Three alcohol dehydrogenase genes in wild and ...

Biochemical Genetics, Vol.22, Nos. 5/6, 1984

Three Alcohol Dehydrogenase Genes in Wild and Cultivated Barley: Characterization of the Products of Variant Alleles A. D. Hanson 1'2 and A. H. D. Brown I

Received 31 Aug. 1983--Final25 Jan. 1984

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adhl locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N2 for 2-3 days. Using N2-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The 35S-labeled monomers induced under iV: in the lines homozygous for Adh 1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was C R M - and that the Adh3 I gene product was smaller than normal. The A d h l and Adh3 variants showed independent segregation. KEY WORDS: alcohol dehydrogenase; isozyrnes;barley; anaerobic induction INTRODUCTION The alcohol dehydrogenase ( A D H ) system of maize is well known in terms of both classical and molecular genetics. This system holds promise (a) for 1CSIRO, Divisionof Plant Industry, G.P.O. Box 1600, Canberra A.C.T. 2601, Australia. 2Permanent address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824. 495 0006-2928/84/0600-0495503.50/0 © 1984 Plenum Publishing Corporation

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understanding the developmental and environmental control of gene expression in plants and (b) as a source of a selectable marker for plant gene transfer systems (Freeling and Birchler, 1981; Gerlach et al., 1982; Peacock et al., 1983). Although far less characterized, the ADH systems of several other plant species are known to have varying degrees of genetic, structural, and functional analogy with that of maize (review, Freeling and Birchler, 1981; see also Ellstrand et al., 1983; Tanksley and Jones, 1981). This analogy can probably be exploited to advantage in investigating (a) above, and can certainly be in studies of molecular evolution. However, the presence of inducible ADHs in other species compromises ADH as a marker for use in interspecific gene transfer. The ADH system of the recipient genotype would presumably first have to be incapacitated, a task which becomes harder as the number of active Adh genes in the recipient increases. A need therefore exists for basic genetic information on other plant ADH systems comparable to that available for maize. There has been considerable progress in characterizing the ADH system of barley (Hordeum vulgate), which is among the better known of plant systems. The closely related species of wild barley (H. spontaneum) was shown by Brown (1980) to have two closely linked Adh loci, functionally resembling the Adhl and Adh2 loci of maize. Mutational analysis of an azide-induced Adhl null by Harberd and Edwards (1982) confirmed the existence of two loci in barley and subsequently implicated a third locus (Harberd and Edwards, 1983). This third gene (Adh3) was, however, only weakly expressed in flooded roots and evidently could escape detection in starch gel systems. In the case of barley, it is possible to turn to the highly diverse wild progenitor H. spontaneum as a source of natural electrophoretic variants, if available, such variants not only save time and effort in assembling a collection of marker genes, but also are of interest in themselves from the standpoint of evolutionary biology. We therefore undertook to develop a convenient, reproducible procedure for obtaining good expression of the three known barley Adh loci, to screen a diverse collection of H. spontaneum lines (known already to carry variant alleles of Adhl and Adh2; see Nevo et al., 1979) for electrophoretic variants at the newly recognized Adh3 locus, and to subject these variants to formal genetic analysis. We also applied sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing in the presence of urea (urea-IEF) to examine the ADH peptides synthesized by genotypes carrying variant alleles at one of the three Adh loci. We reasoned, first, that this should yield information about the genetic changes underlying the electrophoretic variants and, second, that the results might reveal additional Adh genes and shed light on the nature of "conformer" isozyme bands associated with Adhl.

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MATERIALS AND METHODS Seed Samples

The wild and cultivated barley and wheat lines included in this study were as follows. (1) Barley (Hordeum vulgate) cvs. Himalaya, Clipper, Atlas, and five sublines from Iranian land races. These eight lines were those studied for chloroplast DNA variation by Clegg et al. (1983). (2) Samples of 50 seeds from single generations of two barley (H. vulgate) composite crosses: an early generation (F4) from composite cross (CC) 34 and a late generation (F17) from the diverse CC 21. These materials were studied for allozyme variation by Brown and Munday (1982). (3) Barley cv. Betzes, wheat (Triticum aestivum) cv. Chinese Spring, and the six euplasmic wheat-barley chromosome addition lines developed by Islam et al. (1981). (4) Fifty lines of wild barley (H. spontaneum) from several populations in Israel, including those lines studied previously for hordein (Doll and Brown, 1979) and a-amylase variation (Brown and Jacobsen, 1982). Induction of ADH lsozymes

For the experiments involving O2/N 2 mixtures, aleurone layers and whole seedlings of barley cv. Himalaya were prepared and incubated under sterile conditions. Intact grains or deembryonated grains were surface-sterilized for 20 min in l% NaOCI, washed thoroughly in sterile distilled water, and incubated on moist sand for 4 days at 20°C. Whole germinating seedlings were transferred to 25-mi Erlenmeyer flasks (two per flask) containing 1 ml of 10 mU CaCI2 with 10 ~g/ml chloramphenicol (incubation medium). Aleurone layers were stripped from deembryonated grains and placed in 25-ml flasks with 2 ml of incubation medium (10 layers/flask). Pairs of flasks (one each of aleurone layers and seedlings) were placed in sealed 900-ml jars which were purged with high-purity N2 for 1 hr. 02 was then injected, and N2 simultaneously withdrawn, to give the desired gas mixtures (0, 2, 5, 10% 02). Control flasks were incubated in air and were not enclosed in jars. Incubation was in darkness for 2 days at about 24°C on a reciprocal shaker (60 cycles/min). The 02 levels in the flasks were checked by gas chromatography (Turner and Gibson, 1980) 3 hr after 02 injection and just before the end of the 2-day incubation. Measured values at 3 hr were very close to target values (_+5%) and changed little over 2 days. Single aleurone layers or the root tips (2 cm) from a single seedling were harvested for analysis. Conditions for controlledatmosphere experiments with cv. Himalaya embryos differed from the above only in that surface-sterilized half-grains containing embryos (five per flask in

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1 ml of incubation medium) were substituted for aleurone layers or germinating seedlings. Routine induction of embryos of barley, wheat, and wheat-barley chromosome addition lines was carried out under nonsterile conditions as follows. The embryo-containing half of each grain was cut off, dehusked, and imbibed individually in a small vial containing 0.1 ml of water for 1 day at 20-25°C. A further 0.1 ml of water was then added, and tubes were transferred to a 2-1itre desiccator which was purged with high-purity N2 for 30 min. Incubation under N2 continued for 2 or 3 days at 20-25°C, with daily repurging of the desiccator for 15 min. Embryos were dissected from adhering aleurone and starchy endosperm tissues just before analysis. For the wheat-barley chromosome addition lines, sterile aleurone layers were also used as a source of ADH isozymes. Deembryonated grains were imbibed on most sand at 20°C for 3 days under sterile conditions. Aleurone layers were stripped free of starchy endosperm, and pairs of layers of the same genotype were placed in sterile vials containing 0.2 ml of 10 mM CaC12. Vials were incubated under N2 for 3 days as above, and individual layers were taken for analysis. Starch Gel Electrophoresis

Horizontal starch gel electrophoresis was conducted using 12% Connaught starch gels. The electrode buffer was 75 mM LiOH adjusted with solid H3BO3 to pH 8.5. The gel buffer consisted of 6 vol of the electrode buffer plus 94 vol of 9.1 mM citric acid-65 mM Tris. Samples were ground in 100 ul of 50 mM K-phosphate buffer, pH 7.0, with 1 mg • m1-1 dithiothreitol, and the crude extract was absorbed by paper wicks (Beckman, Catalog No. 319329) which were inserted into the gel. After electrophoresis, zones of alcohol dehydrogenase (EC 1.1.1.1; ADH) activity were detected as previously described by Brown et al. (1978). In the studies of joint segregation of other allozyme loci, separate slices of the lithium gel were stained for dipeptidase (EC 3.4.13.11; DIP). Aconitate hydratase (EC 4.2.1.3; ACO), malate dehydrogenase (EC 1.1.1.37; MDH), and glucose phosphate isomerase (EC 5.3.1.9; GPI) were separated in gels made with a histidine buffer system, and N A D H dehydrogenase (EC 1.6.99.3; NDH) in gels made with a Tris-citrate borate system (see Brown, 1983). Isolation of 3SS-Labeled Adh Gene Products by Immunoprecipitation

Immunotitration experiments demonstrated that sheep antiserum raised against ADH1 homodimer purified from maize (Pryor and Huppatz, 1983)

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recognized all barley ADH isozymes. This antiserum was therefore used in combination with 35S labeling of embryo and aleurone proteins under anaerobic conditions (Sachs et al., 1980) to isolate labeled A d h gene products for analysis by gel electrophoresis and fluorography. Sterile embryos and aleurone layers were prepared from 8-16 grains of each genotype as described above, except that the embryos were imbibed for 1 day in air. Imbibed embryos were incubated in one 25-ml flask, aleurone layers in a second flask, for 6 hr under N2. Working under N2, the tissues in each flask were washed thoroughly in sterile distilled water and then received 1.8 ml of fresh incubation medium, to which was added 0.2 ml of a filter-sterilized solution of L- [35S]methionine (80-160 tzCi, >800 Ci/mmol; Amersham). Incubation with label continued for 20 hr under N 2 at 22°C on a rotary shaker (60 cycles/min). Labeled embryos and aleurone layers of each genotype were then pooled, rinsed in ice-cold 1 mM L-methionine, and mixed with 30 unlabeled carrier aleurone layers (cv. Himalaya, incubated under N2 for 1 day). Tissues were extracted by grinding with sand in a pestle and mortar in a total of 8 ml 0.15 M Tris-HC1 buffer, pH 8.0, containing 10 m~ dithiothreitol. Homogenates were centrifuged at 16,000g for 10 min, and supernatants were recentrifuged at 150,000g for 2 hr. To the high-speed supernatants was added 0.1 vol of a solution containing (w/v) 10% NaC1, 1.5% L-methionine, 5% Triton X-100, 2% sodium deoxycholate (× 10 immunoprecipitation medium), followed by a small excess (about 30%) of antiserum. After incubation for 1 hr at 35°C followed by overnight at 4°C, the antigen-antibody complex was collected by centrifugation at 23,000g for 15 min. Proteins in a portion of the supernatant were precipitated by adding 5 vol of cold acetone and holding overnight at - 15°C. The immunoprecipitate was washed three times with X1 immunoprecipitation medium, then twice with water, and stored frozen. Radioactivity was determined by scintillation counting, at an efficiency of about 90%.

S D S - P A G E and Urea-lEF of Labeled A D H Monomers

Samples of immunoprecipitates and corresponding supernatant fractions were analyzed by SDS-PAGE on gradient gels (12.5-20% acrylamide; Spencer et al., 1980). Immunoprecipitate fractions were analyzed also by thin-layer IEF in the presence of 8 M urea (urea-IEF). For this, immunoprecipitate samples were solubilized by vortex mixing for 1 rain in 8 M urea containing (v/v) 2% Nonidet P40, 1%/3-mercaptoethanol, and 9% glycerol. IEF gels (thickness, 1.3 ram; nominal pH range, 3.5-9.5 over a 10-cm width) were prepared according to LKB Application Note 250 except that urea was included to a final concentration of 8 M. Gels were run at 14°C in an LKB Multiphor 2117

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apparatus. Gels were prefocused for 3 hr (about 1.2 kV-hr). Slots (8 × 4 mm) were then cut into the gel 2 cm from the cathode, and samples (50 ~1) were added. Gels were focused for 4 hr (about 3.5 kV-hr). Radioactive proteins were detected in both SDS-PAGE and urea-IEF gels by fluorography (Bonner and Laskey, 1974). Terminology

In the following sections, we use the three-gene model for alcohol dehydrogenase isozymes developed by Harberd and Edwards (1983). Thc alcohol dehydrogcnase loci (Adh 1, Adh2, and Adh3) are italicized as conventional for barley gcnc nomenclature. The polypeptide monomeric products are denoted as capitals (ADH1, ADH2, and ADH3) to distinguish them from the loci. The activc forms of the enzymes are dimers and written as their component monomers" homodimers ADH 1.ADH1, etc., or heterodimcrs ADH1.ADH2, etc. Each distinct allele is signified by a capital letter after the locus name (e.g., Adhl M), and the monomer for which it codes is similarly shown (e.g., ADH1 M). Where this bccomes cumbersome, the ADH label is omitted (e.g., 1M) but should always be understood. Note that the current designation of Adhl as the locus which codcs for the predominant monomer constitutive in seeds supercedcs the locus labels of previous papers (Brown et al., 1978; Ncvo et al., 1979; Brown 1980). These earlier papers had followed the conventions of electrophoretic surveys and labeled the locus coding for the more anodally migrating homodimer Adhl (now called Adh2), whereas the more prominent constitutive locus was labeled Adh2 (now called Adhl ). Barlcy is predominantly self-pollinated, and the alcohol dehydrogcnase genotypes of all the lines listed above were homozygous. Hence we adopt the labeling shorthand of referring to homozygous genotypes as haploids. RESULTS Anaerobic Induction of A D H Isozymes

In barlcy roots, moderate 02 deficiency (3-13% 02) induces higher total ADH activity than severe 02 deficit (Wignarajah et al., 1976). We therefore examincd the effects of a range of 02- tensions on ADH isozyme profiles both in young barley roots and in aleuronc and embryo tissues. We tested the two latter tissues because they are more tolerant of complete anoxia than are roots (Hanson and Jacobsen, 1984; Maync, 1982). Lowering the 0 2 tension provoked changes in the ADH isozymes of aleurone and embryo tissues which differed from those in young root tissues (Fig. 1). In aleurone layers and embryos, a very strong ADH1.ADH1

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homodimer band and a trace of ADH1.ADH2 heterodimer were present under fully aerobic conditions, as noted also by Harberd and Edwards (1982). As the 02 tension fell, isozyme bands associated with Adh2 and Adh3 appeared and strengthened progressively, with maximal development in complete anaerobiosis (N2 atmosphere); Adh3 expression was particularly strong under N2. Additional "conformer" bands flanking the ADH1.ADH1 homodimer are discussed in the next section. Young root tips under aerobic conditions showed weak bands associated with Adhl and Adh2; in response to mild anoxia (5% O2), these bands strengthened and heterodimer bands containing ADH3 monomers appeared. However, more severe anoxia (2% 02) did not elicit any further increases in ADH isozymes in roots, and complete anoxia abolished all ADH isozymes, presumably a result of tissue death. These results led us to adopt embryos or aleurone layers, induced under N2, as a standard system for the work which follows. Time course experiments demonstrated that 2- or 3-day incubation periods were sufficient for maximum expression of the isozymes associated with Adh2 and Adh3 (not shown).

Allozyme Variants at the Three Alcohol Dehydrogenase Loci

Having defined suitable conditions for expression of the three Adh loci, a starch gel electrophoretic survey was made of a set of barley lines known to be genetically diverse. Table I catalogues the variant lines detected. In H. vulgare no variation for ADH isozymes was found in the sample of 50 seeds from either of the composite crosses (CC 21, generation 17; and CC 34, generation 4). Both of these populations are broadly based and polymorphic for about one-third of allozyme loci (Brown and Munday, 1982). Allozyme variation in H. vulgate for the inducible locus Adh2 was known from a survey of Iranian land races. One of these variant lines was included in the present study (see Materials and Methods). All the other cultivars studied had the standard ADH complement. In sharp contrast to the low level of ADH variation in H. vulgate, eight variant composite ADH homozygous phenotypes were detected among the 50 lines of H. spontaneurn. These phenotypes arise from three alleles at the Adhl locus, three at the Adh2 locus, and at least five at the Adh3 locus. The common or normal allele at each locus is designated M. Figure 2 illustrates the type lines for these alleles in comparison with the standard pattern (cv. Himalaya) and the Iranian variant. Considering first the closely linked pair of loci Adhl and Adh2, three variant dilocus phenotypes or ditypes were previously reported in H. spontaneum (Nevo et al., 1979). The Bar Giyyora ditype has an allele designated S

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Table I. Isozyme Allelic Variants for Alcohol Dehydrogenase in Hordeum spontaneum and H. vulgate a Adh locus Variant No.

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aLines are identified by a site name and a family number. The Commonwealth Plant Introduction (CPI) numbers for the sites are Atlit 77129, Bar Giyyora 71283, Damon 71284, Eyzariya 77130, Mehola 77137, Tabigha 77143, Talpiyyot 77144, Wadi Qilt 77135, and Ilam 77158.

at the Adhl locus which codes for an ADH1.ADH1 homodimer with slower mobility. This allele was found in only one population. The Tabigha ditype, with an allele designated S at the Adh2 locus, codes for an ADH2.ADH2 homodimer with slower mobility. This allele was found in several populations in the Hule Valley, Israel. The Talpiyyot ditype had variant alleles at both loci, designated F because they code for faster-migrating ADH1.ADH1 and ADH2.ADH2 homodimers. This ditype was common in the population. It also was found at Atlit as a rare component. In both populations, the Adh 1 F and the Adh 2 F alleles always occur together) Thus although there are three alleles at both loci in H. spontaneum, only four Adhl/Adh2 two locus genotypes (MM, FF, SM, MS) has so far been found. In H. vulgare, a variant at the Adh2 locus from Ilam (Iran)codes for an ADH2.ADH2 homodimer with a mobility slightly faster than that coded by the Adh2 S allele of Tabigha. Although rare in the species, the Ilam allele, designated P, was also found in another land race sample from Shiraz. Figure 2 shows, as dashed lines, three isozymes which usually appear in the N2-treated embryos. One is more anodal and two are less anodal than the 3 The inequality of allele frequencies reported for A d h l F and Adh2 F alleles by Nevo et al. (1979) arose from incomplete typing of some families. Subsequent checking, together with the segregation analysis of the Talpiyyot ditype (Brown, 1980), has shown that so far neither of the fast variants at A d h l and Adh2 has been recovered separately.

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prominent ADH1 homodimer. These bands changed position in parallel with variant ADH1.ADH1 homodimers but were not affected by genetic variation at the other loci. For convenience, these bands are described as conformers in this paper; no hypothesis regarding their origin is implied in our use of this term. The conformer anodal to the ADH1 homodimer was detectable in overstained gels of root samples, but the other two conformers were not. Turning to the Adh3 locus, Table I lists the lines of H. spontaneum which displayed electrophoretic variants at this locus. Four variants were found. The Adh3 S or "slow" allele codes for an ADH3.ADH3 homodimer with a mobility similar to that of the A D H 1 M.ADH3 M heterodimer of the standard pattern. The Adh3 V allele codes for a very slowly migrating ADH3.ADH3 homodimer which appears as a broad band just ahead of the ADH1 M.ADH1 M homodimer. Both S and V variants gave rise to the anticipated shifts in the mobility of the interlocus heterodimers. Thus the IM.3S heterodimer was intermediate in mobility between 1M.3M and 1M.1M, 2M.3S was similar to 3M.3M, 1M.3V is in the broad band 3V.3V + 1M.1M, and 2M.3V is just ahead of 1M.2M.

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As well as these two classical variants in mobility for Adh3, there were two other distinct phenotypes. The isozyme spectrum of the Adh3 n allele was the same as the standard set except that it lacked the three dimers containing the ADH3 M monomer-hence the designation n for a "null" allele. The final variant (Adh3 I) was found in the same population in which the Adhl F, Adh2 F alleles were common (Talpiyyot). Its phenotype was intermediate between that of Adh3 M (normal) and that of Adh3 n (null). The 2M.3I heterodimer was present but with a markedly lower activity than the 2M.3M heterodimer. The 31.31 homodimer was undetectable. It appears that these isozymes have partially or completely impaired ADH activity (hence the designation I), with very little change in mobility. At this stage, the genetic interpretation of the variants is strictly hypothetical. We therefore sought further evidence to support these interpretations in two ways. The first was electrophoretic analysis of the radioactively labeled ADH monomers synthesized in response to N 2 treatment (next section). The second, which was performed only with the clearly codominant variants affecting mobility (Adh3 S and Adh3 V), was the electrophoretic analysis of F2 progenies from crosses among the lines.

Electrophoretic Characterization of Variant ADH Polypeptides Labeled with 3SS

Particular lines with variant numbcrs 2 through 9 (as listed in Tablc I) were analyzed, together with cv. Himalaya and cv. Bctzes. The total spectrum of polypeptides labeled from [35S]methionine under N2 (anaerobic polypeptides; ANPs) was qualitatively similar in all gcnotypes. However, the quantity of [35S]polypcptidcs synthcsized per seed varied considcrably (Fig. 3, horizontal axis). Except in one genotype, thc labclcd ADH peptides precipitated by immune serum constituted at least 4% of the ANP profile; the exception was Wadi Qilt 40 (Adh3 n), in which ADH was only about 1% of the total ANPs (Fig. 3). In SDS-PAGE, the immunoprecipitated ADH [35S]peptides ran as a diffuse doublet with Mr about 42,000 in all genotypes except Talpiyyot 4 (Adh3 I), which contained an additional band migrating just ahead of the doublet (Fig. 3, inset). In urea-IEF, the labeled immunoprecipitates from cv. Himalaya and cv. Betzes (both, with genotype Adhl M, Adh2 M, Adh3 M) were resolved into three major and three minor bands. The latter varied in strength between experiments (compare tracks 1 and 2, Fig. 4A) but were always detectable.

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Fig. 4. (A) Fluorographs of 35S-labeled ADH monomers separated by urea-IEF, for variants at the three Adh loci of barley. On the left, positions of the monomer products of the common alleles for Adh 1, Adh 2, and Adh 3 are marked as solid bars; broken bars show the common positions of minor bands. Key to tracks: Hi, separate samples of cv. Himalaya; Be, cv. Betzes; 3n, Wadi Qilt 40 (.4dh3 n); the remaining tracks are denoted by the variant allele (see Table I). (B) Correlated changes in the behavior of ADH monomers separated by urea-IEF (hatched bars) and that of the corresponding homodimers separated on starch gels. The ordinate shows the change in band position relative to the common allele for each locus; standards for common allele positions were cv. Himalaya and cv. Betzes. Starch gel band shifts were calculated from standards run on the same gel and have been multiplied by 0.6 for easier comparison. Band shifts in urea-IEF were calculated from standards run in adjacent gel tracks and from 1:1 mixtures of standards and variants.

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ADH3, and ADH2 monomers, in order of increasing distance from the cathode (= decreasing pI) The changes in IEF banding patterns in the variants confirm these identifications. Thus, for six of nine variant alleles (1 F, 2F, IS, 2S, 3S, 3V) the corresponding IEF band changed position as predicted from the altered electrophoretic mobility of the corresponding homodimer (Fig. 4B). The three exceptions to this pattern were the 3n, 3I, and 2P alleles, which are now described in turn. In Wadi Qilt 40 (Adh3 n)--the genotype that was usually low in ADH [35S]peptide synthesis--there were only two major bands, with no band in the normal ADH3 monomer position (Fig. 4A, track 4). The Adh3 n allele thus apparently lacks a protein product recognized by the antiserum. In Talpiyyot 4 (Adh3 I) there was a slight increase in the pI of the ADH3 monomer. Since this genotype also had an extra fast-migrating band in SDS-PAGE (Fig. 3), it is likely that the product of the ADh3 I allele differs substantially from that of the normal allele. The behavior of the ADH3 I monomer would be explained if a short section of peptide chain had been deleted, with the deletion including fewer basic than acidic residues. Were such a deletion to affect substrate or cofactor binding, but not dimerization, the apparent lack of active ADH3 I homodimer activity, and weak heterodimer activity, would also be explained. In Ilam 16 (Adh2 P), there was no measurable alteration in the position of the ADH2 monomer, even though this genotype has an ADH2 homodimer in a slower position than normal. That the Adh2 P electrophoretic mobility variant is silent in urea-IEF is hard to explain by invoking a single amino acid substitution (resulting in a single charge difference) in an otherwise unaltered peptide. Such a substitution is expected to alter the pI. No gross change in molecular weight is implicated, because there was no sign of abnormal SDS-PAGE bands with Adh2 P. One way to account for the absence of a pI shift in the ADH2 P monomer would be if a small primary-structure change, involving only uncharged residues, causes alterations in secondary or higherorder structures that bury a negatively charged group within the native homodimer or bring to its surface a positively charged side chain. A second notable point about the ADH2 P monomer is that its pI is unlike that of the ADH2 S monomer (Fig. 4A); this vindicates the distinction drawn between these alleles from the slight difference in their homodimer mobilities in starch gels (Fig. 2).

Minor Urea-IEF Bands. Three minor but quite discrete bands like those in cv. Himalaya and Betzes were clearly discernible in most of the variants tested. They are related to ADH since they were shown to cross-react strongly with highly-purified barley ADH 1 homodimer (Hanson and Jacobsen, 1984), as follows. A 35S-labeled extract of cv. Himalaya, containing all six ADH

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isozymes, was supplemented with 2-, 10-, and 20-fold excesses of unlabeled purified barley ADH1 .ADH1. Sufficient antiserum to titrate only the labeled isozymes was then added. The immunoprecipitates were analyzed by ureaIEF. At increasing antigen excess, the label was competed out of all three minor bands, just as it was out of the major (ADH1, ADH2, ADH3) bands. The pair of minor bands closest to the anode did not change position detectably in any of the variants examined, suggesting that they may be neither posttranslational modifications nor degradation products of any of the three known ADH monomers. However, the third minor band, found normally just beside the ADH3 monomer, was not in this position in Bar Giyyora 3 (Adhl S), but in a position nearer the cathode, paralleling the shift in the major ADH1 monomer band. Since this implies that both major and minor bands are products of the Adhl gene, we suggest the designation ADHI' for the minor band. Present evidence does not indicate whether ADHI' is a posttranslational modification of ADH 1, an in vivo degradation product of the ADH1 peptide, or an extraction artifact (or whether, indeed, ADHI' is a product rather than a precursor of ADH 1). Genetics of the Allozyme Variants at the Adh3 Locus

Segregation of the ADH3 locus and joint segregation of Adh3 with seven other allozyme loci were studied in the F2 generation of five separate crosses. Three crosses were hybridizations between a H. spontaneum line and the cultivar Clipper, and two were between H. spontaneum lines. The Adh3 alleles included were the common (M) allele, the slow (S) allele, and the very slow (V) allele. Four of the five crosses gave F 2 progeny in accord with Mendelian expectations for codominant inheritance, and the fifth departed (X~ = 6.0; 0.01 < P < 0.05), presumably by chance. Two of the five crosses included segregation of the M and S alleles (x~ = 1.2), and the remaining three included the M and V alleles (X~ = 2.07). Joint segregation of the Adh3 locus with the Adhl locus was tested in a cross between lines with Adh variant numbers 3 and 7 (Table I), which included the M and V alleles at the Adh3 locus and the S and M alleles at the Adhl locus. All nine two-locus genotypes in the F2 could be unambiguously determined on starch gels despite the partial overlap of some of the heterodimers. Figure 5 is a typical F 2 progeny array froth this cross, showing the parental types Adhl S, Adhl S; Adh3 M, Adh3 M and Adhl M, Adh 1M; Adh3 V, Adh3 V and an array of F2 phenotypes. The third, fifth, and tenth F2 samples are recombinant double homozygotes, illustrating the free recombination between Adhl and Adh3. As Adhl and Adh3 segregate independently, the F2 progeny of a cross between variant 4 and variant 8 (Table I, Fig. 2) tests whether the Adh3 n

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A

A

A

Fig. 5. ADH zymogram of 18 F2 progeny from the cross between H. spontaneum lines, variants 3 and 7. The first track is the female parent (Adhl S, Adh3 M), and the second is the male parent (Adhl M, Adh3 V). The final track is the standard ADH genotype (here cv. Himalaya). Arrowed tracks denote three double recombinant homozygotes.

allele codes for a product which comigrates with products of the Adh3 locus. In such progeny, the expected ratio of ADH phenotypes (in Fig. 2, type M:type 4:type 8) is 11:4:1 if Adh3 n does not code for a monomer similar to Adh2 M. In contrast, the ratio is 14:1:1 if Adh3 n codes for a comigrating product in starch. In a sample of 65, the observed ratio was 43:16:6, in favor of the hypothesis of a null allele. These results reinforce the conclusion that Adh3 n is a null allele, reached from the urea-IEF data. Table II summarizes the analysis of dihybrid segregations between the Adh3 locus and seven other allozyme loci. The chromosome location of six of these loci was previously known (for details, see Brown 1983). The seventh Table lI. Analysis of Joint Segregation Between the Adh3 Locus and Seven Other A11ozyme Loci in Barley a Joint segregation Chromosome

Locus

N

X~ for second locus

X~

?

SE

4

Adhl Ndhl Gpil Mdhl Dipl Dip2 Acol

82 101 42 70 82 124 42

1.32 9.12" 1.57 2.31 0.54 1.42 2.71

6.61 0.73 4.13 3.70 2.31 8.75 5.22

0.62 0.50 0.44 0.49 0.49 0.59 0.50

0.05 0.05 0.08 0.06 0.04 0.04 0.08

5 6

aN is the number of F2 individuals scored, ×2 is chi-square with i degrees of freedom, x~ tests the single-locus segregation, ×42tests the joint segregation, ? is the maximum-likelihood estimate of recombination, and SE is its standard error. * Rejection of the null hypothesis at the 0.05 level.

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locus, Dip2, is on chromosome 6, flanked by Acol (r = 0.23 + 0.05) and Dipl (r = 0.35 + 0.05). All these loci were expressed in Nz-treated embryos. In all of the joint segregations, Adh3 recombined freely with the other seven allozyme markers. The pairs of markers on barley chromosomes 4 and 5 were known themselves to be freely recombining and, hence, widely spaced on the chromosomes. Overall, the joint segregations of Adh3 with these markers indicate no departure from Mendelian expectations for independent loci. Although this negative probabilistic evidence does not exclude the location of Adh3 on these three chromosomes, it is in accord with the tentative location of Adh3 on chromosome 7 from the addition line studies (next section). Chromosome Location of the Adh Loci Using the Wheat-Barley Addition Lines

An attempt was made to determine the particular chromosomes of barley responsible for the several Adh isozymc loci, using the wheat-barley chromosome addition lines developed by Islam et al. (1981). The active ADH dimcrs produced by N2-induced alcuronc layers or embryos in each of the six chromosome addition lines (1, 2, 3, 4, 6, and 7) were compared with those of the parents Betzcs barley and Chinese Spring whcat after starch gel electrophoresis. The Chinese Spring wheat complement consisted of three prominent and at least four minor bands. The three prominent wheat bands had mobilities similar to those of the ADH1.ADH3, ADH1.ADH2, and ADH3.ADH3 dimers of thc barley common pattern in Fig. 1. The ADH spectra of the addition lines carrying barley chromosomes 1, 2, 3, and 6 were indistinguishablc from that of Chinese Spring wheat. As previously reported by Hart et al. (1980), the addition line with barley chromosome 4 had two extra bands in the positions expected when Adhl was on this chromosome and assuming that barley ADH1 monomers could dimerize with wheat monomers. However it was noted that even with prolonged overstaining or heavy loading of the gels, the addition line 4 spectrum did not display the conformers of barley ADH1 normally found in the Betzes parent. Addition line 7 appeared to differ from Chinese Spring in that the relative intensities of the wheat isozymes were altered toward the position of the ADH3.ADH3 homodimer. This would be consistent with the interpretation that barley chromosome 7 codes for a monomer (presumably barley ADH3) which can dimerize with the wheat monomers. Support for this interpretation came from experiments in which ADH peptides of Betzes barley, Chinese Spring wheat, and the six addition lines were labeled with [35S]methionine under nitrogen, immunoprecipitated, and separated by urea-IEF. The profile of wheat ADH monomers was complex,

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consisting of three major and at least five minor bands. By mixing barley and wheat immunoprecipitates in various proportions, it was found that the barley ADH3 monomer occupied a position exactly filling the gap between two major wheat bands and that the barley ADH1 monomer focused with the least prominent of the wheat bands, well separated from the main cluster. As expected, the urea-IEF profile in addition line 4 showed clear reinforcement of the band corresponding to the barley ADH1 band. Although a discrete additional band was not visible in addition line 7, the interband region corresponding to the barley ADH3 monomer position was denser than in the other lines.

DISCUSSION The Adh3 Locus. Our results substantiate Adh3 as a third locus in barley, demonstrate that it is unlinked to the other two barley Adh loci, and indicate that it is highly variable, with a total of five alleles found in a fairly small-scale survey of H. spontaneum lines. Two of those variant alleles are of particular interest: Adh3 n, which lacks an immunologically active protein product; and Adh3 I, whose product forms enzymically inactive homodimers. Both of these natural variants have striking parallels among the induced and natural variants of maize (Freeling and Birchler, 1981). Since an azide-induced Adhl null is already available in barley (Harberd and Edwards, 1982), an additional round of mutagenesis on the double null recombinant between this and the Adh3 null reported here should lead to a barley lacking Adh activity at all three loci. Such a triple null is a suitable genetic background for gene transfer experiments employing ADH as the selectable marker. Peptide Products of the Adh Genes. The presence of only three major urea-IEF bands under strongly inductive conditions argues against the existence of further major unrecognized active Adh genes. It is worthwhile to note with hindsight that the urea-IEF techniques would have pointed clearly to the existence of Adh3 before its eventual recognition from isozyme analysis. Since antiserum raised against maize ADHI homodimer recognizes ADH isozymes of many other species (Freeling and Birchler, 1981), combined 35S-labeling/immunoprecipitation/urea-IEF techniques could be widely applied to investigate the products of plant Adh genes. The correlated behavior of one of the minor urea-IEF bands (ADHI') with that of the ADH1 S monomer is consistent with, but does not prove, posttranslational modification of the Adhl gene product. Further indication of processing of the Adhl product comes from the absence of "conformer" isozyme bands flanking the ADH 1 homodimer in wheat-barley addition line 4. One interpretation of this would be that genes coding for the ADH1


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processing system are not on barley chromosome 4 and that wheat lacks a compatible processing system. The significance of the two other minor urea-IEF bands is not clear. Since both were present in the ADH3 n variant, neither of them can arise exclusively from modification of the Adh3 gene product. The lack of detectable variation in these minor bands in genotypes carrying variant alleles of Adhl and Adh2 suggests no association with either of these genes. The possibility that the two minor bands are the products of minor Adh genes cannot, therefore, be ruled out. However, an equally valid alternative is that they are modification products of Adhl and/or Adh2 in which the features that generate the pI differences among variant alleles have been obliterated.

The Relationship Between the Adh Loci of Barley and Those of Maize. Our present results, taken with those of Harberd and Edwards (1983), encourage the speculation that the barley Adh3 locus--rather than the Adh2 locus--is analogous to the maize Adh2 locus. The Adh3 locus of barley has in common with Adh2 of maize a lack of linkage to the principal Adh gene expressed in seed tissues (Adhl in both species) and the control of expression by anaerobiosis. Barley Adh2 has this second feature but is tightly linked to Adhl (Brown, 1980). However, maize Adh2 and barley Adh2 are weakly expressed in developing seeds, but barley Adh3 is not (Harberd and Edwards, 1983; Hanson and Jacobsen, 1984; Okimoto et al., 1980), so that the maize Adh2-barley Adh3 analogy is not complete. If barley Adh3 and maize Adh2 are evolutionary homologues as well as functional analogues, then an Adh system comprising two unlinked loci is an ancient condition in the grasses, preceding the divergence of the Maydeae and Hordeae. In this case, barley Adh2 would represent a gene duplication at" Adhl, accompanied or followed by some specialization of function such that Adh2 ceased to share with its ancestral gene the pattern of strong expression in seed tissues. It follows from this that the Adh3 null we report corresponds to a derived or degenerate condition, not a primitive one. In this connection, we note that pearl millet has exact equivalents of barley Adhl and Adh2 but evidently lacks a third (unlinked) Adh locus (Banuett-Bourrillon and Hague, 1979; Banuett-Bourrillon, 1982), so that this species closely resembles the barley Adh3 null. Perhaps a molecular comparison of the genomes of millet and barley Adh3 n variant would reveal similar relicts of a third Adh gene. Why is the Adh3 Locus so Polymorphie? The diversity of Adh3 alleles recovered from natural populations, including two (Adh3 n, Adh3 I) involving drastic loss of activity, is open to two interpretations. (a) Variability is tolerated at the Adh3 locus because it is essentially redundant inasmuch as Adh2 has similar induction properties and similar activity, and both genes are in any case subordinate in significance to Adhl, particularly in seed tissues.

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(b) Adh3, as the Adh gene strongly expressed only in fairly severe hypoxia, is a special case in which maintenance of populations under selective (frequently flooded or waterlogged) conditions leads to conservation of the common M allele, since under such conditions the gene confers a significant adaptive advantage. On the other hand, in environments in which flooding and waterlogging are very rare, the Adh3 gene would almost never be expressed from one generation to another and so could not be subjected to any selection pressure whatever. Th presence of the Adh3 M allele in all cultivated material examined and in the majority of H. spontaneum lines tested argues that the selection pressures of most natural and agronomic environments favor the retention of a functional Adh3 locus. This argument is strengthened by the fact that opportunities have existed through much of the evolution of barley as a crop for exchange with the wild progenitor, in which variant Adh3 alleles are certainly present. The inference that Adh3 has survival value under agronomic conditions is readily testable by using the Adh3 n allele in a backcrossing program with a commercial cultivar. Such a backcrossing effort is simplified by the fact that individual seeds can be analyzed nondestructively from their Adh genotype by inducing a portion of aleurone tissue, leaving the embryo to be grown on.

ACKNOWLEDGMENTS We wish to thank Dr. A. J. Pryor for the gift of antiserum against maize ADH and Dr. J. V. Jacobsen for advice and help with IEF analyses. We also wish to thank Rosemary Metcalf and Jack Munday for technical assistance.

REFERENCES Banuett-Bourrillon, F., and Hague, D. R. (1979). Genetic analysis of alcohol dehydrogenase isozymes in pearl millet (Pennisetum typhoides). Biochem. Genet. 17:537. Banuett-Bourrillon, F. (1982). Linkage of the alcohol dehydrogenase structural genes in pearl millett (Pennisetum typhoides). Biochem. Genet. 20:359. Bonner, W. M., and Laskey, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83. Brown, A. H. D. (1980). Genetic basis of alcohol dehydrogenase polymorphism in Hordeum spontaneum. J. Hered 70:127. Brown, A. H. D. (1983). Barley. In Tanksley, S. D., and Orton, T. J. (eds.), Isozymes in Plant Genetics and Breeding, Elsevier, Amsterdam/New York, Part B. pp. 57-77. Brown, A. H. D., and Jacobsen, J. V. (1982). Genetic basis and natural variation of a-amylase isozymes in barley. Genet. Res. Camb. 40:315. Brown, A. H. D., and Munday, J. (1982). Population-genetic structure and optimal sampling of land races of barley from Iran. Genetica 58:85. Brown, A. H. D., Nevo, E., Zohary, D., and Dagan, O. (1978). Genetic variation in natural populations of wild barley (Hordeum spontaneum). Genetica 49:97.


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Clegg, M. T., Brown, A. H. D., and Whitfeld, P. R. (1983). Chloroplast DNA diversity of wild and cultivated barley. Genet. Res. (in press). Doll, H., and Brown, A. H. D. (1979). Hordein variation in wild (Hordeum spontaneum) and cultivated (H. vulgare) barley. Can. J. Genet. Cytol. 21:391. Ellstrand, N. C., Lee, J. M., and Foster, K. W. (1983). Alcohol dehydrogenase isozymes in grain sorghum (Sorghum bicolor): Evidence of a gene duplication. Biochem. Genet. 21:147. Freeling, M., and Birchler, J. A. (1981). Mutants and variants of the alcohol dehydrogenase-1 gene in maize. In Setlow, J. K., and Hollaender, A. (eds.), Genetic Engineering, Principles and Methods, Plenum Press, New York, Vol. 3, pp. 223-264. Gerlach, W. L., Pryor, A. J., Dennis, E. S., Ferl, R. J., Sachs, M. M., and Peacock, W. J. (1982). eDNA cloning and induction of the alcohol dehydrogenase gene (Adhl) of maize. Proc. Natl. Acad. Sci. USA 79:2981. Hanson, A. D., and Jacobsen, J. V. (1984). Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers. Plant Physiol. (in press). Harberd, N. P., and Edwards, K. J. R. (1982). A mutational analysis of the alcohol dehydrogenase system in barley. Heredity 48:187. Harberd, N. P., and Edwards, K. J. R. (1983). Further studies on the alcohol dehydrogenases in barley: Evidence for a third alcohol dehydrogenase locus and data on the effect of an alcohol dehydrogenase-1 mutation in homozygous and in heterozygous condition. Genet. Res. Camb. 41:109. Hart, G. E., Islam, A. K. M. R., and Shepherd, K. W. (1980). Use of isozymes as chromosome markers in the isolation and characterization of wheat-barley chromosome addition lines. Genet. Res. Camb. 36:311. Islam, A. K. M. R., Shepherd, K. W., and Sparrow, D. H. B. (1981). Isolation and characterization of euplasmic wheat-barley chromosome addition lines. Heredity 46:161. Mayne, R. G. (1982). Ph.D. thesis, University of London, London. Nevo, E., Zohary, D., Brown, A. H. D., and Haber, M. (1979). Genetic diversity and environmental associations of wild barley, Hordeum spontaneum, in Israel. Evolution 33:815. Okimoto, R., Sachs, M. M., Porter, E. K., and Freeling, M. (1980). Patterns of polypeptide synthesis in various maize organs under anaerobiosis. Planta 150:89. Peacock, W. J., Dennis, E. S., Gerlach, W. L., Llewellyn, D., Ltirz, H., Pryor, A. J., Sachs, M. M., Schwartz, D., and Sutton, W. D. (1983). Gene transfer in maize: Controlling elements and the alcohol dehydrogenase genes. Miami Winter Symposium (in press). Pryor, A. J., and Huppatz, J. L. (1983). Purification of maize alcohol dehydrogenase and competitive inhibition by pyrazoles. J. Biochem. Intl. 4:431. Sachs, M. M., Freeling, M., and Okimoto, R. (1980). The anaerobic proteins of maize. Cell 20:761. Spencer, D., Higgins, T. J. V., Button, S. C., and Davey, R. A. (1980). Pulse-labeling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol. 66:510. Tanksley, S. D., and Jones, R. A. (1981). Effect of Oz stress on tomato alcohol dehydrogenase activity: Description of a second ADH coding gene. Biochem. Genet. 19:397. Turner, G. L., and Gibson, A. H. (1980). Measurement of nitrogen fixation by indirect means. In Bergersen, F. J. (ed.), Methods for Evaluating Biological Nitrogen Fixation, John Wiley, New York, pp. 111-138. Wignarajah, K., Greenway, H., and John, C. D. (1976). Effect of waterlogging on growth and activity of alcohol dehydrogenase in barley and rice. New Phytol. 77:585.