Characterization of 10 KDA prolamin genes in Phyllostachys aurea (Bambusoideae, Poaceae)

Prolamin is the dominant class of seed storage protein in grasses (Poaceae). Information on the 10 kDa multigene family coding for prolamins characteristic of the bambusoid-oryzoid grasses is limited. Two genes encoding 10 kDa prolamin were cloned and sequenced in the bambusoid species Phyllostachys aurea to assess the sequence diversity of this gene family in the oryzoid-bambusoid grasses. The genes, ~417 bp in length, were 96% similar at the DNA sequence level, differing in 12 base substitutions dispersed throughout the sequence. Eight of these mutations were nonsynonymous, leading to amino acid substitutions in the coding region, and one was nonsense, producing an amber stop codon. One gene had an open reading frame (ORF) of 139 amino acids, while the other gene had a shorter ORF (106 amino acids) due to the presence of a stop codon in the coding region and, thus, represents a pseudogene. Deduced proteins showed amino acid composition similar to that of rice. The study underscores the overall conserved nature of this multigene family and reflects considerable sequence divergence at the DNA and amino acid levels between the Oryza and the Phyllostachys genes. The systematic implication of the data is discussed in light of the inconsistent placement of Oryza in the Bambusoideae or Oryzoideae.     

Dynamic gene copy number variation in collinear regions of grass genomes

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.     

Cloning and structural analysis of an Indian little millet (Panicum sumatrense) zein-like storage protein: Implications for molecular assembly

Zeins are prolamin storage proteins that accumulate in kernel endosperm of several cereals. For cloning of genes coding for zein-like proteins that accumulate in enhanced quantities in the filling stages of little millet (Panicum sumatrense Roth.) developing grains, RT-PCR was performed using specific primers. A 750-bp cDNA was directly sequenced and in silico analysis showed high identity degree to alpha-prolamins. This family is composed of zeins from Zea mays, coixins from Coix lachryma-jobi, and alpha-kafirins from Sorghum bicolor. The putative conserved domain of zein-like proteins was identified by primary structure comparisons. Furthermore, threading analyses indicated that the millet zein-like protein forms an anti-parallel alpha-helical hairpin with two opposite surfaces: one hydrophobic and the other hydrophilic that probably could be involved in protein storage assembly. Knowledge about zein-like alpha-prolamins in little millet will lead to cloning and transfer of this gene to other major food crops, such as cereals and legumes, with inferior nutritional quality for monogastric animals.     

N-terminal sequences of oat avenins compared to other cereal prolamins

Like the alcohol-soluble seed storage proteins (also called prolamins) of other cereals, avenins, the oat prolamins, are a series of polymorphic molecules belonging to a multigenic family stored within the protein bodies of the starchy endosperm. Nevertheless, they exhibit some pecularities: among the seed storage proteins, their proportion is low compared to prolamins from other cereal species; their net charge is higher; the amount of Gln + Pro only reaches 49 mol%; they are less polymorphic. We have isolated and purified several avenins and sequenced their N-terminal end. The microheterogeneity and the pecularity of avenins are revealed by the comparison of the N-terminal sequences. Like other prolamins, they exhibit tandem repeats; these repetitive peptides are slightly different from those of other prolamins of the Festucoideae, and the repetition begins earlier in the sequence. As for prolamins from other species, their predicted secondary structure reveals successive beta-turns which might be arranged in a pseudo-helix structure.     

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.     

Chromosomal localization of zein genes by in situ hybridization in Zea mays

Summary In order to localize the genes coding for zein, the major storage protein of maize endosperm, zein ¹²⁵I-mRNA and ³H-cDNA labelled at high specific activity were used for in situ hybridization on heterozygous interchanges and paracentric inversions of the KYS strain of Zea mays. The analysis of the diplotene-metaphase I microsporocytes indicated the presence of zein structural genes on the long arm of chromosomes 4 and 5, the short arm of chromosome 7 and the distal segment of the long arm of chromosome 10. The two hybridization sites on chromosomes 7 and 10 are found near opaque-2 and opaque-7 loci which are known to regulate zein synthesis. The present data are discussed in relation to results obtained by other authors using genetical mapping of zein genes.     

Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential

Foxtail millet (Setaria italica), a member of the Poaceae grass family, is an important food and fodder crop in arid regions and has potential for use as a C(4) biofuel. It is a model system for other biofuel grasses, including switchgrass and pearl millet. We produced a draft genome (～423 Mb) anchored onto nine chromosomes and annotated 38,801 genes. Key chromosome reshuffling events were detected through collinearity identification between foxtail millet, rice and sorghum including two reshuffling events fusing rice chromosomes 7 and 9, 3 and 10 to foxtail millet chromosomes 2 and 9, respectively, that occurred after the divergence of foxtail millet and rice, and a single reshuffling event fusing rice chromosome 5 and 12 to foxtail millet chromosome 3 that occurred after the divergence of millet and sorghum. Rearrangements in the C(4) photosynthesis pathway were also identified.     

Simple sequence repeat (SSR) analysis in relation to calcium transport and signaling genes reveals transferability among grasses and a conserved behavior within finger millet genotypes

Being an excellent source of calcium, finger millet crop has nutraceutical importance. Mineral accumulation, being a polygenic trait, becomes essential to target potential candidate genes directly or indirectly involved in the regulation of calcium transport and signaling in cereals and might have influence on grain calcium accumulation. In view of this, genic microsatellite markers were developed from the coding and non-coding sequences of calcium signaling and transport genes viz. calcium transporters (channels; ATPases and antiporters), calcium-binding proteins and calcium-regulated protein kinases available in rice and sorghum. In total, 146 genic "simple sequence repeat" (SSR) primers were designed and evaluated for cross-transferability across a panel of nine grass species including finger millet. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2 %) followed by rice (63.4 %) with an overall average of 68.3 % which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The transfer rate of SSR markers was also correlated with the phylogenetic relationship (or genetic relatedness) of the species. Primers with successful amplification in finger millet were further used to screen for polymorphism across a set of high and low calcium containing genotypes. The results reveal a conserved behavior across the finger millet genotypes indicating that the mineral transport and the storage machinery largely remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution. Single nucleotide polymorphism and differential expression patterns of candidate genes, therefore, might be a plausible reason to explain variations in grain calcium contents among finger millet genotypes.     

N-terminal amino acid sequences of prolamins encoded by the alleles at the Pro1 and Pro2 loci in foxtail millet, Setaria italica (L.) P. Beauv

N-terminal amino acid sequences of six prolamins encoded by seven alleles at two loci, Pro1 and Pro2, of foxtail millet (Setaria italica (L.) P. Beauv.) were analyzed and compared with other prolamins of subfamily Panicoideae. Based on the N-terminal amino acid sequences, band 3 (the prolamin purified from band 3) which is controlled by an allele at the Pro1 locus and bands 1, 2, 4, 5 and 6 which are controlled by alleles at the Pro2 locus could be classified into three groups. Band 3 was found to be homologous to the prolamin of pearl millet (Pennisetum americanum) and is designated as the "pennisetin-like prolamin". Bands 2 and 4, and bands 1, 5 and 6 were subdivided into "x-type prolamin" and "y-type prolamin". Both of the x-type and y-type prolamins showed homology with prolamin of Echinochloa crus-galli and alpha-zein-like prolamins of maize, sorghum and Job's tears. Therefore, these prolamins were designated as "alpha-zein-like prolamin". These results suggest that alleles at the Pro1 locus and those at the Pro2 locus have not arisen from an identical ancestral gene, and that the Pro2 locus comprise two tightly linked genes, which encode similar prolamins. Hypotheses on the diversification of alleles at the Pro2 locus are discussed based on the N-terminal amino acid sequences of the respective bands, combinations of bands controlled by the alleles, and frequencies of the alleles.     

谷子bZIP转录因子的全基因组鉴定及其在干旱和盐胁迫下的表达分析

bZIP蛋白是植物转录因子中最大和最保守的一类转录因子, 参与调控植物生长发育等多种生命活动。谷子(Setaria italica)是一种重要的C4杂粮作物, 其bZIP基因家族与功能报道较少。利用生物信息学工具, 从谷子全基因组中鉴定出73个SibZIP转录因子, 划分为A、B、C、D、E、G、H、I和X等亚家族。与已测序的禾谷类作物相比, 谷子SibZIP基因家族在进化中发生缩减。在谷子SibZIP蛋白中检测到25种不同的保守氨基酸基序。RNA-seq和定量PCR检测结果表明, 在干旱和盐胁迫条件下, 多数SibZIPs基因不同程度地被诱导表达, 预示着部分SibZIP成员在谷子干旱和盐胁迫响应中起重要作用。共表达关联性分析进一步揭示19个谷子SibZIP转录因子可通过与蛋白激酶或NPR1相关调节蛋白等互作介导谷子胁迫响应。研究结果为全面解析谷子SibZIPs基因结构与生物学功能、抗旱分子机制以及分子育种提供了新信息。     

Nutritional control of storage protein synthesis in developing grain of wheat and barley

The availability of nitrogen and sulphur have major effects on thesynthesis of prolamin storage proteins in developing endosperms of wheat andbarley. A high level of available nitrogen results in an increased proportionofprolamin storage proteins. However, changes in the storage protein compositionoccur if additional sulphur is not also provided, with increased proportions ofsulphur-poor prolamins and HMW prolamins and decreased proportions ofsulphur-rich prolamins. In the case of wheat, this results in increasedresistance and decreased extensibility of dough for bread-making, withconsequences for the end-use quality. Further limitation in the availability ofsulphur results in decreased total prolamin synthesis and an increase in freeaspartic acid/asparagine in the grain. Recent studies of the structure andregulation of prolamin genes indicate the presence of regulatory elements inthepromoter regions of genes for S-rich and S-poor prolamins, which could respondto nitrogen levels, although the sensing and signal transduction mechanisms arenot understood. Such elements have not so far been identified in genes for HMWprolamins. Similarly, there is no information on how the availability ofsulphurcould modulate prolamin gene expression.     

Slipped-strand mispairing in a plastid gene: rpoC2 in grasses (Poaceae)

An exception to the generally conservative nature of plastid gene evolution is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous work by others has shown that maize and rice have an insertion in the coding region of rpoC2, relative to spinach and tobacco. To assess the distribution of this extra coding sequence, we surveyed a broad phylogenetic sample comprising 55 species from 17 angiosperm families by using Southern hybridization. The extra coding sequence is restricted to the grasses (Poaceae). DNA sequence analysis of 11 species from all five subfamilies within the grass family demonstrates that the extra sequence in the coding region of rpoC2 is a repetitive array that exhibits more than a twofold increase in nucleotide substitution, as well as a large number of insertion/deletion events, relative to the adjacent flanking sequences. The structure of the array suggests that slipped-strand mispairing causes the repeated motifs and adds to the mechanisms through which the coding sequence of plastid genes are known to evolve. Phylogenetic analyses based on the sequence data from grass species support several relationships previously suggested by morphological work, but they are ambiguous about broad relationships within the family.      

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis