The partial amino-acid sequence of the wheat-germ Ec protein has been published previously. Computer analysis failed to show a significant relation between the amino-acid sequence of Ec and the sequences of other proteins stored in the data base of the National Biomedical Research Foundation, Washington, DC. Visual inspection and comparison of the amino-acid sequence of Ec with the sequences of other proteins rich in half-cystine have revealed that the amino-acid sequence of Ec bears a compelling relation to sequences reported for animal metallothioneins. Experimental evidence is presented which supports the view that Ec is a zinc-containing metallothionein, the first metallothionein from higher plants for which an almost complete amino-acid sequence has been determined.

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P-labeled oligonucleotide probes that encoded parts of the partial amino-acid sequence for the Zn-containing E, protein. Each DNA insert in 11 positives from a screen of lo5 plaques encoded a 5' untranslated and a 3' untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the E, protein. The three different mRNA sequences encoded in the cDNA probably correspond to single-copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P-labeled cDNA and gave a single positive in a screen of 5 x lo5 plaques. A 3.1-kb genomic fragment (gf-3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf-3.1 sequence encodes an intronless mRNA for the E, protein and contains appreciable amounts of 5' and 3' flanking sequences. In addition to a putative TATA box, two inverted-repeat sequences and one direct-repeat sequence, the 5' flank in gf-3.1 contains a sequence similar to the abscisic-acid-responsive element in other higher-plant genes but does not contain sequences similar to the metal-responsive elements in animal metallothionein genes.Consistent with these findings, RNA blotting shows that accumulation of E, mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat E, metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.In 1957, a Cd-containing protein was isolated from horse kidney [I]. The Cd-containing protein was called a metallothionein by Kagi and Vallee [2,3], and over the next 30 years, metallothioneins were isolated from invertebrates, moulds, fungi and prokaryotes [4], but not from higher plants. Characteristically, the metallothioneins ( M , 5000 -10000) contain Cys-Xaa-Cys sequences. As recently as 1987, some three decades after the isolation of the first animal metallothionein, Robinson and his co-workers [5] stated that 'the majority of the cellular Cd ( > 80%) in cadmium-resistant Datum innoxia is bound to a small, metal-induced peptide which is not metallothionein . . . it is not known whether (metallothioneins occur) in higher plants'.Latcr in the same year, this uncertainty was resolved when we reported that the E, (early cysteine-labeled) protein, long known to be encoded by the mRNA conserved in mature wheat embryos [6] and to contain the characteristic Cys-XaaCys sequences found in metallothioneins 171, is a Zn-containCorrespondence to B. G. Lane, Biochemistry Department, UniverFux: + 1 416 978 8548.Ahhveviutions. E,., early cysteine-labclcd protein [6]; ABA, abscisic acid.Note. The novel nuclcotide scquence data published here have bee...

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has bccn named pseudogermin. Pseudogermin accumulates, maximally, between 20 -25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cellwall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potcntials during cmbryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDSjPAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsinresistant protein, but pseudogermin has unprecedented disulphide-independent thermostability propertics that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump : gastric ATPase.If mature embryos ( z 5% water) from Ungerminated wheat grains [I] Ahhrrviation. NaCI/P,, phosphate-buffered salinc.5 -10-h postimbibition, coincident with the onset of a secondary uptake of water, there is nascent synthesis of mRNA for a protein [3 -61 that we have called germin [7, 81 (for review see [9]).Accumulation of germin and its mRNA are conspicuously associatcd with the secondary water uptake that raises the water content of the embryos rrom about 60% at 5-h postimbibition, to about 85% by 24-h postimbibition. Structures for germin (monomer M , z 25000; ...

The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early inhibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0--1 h) of postimbibition development, and easily detected during "lag" phase (1--5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition. The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.

(1) The electrophoretic band patterns (sodium dodecyl sulfate (SDS) – polyacrylamide) for either isotopically labeled or dye-stained proteins in a wash fraction obtained by rinsing germinated wheat embryos with any of a variety of buffers are different from those observed for proteins in either the soluble or pellet fractions of embryo homogenates. Interestingly, the wash fraction is enriched with respect to germin, a marker protein for the onset of growth in germinating wheat embryos.(2) There is selective accumulation of a novel form of germin in the wash fraction. This novel form migrates just ahead of the more usual form of germin during electrophoresis in SDS–polyacrylamide gel, being about 5% smaller, assuming a linear relation between gel mobility and log (relative mass), but it yields the same methionine-labeled peptides when denatured and digested by Staphylococcus aureus protease.(3) Quasi-quantitative analyses of dye-stained proteins suggest that, in the case of embyros cultured in 5% sucrose, which show greatest growth, 50% or more of the total germin is present, in the novel form, in the wash fraction, which contains less than 5% of the total proteins. By way of contrast, germin is barely detected in the wash fraction of embryos excised from germinated grains, unless the excised embryos are incubated in water or 5% sucrose for an additional 24 h.(4) Allied studies of (possibly epiphytic) bacteria, found in wash fractions prepared following germination of preisolated embryos or grains, are described.(5) The significance of the present findings for our continuing efforts to define a phyiological role for germin are discussed at some length.