Anaerobic induction of alcohol dehydrogenase isoenzymes in tetraploid wheats and their diploid relatives

Biochem. Physiol. Pflanzen 17';, 570 ~577 (1980)

Anaerobic Induction of Alcohol Dehydrogenase Isoenzymes in Tetraploid Wheats and Their Diploid Relatives VILVE JAASKA and VELLO JAASKA Laboratory of Biochemistry, Institute of Zoology and Botany, Academy of Sciences of the Estonian SSR, Tartu, Estonian SSR Key Term Index: alcohol dehydrogenase, isoenzymes, anaerobic induction; Triticum spec.,

Aegilops spec.

Sdmary Anaerobic treatment of wheat seedlings induced the formation of aseries of homodimeric and heterodimeric iso enzymes and modificational isoforms of two N AD-dependent alcohol dehydrogenases (ADH, EC 1.1.1.1), ADH-A and ADH-I, which show independent intra- and interspecific variation among wheats (Triticum spec.) and goatgrasses (Aegilops spec.), suggesting their genetic control by two separate gene loci. A homodimeric ADH-A and its two modificational isoforms are present in the germinating embryo of diploid wheats and goatgrasses, whereas a homodimeric ADH-I, its heterodimeric iso enzyme with ADH-A ~ ADH-H, and theit modificational isoforms are additionally induced in the embryo through anaerobiosis. Both ADH-A and ADH-I together with ADH-H are induced in wheat seedlings by anaerobiosis through immersion or in an oxygen-deficient atmosphere (N 2 , Ar, CO 2 ).

Introduction

Polyacrylamide gel electrophoretic analysis has enabled to distinguish, in wheat embryos and seedlings, three types of alcohol dehydrogenase (ADH) of different substrate and coenzyme specificity, developmental activity pattern and evolution-ary variation among the wheat species of different ploidy level (JAASKA and JAASKA 1978). These are: 1), a NAD-depelldent ADH (EC 1.1.1.1), catalyzing the oxidation of different aliphatic alld aromatic alcohols and certain organic compounds with several hydroxyl groups (tris-hydroxymethyl-aminomethane, triethanolamine ); 2), a NADP-dependent aromatic ADH(EC 1.1.1.91); and 3), a coenzyme non-specific aromatic ADH active with both NAD alld NADP. The NAD-dependent ADH is highly active in the ungerminated and germinating wheat embryo but its activity decreases rapidly to a scarcely detectable levels in the seedling tissues (JAASKA 1976a). Electrophoretic analysis reveals (HART 1971a; MITRA and BHATIA 1971; JAASKA 1976 b) one major and one or two minor isoenzymes of this ADH in diploid wheat alld Aegilops species and in a tetraploid wheat Triticum timopheevii ZHUK. s.l., whereas a triplet of major isoenzymes is characteristic of tetraploid wheats of the emmer group (T. turgidum L. s.l.) and of hexaploid bread wheats (T. aestivum L. s.l.). It has been shown (JAASKA 1976a) that aseries of additional, electrophoretically fastermoving ADHisoenzymes may be induced in wheat andrye embryos as a response to

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partial anaerobiosis by immersion one-day-old germinating seeds for 24 hin water. The presence of actinomycin D (50,ug(ml), a transcription inhibitor, in the immersion medium had no effect on the appearance of new ADH isoenzymes in the germinating embryo but strongly inhibited the anaerobic ADH induction in a submerged leaf of rye. On this basis it has been suggested (JAASKA 1976a) that anaerobiosis induces the transcription of the gene which codes for the biosynthesis of the basic ADH iso enzyme present in the embryo, whereas the set of faster-moving ADH iso enzymes represent epigenetic isoforms formed through so me kind of post-translational modification ofthe basic iso enzyme under anaero bic conditions. In this paper we present the data which revise the above interpretation and favours a two-Iocus model of the genetic control of the NAD-ADH isoenzymes induced anaerobically in wheat embryos and seedlings. Polyacrylamide gel electrophoresis is applied to study the evolutionary variation of the anaerobically induced NAD-ADH among the species of tetra- and diploid wheats (Triticum spp.) and goatgrasses (Aegilops spp.) and the effect of some conditions on the anaerobic ADH induction.

Materials and Methods Plant Material 1. Diploid wheats: T. monococcum L. s.l. (48), including the cultivated einkorn T. monococcum L. s.str. (20) and its wild-growing relative T. boeoticurn ROISS. (28), and T. urartu THUM. ex Gandilian (12). 2. Diploid goatgrasses: Aegilops speltoides TAUSCH s.l. (45), including the awnless var. speltoides (21) and the awned var. ligustica BORNM. (24); Ae. longissima SCHWEINF. et 11USCHL. S.str. (6); Ae. sharonensis EIG (6); Ae. bicornis (FORSK.) J AUB. et SP. (5); Ae. caudata L. (6); Ae. comosa SIBTH. et SM. s.l. (6); in cl. Ae. heldreichii HOLZ~I. (3); Ae. uniaristata Vrs. (3); Ae. umbellulata ZHUK. (6). 3. Wild-growing tetraploid wheats: T. dicoccoides (KOERN.) AARONSOIlN (12) and T. araraticurn JAKUBZ. (12).4. Cultivated tetraploids: T. turgidum L. s.l. (32) and T. timopheevil: ZHUK. (5). The number of accessions studied is indicated in brackets. Seeds were germinated in Petri dishes or in glass vessels on two layers of filter paper saturated with water or 0.0511 phosphate buffer, pH 7.4-8, in a thermostate at 26°C in the dark for 1-4 days. One-day-old germinating seeds or 3 -4 days old seedlings were subjected to anaerobiosis for 24-48 h at 26°C either by soaking in water or in a 0.0511 buffer, pH 6.8-7.2, or in the atmosphere of N2 , Ar, or CO 2 •

Biochernical lrJ ethods Enzyme extracts were prepared by grinding of the excised tissue (the embryo with scutellum, coleoptile, primary leaf, primary roots) of individual seedlings separately in 0.1-0.2 ml aliquotes of a cold buffer containing 0.05 11 tris-hydroxymethyl-aminomethane (Tris), 0.0111 EDTA and 5 m11 cysteine hydrochloride. After removal of the rell debris, a suitable amount (20-50 mg) of a sucroseSephadcx G-200 4: 1 mixture was added to the enzyme extract to inerease its viscosity. The extraets were immediately subjected to electrophoresis in a polyacrylamide gel slab (60 x 45 x 3 mm) made in a vertieal eathode chamber by photopolymerizing between two fluorescent lamps of a freshly prepared mixture composed of 10 per cent acrylamide, 0.12 per cent N,N'-methylene-bisacrylamide, 0.126 M Tris, 0.1 M HCl, 0.2 per cent triethanolamine and 0.5 mg per cent riboflavine. The upper cathode buffer contained 0.01 :J'r tris
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After electrophoresis, the gels were stained for the NAD-ADH activity in a histochemical reaction mixture made as folIows: 25 ml of 0.1 M Tris-HCl buffer (pH 8.8),1 ml of 96 % ethanol, 2 ml of NAD (5 mg/mI), :2 ml of tetmnitrotetrazolium blue (2 mg/mI) and 0.2 ml of phenazine methosulphate (2.5 mg/mI).

Results

Polyam'ylamide gel electrophoretic euzymograms of ADH in the seed embryos of tetraploid wheats and their diploid relatives germinated for oue day and then immersed in water or in a phosphate-ascorbate buffer(pH 7.2 -7.8) for 24 h are presented in Fig. J. Tetraploid wheats of the emmer group show 7 -8 closely spaced NAD-ADH bands (1-2, Fig. 1). The thrcc successivc major bands labelled AI, A2 and A3 are also present on thn enzymograms of ungerminated and aerobically germinating embryos, whereas the four faster-moving isoenzymes are induced in response to anaerobiosis by submersion of germinating seeds. From the studies of HART (1969, 1970, 1971 b) it is known that isoenzymes Al and .A3 are homo dimers of two different polypeptides encoded by homoeoallelic genes in the chromosomes 4A and 4 B of tetraploid wheats, respectively, and A2 is a hybrid heterodimer combining both types of polypeptides. The same pattern of aerobic and anaerobic ADH isoenzymes is characteristic of wild and cultivated emmers, comprising 1'. turgidum s.l. and involving the linneons 1'. dicoccoides, 1'. dicoccon, 1'. tUl'gidum s. str., 1'. durum, etc. Only one accession of T. dicoccon, among those studied, revealed a mutant phenotype with the A-genome-specified isoenzyme shifted towards higher electrophoretic mobility in comparison with ADH-AI, as also described by HART (1969). However, no change in the mobility of the anaerobicaBy inducible ADH isoenzymes was observed in this accession of T. dicoccon. Comparison of enzymograms 3-9 in Fig. 1 shows that tetraploid wheats of thc timopheevii group, comprising T. timopheevii s.l. and involving the cultivated T. timopheevii s.str. and its wild relative T. aramticum, as weil as aB taxa of diploid wheats, the 1

2

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Fig.1. Electrophoretic enzymograms of N AD-.1DH in submerged embryos of gerrninating seeds of tetmploid wheats and their diploid relat'i1ies. 1 - Triticum dicoceon, :2 - - T. dicoccoides, Cl - T. araraticum, 4 - T. timopheevii, 5 - T. urartu, G-8 - T. boeoticulII, 9 - T. umrtu, 10 - Aegilops caudata, 11 - Ac. comosa, 12 ,- Ac. heldreichi'i, 13 - Ae. longissima, 14 - Ae, umbellulata, 15 -18 - Ae. speltoides. The migration is from the origin at th e top towards the anode at the bottOIl1,

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cultivated T. monococcum s.str. and the wild '}'. boeoticum and T. urartu, aB share the major aero bic isoenzyme ADH-A3 but differ with respect to the number and electrophoretic mobility of faster-moving iso enzymes induced by anaerobiosis. The independent nature of intra- and inter-specific variation in the aerobic and anaero bic AD H isoenzymes among the wheat biotypes and species provides an indirect evidence in favour of their genetic control by two different gene loci. Thc anaerobically induced iso enzymes should therefore be related to a different enzyme laboled ADH-I (inducible) to distinguish t hem from the constitutive embryonic enzyme ADH-A. The cultivated diploid wheat 1'. monococcum s. str. and its wild-growing relative T. boeoticum share two electrophoretic phenotypes of ADH-I, both consisting of three closely-spaced isoenzymes (7 - 8, Fig. 1) which frequently appear on the enzymograms as fused broad bands. The ADH-I isoenzymes of the fast er triplet are designated F, 12 and 13 in the decreasing order of their electrophoretic mobility, whereas 13 and J5 specify the outer isoenzymes of the slower trip let. Most accessions of T. boeoticum were characterized by a slower triplet, whereas both trip let phenotypes of ADH-I were equally frequently distributed among the accessions of T. monococcum s.str. No correlation between the ADH- I phenotype and morphological variety or geographie origin of t he T. monococcum accessions could be established. An additional ADH-1 phenotype (6, Fig. 1) with a single band labeled 14 was rarely encountered in T. boeoticum. T. urartu revealed three electrophoretic phenotypes of ADH-I - one three-banded and two single-banded. The ADH-1 triplet of T. urartu corresponds to the fastest triplet of T. boeoticum and T. monococcum and was observed in two accessions of T. urartu both originating from Iran. The acccssions of T. urartu originating from the Armenian SSR showed all a single band of ADH-1 which corresponded to the fastest iso enzyme of thc triplet, AD H- P. A phenotype with only the slowest iso enzyme of the triplet, AD H-13, was encountered in two Turkish accessions of T. urartu. The anaerobic ADH enzymogram of T. timopheevii s.str. showed the fast triplet of ADH-I (4, Fig. 1) in common with T. monococcum s. I. and differed from that of T. araraticum (3, Fig. 1) which revealed the presence of ADH-P and AD H-14. Diploid goatgrasses Ae. caudata, Ae. comosa s.1. (incl. Ae. heldreichii), Ae. umbellulata, Ae. uniaristata, Ae. bicornis and Ae.longissima s.l. (incl. Ae. sharonensis) revealed qualitatively similar six-banded enzymograms of anaerobic ADH (10 - 14, Fig. 1) with ADH-A3 and ADH-14 shared by aB species. The only diffcrence noticed was in thc intensity of thc anaerobica.Ily induced fastest band 14 which was of rcduced intensity in the diploids of thc scction Sitopsis. Enzymogram 15 -18 in Fig. 1 demonstrate intrapopulational genetic polymorphism of ADH-A in Ae. speltoides with three electrophoretic phenotypes, two homozygous and onc heterozygous, as reported previously (JAASKA, 1976 b), and show invariance of ADH-14. ADH-A homozygotes reveal one of the two alloenzymc electromorphs, either A3 or A5, but each with two additional minor bands of low staining intensity. Concominant and similar changcs in the electrophorctic mobility of the major and two minor bands of ADH-A suggest that they are due to a single mutatioll. The two minor 39*

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Fig.2. Electrophoretic enzymograms of N AD-ADH monococcum.

Ül

anaerobicaUy treated seedlings of Triticum

1-2 whole seedling subrnerged in water ; 1 - coleoptile, 2 - prirnary leaf; 3-5 - only roots of a seedling immersed in water: 3 - TootS, 4 - coleoptile, 5 - primary leaf; 6-9 the primary leaf of a seedling grown in air (6) and treated for 24 h in the atmospllere of nitrogen (7), argon (8), or earbon dioxide (9). The migration is from the origin at the top towards thc anode at the bottom.

bands of ADH-A, one being slightly faster than the major band and the second - of slightly lower mobility, evidently represent epigenetic modifieatioual isoforms formed from the basic isoenzymes, N and N, in the resuIt of two different kinds of post-trauslational modification. One of the modifications pro duces an isoform of slower electrophoretic mobility and the second - an increase in the mobility of the basic isoenzyme. Heterozygotes üf Ae. speltoides for ADH-A rcveal atriplet of major isoenzymes and a triplet of minor modificational isoforms of higher mobility. The fastest and the slowest bands correspond to homodimers A3 and A5 of homozygotes, whereas the baud A4 of intermediary mobility evidently represents a hybrid heterodim er. Frequeneies of the thrcc ADH-A phcnotypcs in two polymorphie aecessions of Ae. speltoides were found to fit eloscly the requirements of Hardy-Weinberg equilibrium for two eodominant alleles at a single loeus. Anaerobie enzymograms of diploids reveal a band always positioned at a mid-way between the bands of ADH-A and ADH-I. This band which rcfleets changes in the eleetrophoretie mobilit.\, of both ADH-A and ADH- I prcsumably represents their heterodimerie hybrid isoen/lym e whieh we designatc ADH-H. Thu8, a band labelcd ADH-Hl in Fig. 1 may be a hcterodimer eombining subunits of ADH-A3 and ADH-I4. A band positioned at a mid-way between AD H-A5 and ADH-J4 on the enzymogram 16 of Ae. speltoides presumably is their heterodimerie hybrid whieh oeeasionally eoilleides homodimerie ADH-N. It has previously been 8how11 (JAASKA 1976) that thc activity of ADH-A decreased rapidl~' during the seedling dcvelopmcnt to a low or undeteetable level in wheat seedlings Ö-6 days old. Submersion of 4-5-days-old wheat seedlings in water or in a buffer,

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tris or phosphate, at a pH range from 6 to 8 induces a full spectrum of ADH isoenzymes (1-2, Fig. 2) similar to that of immersed embryos. This result implies that both ADH-A and ADH-I are induced by anaerobiosis, i.e. both are the enzymes characteristic of anaerobic metabolism. Immersion of only the seedling roots induced the full ADH isoenzyme spectrum in roots (3, Fig. 2), caused a partial induction in the coleoptile (4, Fig. 2) and no induction in the primary leaf (5, Fig. 2). Anaerobic treatment of wheat seedlings in the atmosphere of nitrogen 01' argon caused the induction of both ADH-A and ADH-I (7 -8, Fig. 2), whereas preferential induction of ADH-A was observed in the atmosphere of carbon dioxide (9, Fig. 2). Discussion

The results of the present study described above show that anaerobic treatment of germinating seeds 01' seedlings of wheat and goatgrass species caused the illduction of a complex pattern of homodimeric and heterodimeric isoellzymes and modificational isoforms of two NAD-dependent alcohol dehydrogenases, ADH-A and ADH-I. The observed independent intra- and interspecific variation pattern of ADH-A and ADH-I suggests that they are encoded by two separate gene loci. The appearance of hybrid heterodimeric iso enzymes labeled ADH-H and combilling subunits of ADH-Aand ADH-I implies their structural affinities. This indicates their possible commOll evolutionary origin which might have occurred through the duplication of a common ancestral gene. A similar two-locus system of the genetic control of ADH iso enzymes was shown to be operating in maize Zea mays L. (FREELING 1973; FREELING and SCHWARTZ 1973; SCANDALIOS 1967,1969; SCHWARTZ 1967, 1969a), soft bromegrass BromusmollisL.(BRowN et al. 1974), safflower Carthamus tinctorus L. (EFRO:'< et al. 1973), narrow-Ieafed lupin Lupinus angustifolius L. (MARS HALL et al. 1974), sunflower Helianthus annuus L. (TORRES 1974a, b, c), and soybean Glycine max L. (BEREMAND 1975). This indicates that the assumed duplication of the ancestral ADH gene has occurred at early stages of higher plant evolution, at least before the divergence of monocotyledonous and dicotyledonous lineages. There are, however, significant differences between plant species in the degree of expression of the two ADH loci. Thus, the fastest-migrating set of ADH isoenzymes comprise about 70 % of the total ADH activity in the cotyledons of sunflower seeds (TORRES 1976) but is essentially lacking in ungerminated seeds of grass es (wheat, rye, maize). It is induced as a minor component in anaerobically treated maize seedlings (FREELING 1973) and remains undetectable in submerged seedlings of rice (JAASKA 1976). A special discussion deserves the occurrellce of triplet electrophoretic phenotypes of ADH-I in diploid wheats. Triplet phenotypes ordinarily reflect genetic heterozygosity at a locus of a dimeric enzyme. Diploid wheats are, however, self-pollinating plants, and the accessiolls studied were monomorphic and homozygous for other enzymes. One of the possible explanations is to assume a duplication of the ADH-I locus in an ancestor of diploid wheats followed by two different mutations fixed in one of the duplicated loci. The second, alternative explanation assumes that diploid wheats have acquired a

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genetically controlIed abilit~· for two different kinds of post-translational modifications, resuIting in the formaton of ADH-P and ADH-!5 from ADH-P. Of additional interest is that no hybrid iso enzyme is observed between the basic AD H-A isoenzyme and its two minor isoforms. At the same time, hybrid heterodimers are formed between the two basic ADH-A allelomorphs of Ae. speltoides, as weIl as between their faster-moving minor isoforms. This fact may indicate different kinds of modificational changes. Thc ADH-A data reported previously (JAASKA 1976b) have allowed to differentiate the two genetic groups of tetraploid wheats comprising T. turgidum s.l. (the emmer group) and T. timopheevii s.l. (incl. ']'. araraticum). The two groups, as shown in the present paper, differ also in their ADH-I electrophoretic spectra. The ADH-I data, however, show further genetic differentiation within T. timopheevii s.l. and distinguish the cultivated T. timopheevii s.str. from its wild relative T. araratium. The anaerobic ADH enzymogram of T. timopheevii s.str. is essentially similar with that of T. monococcum s.l. with a fast er trip let of ADH-I. The enzymogram of T. araraticum shows ADH-P characteristic of T. urartu of Armenian origin andADH-I4 presumably controlled by the second genome related to Ae. speltoides. The coleoptile acid phosphatase of ']'. araraticum was previously found (JAASKA 1976c) to comprise the sum of isophosphatases characteristic of T. ur ar tu and Ae. speltoides. No difference in the ADH-I was founel in the emmer group between the wild T. dicoccoides and its cultivated relatives T. dicoccon, T. du rum, T. turgidum s.str., T. carthlicum anel ']'. polonicum. All the species of the rye genus were previously shown (JAASKA 1975) to share similar electrophoretic phenotypes of both ADH-A and ADH-I. The rye ADH-A was found to be of lower electrophoretic mobility in comparison with ADH-N of wheats, while the rye ADH-I is clectrophoretically similar to ADH-P of wheats. The elata demOllstrate independent find mosaic evolutionary divergence of ADH-A anel ADH-I among the grasses of the wheat group.

References DEHEMAND, .M. L.: Soybean alcohol dehydrogenase: dimeric strueture and subunit composition of multIple isozymes. Genetics 81l, Suppl. 12 -13 (1975). DROW", A. H. D., l\1ARSIIALL, D. R., and ALBHECHT, L.: The maintenanee of alcohol dehy(lrogenase polymorphism in Brornus mollis L. Austral. J. Biol. Sei. 27, 545-559 (1974). EFllOX, Y., PE LEG, .M., and ASJIlU, A.: Aleohol dehydrogenase allozymes in the safflower genus Carthamus L. Diochem. Genet. 9, 299-308 (1973). FHEELl:\G, 11.: Simultaneous indudion by anaerobiosis or 2,4-D of multiple enzymes specified by two unliked genes: Differential Adhl-Adh:2 expression in maize . .Mol. Gell. Genet.127, 215-227 (1973). - and SCII\nwrz, D.: Genetie relationships between the multiple alcohol dehydrogenases of maize. Biochem. Genet. 8, :n -ilG (HI73). HART, G. E.: Geneti(' ('(mtrol of akohol dehydrogenase isozymes in Triticum dicoccum. Biochem. Genet. 3, 617 -(i:26 (19G9). Evidelll'e of triplil'ate genes for aleohol dehy(lrogenase in hexaploid wheat. Prol'o Nat. Acad. Sl'i. USA eil, 11,)(;-IHO (1D70).

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Evolution of alcohol dehydrogenase isozymes in Triticum. Isozyme Bull. 4, 15 (1971 a). Alcohol dehydrogenase isozymes in Triticum: dissoeiation and recombination of subunits. Mol. Gen. Genet. 111, 61- 65 (1971 b). JAASKA, VELLO: Evolutionary variation of enzymes and phylogenetic relationships in the genus Secale L. Eesti NSV Tead. Akad. Toimet. Bioloogia 24, 179-198 (1975). JAASKA, VILVE: The effert of germination environment on the alcohol dehydrogenase isoform pattern in wheat, rye and rice seedlings. Eesti NSV Tead. Akad. Toimet. Bioloogia 25,237 -244 (1976a). JAASKA, V. E.: Alcohol dehydrogenase of polyploid wheats and their diploid relatives. On the phylogeny of tetraploid wheats. Genetika (Moscow) 12, 22-28 (1976b). JAASKA, VELLO: Genome- and tissue-specific regulation of esterase and acid phosphatase isoenzymes in tetraploid wheats. Eesti NSV Tead. Akad. Toim. Bioloogia 25,132-145 (1976c). JAASKA, V. H., and JAASKA, V. E.: Electrophoretic analysis of substrate specificity of wheat alcohol dehydrogenases. Biokhimiya (Moscow) 43,2011-2015 (1978). MARSIlALL, D. R, BROUE, P., and ORAM, RN.: Genetic control of alcohol dehydrogenase isozymes in narrow-Ieafed lupins. J. Hered. 65, 198-203 (1974). MITRA, R., and BHATIA, C. R: Isoenzymes and polyploidy. 1. Qualitative and quantitative iso enzyme studies in the Triticinae. Genet. Res. 17, 57-69 (1971). SCANDALIOS, J. G.: Genetic control of alcohol dehydrogenase in maize. Biochem. Genet. 1, 1- 8 (1967). - Alcohol dehydrogenase in maize. Genetic basis for isoenzymes. Science 166, 623 (1969). SCHWARTZ, D.: The genetic control of alcohol dehydrogenase in maize: gene duplication and repression. Proc. Nat. Acad. Sci. USA 56,1431-1436 (1966). Alcohol dehydrogenase in maize: Genetic basis for multiple isoenzymes. Science 164, 585-586 (1969a). - An example of gene fixation resulting from selective advantage in sub optimal conditions. Amer. Natur. 103, 479-481 (1969b). TORRES, A. M.: Sunflower alcohol dehydrogenase: ADH 1 genetics and dissociation-recombination. Biochem. Genet. 11, 17 -24 (1974a). An intergenic alcohol dehydrogenase isozyme in sunflowers. Biochem. Genet. 11, 301-308 (1974b). Genetics of sunflower alcohol dehydrogenase: Adh 2 , nonlinkage to Adh1 , and Adh1 early alleles. lliochem. Genet. 12, 385-392 (1974c). Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities. Biochem. Genet. 14, 87 - 98 (1976).

Received February 18, 1980. Authors' address: VILVE JAASKA and VELLO JAASKA, Laboratory of Biochemistry, Institute of Zoology and Botany, 21 Vanemuise St., 202400 Tartu, Estonian SSR (USSR).