Uniparental and transgressive expression of α-zeins in maize endosperm of o2 hybrid lines.

The α-zein gene family encodes the most abundant storage proteins of maize (Zea mays) endosperm. Members of this family are expressed in a parent-of-origin manner. To characterize this phenomenon further, we investigated the expression of a subset of α-zein polypeptides in reciprocal crosses between o2 lines that were characterized by a simplified α-zein pattern. Maize lines that suppressed the expression of α-zeins when used as female parents were identified. The suppression was cross-specific, occurring only when specific genetic backgrounds were combined. Four α-zein sequences that were sensitive to uniparental expression were isolated. Molecular characterization of these α-zeins confirmed that their expression or suppression depended on the genetic proprieties of the endosperm tissue instead of their parental origin. DNA methylation analysis of both maternally and paternally expressed α-zeins revealed no clear correlation between this epigenetic marker and parent-of-origin allelic expression, suggesting that an additional factor(s) is involved in this process. Genetic analyses revealed that the ability of certain lines to suppress α-zein expression was unstable after one round of heterozygosity with non-suppressing lines. Interestingly, α-zeins also showed a transgressive expression pattern because unexpressed isoforms were reactivated in both F2 and backcross plants. Collectively, our results suggest that parent-of-origin expression of specific α-zein alleles depends on a complex interaction between genotypes in a manner that is reminiscent of paramutation-like phenomena.

Epigenetic variation, inheritance, and parent-of-origin effects of cytosine methylation in maize (Zea mays).

Pure epigenetic variation, or epigenetic variation that is independent of genetic context, may provide a mechanism for phenotypic variation in the absence of DNA mutations. To estimate the extent of pure epigenetic variation within and across generations and to identify the DNA regions targeted, a group of eight plants derived from a highly inbred line of maize (Zea mays) was analyzed by the methylation-sensitive amplified polymorphism (MSAP) technique. We found that cytosine methylation (mC) differences among individuals accounted for up to 7.4% of CCGG sites investigated by MSAP. Of the differentially methylated fragments (DMFs) identified in the S0 generation, ∼12% were meiotically inherited for at least six generations. We show that meiotically heritable mC variation was consistently generated for an average of 0.5% CCGG sites per generation and that it largely occurred somatically. We provide evidence that mC variation can be established and inherited in a parent-of-origin manner, given that the paternal lineage is more prone to both forward and reverse mC changes. The molecular characterization of selected DMFs revealed that the variation was largely determined by CG methylation changes that map within gene regions. The expression analysis of genes overlapping with DMFs did not reveal an obvious correlation between mC variation and transcription, reinforcing the idea that the primary function of gene-body methylation is not to control gene expression. Because this study focuses on epigenetic variation in field-grown plants, the data presented herein pertain to spontaneous epigenetic changes of the maize genome in a natural context.

Wild-type opaque2 and defective opaque2 polypeptides form complexes in maize endosperm cells and bind the opaque2-zein target site.

The Opaque2 (O2) basic leucine (Leu)-zipper transcriptional activator controls the expression of several genes in maize (Zea mays). We investigated the phosphorylation extent of wild-type O2 and mutant-defective or mutant-truncated o2 polypeptides in endosperm cells, their subcellular localization, participation in complex formation, and involvement in functional activity. Besides wild type, four mutant alleles (o2T, o2-52, o2It, and o2-676) producing o2 polypeptides and a null transcript allele (o2R) were considered. Observing the effects of these mutations, multiphosphorylation events in O2 or o2 proteins were confirmed and further investigated, and the involvement of both the nuclear localization signal (NLS)-B and Leu-zipper domains in proper targeting to the nucleus was ascertained. The absence of these domains in the o2T and o2It-S mutant-truncated forms holds them within the cytoplasm, where they are partially phosphorylated, whereas the presence of NLS-B and a partial Leu-zipper domain in o2-52 distributes this mutant-truncated form in both cytoplasm and nucleus. Although mutated in the NLS-B domain, the o2It-L and o2-676 mutant-defective forms are, respectively, partially or completely distributed into the nucleus. Only wild-type O2 and mutant-defective o2 polypeptides bearing the Leu-zipper are able to form complexes whose components were proven to bind the O2-zein target site by in vitro analyses. The transcription of a subset of H-zein genes as well as H-zein polypeptide accumulation in several o2-mutant-defective genotypes indicate the in vivo involvement of o2-mutant-defective proteins in O2-zein target site recognition. The gathered information broadens our knowledge on O2 functional activity and our view on possible quality protein maize trait manipulation or plant transformation via the utilization of cisgenic elements.

ESTuber db: an online database for Tuber borchii EST sequences.

BACKGROUND: The ESTuber database (http://www.itb.cnr.it/estuber) includes 3,271 Tuber borchii expressed sequence tags (EST). The dataset consists of 2,389 sequences from an in-house prepared cDNA library from truffle vegetative hyphae, and 882 sequences downloaded from GenBank and representing four libraries from white truffle mycelia and ascocarps at different developmental stages. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts. Data were collected in a MySQL database, which can be queried via a php-based web interface. RESULTS: Sequences included in the ESTuber db were clustered and annotated against three databases: the GenBank nr database, the UniProtKB database and a third in-house prepared database of fungi genomic sequences. An algorithm was implemented to infer statistical classification among Gene Ontology categories from the ontology occurrences deduced from the annotation procedure against the UniProtKB database. Ontologies were also deduced from the annotation of more than 130,000 EST sequences from five filamentous fungi, for intra-species comparison purposes. Further analyses were performed on the ESTuber db dataset, including tandem repeats search and comparison of the putative protein dataset inferred from the EST sequences to the PROSITE database for protein patterns identification. All the analyses were performed both on the complete sequence dataset and on the contig consensus sequences generated by the EST assembly procedure. CONCLUSION: The resulting web site is a resource of data and links related to truffle expressed genes. The Sequence Report and Contig Report pages are the web interface core structures which, together with the Text search utility and the Blast utility, allow easy access to the data stored in the database.

Extensive maternal DNA hypomethylation in the endosperm of Zea mays.

A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.

The nitrogen-induced recovery of alpha-zein gene expression in in vitro cultured opaque2 maize endosperms depends on the genetic background.

The effect of nitrogen nutrition on the accumulation of seed storage proteins has been studied in vitro by cultivating on agar media maize (Zea mays L.) endosperm explants from seeds at 10 days after pollination. The experiments were performed on various genetic backgrounds bearing different opaque2 (o2) mutant alleles and on the corresponding wild-type lines. In the seed of the o2 genotypes the high molecular weight alpha-zein polypeptides (zHs), whose transcription is Opaque2 (O2) regulated, are absent or extremely reduced. The endosperms were incubated on basal agar medium with amino acid supply. In these growth conditions, fresh and dry weights increased in both wild-type and o2 endosperms, irrespective of the genetic background. In 4 out of the 5 o2 mutant genotypes analysed we detected an accumulation of the zHs similar to the corresponding wild-type explants or seeds. However, in one of these mutants, Mo17o2R, the addition of amino acids to the culture media had no effect on the zH accumulation. We showed that the Mo17o2R behaviour is not due to a negative regulation but to the absence of putative transcription factor(s) able to regulate the zH transcription occurring in the other o2 mutants.