Targeting Induced Local Lesions in the Wheat DEMETER and DRE2 Genes, Responsible for Transcriptional Derepression of Wheat Gluten Proteins in the Developing Endosperm

Abstract

Wheat is a major source of energy and nutrition worldwide, but it is also a primary cause of frequent diet-induced health issues, specifically celiac disease, for which the only effective therapy so far is strict dietary abstinence from gluten-containing grains. Wheat gluten proteins are grouped into two major categories: high-molecular-weight glutenin subunits (HMWgs), vital for mixing and baking properties, and gliadins plus low-molecular-weight glutenin subunits (LMWgs) that contain the overwhelming majority of celiac-causing epitopes. We put forth a hypothesis that eliminating gliadins and LMWgs while retaining HMWgs might allow the development of reduced-immunogenicity wheat genotypes relevant to most gluten-sensitive individuals. This hypothesis stems from the knowledge that the molecular structures and regulatory mechanisms of the genes encoding the two groups of gluten proteins are quite different, and blocking one group's transcription, without affecting the other's, is possible. The genes for gliadins and LMWgs have to be de-methylated by 5-methylcytosine DNA glycosylase/lyase (DEMETER) and an iron-sulfur (Fe-S) cluster biogenesis enzyme (DRE2) early during endosperm development to permit their transcription. In this study, a TILLING (Targeting Induced Local Lesions IN Genomes) approach was undertaken to identify mutations in the homoeologous DEMETER (DME) and DRE2 genes in common and durum wheat. Lines with mutations in these genes were obtained that displayed reduced content of immunogenic gluten proteins while retaining essential baking properties. Although our data at first glance suggest new possibilities for treating celiac disease and are therefore of medical and agronomical interest, it also shows that inducing mutations in the DME and DRE2 genes analyzed here affected pollen viability and germination. Hence there is a need to develop other approaches in the future to overcome this undesired effect.

Reduced-Immunogenicity Wheat and Peanut Lines for People with Foodborne Disorders

“…We observed that the nonsense mutations in wheat DEMETER genes have significantly reduced the anther locule size the number of viable pollen and negatively affected the pollen germinate. Thus, this analysis also suggested a vital role of DEMETER in pollen development and germination [31,32,37].…”

“…in order to develop celiac-safe wheat cultivars, we undertook two approaches: genetic engineering and germplasm screening. The latter approach consists of two different strategies: (i) epigenetic elimination of gliadins and low-molecular-weight glutenins by silencing the wheat genes encoding a DNA glycosylase DEMETER and an iron-sulfur biogenesis enzyme Dre2, which are responsible for transcriptional de-repression of prolamin genes in the developing endosperm [29][30][31][32] and (ii) post-translational detoxification of 'gluten' proteins by ectopic expression of glutenases in wheat endosperm [33,34].…”

“…Based on the knowledge that the enzyme coded by the wheat DEMETER gene is responsible for the transcriptional activation of the gluten genes in developing wheat grains [29], we screened two TILLING populations, one each in the 'Kronos' and 'Express' backgrounds, for mutations in the DEMETER genes [31,32]. To reduce the workload, we confined our search for the mutants to one kb region in the active site of the DEMETER genes.…”

“…Screening for mutations in the 'Kronos' and 'Express' populations, respectively, yielded 77 and 149 mutations. The single mutations in the DEMETER homoeologues showed reductions in specific prolamins [32]. Crosses were made between the mutants carrying substitutions, premature stop codons, and splice-site variations in A and B subgenome DEMETER homoeologues, to pyramid the effect of the individual mutations in a single genotype.…”

“…Crosses were made between the mutants carrying substitutions, premature stop codons, and splice-site variations in A and B subgenome DEMETER homoeologues, to pyramid the effect of the individual mutations in a single genotype. Unfortunately, the crosses between the mutants carrying the premature stop codons or splice-site variations never yielded a double mutant [31,32]. To determine the cause of the observed segregation distortion, we closely monitored the anther morphology, number of viable pollens, and pollen germination.…”