Molecular characterization of oat seed globulins

We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).     

Placement of loci for avenins and resistance to Puccinia coronata to a common linkage group in Avena strigosa.

Alcohol-soluble seed storage proteins of oat (avenins) were extracted from two diploid accessions representing the A genome and separated by high-resolution acid polyacrylamide gel electrophoresis. Polymorphisms were detected for three clearly resolved protein bands. Linkage analysis of 88 F2:3 families mapped the three bands to a single locus. Integration of avenin segregation data with an RFLP linkage map constructed from the same population, mapped the avenin locus to a linkage group containing a locus conferring resistance to nine isolates of Puccinia coronata. Linkage between genes encoding alcohol-soluble seed proteins and genes for resistance to Puccinia species was also observed for the homoeologous group 1 chromosomes of barley (1H), rye (1R), wheat (1A, 1B, 1D), and chromosomes 4 and 10 of maize.     

Globulins are the main seed storage proteins in Brachypodium distachyon

Brachypodium distachyon is being developed as a model system to study temperate cereals and forage grasses. We have begun to investigate its utility to understand seed development and grain filling by identifying the major seed storage proteins in a diploid accession Bd21. With the use of ID SDS-PAGE and mass spectrometry we detected seven major storage protein bands, six of which were identified as globulins. A subset of the major seed proteins isolated from three hexaploid accessions, Bd4, Bd14 and Bd17 were also identified as globulins. Several Brachypodium cDNAs clones encoding globulin were completely sequenced. Two types of globulin genes were identified, Bd.glo1 and Bd.glo2, which are similar to maize 7S and oat 12S globulins, respectively. The derived polypeptide sequences of the globulins contain a typical signal peptide sequence in their polypeptide N-termini and two cupin domains. Bd.glo1 is encoded by a single copy gene, whereas, Bd.glo2 belongs to a gene family.     

Isolation and characterization of cDNAs encoding oat 12S globulin mRNAs

Summary A cDNA library was made from poly(A⁺) RNA isolated from developing oat seeds, and oat globulin cDNA clones were identified by hybridization with synthetic oligonucleotides. Globulin clones were characterized by restriction enzyme mapping and cross-hybridization analysis. Based on these comparisons, four classes of globulin clones were distinguished. These clones hybridized to multiple DNA fragments in restriction enzyme digests of oat genomic DNA, indicating that the genes exist in a multigene family. The nucleotide sequence of one of the globulin cDNA clones was determined. The amino acid sequence derived from the DNA sequence verified its identity as an oat globulin and confirmed that the protein is synthesized as a precursor similar to legume 11S storage globulins. The basic polypeptide encoded at the 3 end of the mRNA was found to be homologous to the basic polypeptides of other 11S seed globulins.Abbreviations ds double stranded - kb kilobase Author to whom correspondence should be addressed. Journal paper number 10460 of the Purdue Agricultural Experimental Station.     

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.     

Sequence and characterization of 6 Lea proteins and their genes from cotton

Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins.The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.     

Evolution of seed storage protein genes: Legumin genes of Ginkgo biloba

Legumin-like seed storage proteins have been intensively studied in crop plants. However, little is known about the molecular evolution of these proteins and their genes and it was assumed that they originated from an ancestral gene that already existed at the beginning of angiosperm evolution. We have evidence for the ubiquitous occurrence of homologous proteins in gymnosperms as well. We have characterized the major seed storage globulin from Ginkgo biloba by amino acid sequencing, which reveals clear homology to legumin-like proteins from angiosperms. The Ginkgo legumin is encoded by a gene family; we describe two of its members. The promoter regions contain sequence motifs which are known to function as regulatory elements involved in seed-specific expression of angiosperm legumins, although the tissues concerned are different in gymnosperms and angiosperms. The Ginkgo legumin gene structure is divergent from that of angiosperms and suggests that the evolution of legumin genes implicated loss of introns. From our data and from functional approaches recently described it becomes obvious that the posttranslational processing site of legumin precursors is less conserved than hitherto assumed. Finally, we present a phylogenetic analysis of legumin encoding sequences and discuss their utility as molecular markers for the reconstruction of seed plant evolution.Correspondence to: K.-P. Häger     

Identification of legumin-like proteins in wheat

We have obtained several amino acid sequences from purified polypeptides of a wheat endosperm storage globulin previously described as triplet protein. The amino acid sequence data supported by immunochemical analysis using anti-oat 12S globulin antibodies, provide definitive evidence that the triplet protein is homologous to pea legumin and related seed storage proteins of oats, rice and several dicotyledonous species. Thus, it is now proposed that the triplet protein of wheat be renamed triticin. The oat globulin antibodies also cross-reacted strongly with the high-molecular-weight (HMW) glutenin subunits which have been implicated in bread-making quality.     

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

Histochemical Studies of Scutellum During Germination of Oat Seed by Light Microscopy and Immunofluorescent Localization of Oat Globulin

The protein, phytin, lipid and starch contents of the scutellum of oats showed marked changes during the first three days after seed germination. Protein and phytin disappeared almost completely during the first two days after seed germination. The degradation at lipid was much slower. In the scutellum of the ungerminated seed very few starch granules were seen. At days-1 and -2 after germination the starch contents increased, but at day-3 the starch contents decreased. Immunofluorescent localization of oat globulin indicated that the oat globulin was sequestered mostly, if not exclusively, in the protein bodies. The degradation of the oat globulin inside the protein bodies was very rapid. At day-3 after seed germination it disappeared almost completely. When excited by the ultraviolet light the walls of both the epithelial and parenchyma cells of the scutellum autofluoresced intensely. As germination progressed further, the autofluorescence in the walls of the epithelial cells gradually faded away, whereas the autofluorescence in the walls of the parenchyma cells did not.     

Structural relationship between barley (Hordeum vulgare) trypsin inhibitor and castor-bean (Ricinus communis) storage protein.

A significant sequence homology was found between barley (Hordeum vulgare) trypsin inhibitor and castor-bean (Ricinus communis) seed glutamine-rich storage protein. This appears to suggest a divergent evolution of the two different classes of seed proteins and to support a view that plant proteinase inhibitors may also act as storage proteins.