Differential Gene Expression and Epiregulation of Alpha Zein Gene Copies in Maize Haplotypes

Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80–500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members

Regulation of gene expression during maize endosperm development

My dissertation consists of two chapters that have been formatted into two manuscripts. Both of them relate to endosperm-specific gene expression in maize by a multigene family. Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait and one of its factors is the composition and relative expression of storage protein genes, known as zeins in maize. They represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds were selected to analyze allelic and non-allelic variability among the α-zeins. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype specific manner. Once genes were copied, expression of donor genes was reduced relative to new copies. Epigenetic regulation appeared to have contributed to silencing older copies because some of them could be reactivated when endosperm was maintained as cultured cells. Bisulfite sequencing of the promoter region also showed distinct methylation patterns of separate gene clusters as well as different tissues, possibly reflecting position effects on regulatory mechanisms as a result of inserting copies at unlinked locations. Seed mutants with non-vitreous endosperm play an important role in the regulation of α-zein gene expression. Although a number of genes affecting endosperm vitreousness have already been isolated, it has been difficult to clone opaque7 (o7) – the last in the series of three high-lysine corn mutants – mainly because of its low penetrance in many genetic backgrounds. To overcome these difficulties, a combination of map-based cloning and transposon tagging was undertaken. An o7 candidate gene, based on map position, contained a 12-bp deletion in the second exon of an acyl-CoA synthetate (ACS)-like gene. A second allele was generated by transposon tagging, with a Dissociation (Ds) element inserting ~500 bp downstream of the deletion, in the same exon. Although zein synthesis appeared to be unaltered in o7-ref and protein accumulation changed only slightly, PBs exhibited striking membrane invaginations. A plausible model consistent with these observations is that the ACS enzyme plays a key role in membrane biogenesis and that altered PBs render the seed non-vitreous.Ph. D.Includes bibliographical referencesIncludes vitaby Mihai Miclau

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

<div><p>An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), <i>z1A2</i>-1 and <i>z1B4</i>, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the <i>z1A</i> and <i>z1B</i> gene clusters. The expression of <i>z1B</i> gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas <i>z1A</i> gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.</p></div

Analysis of a common spoken language in Staňkov

The aim of this dialectological bachelor's thesis was to found out if and which characterictics features are appearing in the everyday speech of native speakers in Staňkov. In a brief way it described the specifics and general distribution of the Southwest bohemia dialect. The practical part focused on the observation of local language in the context of three generations

Maize Cytolines Unmask Key Nuclear Genes That Are under the Control of Retrograde Signaling Pathways in Plants

The genomes of the two plant organelles encode for a relatively small number of proteins. Thus, nuclear genes encode the vast majority of their proteome. Organelle-to-nucleus communication takes place through retrograde signaling (RS) pathways. Signals relayed through RS pathways have an impact on nuclear gene expression but their target-genes remain elusive in a normal state of the cell (considering that only mutants and stress have been used so far). Here, we use maize cytolines as an alternative. The nucleus of a donor line was transferred into two other cytoplasmic environments through at least nine back-crosses, in a time-span of > 10 years. The transcriptomes of the resulting cytolines were sequenced and compared. There are 96 differentially regulated nuclear genes in two cytoplasm-donor lines when compared with their nucleus-donor. They are expressed throughout plant development, in various tissues and organs. One-third of the 96 proteins have a human homolog, stressing their potential role in mitochondrial RS. We also identified syntenic orthologous genes in four other grasses and homologous genes in Arabidopsis thaliana. These findings contribute to the paradigm we use to describe the RS in plants. The 96 nuclear genes identified here are not differentially regulated as a result of mutation, or any kind of stress. They are rather key players of the organelle-to-nucleus communication in a normal state of the cell

Methylation status of promoter regions of <i>z1A</i> genes.

<p>Panel (A) displays average DNA methylation, and (B) CG methylation in leaf, endosperm and tissue-cultured endosperm (TC) as graphic bars. The universal primers of <i>z1A</i> genes, described in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146416#pone.0146416.ref030" target="_blank">30</a>], were used for bisulfite PCR amplification. Ninety-six colonies were sequenced, and each sequence was matched to individual <i>z1A</i> genes based on sequence similarity. The average methylation levels were calculated for individual genes with all three contents (A) and CG (B). The color code for individual gene copies is displayed as an insert.</p

Methylation status of promoter regions of <i>z1B</i> genes.

<p>The average DNA methylation of <i>z1B</i> genes in leaf, endosperm and tissue-cultured endosperm is displayed as bar graphs. The universal primers of <i>z1B</i> genes described in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146416#pone.0146416.ref030" target="_blank">30</a>] were used for bisulfite PCR amplification. The color code for individual gene copies is displayed as an insert.</p

C methylation at CCG sites.

<p>Methylation levels of <i>z1A</i> (A, C) and <i>z1B</i> (B, D) gene coding regions in leaf (C, D) and endosperm (A, B) is shown. The methylation of outer and inner C is marked with mCCG (blue) and CmCG (red), respectively.</p