Barley embryo globulin 1 gene, Beg1: Characterization of cDNA, chromosome mapping and regulation of expression

We report identification of a 2189 by cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar proteins are present in the barley embryo. Accumulation of Beg1 mRNA was noted beginning 15–20 days post-anthesis in both the aleurone layer and embryo of the developing barley grain but not in the starchy endosperm. A high level of Beg1 mRNA is also present in the mature imbibed aleurones, which can be repressed by treatment with gibberellic acid. This repressive effect of gibberellin on the levels of Beg1 mRNA is confirmed in the gibberellin response-constitutive mutant, slender, whose aleurone layers do not accumulate Beg1 mRNA even in the absence of applied gibberellic acid. The deduced primary translation product of the Beg1 mRNA is a 637 amino acid (72 kDa) protein with homology to maize embryo globulin 1 (GLB1) and a partial sequence of a wheat 7S globulin. The internal amino acid sequence of BEG1 closely matches the N-terminal sequence of isolated barley aleurone globulin. Seven imperfect tandem repeats of 16 amino acids each are present near the N-terminus of BEG1, which conform to the consensus HGEGEREEEXGRGRGR, and contribute to the observed unusual amino acid composition of this protein. A second, distinct barley globulin gene, Beg2, which is homologous to maize Glb2, was detected by Northern and Southern analysis. Beg-2 and Beg1 are regulated differently which may indicate variation in storage or utilization properties among the barley globulins.     

Barley embryo globulin 1 gene,Beg1: Characterization of cDNA,chromosome mapping and regulation of expression

We report identification of a 2189 by cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar proteins are present in the barley embryo. Accumulation of Beg1 mRNA was noted beginning 15–20 days post-anthesis in both the aleurone layer and embryo of the developing barley grain but not in the starchy endosperm. A high level of Beg1 mRNA is also present in the mature imbibed aleurones, which can be repressed by treatment with gibberellic acid. This repressive effect of gibberellin on the levels of Beg1 mRNA is confirmed in the gibberellin response-constitutive mutant, slender, whose aleurone layers do not accumulate Beg1 mRNA even in the absence of applied gibberellic acid. The deduced primary translation product of the Beg1 mRNA is a 637 amino acid (72 kDa) protein with homology to maize embryo globulin 1 (GLB1) and a partial sequence of a wheat 7S globulin. The internal amino acid sequence of BEG1 closely matches the N-terminal sequence of isolated barley aleurone globulin. Seven imperfect tandem repeats of 16 amino acids each are present near the N-terminus of BEG1, which conform to the consensus HGEGEREEEXGRGRGR, and contribute to the observed unusual amino acid composition of this protein. A second, distinct barley globulin gene, Beg2, which is homologous to maize Glb2, was detected by Northern and Southern analysis. Beg-2 and Beg1 are regulated differently which may indicate variation in storage or utilization properties among the barley globulins.     

A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions

Legumes, and a very few non-legume plant species, are known to possess functioning haemoglobin genes. We describe here the characterization of a haemoglobin cDNA isolated from barley. The deduced amino acid sequence shows 71% amino acid identity with a non-legume haemoglobin gene, a further 16% of the residues being conservative replacements. The barley cDNA also hybridizes to genomic sequences in rye, maize and wheat. The demonstration of a gene from a monocotyledon with close sequence homology to the known non-legume plant haemoglobins fills a major gap in the known distribution of haemoglobin genes in the plant kingdom. The expression of the gene is induced in isolated barley aleurone layers exposed to anaerobic conditions, and the roots of flooding-stressed barley plants. The expression of the RNA under anoxic conditions is similar to that of a known anaerobic response gene, alcohol dehydrogenase. Our results suggest that the increased expression of haemoglobin RNA is an integral part of the normal anaerobic response in barley. The findings are discussed in the light of current theories of haemoglobin function and evolution.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Cloning and sequence analysis reveal structural variation among related zein genes in maize

We have isolated a gene encoding one of the 19,000 dalton zein proteins from a maize genomic library constructed in Charon 4A. This gene occurs on a 7.7 kb Eco RI fragment, and based on Southern hybridization analysis, represents one of several homologous sequences present in the maize genome. The nucleotide sequence of the gene predicts a protein composed of 235 amino acids, including a signal peptide of 21 amino acids. There are no intervening sequences in the gene. By comparing the nucleotide sequence of this gene with that of a homologous cDNA clone, we have identified a basis for microheterogeneity within the gene family. The 5′ nucleotide sequences of the genomic and cDNA clones are identical, but they differ in the center of the protein, where repeated amino acid sequences occur. A nucleotide sequence encoding a conserved peptide of 20 amino acids is repeated nine times in the center of both of these clones.     

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.     

Isolation and sequence analysis of a barley alpha-amylase cDNA clone

We have isolated a cDNA clone derived from poly(A+) RNA from barley aleurone cells stimulated with gibberellic acid. This cDNA clone contains one open reading frame coding for 438 amino acids. The cloned DNA hybridizes to a poly(A+) RNA species 1550 bases in size, the same size as the most abundant poly(A+) RNA molecules in stimulated cells. RNA complementary to this clone can be translated to make immunoprecipitable alpha-amylase in the wheat germ system and increases about 5-fold in quantity after gibberellic acid stimulation of aleurone cells. In contrast, hybridization experiments using a total cDNA probe demonstrate that the most abundant mRNA population, identical in size with our cloned sequence and presumably that for alpha-amylase, increases at least 17-fold after gibberellic acid stimulation. We therefore infer that there must be at least two populations of alpha-amylase mRNA molecules derived from separate structural genes differently influenced by gibberellic acid in aleurone cells.     

Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000

We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine-containing barley B-hordein.     

Primary structure and differential expression of beta-amylase in normal and mutant barleys

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Ris? mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.     

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.