Methodology Fahselt 1980 Hypogymnia physodes Isozyme var. : pop. Fahselt 1988 Lasa//ia papulosa lsozyme: repr. strat. Hageman & Fahselt 1990b Parmelia caperata Symbiosis. Martin 1973 Parmelia conspersa Taxonomy: sp., ssp. Skult et al. 1986 Parmelia cumberlandia Methodology Fahselt 1980 Parmelia hypoleucina Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia hypotropa Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia omphalodes Taxonomy: sp., ssp. Skult et al. 1986 Parme/ia omphalodes Taxonomy: ssp., pop. Skult et al. 1990 Parmelia perforata Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia rigida Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia saxatilis Taxonomy: sp., ssp. Skult et al. 1986 Parmelia sulcata Taxonomy: sp., ssp. Skult et al. 1986 Peltigera canina Methodology Fahselt 1980 Peltigera rufescens Physiology. Brown & Kershaw 1985 Platismatia tuckermannii Isozyme var.: pop. Fahselt 1988 Ramalina cuspidata Taxonomy: sp., v., pop. Mattsson & Klirnefelt 1986 Ramalina siliguosa Taxonomy: sp., v., pop. Mattsson & Karnefelt 1986 Stereocaulon saxatile Taxonomy: sp., v. Fahselt 1991 Umbilicariaceae Taxonomy: sp., v., pop. Hageman & Fahselt 1992 238 J. Hattori Bot. Lab. No. 76 I 9 9 4 Table I. (Continued) Species Topic Reference Umbilicaria decussata Isozyme var. : pop. Fahselt 1989 Umbilicaria deusta Isozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria hirsuta lsozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria hyperborea Isozyme var. : pop. Fahselt 1989 Umbi/icaria mammulata lsozyme var.: sp. Hageman & Fahselt 1984 Umbilicaria mammulata Isozyme: Seasonal var. Hageman & Fahselt l 986b Umbilicaria mammulata Physiolog. var.: ind. Larson & Carey 1986 Umbilicaria mammulata Isozyme; repr. strat.: sp. Hageman & Fahselt 1990b Umbi/icaria muhlenbergii Isozyme; morph.: sp., pop. Hageman & Fahselt l 986a Umbilicaria muhlenbergii Isozyme; repr. strat. Hageman & Fahselt 1990b Umbilicaria vellea Physiolog. var.: ind. Larson & Carey 1986 Umbilicaria vellea Functional sexuality. Hageman & Fahselt l 990a Umbilicaria vellea Isozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria virginis lsozyme var. : pop. Fahselt 1989 Usnea subjioridana Isozyme var.: pop. Fahselt 1988 Xanthoria elegans Isozyme var.: morph, pop. Fahselt & Krol 1989 Abbreviations: g. = genus, ind. = individual, morph.= morphology, pop.= population, repr. strat. = reproductive strategies, sp. = species, ssp. = subspecies, v. = variety, var. = variation. closely related taxa from