Slower Moving to the Anode Malate Dehydrogenase Isozymes of Rye, Wheat and Triticale Seeds

Slower Moving to the Anode Malate Dehydrogenase Isozymes of Rye, Wheat and Triticale Seeds

Biochem. Physio!. Pflanzen 187, 409-416 (1991) Gustav Fischer Verlag lena Slower Moving to the Anode Malate Dehydrogenase Isozymes of Rye, Wheat and ...

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Biochem. Physio!. Pflanzen 187, 409-416 (1991) Gustav Fischer Verlag lena

Slower Moving to the Anode Malate Dehydrogenase Isozymes of Rye, Wheat and Triticale Seeds ROUMYANA VLADOVA Doncho Kostoff Institute of Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria Key Term Index : electrophoretic analysis, malate dehydrogenase , isozymes , slower moving to the anode; rye, triticale , wheat

Summary The author's own modification of Davis' method (1964) for protein electrophoretic separation was used in an analysis of protein extracts from seeds of tetraploid and hexaploid forms of triticale, diploid and tetraploid rye, T. aestivum, T. durum. Results substantiated the conclusion that the slower moving to the anode isozymes were dimers. This corresponded to data from the dissociation - recombinant analysis of the mixture of T. durum and rye extracts . It was found that the two groups of isozymes separated specially on the gel differed in their relation to 2 M urea in the protein extract - those moving fast lost their activity completely , while the ones moving slower remained almost unchanged. This fact is connected with the known reference data that the most anodic isozymes in wheat and barley are from the fraction of cytoplasmic malate dehydrogenase (McDANIEL 1969; NEWTON 1983). It was presumed that malate dehydrogenase isozymes of rye, wheat and triticale seeds moving slower to the anode belong to the mitochondrial fraction. It is pointed out that the reaction of malate dehydrogenase isozymes to urea in suitable concentrations could be used as an easily applied criterion differentiating the two basic types of malate dehydrogenase isozymes in a total protein extract.

Introduction NAD-dependent malate dehydrogenase (MDH, Ee 1.1 . 1.37) catalyses the interconversion of malate and oxalacetate . This is a key reaction in the malate oxalacetate aspartate "shuttle" through the mitochondrial membrane and in the cycle of tricarbonic acids, which takes place in the mitochondrial matrix. Two types of malate dehydrogenase exist, which catalyse one and the same chemical reaction occurring in two differentiated cell areas - supernatant or cytosolic malate dehydrogenase (s MDH) and mitochondrial malate dehydrogenase (m MDH). Both enzymes are coded by nuclear genes and are represented with several isozymes. Usually they are dimers of 60 ,000-80,000 mol· wt. (McALISTER-HENN 1988). Heterodimers are formed in vivo only between subunits of the same type of malate dehydrogenase. Differences in kinetic and physico-chemical properties, in the kind of variation between the individual species etc., exist between the two isozyme types. They are controlled by genes situated in different gene loci (WHITT 1970; WHEAT and WHITT 1971). Electrophoresis of a protein extract containing both types malate dehydrogenases in PAAG with pH about 7 showed that wheat and rye isozymes produce two zones clearly BPP 187 (1991) 6

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differentiated in their electrophoretic mobility - zone I, consisting of isozymes moving faster to the anode, and zone II, consisting of isozymes moving slower to the anode. BENITO and SALINAS (1983) discovered 3 isozymes in zone II of Triticum aestivum, and while investigating aneuploid lines of cv. Chinese Spring found that they are dimers, coded by the long arms of the chromosomes from the first homoeologous group of wheat. DIAZ et al. (1986) confirmed the localization of MDH genes from zone II on the chromosomes from the first homoeologous group. However, they had used another gel system in their electrophoretic analysis and obtained 4, not 3 isozymes, explaining these results by accepting that the isozymes of zone II were monomers. They substantiated this presumption by the hypothesis proposed before about the monomeric type of rye isozymes from zone II (SALIN AS and BENITO 1985). LJU and GALE (1989) assessed by isoelectrofocusing the localization of dimeric malate dehydrogenase isozymes on the short arms of wheat chromosomes of the fifth homoeologous group. The primary aim of this investigation was on the basis of MDH electrophoretic analysis of tetraploid triticale form seeds to confirm the presence of chromosome 1 B in their wheat genomes. The results obtained in the course of the investigation provided possibilities of revealing interesting facts about MDH performance in the triticale forms studied and of explaining the question about the subunit composition (monomers or dimers) of isozymes from zone II in wheat and rye .

Material and Methods The investigation included the following plant material : (1) five tetraploid triticale forms, AD 248-33, AD 248-22, AD 248-22-10, AD 248-22-14 and AD 248-22-15, developed by Z. SABEVA at the Institute of Genetics, Sofia. These forms contain a whole rye genome and a mixture of chromosome pairs belonging to A and/or B wheat genomes (SABEV A 1983; SABEV A 1985). It was found that their wheat genome contains chromosomes 1B, 3 A and 4 B (VLADOV A and SABEVA 1987; VLADOVA 1987; VLADOVA 1988); (2) the initial parental forms - hexaploid triticale 102 NAD 137 , 2n = 42 , AABBRR and tetraploid rye, Secale cereale L. cv. Vladimirovka, 2n = 28, RRRR ; (3) the primary hexaploid triticale AD C I 42, 2n = 42, AABBRR and the forms, from which it was developed Triticum durum Desf. cv. No . 7442 , 2n = 28, AABB and diploid rye, cv. BNR Isol , 2n = 14, RR ; (4) Triticum durum Desf. cvs . Zagorka and Parus, 2n = 28, AABB and Triticum aestivum L. cvs. Sadovo I and Bezostaya I, 2n = 42, AABBDD; (5) 6 x triticale St. 7291, 2n = 42 , AABBRR. Seed protein extracts were analysed, using 0.05 M tris-HCl buffer, pH 7.2, containing protective supplements 6 mM cysteine hydrochloride, 6 mM ascorbic acid and 0.5 M saccharose (RYCHTER and LEVAK 1969). The ratio sample weight (g): buffer volume (ml) was I: 10. Average sample weights varied within the range of 0.2 to 1.0 g. Vertical block polyacrylamide gel electrophoresis was used with gel size 1/90170 mm and 1/90/170 mm . Two gel systems for electrophoretic separation were applied: (1) electrophoresis after DAVIS (1964) in 7.5 % gel without upper gel; (2) 6% gel after DAVIS (1964), the separating gel and the upper electrode buffer containing 0 .03 M Na2 EDTA . MDH isoenzymes were visualized after the method of SHAW and PRASAD (1970). The dissociation-recombinant analysis was made after the method of HART (1971) modified by NAGY et al. (1980). The effect of 2 M urea on malate dehydrogenase isoenzyme activity was also tested using two procedures : (1) adding urea of final concentration 2 M to the extraction buffer before grinding; (2) adding a respective quantity of urea to the ready, centrifugated, clear, protein extracts. In both cases the results were the same. 410

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Results and Discussion In both gel systems used for electrophoretic separation MDH isoenzymes occupied two zones. Fig . 1shows MDH isozymes separated by the gel system No.1. In zone J each one of the species investigated contained one isozyme. In zone II rye and the tetraploid

+

a

--------a

b

b

c d

c

d

-1

-2 n -3

e

Fig. 1. Diagrammatic representation (A) and zymograms (8) of the MDH isozymes. separated on 70mm long 7.5% PAAG after DAVIS (1964). S. cereale (lA-a) cvs. Vladimirovka (IB-j) and Imel ( IB-k); tetraploid triticale forms (lA-b), AD 248-33 (lB-a), 248-22 (lB-b) . 248-22-10 (1 B-e). 248-22-14 (1 B-h) and 248-22-15 (1 B-i) ; hexaploid triticale forms (1 A-c), 102 NAD 137 (IB-g); T. durum (lA-d) cv. Zagorka (lB-f) ; T. aestivum (lA-e) cvs. Sadovo 1 (IB-c) and Bezostaya 1 (I B-d) .

triticale forms contained two isozymes each - No.2 and No.3. Isozyme No.3 in rye was with very low intensity of staining, but in the tetraploid triticale forms staining was well expressed. Each one of the hexaploid triticale forms , T. durum and T. aestivum contained 3 isozymes in zone II. Isozyme No . 1, which according to the hypothesis of BENITO and SALINAS (1983) is coded by genes localized in chromosome 1 Awas not found in the spectra of tetraploid triticale forms. BPP 187 (1991) 6

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- - - --- --- 3,

+

a

b

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-23

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Fig. 2. Diagrammatic representation of slower moving to the anode MDH isozymes, separated on 70 mm long 6% PAAG after DAVIS (1964) with modifications - the upper, cathode buffer and the gel contain 0.03 M Na2 EDTA. Denotations are the same as in Fig. 1 A.

Addition of 0.03 M Na2 EDTA in the separating gel and in the upper electrode buffer enhanced considerably the quality of separation (Fig. 2). In zone II T. durum and T. aestivum contained each one 4 isozymes, which according to the hypothesis of DIAZ et al. (1986) were controlled as follows: isozymes Nos. 1 and 3 by chromosomes 1 A and I D, isozymes Nos. 2 and 4 - by chromosome 1 B. The tetraploid and hexaploid triticale forms contained each one, two, respectively three, main isozymes in zone II. Isozyme No.1 was included only in hexaploid triticale forms, a fact in conformity with its genetic control (chromosome 1 A). Isozyme No.2 was found in T. durum, T. aestivum and in rye and its presence in the spectra of the investigated forms of triticale was easily explained. An unexpected result, if we accept the hypothesis of DIAZ et al. (1986) about the monomeric composition of isozymes from this zone, seemed to be the appearance of a new isozyme with high relative staining intensity in the spectra of the triticale forms investigated. This was isozyme No.3', localized according to its relative electrophoretic mobility between isozymes No.3 and No.4 in the spectra of T. durum and T. aestivum. Its presence could be explained in several ways: - 102 NAD 137 is a secondary hexaploid triticale. The forms included in it are not available and have not been investigated, so that the possibility of isozyme No.3' being an allelic variant, participating in one of them, cannot be excluded. Data of the electrophoretic analysis of one primary hexaploid plant, AD C I 42, and its initial forms T. durum and diploid rye do not differ from the data concerning 102 NAD 137,

Fig. 3. Diagrammatic representation (A) and zymograms (B and C) of MDH isozymes separated on 170 mm long 6% gel after DAVIS (1964) with modifications, indicated in the text of Fig. 2. S. cereale (3A-a), cvs. Vladimirovka (3B-d) and Imel (3C-c); tetraploid triticale forms (3A-b), AD 248-33 (3B-a and 3C-e) AD 248-22 (3B-i and 3C -f), 248-22-10 (3B-j), 24822-14 (3B-k), 248-22-15 (3B-l); hexaploid triticale forms (3A-c), 102 NAD 137 (3B-e), AD C I (3C-d); T. durum (3A-d) cvs. Parus (3B-b), Zagorka (3B-f) and N 7472 (3C-b; T. aestivum (3A-e) cvs. Sadovo 1 (3B-c) and Bezostaya 1 (3C-a). Mixtures of seed extracts from T. durum cv. Zagorka and S. cereale cv. Vladimirovka (3A-f and 3B-g); the same mixture after in vitro hybridization (3A-g, 3B-h and 3C-h). Isozyme band patterns after treatment with 2M urea: T. durum cv. Zagorka (3C-i), S. cereale cv. Vladimirovka (3C-j), hexaploid triticale 102 NAD 137 (3C-k) and tetraploid triticale form AD 248-33 (3C-l). 412

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.. - -- - -- -- •- ----

+

a

b

c

d

e

-1

-2 -3 -3' -4

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T. durum cv. Zagorka and tetraploid rye cv. Vladimirovka (Fig. 2), which is a sufficiently sure ground for rejecting this possibility. - Modifier genes for the electrophoretic mobility of mitochondrial malate dehydrogenase isozymes have been discovered in some plant species (DOEBLEY et al. 1986; NEWTON 1983). These genes, however, change the electrophoretic mobility of all mitochondrial isozymes and are recessive. So that the possibility of isozyme No.3' being "displaced" by isozyme No.3 or No.4 as an effect of similar genes, included in the rye genome, seems slightly probable. - The third possible explanation requires rejection of the hypothesis about the monomeric structure of malate dehydrogenase isozymes from zone II in wheat and rye (DIAZ et al. 1986; SALINAS and BENITO 1985). If accepted that this is a case of dimers of been fixed, because a whole rye genome is contained in the investigated triticale forms. Isozyme No.3' is a heterodimer, consisting of Wand p subunits. WW

+

pp ~ 2Wp

(1)

The equilibrium No. 1 in vivo is drawn considerable to the right, therefore the concentrations of the respective homodimer (WW and pp) have been greatly reduced. Isozyme No.4 (WW) "vanished" almost completely while isozyme No.2 remained well expressed also in the spectra of the triticale forms investigated, since at the same place in the gel the homodimer ~~ was situated. The picture of malate dehydrogenase isozymes was further worked out in detail by the use of a longer gel - 170 mm (Fig. 3). Isozyme No.4 was present in insignificant quantities in the spectra of hexaploid and tetraploid triticales, which was quite natural because chromosome 1 B, responsible for the synthesis of this isozyme, was contained in their wheat genomes. It turned out, however, that isozyme No.3 was observed not only in hexaploid triticale forms, which include chromosome 1 A, but also in tetraploid triticale forms which had not been found to contain chromosome 1 A. The most probable explanation for this fact - development of heterodimers composed of ~W, led again to the assumption that wheat and rye isozymes in zone II are dimers, not monomers.

(2) The relative staining intensities of isozymes No.3 and No.4 in tetraploid triticale forms were higher than in hexaploid triticale forms, a fact substantiating the opinion that in the investigated triticale forms equilibrium No. 2 was under a complex genetic control, in which the rye genome undoubtedly took part. Thus, if accepted that MDH isozymes from zone II were dimers, the fact that the ~ and p subunits, with homodimers of the same electrophoretic mobility, using a system of electrophoretic separation No.2 produced heterodimers with W subunits, differing in electrophoretic mobility, proved the existence of definite structural differences between them. Besides that, to the proposed subunit formulae (X' (X' and 6'6' for isozyme No.3 could be added also ~W. Dissociation-recombinant analysis is a relatively direct method proving the presence of quarternary structure for a given isozyme. After processing a mixture of T. durum 414

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and rye cv . Vladimirovkaprotein extracts (Fig. 3A-f and 3B-g) the isozyme No.3' was developed (Fig. 3A-g, 3B-h and 3C-h). It should be noted that in all objects investigated, both of plant and animal origin, malate dehydrogenase appeared to be with quarternary structure and it was hardly probable that monomeric malate dehydrogenase would exist only in wheat and rye. Isozymes from zone I and zone II had different physico-chemical properties. Adding 2 M urea to the extracting buffer or to the ready protein extract resulted in complete loss of activity in isozymes from zone I, while those from zone II remained almost unchanged (Fig. 3C - i, j, k and I). Since urea destroyed hydrogen bonds it was clear that the subunits of isozymes from group I were bound with one another, mainly by hydrogen bonds. The latter played a certain role also in the molecules of isozymes from zone II , but it was not a decisive one and the isozymes preserved their activity almost unchanged. Addition of NaCI led to considerable changes in both isozymes groups. After dialysis, however, isozymes fram group I restored faster and more fully their activity. Evidently, definite structural differences existed between the isozymes of the two zones, a fact substantiating the presumption that both electrophoretically conditioned groups of malate dehydrogenase isozymes represented the two malate dehydrogenase types separated by space, namely supernatant and mitochondrial. Taking into account the assumption for malate dehydrogenase isozymes in barley (McDANIEL 1969) and in wheat (NEWTON 1983) that the fraction moving faster to the anode consists of supernatant malate dehydrogenase , while the slower moving - of mitochondrial, it could be assumed that malate dehydrogenase isozymes of zone II are mitochondrial. It was reported in a recently published article (LIU and GALE 1989) that 9 isozymes with isoelectric points in the area of pH 7.0-7.9 were discovered, which were coded by the fifth homoeologous group of wheat. Within the range of pH 3-8 the authors found a total of 25 isozymes, which means that a large part of malate dehydrogenase activity was represented by isozymes with lower pI. Among them an isozyme coded by the chromosome 1 B was discoverd. Therefore, it could be assumed that malate dehydrogenase isozymes coded by the 5th homoeologous group are those from, zone I - supernatant malate dehydrogenases, for which no variation between the aneuploid lines of Chinese Spring was revealed by application of ordinary electrophoresis . In conclusion, data from this investigation confirmed the presence of chromosome 1 B in the wheat genomes of the tetraploid triticale forms studied and proved the dimeric type of the slower moving to the anode mitochondrial malate dehydrogenase isozymes.

Acknowledgement The author expresses her gratitude to ZDRA VKA SABEVA, Senior Research Associate at the Institute of Genetics, Sofia, for kindly supplying the plant material.

References BENITO, C., and SALINAS , J . : Chromosomal location of malate dehydrogenase isozymes in hexaploid wheat (Triticum aeslivum L.). Theor. App!. Genet. 64, 255-258 (1983). DAVIS, B. L.: Disk electrophoresis. II. Method and application to human serum proleins. Ann . N. Y. 121,404-427 (1964).

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DIAZ, F., FERNANDES, J. A., and JOUVE, N.: Structure and chromosomal location of malate dehydrogenase (zone 2) isozymes of common and durum wheats. Euphytica 35, 509-513 (1986). DOEBLEY, J., MORDEN, C. W., and SCHERTZ, K. F.: A gene modifying mitochondrial malate dehydrogenase isozymes in sorghum (Gramineae). Biochem. Genet. 24, 813-819 (1986). HART, G. E.: Alcohol dehydrogenase isozymes of Triticum: Dissociation and recombination of subunits. Molec. Gen. Genet. 111,61-65 (1971). LIU, C. J., and GALE, M. D.: Chromosomal location of a third set of malate dehydrogenase loci, Mdh-3, in wheat, barley and related species. Theor. App!. Genet. 78, 349-352 (1989). McAuSTER-HENN, L.: Evolutionary relationships among the malate dehydrogenases. Trends Biochem. Sci. 13, 178-181 (1988). McDANIEL, R. G.: Mitochondrial heterosis in barley. In: NILAN, R. A. (Ed.) Barley Genetics II. pp. 323-339. Washington State Univ. Press, Pullman, Washington 1969. NAGY, A. H., SIDDIQUI, M. 0., KOCSIS, Z. G., and VIDA, G.: In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis salida. Theor. App!. Genet. 58, 75-78 (1980). NEWTON, K. J.: Genetics of mitochondrial isozymes. In: TANKSLEY, S. D., and ORTON, T. Y. (Eds.). Isozymes in Plant Genetics and Breeding, Part A, pp. 157-174. Elsevier Science Publishers, B.V., Amsterdam 1983. RYCHTER, A., and LEVAK, S.: Polyacrylamide gel electrophoresis of apple seed enzymes. Acta Biochemica Polonica 16, 333- 338 (1969). SABEVA, Z.: Genetic and cytological investigations of tetraploid triticale forms. Genetics and plant breeding (Sofia) 16, 334-338 (1983). SABEVA, Z.: Obtaining and investigation of tetraploid triticale forms. Cereal Res. Commun. 13, 71-76 (1985). SHAW, C. R., and PRASAD, R.: Starch gel electrophoresis of enzymes - a compilation of recipes. Biochem. Genet. 4, 297-320 (1970). VLADOVA, R.: Alcohol dehydrogenase-I isozymes in seeds of tetraploid triticale forms. Biochem. Physio!. Pflanzen 182, 293-297 (1987). VLADOVA, R.: Biochemical markers of chromosome 1 B in tetraploid triticale forms. Proc. 4th Nat!. Congo Biochem. Biophys. Varna, Bulgaria, 3-9 May, Abstracts II.!.6. (1988). VLADOVA, R., and SABEVA, Z.: Use of esterase isozymes as chromosome markers in the study of tetraploid triticale forms. Cereal Res. Commun. 14,177-184 (1986). WHEAT, T. E., and WHITT, G. S.: In vivo and in vitro molecular hybridization of malate dehydrogenase isozymes. Experientia 27, 647-648 (1971). WHITT, G. S.: Genetic variation of supernatant and mitochondrial malate dehydrogenase isozymes in the teleost Fundulus heterocilitus. Experientia 26, 734-735 (1970). Received August 15, 1990; revised form accepted April 25, 1991 Author's address: Dr. ROUMYANA VLADOVA, Department of Biochemistry, Doncho Kostoff Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

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