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Isoenzymes of Superoxide Dismutase in Wheats and Their Relatives: Alloenzyme Variation
Biochem. Physiol. Pflanzen In, 747-755 (1982)
Isoenzymes of Superoxide Dismutase in Wheats and Their Relatives: Alloenzyme Variation V ELLO
J AASKA
Laboratory of Hioehemistry, Institute of Zoology and Botany, Academy of Sciences of the Estonian SSR, Tartu, Estonian SSR Key Ter III I n de x: superoxide dismutase, isoenzymes, wheat phylogeny; Triticum spec., Aegilops spec., SCC!lle spec.
Summary Polya(,[ylamide gel electrophoresis and isoeledric focusing have been applied to study the alloenzymic variation of three genetieally independent isoenzymes of superoxide dismutase (SOD) in wheat (TritiC/lIII L.), goatgrass (Acyilops L.) and rye (,'lecale L.) speeies. SOD-A, a diethyl dithioearbamate (DlECA)-sensitive chloroplastie isoenzmye, revealed three alloenzymes. The common allocnzyme with pI 4.5:) was shared by all wheat, goatgrass and rye speeies, except SI'cu/eril/osa L. [= Dllsypyrulll viliosu/ll (L.) HOlm.] which carries a rare alloenzyme with pI 4A5. The appearance of a three-bandel[ heterozygous phenotype in AegiloJis maticll BOISS. suggests the dimcric struI·ture of SOD-A. SO D- Il, a DIECA-sensitive I'ytosolie isoenzyme, showed three alloenzymes, Bu, Urn and Br with pI values liAi'>, liJ) and 0.7, respeetively. All goatgrass speeies of different ploidy level, all polyploid wheats and the diploid wheat T. umrtu Tlln!. ex GAXDIL. shared a tommon alloenzyme Bu. The wild diploil[ wheat T. boeoticum BOiss. and its I'llltivatel[ relative T. mOilOCOCCUIII L. s.str. have ,L divergent alloenzyme Hm. Alloenzyme Br is eharaeteristi~ of rye species, ex~ept S. villosa L. whieh earries the common wheat alloenzyme Bu. The wheat-rye amphidiploids, tritieules, show codominant expression of parental alloenzym8s together with a hybrid isoenzyme in a three-banded isoeleetric phenotype of SOD-B, as eharaeteristic of a dimerie enzyme. SOD-C, a DIECA-insensitive mitol'hondrial isoenzyme, revealed several rare isoelectric variants and one {'ommon alloenzyme (pI ~ 6.2) shared by all wheat and rye species and by most goatgrass species. The appearanl'e of a five-banded isoelectrie phenotype in some accessions of the polyploid goatgrass .:ie. OI;allt s.1. indil'ates the tetrameric strlleture of SOD-C. Implieations of the SOD alloenzyme dab on the phylogeny of wheats and their relatives are disellssed.
Introduction
With a previous work (JAASKA and JAASKA 1982) we initiated a systematic study of the evolutionary variation of isoenzymes of superoxide dismutase (SOD, EC 1.15.1.1) among the grass species. Two major and two minor genetically ind(~pendent isoenzymes of SOD of different tissue specificity and inhibitor sensitivity were specified in the seedlings of barley, wheat and some related species by polyacrylamide gel elcctrophoretic and isoelectric focusing techniques. The two major isoenzymes, SOD-A and SOD-B, Abbreviations: SOD, supcroxide dislllutase; DIECA, diethyl dithiocarbamate; pI, isoelectric point. 49*
748
V. JAASKA
were inhibited by cyanide and diethyldithiocarbamate (DIECA) as characteristic of Cu-Zn-SOD, whereas the two minor isoenzymes, SOD-C and SOD-D, were insensitive to the two inhibitors as characteristic of Mn-SOD. The isoenzymes of SOD proved evolutionarily rather conservative showing only rare cases of variation in their electrophoretic and isoelectric properties among the barley species. Even such distinct species as the cultivated barley Hordeum vulgare 1. and the cultivated hexaploid wheat Triticum aestivum 1. had identical electrophoretic enzymograms of SOD and only isoelectric focusing revealed a shift in the isoelectric points of their SOD-B. The present paper extends the study of the evolutionary variation of SOD isoenzymes among the grasses of the tribe Triticeae DUM. and describes the electrophoretic and isoelectric enzymograms of SOD among the species of the subtribe Triticinae Trin. ex Griscb. belonging to the genera Triticum 1., Aegilops 1. and Secale L.
Materials and Methods Plant Material Seed accessions were received from the Vavilov World Collection of the Institute of Plant Industry in IJeningrad, from botanical gardens and other eollections or eollected by the author in nature. The number of accessions studied is indicated in brackets.
1. The wheat genus, Triticum L. 1 a. Diploid wheats: T. monococcum L. s.l. (27), involving the cultivated einkorn T. monococcum 1. s.str. (6) and its wild-growing relative T. boeticum BOISS. (21); T. urartu THuM. ex GANDILIAN (7). 1 b. Tetraploid wheats of the Emmer group eomprising T. turgidum 1. emend. THELL. (14), involving the cultivated T. turgidum L. s.str. (1), T. durum DEsF. (1), T. polonicum L. (1), T. carthl/cum NEVSKI (1), T. dicoccon (SCHRANK) SCIIUEBL. (1) and the wild emmer T. dicoccoides (KOERX.) SCHWEINF. (9). 1 c. Tetraploid wheats of the Timopheevii group comprising T. timopheevii ZlIUK. emend. MAC KEY and involving the cultivated T. timopheevii ZHUK. s.str. (2) and the wild T. araraticum JAKUBZ.
(8). 1 d. Hexaploid bread wheats comprising T. aestivum 1. emend. THELL. (7) and involving T. aestivum L. s.str. (2), T. compactum HOST (1), T. sphacrococcum PERC. (1), T. spc/ta L. (2) and T. macha DEli:. et ~rEN. (1). 1e. A spontaneous allohexaploid T. ti1l10pheevii ZHUK. x T. monococcum L. (= T. zhukovskyi MEN. et EIUZ., 1) and a synthetic allohexaploid T. carthlicum NEVSKI x T. monococcum 1. (1). 2. The goatgrass genus, Aegilops L. 2a. Diploid goatgrasses: Aegilops tauschii Coss. (4), Ae. speltoides TAUSCH s.!. (5), Ae. mutica BOISS. (1), Ae. bicornis (FO]{SK.) JAvn. et Sp. (4), Ae. longissima SCHWEINF. et MuscHL. s.l. (5), Ae. searsii FELDMAN et KISU:V (1), 11e. cauda/a L. (f)), At'. umbellulafa ZHUK. (4), Ae. c01l10sa SIBTH. et Sm. s.L (3), Ae. /il/iaristala VIS. (2). 2b. Tetraploid goatgmsses: Jje.1'f'lItricosa TAUSCIl (1), Ae. cylindrica HOST (1), Ae. crassa BOISS. ssp. macrathera (BOISS.) ZIIVK. (1), Ae. friuucia/is L. (1), Ae. biuncialis VIS. (1), Ae. ovata L. (G), Ar. coluiililaris ZHUK. (1). 2 e. Hexaploid goatgrusscs: Ae. cmssa BOlSS. ssp. crass a (1), Ae. juvenalis (THELL.) Eig (1). :l. The rye genus, 8ecale 1.: the perennial rye, S. monlanum Guss. ssp. analolicum(HOIss.) TzvEL. (2), the annual r1eistogamous rye, 8. sylvestre HOST (2), the annual self-incompatible rye, 8. ctrcale 1.. s.l. (il), involving the eultivated 8. cercale ssp. cercale (1) and the wild 8. cere ale ssp. allcestrale ZHUK.
Isoenzymes of Superoxide Dismutase in Wheat
749
s.l. (4), a 1illnean species Secale villosa 1. (2), presently treated as Dasypyrum villosum (1.) BORB., syn. Haynaldia villosa (1.) SCHUR and Triticum villosum (1.) BlEB. 4. Wheat-rye aIIohexaploids Triticum turgidum 1. sol. x Secale cereale 1. ssp. cereale - hexaploid tritirales (18) and wheat-rye chromosomal addition lines (7).
Biochemical Methods Enzyme extracts were prepared from the primary leaf 4-8 d old etiolated seedlings and subjected to electrophoresis or isoelectric focusing in polyacrylamide gel slabs to stain thereafter for the SOD activity as described previously (hASKA and JAASKA 1972). The genetically independent isoenzymes are designated by capital letters (SOD-A, SOD-B, SOD-C, etc.), and the corresponding gene loci by arabic numbers (Sod-l, Sod-2, Sod-3, respectively). The letter or numerical superscripts label the genetic variants of isoenzymes (alloenzymes, homoeoallelic isoenzymes of polyploids, hybrid isoenzymes) whereas the numerieal underscripts specify the modifirational isoforms of epigenetic nature. The numerical superscripts reflect either the relative electrophoretic mobility values in Rrunits or the pI values of electrophoretie or isoelectric electromorphs, respectively. The letter superscripts specify alloenzymes or alleles.
Results and Discussion Figs. 1 and 2 present a set of the SOD enzymograms obtained by means of polyacrylamide gel slab electrophoretic and isoelectric focusing techniques, respectively, coupled with the photochemical staining method. On both types of enzymograms three regions of unstained achromatic bands of independent interspecific variation can be distinguished. This suggests three genetically independent isoenzymes of SOD which were labelled (JAASKA and JAASKA 1982) SOD-A, SOD-B and SOD-C in the order of their decreasing electrophoretic mobility and increasing isoelectric points. SOD-A, acyanide- and DIECA-sensitive chloroplastici soenzyme (JAASKA and JAASKA 1982), is seen on the enzymograms as a major achromatic band of fastest mobility and of lowest isoelectric point and reveals, among the Triticeae grasses studied, two genetic variants. The variant of lower mobility (RF 0.83) and higher isoelectric point (pI,....., 4.55) labelled A4.55 according to its pI value is common to all wheat, goatgrass and rye species which showed no intra- or interspecific variation in SOD-A. The same variant was previously reported (JAASKA and JAASKA 1982) shared by species belonging to the genera Hordeum L., Taeniatherum NEVSKI and Heteranthelium HOCHST. of the tribe Triticeae DUM. The variant of higher electrophoretic mobility (RF 0.88) and lower isoelectric point (pI,....., 4.45), A4.45, was found in only one species of the subtribe Trifieinae, in Dasypyrum villosum (= Secale villosa). Previously (JAASKA and JAASKA 1982), this variant was also reported for Hordeum geniculatum All. of the subtribe Hordeinae DUM. Aegilops mutica, an outcrossing diploid, exhibited intrapopulational polymorphism with two pI-variants. In some individuals, the common variant AU& and a rare unique variant AU showed a symmetrical three-banded phenotype (6, Fig. 3) as characteristic of heterozygotes for a dimeric enzyme. SOD-.B, a cyanide- and DIECA-sensitive cytosol isoenzyme (JAASKA and JAASKA 1982), reveals on the electrophoretic enzymograms (Fig. 1) of the Tritieinae species two electromorphs. The fast electromorph with RF 0.58, SOD-Bo.58, is common to all poly-
750
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JAASKA
Fig. 1. Eleclrophorehc enzyrnograrns of superoxid(~ dismulase ill various specj:es. ], Triticum rnonococcuill vaT. horuemannii; :;! ·- 4, T. bo('olicurII va.T. thaoudar (:!), va.r. fuscurn (Cl) and var. baydaricum (4); 5, T. urartu; 6,1'. dicoceoides vaT. kotsellyi; 7-9, 1'. flraralicum var. kurdistallicwm (8), and var. araxicum (9); 10- 11, 1'. dieoccoides var. arabicu'lli (10) and var. dicoccoides (11); 12, Secale cereale ssp. ancestrale; 13, S. montallulll ssp. allatoijeulli; 14, 1(i, Dasypyrurn villosum ( = Secale t'1:11osa); 15, S. sylvestre. The migration is from the origin at the top towards the anode at the bottom.
A"·S.[
-A If'
Fig. :!. pl-Nllzymograll/s of superoxide dis mutase obtained by means of isocleetric focusillg with tlte LKR ampholyte Ampholine pH 4--6. 1, Triticum monococcum; 2, 1'. bocotfeu-III; 3, T. u-mrtu; 4, T. dicocco'ides; 5, '1'. araraticum; G, a synthetic allohexaploid T. carthlicum x 1'. monococcwlI; 7, T. monococewn; 8, 1'. tirnop/ieevil:; 9, T. zhukovskyi ( = T. timopheevii x 1'. mo'//ococcum); 10, T. sp('/ta; 11 , T. carthlicum x 1'. '///onococcum; 12, Beeale are Ill!' ssp. rereale; 13, Hordeu.m l/Iuriuum ssp. glllucum; 14, J)nsypyrum vil/osum ( - Seeale villosa); 15, 8. cereah~ ssp. /ll/cl'stra/f; 16, a synthctie allohexaploid .T. i'UrgidwJI x 8. (:('I"ea/(' (= .Triticale t'Urgidoc('reale); 17, '1'. turyid'Ulll. The ra,tholyte (fUll M ethylcnediaminc) is at the top, the nnolyte (0.01 M H Z:;;04) at the bottom.
Isoenzymes of Superoxide Dismutase in Wheat
751
Table 1. Electrophoretic properUes and the distribution of the SOD-B alloenzymes among the TriticelJe grasses
AHo-
pI
Speeies
SOD-Bu 0.58
0.40
SOD-Bm 0.6:3
oJ)
SOD-IY 0.53 SOD-1Jb 0.08
0.7 5.7
T. martu, T. turgidum s.l., T. timopheevii s.l., T. aesfivulil s.1., Aegilops spec., Elytrigia spec. T.lllonococcuHI s.l., Elytrigia spec., Hordeum brevisubulatum 8('clrie )llOntanurn s.l., S. cereale s.l., S. sylvestre
RF
enzyme
Hordl'll})) spec, Taeniatherwn spec.
ploid wheats, diploid and polyploid goatgrasses, to a diploid wheat T. urartu and to Dasypyrum villosum. The diploid wheats comprising '1'. monococcum s.1. (incl. the wild T. boeoticum) and all the rye species shared the slow electromorph SOD-Bo.53. Some allopolyploids, such as T. timopheevii X T. monococcum (= T. zhukovskyi), T. turgidum x T. monococcum and hexaploid triticales - T. turgidum x S. cereale showed a broad band of SOD-B comprising both electromorphs, BO.58 and BO.53, as expected in the case of codominant expression of homoeoallelic genes of parental species of these polyploids. Isoelectric pI-enzymograms (Fig. 2) distinguish, however, among the Triticinae species, thrce genetic variants of SOD-B. The two wheat SOD-B electromorphs, BO.58 and BO.53, differ also slightly in their pI values estimated 5.45 and 5.5, respectively. A third pIvariant of SOD-B with a distinctly different pI value 5.7 was found to be characterstic of the rye species and, previously (JAASKA and JAASKA 1982), - of barley species. Since the rye cond barley species have different SOD-B electromorphs, BO.53 and BO.58 respectively, a total of four alloenzymes of SOD-B should be specified. The distribution of the four SOD-B alloenzymes labelled by letter superscripts as Bu, Bm, Br and Bb among the Triticeae grasses and their electrophoretic properties are summarized in the Table, which includes our previous (I.c.) and some preliminary unpublished data. It can be seen that the most wide distribution among the Triticeae genera have alloenzymes Bu and Bm. Presumably, they are the ancestral alloenzymes from which the other two alloenzymes have arisen in the course of the evolutionary differentiation of Triticeae grasses. The two allohexaploid wheats, T. timopheevii x T. monococcum (= T. zhukovskyi) and T. turgidum x '1'. monococcum, show (Fig. 2) codominant expression of B-alloenzymes of both parental species. Hexaploid triticales reveal (Fig. 2) a distinct three-banded (triplet) pI-phenotype of SOD-B. In addition to the alloenzymes of the parental species, Bu and Br, a band of intermediate pI-value, as characteristic of heterodimeric hybrid isoenzymes, is clearly seen. This result suggests that SOD-B is a dimeric enzyme which exhibits codominant expression of parental homodimers and a hybrid heterodimn in hexaploid triticales. Among the series of seven wheat-rye chromosomal addition lines analyzed, only the line with a pair of rye chromosome 2R revealed the triplet pI-phenotype of SOD-B
752 I
2
3
~
5
6
7
8
9 10
11
_ B 5.ltj _A~·e _ A ft.. ItS I"ig.3. pI-Enzymograms of superoxide dismutase obtained by means of isoelectric focussing with the LKB ampholyte Ampholine pH 5-7. 1, Aegilops triuncialis; 2, Ae. ovata; 3, Ae. umbellulata; 4, Ae. ovata; 5, Ae. umbellulata; 6, Ae. mutica; 7, Ae. biuncialis; 8, Ae. comosa; 9, Ae. longissima; 10, Ae. tauschii; 11, Dasypyrum villosum. The eatholyte (0.01 A{ ethylenediamine) is at the top, the anolyte (0.01 M H2S0 4 ) at the bottom.
characteristic of triticales. This implies that the gene controlling the rye alloenzyme Br is located in the rye chromosome 2R. All the accessions of primary triticales analyzed showed an identical triplet phenotype of SOD-B. Among the secondary triticales, two accessions out of eight analyzed lacked the rye alloenzyme Br and the hybrid heterodimer Bur, showing only the wheat alloenzyme Bu. SOD-B was reported (JAASKA and JAASKA 1982) to exist in three epigenetic isoforms, Bl , B2 and B3, interconverted by the treatment with sulphydryl reagents, such as cysteine, p-chloromercuribenzoate, etc. The three epigenetic isoforms of SOD-B can be distinguished on the pI-enzymograms by isoelectric focusing. In the presence of a SHreductant (cysteine, dithiothreitol) in the homogenization buffer the isoforms of lower pI, Bl and B2 , are converted partially or totally, depending on the treatment conditions, into the isoform B3 which becomes dominating on the pI-enzymograms, as seen in Fig. 2. The point of interest is that SOD-B of 1'. monococcum s.I. (inci. T. boeoticum) :.tnd of all three rye species revealed only the isoform B3, whereas the two isoforms of lower isoelectric points, Bl and B2 , were totally absent even when the enzyme extract was made in a buffer without the sulphydryl reductant (cysteine) added. At the same time, D. villosum, 1'. urartu, all polyploid wheats and all goatgrass species showed three epigenetic isoforms of SOD-Bu. SOD-B3m of T. monococcum andSOD-Bllf rye, in contrast to SODBau , could not be converted into the fast isoform by a treatment with p-chloromercuribenzoate ill the extraction buffer. Presumably the alloenzymes Bm and Br lack a free sulphydryl group capable to react with p-chloromercuribenzoate.
Isoenzymes of Superoxide Dismutase in Wheat
753
SOD-C, a cyanide- and DIECA-insensitive mitochondrial isoenzyme (JAASKA and 1982), revealed on both the electrophoretic and isoelectric enzymograms (Figs. 1 and 2) a weak achromatic band of slower mobility and higher pI than SOD-B. SOD-C exhibited only a slight variation in its electrophoretic mobility and isoelectric point which was more distinct on the pI-enzymograms obtained with the use of the pH 5-7 ampholyte (Fig. 3). All wheat and rye species had one SOD-C band of closely similar or identical electrophoretic mobility and isoelectric point (pI about 6.2). Among the polyploid goatgrasses, some accessions of Ae. OVcttct s.1. revealed a symmetrical five-banded phenotype (2 and 4, Fig. 3) as characteristic of a tetrameric enzyme, consisting of two subunits of different isoelectric points (pI values about 6.2 and 6.8). All other polyploid goatgrass species showed one invariant band of SOD-C, labelled C6.2 according to its pI value, in common with wheat and rye species. In some diploids, such as D. villo8um, Ae. comosct and Ae. searsii, SOD-C was, however, distinctly shifted twoards lower isoelectric points. Intraspecific variation with two pI-variants of SOD-C was observed among the accessions of the diploid Ae. longissima, s.l. The five-banded SOD-C phenotype of Ae. ovata is presumed to combine the two outer homotetramers, C6.2 and C6.8, with three hybrid heterotetramers of intermediate isoelectric points. SOD-C6.2 was also characteristic of most diploid goatgrasses - genome donors and precursors of polyploid goatgrasses. SOD-C6.8 was, however, not encountered among the diploids and may thus be presumed to have been arisen at the polyploid level in Ae.ovatct. Phylogenetic implications. The SOD-B alloenzyme data present a new significant evidence on the origin of tetraploid wheats. Of phylogenetic importance is the finding that all tetraploid wheats of both genetic groups, including their wild representatives T. dicoccoides and T. arnratiwm, shared the alloenzyme SOD-Bu characteristic of 1'. urartu and goatgrass species, while the alloenzyme SOD-Bm specific to T. boeoticum was never encountered in tetraploid wheats. The two wild diploid wheats, T. urartu and T. boeoticum, revealed a distinct diagnostic divergence ill SOD-B, being fixed for different SOD-B alloenzymes, Bu and Bm, respectively. The SOD-B alloenzyme data, thus, clearly argue against the theory of separate diphyletic origin of 7'. di(:occoide,~ and 7'. ctramticum with T. urartu and T. monococcum s.l. as their A-genome donors, respectively, proposed (KONAREV 1975; KONAREV et a1. 1976) on the basis of immunoehemical properties of seed proteins. Instead, a new support to the concept of monophyletic origin of tetraploid wheats is presented. Our previous studies on acid phosphatase, alcohol dehydrogenase and esterase isoenzymes have discovered (discussed in JAASKA 1980) distinct genetic differences bettween the A genomes of contemporary 7'. boeoticum, T. urartu and tetraploid wheats. This means that contemporary diploid wheats '1'. umrtu and T. boeoticum separately do not fit the A genome donor of tetraploid wheats. To explain the alJocnzyme data it has been suggested (JAASKA 1976, 1980) that both T. boeoticum and T. urartu, might have contributed, together with Aegilops speltoides, JAASKA
754 to the origin of a primitive allotetraploid wheat from which 1'. araraticum and T. dicoccoides then diverged. The involvement of a male-sterile hybrid between two of the three diploids (e.g. between 1'. urartu and 'l'. boeoticurn, Ae. speltoides and 'l'. urartu or Ae. speltoides and 'l'. boeoticurn) pollinated by the third diploid is an attractive hypothesis of the origin of the primitive tetraploid wheat. Alternatively, the participation of a common anc('stor of 7'. ufarlu and 7'. boeoticum with a suitable combination of alloenzymes in the origin of tetraploid wheat should still be seriously considered. Although such ancestral biotype has not yet found among the accessions of contemporary diploid wheats, its existence, at least in the past times, has theoretically good grounds. The Linnean rye species, Secale 'Oill(),~n 1,., was found to differ from the rem,lincier rye species in allocnzymes of all the three SOD isoenzymes. With wheats it has a common allopnzyme, SO 0- Bll, but differs from them in the alloenzymes of SOD-A and SOD-C. Previously it has been reported (JAASKA 1975) that S. 'Oillosa has allocnzymes of alcohol dehydrogenase ADH-A and of aspartate aminotransferasesAAT-A and AAT-B in common with rye species and different from those of wheats. However, its 6-phosphogluconate dehydrogenase and fast-moving isoperoxidase proved, reversely, different from the rye alloenzymes and in common with wheats. The alloenzyme data as a whole, thus, support the taxonomic treatment of the Linnean Secale 'Oillosa in a separate genus Dasypyrum (Coss. <'t DUR.) BORB. as J). villosum (L.) BORB. Qua ternary structure. Codominant inheritance of evolutionarily divergent parental isoenzymes in a three-banded isoelectric phenotype of SOD-B in the wheat-rye amphidiploids (triticales) provides an indirect evidence on the dimeric structure of the cytosolic isoenzymes SOD-B. Similarly, the appearance of a five-banded isoelectric phenotype of SOD-C in some biotypes of a polyploid goatgrass Aegilop8 ovala indicatps the tptrameric structure of the mitochondrial isoenzyme SOD-C. The dimerie nature of the cytosolic CujZn-SOD and the tetrameric nature of the mitochondrial Mil-SOD is known (review: FRIDOVICH 1975) for diverse cukaryotic organisms. TIl(' same was recently reported (BAUM and SCANDALIOS 1981) for the SOD isop!1zymes ill maize. The quaternary structure of the SOD iwenzymes has evidently arisen at initial stages of f'volution and has become conserved during the eukaryotie evolution. The discovpry of a. three-banded heterozygous phenotype of SOD-A in some individuals of an outbreeding diploid Ae. rnulica suggests that the chloroplastic isoenzyme has a dimf'ric structure. Our previous rpsu't (JAASKA and JAASKA 1982) on a two-banded electrophoretic phenotype of SOD-A without a distinct hybrid band in a tetraploid barley H. geni(:j,(,latum indicates that hybrid heterodimer may not be formed between every type of alloenzymic subunits of the chloroplastic isoenzyme. The absence of the heterodimeric hybrid between the chloroplastic SOD-A and the cytosolic SOD-B implies that the two IHECA-sensitive Cu/Zn-isoenzymes arc structurally divergent proteins. References BAUM, J. A" and SCANDALIOS, J. E.: Isolation and eha.raderization of the cytosolic and mitochondrial superoxide dislIlutases in maize. Areh. Hioehem. Biophys. 208, 249-264 (1981).
Isoenzymes of Superoxide Dismutase in Wheat
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1.: Superoxide dismutases. Annu. Rev. Hiorhem. 44, 147-159 (1975). V.: Evolutionary variation of enzymes and phylogenetic relationships in the genus 8ecale L. (russ.). Eesti NSV TA Toimetised, Bioloogia 24, 179-198 (1975) . .JH~K'\, V.: Genome- ancl tissue-specific regulation of esterase and add phosphatase isoenzymes in tetraploid wheats (russ.). Eesti NSV 'fA Toimetised, Bioloogia 2;), 132-145 (197G) . •LIASKA, V.: Eleetrophoretie survey of seedling esterases in wheats in relation to their phylogeny. Theor. App!. (ienet. ;)6, 273~-284 (1980) . .1,118K;\, VILVE, anll .LLlS hI, V.: lsoenzymes of superoxide dismlltase in barley. Biol'hem. Physiol. Pflanzen 177, (1982). l\oNAHEV, A. V.: Differentiation of the first genomes of polyploid wheats based on data from immunoehemieal analysis of ethanol fral,tion of grain proteins (russ.). Hnll. All-Union Vavilov Inst. PI. Indllstr. 47, 811 (1fJ7i'»). KONAREY, V. G., ()AI'IUL,ll:K, I. P., PENEVA, T. I., [(ONAREV, A. V., [(BAKIMOVA, A. G., l'iIIGUSIJOVA, E. F.: On the nature anll origin of wheat genomes on the basis of biochemistry anc! immunochemistry of the grain proteins (russ.). Agric. BioI. (USSR)ll, G5G-G65 (197G). FHlDOYH'I1,
JAA~h'\,
Receit'ed .J nne 3, 1982 Author's address: VELLO ,LushA, Laboratory of Biochemistry, Institute of Zoology and Botany, 21 Vanemnise St., USSR - 202400 Tartn, Estonian SSR (USSR).
Isoenzymes of Superoxide Dismutase in Wheats and Their Relatives: Alloenzyme Variation V ELLO
J AASKA
Laboratory of Hioehemistry, Institute of Zoology and Botany, Academy of Sciences of the Estonian SSR, Tartu, Estonian SSR Key Ter III I n de x: superoxide dismutase, isoenzymes, wheat phylogeny; Triticum spec., Aegilops spec., SCC!lle spec.
Summary Polya(,[ylamide gel electrophoresis and isoeledric focusing have been applied to study the alloenzymic variation of three genetieally independent isoenzymes of superoxide dismutase (SOD) in wheat (TritiC/lIII L.), goatgrass (Acyilops L.) and rye (,'lecale L.) speeies. SOD-A, a diethyl dithioearbamate (DlECA)-sensitive chloroplastie isoenzmye, revealed three alloenzymes. The common allocnzyme with pI 4.5:) was shared by all wheat, goatgrass and rye speeies, except SI'cu/eril/osa L. [= Dllsypyrulll viliosu/ll (L.) HOlm.] which carries a rare alloenzyme with pI 4A5. The appearance of a three-bandel[ heterozygous phenotype in AegiloJis maticll BOISS. suggests the dimcric struI·ture of SOD-A. SO D- Il, a DIECA-sensitive I'ytosolie isoenzyme, showed three alloenzymes, Bu, Urn and Br with pI values liAi'>, liJ) and 0.7, respeetively. All goatgrass speeies of different ploidy level, all polyploid wheats and the diploid wheat T. umrtu Tlln!. ex GAXDIL. shared a tommon alloenzyme Bu. The wild diploil[ wheat T. boeoticum BOiss. and its I'llltivatel[ relative T. mOilOCOCCUIII L. s.str. have ,L divergent alloenzyme Hm. Alloenzyme Br is eharaeteristi~ of rye species, ex~ept S. villosa L. whieh earries the common wheat alloenzyme Bu. The wheat-rye amphidiploids, tritieules, show codominant expression of parental alloenzym8s together with a hybrid isoenzyme in a three-banded isoeleetric phenotype of SOD-B, as eharaeteristic of a dimerie enzyme. SOD-C, a DIECA-insensitive mitol'hondrial isoenzyme, revealed several rare isoelectric variants and one {'ommon alloenzyme (pI ~ 6.2) shared by all wheat and rye species and by most goatgrass species. The appearanl'e of a five-banded isoelectrie phenotype in some accessions of the polyploid goatgrass .:ie. OI;allt s.1. indil'ates the tetrameric strlleture of SOD-C. Implieations of the SOD alloenzyme dab on the phylogeny of wheats and their relatives are disellssed.
Introduction
With a previous work (JAASKA and JAASKA 1982) we initiated a systematic study of the evolutionary variation of isoenzymes of superoxide dismutase (SOD, EC 1.15.1.1) among the grass species. Two major and two minor genetically ind(~pendent isoenzymes of SOD of different tissue specificity and inhibitor sensitivity were specified in the seedlings of barley, wheat and some related species by polyacrylamide gel elcctrophoretic and isoelectric focusing techniques. The two major isoenzymes, SOD-A and SOD-B, Abbreviations: SOD, supcroxide dislllutase; DIECA, diethyl dithiocarbamate; pI, isoelectric point. 49*
748
V. JAASKA
were inhibited by cyanide and diethyldithiocarbamate (DIECA) as characteristic of Cu-Zn-SOD, whereas the two minor isoenzymes, SOD-C and SOD-D, were insensitive to the two inhibitors as characteristic of Mn-SOD. The isoenzymes of SOD proved evolutionarily rather conservative showing only rare cases of variation in their electrophoretic and isoelectric properties among the barley species. Even such distinct species as the cultivated barley Hordeum vulgare 1. and the cultivated hexaploid wheat Triticum aestivum 1. had identical electrophoretic enzymograms of SOD and only isoelectric focusing revealed a shift in the isoelectric points of their SOD-B. The present paper extends the study of the evolutionary variation of SOD isoenzymes among the grasses of the tribe Triticeae DUM. and describes the electrophoretic and isoelectric enzymograms of SOD among the species of the subtribe Triticinae Trin. ex Griscb. belonging to the genera Triticum 1., Aegilops 1. and Secale L.
Materials and Methods Plant Material Seed accessions were received from the Vavilov World Collection of the Institute of Plant Industry in IJeningrad, from botanical gardens and other eollections or eollected by the author in nature. The number of accessions studied is indicated in brackets.
1. The wheat genus, Triticum L. 1 a. Diploid wheats: T. monococcum L. s.l. (27), involving the cultivated einkorn T. monococcum 1. s.str. (6) and its wild-growing relative T. boeticum BOISS. (21); T. urartu THuM. ex GANDILIAN (7). 1 b. Tetraploid wheats of the Emmer group eomprising T. turgidum 1. emend. THELL. (14), involving the cultivated T. turgidum L. s.str. (1), T. durum DEsF. (1), T. polonicum L. (1), T. carthl/cum NEVSKI (1), T. dicoccon (SCHRANK) SCIIUEBL. (1) and the wild emmer T. dicoccoides (KOERX.) SCHWEINF. (9). 1 c. Tetraploid wheats of the Timopheevii group comprising T. timopheevii ZlIUK. emend. MAC KEY and involving the cultivated T. timopheevii ZHUK. s.str. (2) and the wild T. araraticum JAKUBZ.
(8). 1 d. Hexaploid bread wheats comprising T. aestivum 1. emend. THELL. (7) and involving T. aestivum L. s.str. (2), T. compactum HOST (1), T. sphacrococcum PERC. (1), T. spc/ta L. (2) and T. macha DEli:. et ~rEN. (1). 1e. A spontaneous allohexaploid T. ti1l10pheevii ZHUK. x T. monococcum L. (= T. zhukovskyi MEN. et EIUZ., 1) and a synthetic allohexaploid T. carthlicum NEVSKI x T. monococcum 1. (1). 2. The goatgrass genus, Aegilops L. 2a. Diploid goatgrasses: Aegilops tauschii Coss. (4), Ae. speltoides TAUSCH s.!. (5), Ae. mutica BOISS. (1), Ae. bicornis (FO]{SK.) JAvn. et Sp. (4), Ae. longissima SCHWEINF. et MuscHL. s.l. (5), Ae. searsii FELDMAN et KISU:V (1), 11e. cauda/a L. (f)), At'. umbellulafa ZHUK. (4), Ae. c01l10sa SIBTH. et Sm. s.L (3), Ae. /il/iaristala VIS. (2). 2b. Tetraploid goatgmsses: Jje.1'f'lItricosa TAUSCIl (1), Ae. cylindrica HOST (1), Ae. crassa BOISS. ssp. macrathera (BOISS.) ZIIVK. (1), Ae. friuucia/is L. (1), Ae. biuncialis VIS. (1), Ae. ovata L. (G), Ar. coluiililaris ZHUK. (1). 2 e. Hexaploid goatgrusscs: Ae. cmssa BOlSS. ssp. crass a (1), Ae. juvenalis (THELL.) Eig (1). :l. The rye genus, 8ecale 1.: the perennial rye, S. monlanum Guss. ssp. analolicum(HOIss.) TzvEL. (2), the annual r1eistogamous rye, 8. sylvestre HOST (2), the annual self-incompatible rye, 8. ctrcale 1.. s.l. (il), involving the eultivated 8. cercale ssp. cercale (1) and the wild 8. cere ale ssp. allcestrale ZHUK.
Isoenzymes of Superoxide Dismutase in Wheat
749
s.l. (4), a 1illnean species Secale villosa 1. (2), presently treated as Dasypyrum villosum (1.) BORB., syn. Haynaldia villosa (1.) SCHUR and Triticum villosum (1.) BlEB. 4. Wheat-rye aIIohexaploids Triticum turgidum 1. sol. x Secale cereale 1. ssp. cereale - hexaploid tritirales (18) and wheat-rye chromosomal addition lines (7).
Biochemical Methods Enzyme extracts were prepared from the primary leaf 4-8 d old etiolated seedlings and subjected to electrophoresis or isoelectric focusing in polyacrylamide gel slabs to stain thereafter for the SOD activity as described previously (hASKA and JAASKA 1972). The genetically independent isoenzymes are designated by capital letters (SOD-A, SOD-B, SOD-C, etc.), and the corresponding gene loci by arabic numbers (Sod-l, Sod-2, Sod-3, respectively). The letter or numerical superscripts label the genetic variants of isoenzymes (alloenzymes, homoeoallelic isoenzymes of polyploids, hybrid isoenzymes) whereas the numerieal underscripts specify the modifirational isoforms of epigenetic nature. The numerical superscripts reflect either the relative electrophoretic mobility values in Rrunits or the pI values of electrophoretie or isoelectric electromorphs, respectively. The letter superscripts specify alloenzymes or alleles.
Results and Discussion Figs. 1 and 2 present a set of the SOD enzymograms obtained by means of polyacrylamide gel slab electrophoretic and isoelectric focusing techniques, respectively, coupled with the photochemical staining method. On both types of enzymograms three regions of unstained achromatic bands of independent interspecific variation can be distinguished. This suggests three genetically independent isoenzymes of SOD which were labelled (JAASKA and JAASKA 1982) SOD-A, SOD-B and SOD-C in the order of their decreasing electrophoretic mobility and increasing isoelectric points. SOD-A, acyanide- and DIECA-sensitive chloroplastici soenzyme (JAASKA and JAASKA 1982), is seen on the enzymograms as a major achromatic band of fastest mobility and of lowest isoelectric point and reveals, among the Triticeae grasses studied, two genetic variants. The variant of lower mobility (RF 0.83) and higher isoelectric point (pI,....., 4.55) labelled A4.55 according to its pI value is common to all wheat, goatgrass and rye species which showed no intra- or interspecific variation in SOD-A. The same variant was previously reported (JAASKA and JAASKA 1982) shared by species belonging to the genera Hordeum L., Taeniatherum NEVSKI and Heteranthelium HOCHST. of the tribe Triticeae DUM. The variant of higher electrophoretic mobility (RF 0.88) and lower isoelectric point (pI,....., 4.45), A4.45, was found in only one species of the subtribe Trifieinae, in Dasypyrum villosum (= Secale villosa). Previously (JAASKA and JAASKA 1982), this variant was also reported for Hordeum geniculatum All. of the subtribe Hordeinae DUM. Aegilops mutica, an outcrossing diploid, exhibited intrapopulational polymorphism with two pI-variants. In some individuals, the common variant AU& and a rare unique variant AU showed a symmetrical three-banded phenotype (6, Fig. 3) as characteristic of heterozygotes for a dimeric enzyme. SOD-.B, a cyanide- and DIECA-sensitive cytosol isoenzyme (JAASKA and JAASKA 1982), reveals on the electrophoretic enzymograms (Fig. 1) of the Tritieinae species two electromorphs. The fast electromorph with RF 0.58, SOD-Bo.58, is common to all poly-
750
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JAASKA
Fig. 1. Eleclrophorehc enzyrnograrns of superoxid(~ dismulase ill various specj:es. ], Triticum rnonococcuill vaT. horuemannii; :;! ·- 4, T. bo('olicurII va.T. thaoudar (:!), va.r. fuscurn (Cl) and var. baydaricum (4); 5, T. urartu; 6,1'. dicoceoides vaT. kotsellyi; 7-9, 1'. flraralicum var. kurdistallicwm (8), and var. araxicum (9); 10- 11, 1'. dieoccoides var. arabicu'lli (10) and var. dicoccoides (11); 12, Secale cereale ssp. ancestrale; 13, S. montallulll ssp. allatoijeulli; 14, 1(i, Dasypyrurn villosum ( = Secale t'1:11osa); 15, S. sylvestre. The migration is from the origin at the top towards the anode at the bottom.
A"·S.[
-A If'
Fig. :!. pl-Nllzymograll/s of superoxide dis mutase obtained by means of isocleetric focusillg with tlte LKR ampholyte Ampholine pH 4--6. 1, Triticum monococcum; 2, 1'. bocotfeu-III; 3, T. u-mrtu; 4, T. dicocco'ides; 5, '1'. araraticum; G, a synthetic allohexaploid T. carthlicum x 1'. monococcwlI; 7, T. monococewn; 8, 1'. tirnop/ieevil:; 9, T. zhukovskyi ( = T. timopheevii x 1'. mo'//ococcum); 10, T. sp('/ta; 11 , T. carthlicum x 1'. '///onococcum; 12, Beeale are Ill!' ssp. rereale; 13, Hordeu.m l/Iuriuum ssp. glllucum; 14, J)nsypyrum vil/osum ( - Seeale villosa); 15, 8. cereah~ ssp. /ll/cl'stra/f; 16, a synthctie allohexaploid .T. i'UrgidwJI x 8. (:('I"ea/(' (= .Triticale t'Urgidoc('reale); 17, '1'. turyid'Ulll. The ra,tholyte (fUll M ethylcnediaminc) is at the top, the nnolyte (0.01 M H Z:;;04) at the bottom.
Isoenzymes of Superoxide Dismutase in Wheat
751
Table 1. Electrophoretic properUes and the distribution of the SOD-B alloenzymes among the TriticelJe grasses
AHo-
pI
Speeies
SOD-Bu 0.58
0.40
SOD-Bm 0.6:3
oJ)
SOD-IY 0.53 SOD-1Jb 0.08
0.7 5.7
T. martu, T. turgidum s.l., T. timopheevii s.l., T. aesfivulil s.1., Aegilops spec., Elytrigia spec. T.lllonococcuHI s.l., Elytrigia spec., Hordeum brevisubulatum 8('clrie )llOntanurn s.l., S. cereale s.l., S. sylvestre
RF
enzyme
Hordl'll})) spec, Taeniatherwn spec.
ploid wheats, diploid and polyploid goatgrasses, to a diploid wheat T. urartu and to Dasypyrum villosum. The diploid wheats comprising '1'. monococcum s.1. (incl. the wild T. boeoticum) and all the rye species shared the slow electromorph SOD-Bo.53. Some allopolyploids, such as T. timopheevii X T. monococcum (= T. zhukovskyi), T. turgidum x T. monococcum and hexaploid triticales - T. turgidum x S. cereale showed a broad band of SOD-B comprising both electromorphs, BO.58 and BO.53, as expected in the case of codominant expression of homoeoallelic genes of parental species of these polyploids. Isoelectric pI-enzymograms (Fig. 2) distinguish, however, among the Triticinae species, thrce genetic variants of SOD-B. The two wheat SOD-B electromorphs, BO.58 and BO.53, differ also slightly in their pI values estimated 5.45 and 5.5, respectively. A third pIvariant of SOD-B with a distinctly different pI value 5.7 was found to be characterstic of the rye species and, previously (JAASKA and JAASKA 1982), - of barley species. Since the rye cond barley species have different SOD-B electromorphs, BO.53 and BO.58 respectively, a total of four alloenzymes of SOD-B should be specified. The distribution of the four SOD-B alloenzymes labelled by letter superscripts as Bu, Bm, Br and Bb among the Triticeae grasses and their electrophoretic properties are summarized in the Table, which includes our previous (I.c.) and some preliminary unpublished data. It can be seen that the most wide distribution among the Triticeae genera have alloenzymes Bu and Bm. Presumably, they are the ancestral alloenzymes from which the other two alloenzymes have arisen in the course of the evolutionary differentiation of Triticeae grasses. The two allohexaploid wheats, T. timopheevii x T. monococcum (= T. zhukovskyi) and T. turgidum x '1'. monococcum, show (Fig. 2) codominant expression of B-alloenzymes of both parental species. Hexaploid triticales reveal (Fig. 2) a distinct three-banded (triplet) pI-phenotype of SOD-B. In addition to the alloenzymes of the parental species, Bu and Br, a band of intermediate pI-value, as characteristic of heterodimeric hybrid isoenzymes, is clearly seen. This result suggests that SOD-B is a dimeric enzyme which exhibits codominant expression of parental homodimers and a hybrid heterodimn in hexaploid triticales. Among the series of seven wheat-rye chromosomal addition lines analyzed, only the line with a pair of rye chromosome 2R revealed the triplet pI-phenotype of SOD-B
752 I
2
3
~
5
6
7
8
9 10
11
_ B 5.ltj _A~·e _ A ft.. ItS I"ig.3. pI-Enzymograms of superoxide dismutase obtained by means of isoelectric focussing with the LKB ampholyte Ampholine pH 5-7. 1, Aegilops triuncialis; 2, Ae. ovata; 3, Ae. umbellulata; 4, Ae. ovata; 5, Ae. umbellulata; 6, Ae. mutica; 7, Ae. biuncialis; 8, Ae. comosa; 9, Ae. longissima; 10, Ae. tauschii; 11, Dasypyrum villosum. The eatholyte (0.01 A{ ethylenediamine) is at the top, the anolyte (0.01 M H2S0 4 ) at the bottom.
characteristic of triticales. This implies that the gene controlling the rye alloenzyme Br is located in the rye chromosome 2R. All the accessions of primary triticales analyzed showed an identical triplet phenotype of SOD-B. Among the secondary triticales, two accessions out of eight analyzed lacked the rye alloenzyme Br and the hybrid heterodimer Bur, showing only the wheat alloenzyme Bu. SOD-B was reported (JAASKA and JAASKA 1982) to exist in three epigenetic isoforms, Bl , B2 and B3, interconverted by the treatment with sulphydryl reagents, such as cysteine, p-chloromercuribenzoate, etc. The three epigenetic isoforms of SOD-B can be distinguished on the pI-enzymograms by isoelectric focusing. In the presence of a SHreductant (cysteine, dithiothreitol) in the homogenization buffer the isoforms of lower pI, Bl and B2 , are converted partially or totally, depending on the treatment conditions, into the isoform B3 which becomes dominating on the pI-enzymograms, as seen in Fig. 2. The point of interest is that SOD-B of 1'. monococcum s.I. (inci. T. boeoticum) :.tnd of all three rye species revealed only the isoform B3, whereas the two isoforms of lower isoelectric points, Bl and B2 , were totally absent even when the enzyme extract was made in a buffer without the sulphydryl reductant (cysteine) added. At the same time, D. villosum, 1'. urartu, all polyploid wheats and all goatgrass species showed three epigenetic isoforms of SOD-Bu. SOD-B3m of T. monococcum andSOD-Bllf rye, in contrast to SODBau , could not be converted into the fast isoform by a treatment with p-chloromercuribenzoate ill the extraction buffer. Presumably the alloenzymes Bm and Br lack a free sulphydryl group capable to react with p-chloromercuribenzoate.
Isoenzymes of Superoxide Dismutase in Wheat
753
SOD-C, a cyanide- and DIECA-insensitive mitochondrial isoenzyme (JAASKA and 1982), revealed on both the electrophoretic and isoelectric enzymograms (Figs. 1 and 2) a weak achromatic band of slower mobility and higher pI than SOD-B. SOD-C exhibited only a slight variation in its electrophoretic mobility and isoelectric point which was more distinct on the pI-enzymograms obtained with the use of the pH 5-7 ampholyte (Fig. 3). All wheat and rye species had one SOD-C band of closely similar or identical electrophoretic mobility and isoelectric point (pI about 6.2). Among the polyploid goatgrasses, some accessions of Ae. OVcttct s.1. revealed a symmetrical five-banded phenotype (2 and 4, Fig. 3) as characteristic of a tetrameric enzyme, consisting of two subunits of different isoelectric points (pI values about 6.2 and 6.8). All other polyploid goatgrass species showed one invariant band of SOD-C, labelled C6.2 according to its pI value, in common with wheat and rye species. In some diploids, such as D. villo8um, Ae. comosct and Ae. searsii, SOD-C was, however, distinctly shifted twoards lower isoelectric points. Intraspecific variation with two pI-variants of SOD-C was observed among the accessions of the diploid Ae. longissima, s.l. The five-banded SOD-C phenotype of Ae. ovata is presumed to combine the two outer homotetramers, C6.2 and C6.8, with three hybrid heterotetramers of intermediate isoelectric points. SOD-C6.2 was also characteristic of most diploid goatgrasses - genome donors and precursors of polyploid goatgrasses. SOD-C6.8 was, however, not encountered among the diploids and may thus be presumed to have been arisen at the polyploid level in Ae.ovatct. Phylogenetic implications. The SOD-B alloenzyme data present a new significant evidence on the origin of tetraploid wheats. Of phylogenetic importance is the finding that all tetraploid wheats of both genetic groups, including their wild representatives T. dicoccoides and T. arnratiwm, shared the alloenzyme SOD-Bu characteristic of 1'. urartu and goatgrass species, while the alloenzyme SOD-Bm specific to T. boeoticum was never encountered in tetraploid wheats. The two wild diploid wheats, T. urartu and T. boeoticum, revealed a distinct diagnostic divergence ill SOD-B, being fixed for different SOD-B alloenzymes, Bu and Bm, respectively. The SOD-B alloenzyme data, thus, clearly argue against the theory of separate diphyletic origin of 7'. di(:occoide,~ and 7'. ctramticum with T. urartu and T. monococcum s.l. as their A-genome donors, respectively, proposed (KONAREV 1975; KONAREV et a1. 1976) on the basis of immunoehemical properties of seed proteins. Instead, a new support to the concept of monophyletic origin of tetraploid wheats is presented. Our previous studies on acid phosphatase, alcohol dehydrogenase and esterase isoenzymes have discovered (discussed in JAASKA 1980) distinct genetic differences bettween the A genomes of contemporary 7'. boeoticum, T. urartu and tetraploid wheats. This means that contemporary diploid wheats '1'. umrtu and T. boeoticum separately do not fit the A genome donor of tetraploid wheats. To explain the alJocnzyme data it has been suggested (JAASKA 1976, 1980) that both T. boeoticum and T. urartu, might have contributed, together with Aegilops speltoides, JAASKA
754 to the origin of a primitive allotetraploid wheat from which 1'. araraticum and T. dicoccoides then diverged. The involvement of a male-sterile hybrid between two of the three diploids (e.g. between 1'. urartu and 'l'. boeoticurn, Ae. speltoides and 'l'. urartu or Ae. speltoides and 'l'. boeoticurn) pollinated by the third diploid is an attractive hypothesis of the origin of the primitive tetraploid wheat. Alternatively, the participation of a common anc('stor of 7'. ufarlu and 7'. boeoticum with a suitable combination of alloenzymes in the origin of tetraploid wheat should still be seriously considered. Although such ancestral biotype has not yet found among the accessions of contemporary diploid wheats, its existence, at least in the past times, has theoretically good grounds. The Linnean rye species, Secale 'Oill(),~n 1,., was found to differ from the rem,lincier rye species in allocnzymes of all the three SOD isoenzymes. With wheats it has a common allopnzyme, SO 0- Bll, but differs from them in the alloenzymes of SOD-A and SOD-C. Previously it has been reported (JAASKA 1975) that S. 'Oillosa has allocnzymes of alcohol dehydrogenase ADH-A and of aspartate aminotransferasesAAT-A and AAT-B in common with rye species and different from those of wheats. However, its 6-phosphogluconate dehydrogenase and fast-moving isoperoxidase proved, reversely, different from the rye alloenzymes and in common with wheats. The alloenzyme data as a whole, thus, support the taxonomic treatment of the Linnean Secale 'Oillosa in a separate genus Dasypyrum (Coss. <'t DUR.) BORB. as J). villosum (L.) BORB. Qua ternary structure. Codominant inheritance of evolutionarily divergent parental isoenzymes in a three-banded isoelectric phenotype of SOD-B in the wheat-rye amphidiploids (triticales) provides an indirect evidence on the dimeric structure of the cytosolic isoenzymes SOD-B. Similarly, the appearance of a five-banded isoelectric phenotype of SOD-C in some biotypes of a polyploid goatgrass Aegilop8 ovala indicatps the tptrameric structure of the mitochondrial isoenzyme SOD-C. The dimerie nature of the cytosolic CujZn-SOD and the tetrameric nature of the mitochondrial Mil-SOD is known (review: FRIDOVICH 1975) for diverse cukaryotic organisms. TIl(' same was recently reported (BAUM and SCANDALIOS 1981) for the SOD isop!1zymes ill maize. The quaternary structure of the SOD iwenzymes has evidently arisen at initial stages of f'volution and has become conserved during the eukaryotie evolution. The discovpry of a. three-banded heterozygous phenotype of SOD-A in some individuals of an outbreeding diploid Ae. rnulica suggests that the chloroplastic isoenzyme has a dimf'ric structure. Our previous rpsu't (JAASKA and JAASKA 1982) on a two-banded electrophoretic phenotype of SOD-A without a distinct hybrid band in a tetraploid barley H. geni(:j,(,latum indicates that hybrid heterodimer may not be formed between every type of alloenzymic subunits of the chloroplastic isoenzyme. The absence of the heterodimeric hybrid between the chloroplastic SOD-A and the cytosolic SOD-B implies that the two IHECA-sensitive Cu/Zn-isoenzymes arc structurally divergent proteins. References BAUM, J. A" and SCANDALIOS, J. E.: Isolation and eha.raderization of the cytosolic and mitochondrial superoxide dislIlutases in maize. Areh. Hioehem. Biophys. 208, 249-264 (1981).
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1.: Superoxide dismutases. Annu. Rev. Hiorhem. 44, 147-159 (1975). V.: Evolutionary variation of enzymes and phylogenetic relationships in the genus 8ecale L. (russ.). Eesti NSV TA Toimetised, Bioloogia 24, 179-198 (1975) . .JH~K'\, V.: Genome- ancl tissue-specific regulation of esterase and add phosphatase isoenzymes in tetraploid wheats (russ.). Eesti NSV 'fA Toimetised, Bioloogia 2;), 132-145 (197G) . •LIASKA, V.: Eleetrophoretie survey of seedling esterases in wheats in relation to their phylogeny. Theor. App!. (ienet. ;)6, 273~-284 (1980) . .1,118K;\, VILVE, anll .LLlS hI, V.: lsoenzymes of superoxide dismlltase in barley. Biol'hem. Physiol. Pflanzen 177, (1982). l\oNAHEV, A. V.: Differentiation of the first genomes of polyploid wheats based on data from immunoehemieal analysis of ethanol fral,tion of grain proteins (russ.). Hnll. All-Union Vavilov Inst. PI. Indllstr. 47, 811 (1fJ7i'»). KONAREY, V. G., ()AI'IUL,ll:K, I. P., PENEVA, T. I., [(ONAREV, A. V., [(BAKIMOVA, A. G., l'iIIGUSIJOVA, E. F.: On the nature anll origin of wheat genomes on the basis of biochemistry anc! immunochemistry of the grain proteins (russ.). Agric. BioI. (USSR)ll, G5G-G65 (197G). FHlDOYH'I1,
JAA~h'\,
Receit'ed .J nne 3, 1982 Author's address: VELLO ,LushA, Laboratory of Biochemistry, Institute of Zoology and Botany, 21 Vanemnise St., USSR - 202400 Tartn, Estonian SSR (USSR).