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).     

Gene Families Encoding 11S Globulin and 2S Albumin Isoforms of Jelly Fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS–PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

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     

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.     

Sequence Peculiarity of Gnetalean Legumin-Like Seed Storage Proteins

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia—morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms. Received: 5 June 1997 / Accepted: 31 March 1998     

An unstable domain in the vicilin genes of higher plants

The genes for the vicilin seed storage proteins of higher plants code for proteins that fall roughly into two size classes. All the vicilin genes descended from a single ancestral gene, and there is considerable homology among the amino acid sequences of all vicilin proteins except in an NH2-terminal domain that is present only in the vicilins of the large size class. This domain, composed largely of charged residues, has diverged greatly. Its nucleotide composition in all species is high in G + A (sense strand) and the domain contains long polypurine.polypyrimidine tracts. Slippage in replication due to non-B DNA structures (for example, H-DNA) resulting from these tracts is considered to be the cause of the rapid divergence of this domain.     

Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification of PP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.     

Gene families encoding 11S globulin and 2S albumin isoforms of jelly fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS-PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes

We have identified cDNA clones coding for the major sulphur-rich and sulphur-poor groups of barley storage proteins (the B- and C-hordeins, respectively). Hybridization studies have revealed unexpected homologies between B- and C-hordein mRNAs. Using a deletion mutant (Risø 56), we have mapped some C-hordein-related sequences within, or closely associated with, B-hordein genes at the Hor 2 locus. Nucleotide sequencing has shown that the primary structure of B-hordein polypeptides can be divided into at least two domains: domain 1 (repetitive, proline-rich, sulphur-poor), which is homologous to C-hordein sequences, and domain 2 (non-repetitive, proline-poor, sulphur-rich), which makes up two-thirds of the polypeptide and is partially homologous to a 2S globulin storage protein found in dicotyledons. The coding sequences that are homologous in B- and C-hordein mRNAs have an asymmetric base composition (>80% C-A) and are largely composed of a degenerate tandem repeat based on a 24 nucleotide consensus that encodes Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. We discuss the evolutionary implications of the domain structure of the B-hordeins and the unusual relationship between the two groups of barley storage proteins.     

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.      

Characterization of ZmCOLD1, novel GPCR-Type G Protein genes involved in cold stress from Zea mays L. and the evolution analysis with those from other species

Maize is one of the most vital staple crops worldwide. G proteins modulate plentiful signaling pathways, and G protein-coupled receptor-type G proteins (GPCRs) are highly conserved membrane proteins in plants. However, researches on maize G proteins and GPCRs are scarce. In this study, we identified three novel GPCR-Type G Protein (GTG) genes from chromosome 10 (Chr 10) in maize, designated as ZmCOLD1-10A, ZmCOLD1-10B and ZmCOLD1-10C. Their amino acid sequences had high similarity to TaCOLD1 from wheat and OsCOLD1 from rice. They contained the basic characteristics of GTG/COLD1 proteins, including GPCR-like topology, the conserved hydrophilic loop (HL) domain, DUF3735 (domain of unknown function 3735) domain, GTPase-activating domain, and ATP/GTP-binding domain. Subcellular localization analyses of ZmCOLD1 proteins suggested that ZmCOLD1 proteins localized on plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, amino acid sequence alignment verified the conservation of the key 187th amino acid T in maize and other wild maize-relative species. Evolutionary relationship among plants GTG/COLD1 proteins family displayed strong group-specificity. Expression analysis indicated that ZmCOLD1-10A was cold-induced and inhibited by light. Together, these results suggested that ZmCOLD1 genes had potential value to improve cold tolerance and to contribute crops growth and molecular breeding.     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

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.