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The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:271-274

Brief Communication

Inheritance and Subcellular Localization of Triose-Phosphate Isomerase in Dwarf Mountain Pine (Pinus mugo)

I. J. Odrzykoski

From the Department of Genetics, A. Mickiewicz University, 60–371 Poznan, Miedzychodzka 5, Poland.

Address correspondence to I. J. Odrzykoski at the address above or e-mail: ireko{at}main.amu.edu.pl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TpiB. Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inheritance of electrophoretic variants of many enzymes has been studied in pines and other gymnosperms since the beginning of the 1970s and especially during the 1980s (El-Kassaby and White 1985; Paule 1990 for a bibliography). Allelic variants of different enzyme loci can be easily observed in these plants in a sample of haploid megagametophytes of heterozygous trees and they usually segregate in proportions expected in a test cross. Many reports have been published since the first studies of this kind (e.g., Bartels 1971; Rudin 1975) and some provided the first evidence for linkage groups and showed evidence for conservation of gene blocks between different conifer genera (Conkle 1981; Guries et al. 1978; Rudin and Ekberg 1978). Polymorphic isozyme loci still provide a set of useful genetic markers in many population studies due to their technical simplicity of detection and the relatively low cost of data collection in comparison with DNA markers.

Inheritance data for about 40 enzymes have been published to date and as electrophoretic phenotype is usually made up of the products of one to five separate genes, inheritance of roughly 80 loci has been studied in the genus as a whole (Ledig 1998). Despite the large number of enzymatic systems available, not all enzymes frequently studied in other plant groups have a documented mode of inheritance in this genus. During the isozyme studies on populations of dwarf mountain pine (Pinus mugo Turra), I found several apparently heterozygous trees based on sporophytic phenotypes expected for the dimeric enzyme triose-phosphate isomerase (TPI, EC 5.3.1.1). Because a formal inheritance study of this enzyme has never been published for pines, seeds from several heterozygous trees were collected and used to study allelic segregation and linkage relationships.

Triose-phosphate isomerase is a glycolytic enzyme usually present in diploid homozygous plants as two isozymes with different subcellular localization (Wendel and Weeden 1989). The product of one nuclear gene is active in the cytosol and the other one, after translation, is actively transported into plastids (Pichersky and Gottlieb 1984). These two isozymes can be distinguished by comparison of the total cellular extract and the extract from a purified plastid preparation.

The purpose of this study was to test the inheritance and linkage relationships of the two TPI isozymes found in buds and megagametophytes of P. mugo. Also, the subcellular localization of the two isozymes was inferred by comparing the phenotype from crude extracts with those enriched for plastids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plant Material
Winter buds and seeds from eight trees were taken from the "Bór na Czerwonem" peat bog population (located near Nowy Targ, southern Poland) in late autumn. Small apical portions of the stem with winter buds and cones were collected from a single branch of each polycormic plant, and the buds were stored at -20°C prior to enzyme extraction. Seeds were extracted from cones at room temperature and after desiccation stored at -20°C.

Isozyme Analyses
Enzyme extraction. Three kinds of tissue were used for enzyme extraction: (1) a fragment of sporophytic tissue dissected from a winter bud, (2) seedlings, and (3) a sample of megagametophytes extracted from seeds germinated for about 10 days (to the moment when the radical of the seedling was 5 mm long). The tissue was homogenized in 0.1 M Tris-HCl buffer (pH 7.5), with the addition of 1 mM EDTA Na2, 10 mM KCl, 10 mM MgCl2 and 0.1% Triton X-100. Just before the extraction, 10 µl of 2-mercaptoethanol were added per 10 ml of extraction buffer. The extraction buffer for the bud tissue was modified by addition of 3% (w/v) PVP K-25. The homogenate was filtrated through a small piece of Miracloth tissue and absorbed into 2–2.5 mm wide Whatmann 3MM strips.

Electrophoretic separation and isozyme detection. Isozymes were separated using horizontal 10% starch-gel electrophoresis and a modification of the discontinuous buffer system A (Conkle et al. 1982) using 190 mM boric acid and 40 mM lithium hydroxide (pH 8.3) as the electrode buffer and 50 mM Tris, 6 mM citric acid with the addition of 10% of the electrode buffer (pH 8.2) as a gel buffer. Electrophoresis was performed in a refrigerated chamber under constant voltage (280 V) for about 4.5 h, to the moment when the "borate front" moved 8 cm from the origin. TPI isozymes were localized on top of the gel slices using the agar overlay technique (Manchenko 1994). The staining mixture contained 7.5 ml 50 mM Tris-HCl buffer (pH 7.5) with 1 mg of lithium salt of dihydroxyacetone phosphate (Sigma D7137) or a substrate prepared by hydrolysis of dihydroxyacetone phosphate dimethyl ketal (Sigma D7878), according to the supplier's instructions, 2 mg NAD, 45 mg sodium arsenate, 2 mg MTT, 0.5 mg PMS, and 7.5 ml 1.8% of warm-water agar solution. The volume of staining assay was occasionally reduced to about 10 ml, when the location of the isozymes could be deduced from migration of pigment markers. For detection of the products of other enzyme loci (PGD, 6-phosphoglucose dehydrogenase; GOT, glutamic-oxaloacetic transaminase; GDH, NAD glutamate dehydrogenase; ADH, alcohol dehydrogenase; SDH, shikimate dehydrogenase; FLE, "fluorescent" esterase; ACO, aconitase; DIA, NADH-diaphorase; LAP, leucine aminopeptidase; MDH, NAD malate dehydrogenase; PGM, phosphoglucomutase; and ACP, acid phosphatase), the standard technique was used (Conkle et al. 1982) and designation of enzyme loci follows standards for Scots pine (Niebling et al. 1987; Szmidt and Muona 1989).

Chloroplast preparation. A small amount of purified plastids were obtained from cotyledons of 2-week-old seedlings by a density gradient centrifugation in buffered Percoll (Pharmacia) using a modified method described by Odrzykoski and Gottlieb (1984). The procedure involves a few simple steps. First, a pulp obtained by gentle maceration of cotyledons in the extraction buffer was centrifuged for 1 h in a Percoll gradient buffered with 25 mM HEPES-KOH buffer (pH 7.5) with the addition of 1 mM EDTA, 330 mM sorbitol, 1% ficol, 0.2 % BSA, 10 mM KCl, and 48 mM 2-mercaptoethanol using SW41Ti rotor and a Beckman ultracentrifuge at 15,000 rpm (36,000g). After centrifugation the layer of intact chloroplasts was transferred into a 1.5 ml Eppendorf tube, washed twice in a washing buffer, and the resulting pellet was homogenized in the same extraction buffer as used for seedlings. Isozymes extracted from seedlings and those from the plastid-enriched fraction were compared side by side in the same gel.

Statistical Analysis
For a statistical evaluation of segregation data and linkage relationships, the chi-squared goodness-of-fit test was used, as described by Mather (1951). The data for the same locus pair from individual trees were pooled, if the tests results were homogeneous. Recombination values (r = R/n) and their standard error SEr = (r(1 -r)/r)1/2 were estimated for pairs of loci with significant linkage only (R = the number of recombinant types observed in a sample of n megagametophytes).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enzyme Phenotype for Winter Buds
The most common phenotype of TPI is composed of two isozymes: TPI-A and TPI-B (Figure 1A, line 1). In the studied population some other phenotypes were detected, one for TPI-A (labeled TPI-A12; Figure 2A, line 1) and three for TPI-B (labeled TPI-B22, TPI-B12, and TPI-B13 (Figure 1A, lines 2, 7, and 9, respectively).



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  		Figure 1.. (A) Electrophoretic TPI phenotypes for buds of several individuals of P. mugo. TPI-A is invariable in all trees. The most common phenotype is TPI-A11/TPI-B11 (e.g., line 1). The other phenotypes are TPI-B22 (line 2), TPI-B12 (line 7), TPI-B13 (line 9). (B) TPI phenotype from seedlings with the most common phenotype (lines 1, 2, 5, and 6), and from the chloroplast-enriched fraction obtained from the same seedlings (lines 4 and 5 marked with "p").

 


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  		Figure 2.. (A) Segregation of TpiA allozymes in megagametophytes of a tree with the sporophytic phenotype TPI-A12/TPI-B11 (line 1). (B) Segregation of TpiB allozymes in megagametophytes of a tree with the sporophytic phenotype TPI-A11/TPI-B12 (line 1).

 
Subcellular Localization of Isozymes
The seedlings of trees with the sporophytic phenotype TPI-A11/TPI-B11 had the same phenotype as the mother tree. In extracts from three independent preparations enriched in chloroplasts, only TPI-B isozyme was found (Figure 1B, lines 3 and 4 marked with "p").

Segregation in Heterozygous Trees
Segregation was studied in three trees with the sporophytic phenotype TPI-A12 (Figure 2A) and the results were homogeneous, showing the 1:1 ratio in all trees ({Sigma}n = 166, {chi}2(1:1) = 0.60, {chi}2(het) = 0.299, df = 2). Similarly five trees with the phenotype TPI-B12 (Figure 2B) showed a segregation ratio expected from the allelic variants ({Sigma}n = 479, {chi}2(1:1) = 0.012, {chi}2(het) = 3.829, df = 4). These results suggest that the TPI phenotype in dwarf mountain pine is composed of the product of two genes: TpiA and TpiB.

Linkage Studies
Trees heterozygous for TpiA were also heterozygous for nine other enzyme loci, therefore studies of joint segregation were possible (Table 1). The evidence for linkage was detected between TpiA and PgdA [6-phosphoglucose dehydrogenase (EC 1.1.1.44), locus A] in one tree tested for this combination (r = 0.10, SEr = 0.04). A weak linkage was also detected between TpiA and GotC [glutamate oxaloacetate transaminase (EC 2.6.1.1), locus C] also in one tree (r = 0.34, SEr = 0.05), and between TpiA and Fle ["fluorescent" esterase (EC. 3.1.1)] in one of two trees tested for these combinations (r = 0.33, SEr = 0.07). One or more trees were tested for a joint segregation of TpiB with 17 other markers, and the linkage was detected only for TpiB and DiaD [diaphorase (EC 1.6.4.3), locus D] in one from two trees tested for this combination (r = 0.36, SEr = 0.04).


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  		Table 1.. Two locus data and chi-squared analysis of allelic variants segregation at locus A ({chi}2A), locus B ({chi}2B), and joint segregation for the detection of linkage ({chi}2AB) for trees heterozygous for TpiA or TpiB

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Little is known about the genetic control and variation of the important glycolytic enzyme TPI in pines and other gymnosperms. Recently the enzyme was surveyed for variation in population genetics studies of Pinus echinata, P. virginiana,{-} and {+}P. palustris (Edwards and Hamrick 1995; Parker et al. 1997; Schmidtling and Hipkins 1998), however, no formal validation by segregation analysis was conducted. Nearly 550 plants from a peat bog population of P. mugo were tested for variability of this enzyme and some expressed an apparently heterozygous phenotype in the sporophytic tissue. The presumably heterozygous phenotype for fast-migrating TPI-A (a diffuse broad band of enzyme activity) was detected in 12 trees. Segregation studies showed that trees with this phenotype are indeed heterozygous, and in a sample of haploid megagametophytes the variants segregate in the 1:1 ratio. A three-banded phenotype was found in 85 trees for the more slowly migrating TPI-B, and segregation studies confirmed the heterozygosity of five of these trees. The enzyme phenotype of TPI in dwarf mountain pine is therefore composed of the products of two genes, a result commonly found also in diploid angiosperms (Gottlieb 1982; Weeden and Wendel 1989), excluding rare cases of duplication (or sometimes triplications) found in some genera (e.g. Pichersky and Gottlieb 1984; Wendel et al. 1989; and reports discussed by Weeden and Wendel 1989).

The results of linkage studies showed that TpiA is probably linked to PgdA. This may locate TpiA in the well-established "linkage group E" (Conkle 1981), known in numerous pine species including the closely related P. sylvestris (Goncharenko et al. 1994; Niebling et al. 1987; Szmidt and Muona 1989). The other enzyme loci from this group are GotC, SdhB, and MdhA. A weak linkage of TpiA with GotC was also detected in P. mugo, therefore the location of TpiA between PgdA and GotC is likely, but this should be confirmed with more detailed analysis. The evidence for linkage between TpiB and DiaD was also found in one tree. The second locus has been mapped in "linkage group A" (Conkle 1981), also in P. mugo, where DiaD and AdhA are tightly linked, with a recombination frequency of less than 0.10 (Odrzykoski IJ, unpublished data). The linkage of TpiB with loci from this group should be further tested, because at the same time no linkage was detected between TpiB and two other loci (GotA, AdhA) located close to DiaD.

The subcellular localization experiment showed that the more slowly migrating TPI-B isoenzyme is the only one present in the plastid-enriched fraction. The fast-migrating TPI-A is likely to be a cytosolic form of this enzyme. This is a similar situation to that found in diploid flowering plants, where TPI is usually present as two isozymes encoded by separate nuclear genes (Pichersky and Gottlieb 1984; Wendel et al. 1989). Only a little is known about the subcellular localization of enzymes used as genetic markers in population genetics studies in gymnosperms. Cytosolic and plastid isozymes of CuZn-superoxide dismutases from needles of Scots pine (Karpinski et al. 1992; Wingsle et al. 1991) and Norway spruce (Kroniger et al. 1992) are well characterized and the mitochondrial Mn-superoxide dismutase isozyme is known from the second species (Sehmer and Dizengremel 1998). Another example is provided by two NAD-dependent malate dehydrogenases (MDH2 and MDH3) resistant to ascorbic acid treatment, which suggests their mitochondrial localization, similar to the situation found in maize (Breitenbach-Dorfer and Geburek 1995). The procedure of subcellular localization tested for pines in this report may be useful for better characterization of isozyme markers. It should help to identify products of homologous genes, which may be important especially in comparative studies (Conkle 1991).


   Acknowledgments
 
I appreciate the comments and suggestions of Dr. D. B. Wagner and by the anonymous reviewers.


   Footnotes
 
Corresponding Editor: David B. Wagner

Received February 16, 2000
Accepted January 15, 2001


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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