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. 2012 Dec 11;109(50):20543-8.
doi: 10.1073/pnas.1217927109. Epub 2012 Nov 26.

Structural Genes of Wheat and Barley 5-methylcytosine DNA Glycosylases and Their Potential Applications for Human Health

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Free PMC article

Structural Genes of Wheat and Barley 5-methylcytosine DNA Glycosylases and Their Potential Applications for Human Health

Shanshan Wen et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cytogenetic deletion map of wheat and barley DME homologs on wheat group 5 (5A, 5B, and 5D) and barley 5H chromosomes. The green bars show gross chromosomal locations (based on homology with mapped wheat ESTs and/or synteny and colinearity in case of barley), and the red bar shows precise location (based on wheat nullitetrasomic and deletion lines). Subchromosomal localization of wheat DME homeolog on 5B was determined using subgenome specific primers 5B_1946_2 forward (F) and reverse (R) (Table S2) on the genomic DNA of wheat cultivar CS (control), nullitetrasomic lines for group 5 chromosomes, deletion lines for long and short arms of chromosome 5B, and an interstitial deletion line ph1b. TaDME-5B localizes to the subcentromeric bin of the long arm of chromosome 5B (5BL). Specific product indicated by arrow head. M, 100-bp ladder.
Fig. 2.
Fig. 2.
Bar diagrams showing observed and expected (dotted line) frequencies of HSVs (synonymous changes in green and nonsynonymous changes in red) across the length of wheat DME homeologs. Location of different exons and four functional domains were shown below each bar diagram. PCR amplification profiles obtained using subgenome specific primers on the genomic DNAs extracted from diploid wheat progenitors (T.u, T. urartu “A” subgenome donor; Ae.s, Ae. speltoides most likely “B” subgenome donor; and Ae.t, Ae. tauschii “D” subgenome donor) and CS (AABBDD) was shown at the top of each bar diagram. To confirm assignment to wheat DME homeologs two primer pairs each from BAC clones 2159B03, 1946D08, and 2160P11 were designed and tested (Table S2). The primers for 1946D08 were additionally validated on wheat nullitetrasomic lines for group 5 chromosomes.
Fig. 3.
Fig. 3.
(A) Phylogenetic analysis of DME homologs from Arabidopsis (REFSEQ accession no. NP_001078527.1), rice (GenBank accession no. BAF04322.1), sorghum (GenBank accession no. JF683319), barley (GenBank accession no. CAQ58412.1), and wheat (GenBank accession nos. JF683316–JF683318) showing high level of conservation at sequence, as well as structural, levels. (B) Diagrammatic representation of DME protein showing four conserved domains with a magnified view of helix–hairpin–helix domain and iron sulfur cluster underlying the active site of the enzyme showing homology among 5-methylcytosine DNA glycosylases obtained from different organisms. (C) Three-dimensional structural model of the glycosylase domain of EndoIII in complex with DNA to show close functional conservation among different glycosylases.
Fig. 4.
Fig. 4.
RP-HPLC and SDS/PAGE of gliadins and glutenin fractions extracted from the T2 grains of the two progeny plants (P31D12 and P32F2) of transformant 10-728 (Table S6) expressing DME silencing hpRNA and the wild-type “Brundage 96” (B96). A random sample of T2 grains with their respective thousand kernel weights (TKWs) is shown at right.

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