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

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.

A Bitter-Sweet Story: Unraveling the Genes Involved in Quinolizidine Alkaloid Synthesis in Lupinus albus.

Alkaloids are part of a structurally diverse group of over 21,000 cyclic nitrogen-containing secondary metabolites that are found in over 20% of plant species. Lupinus albus are naturally containing quinolizidine alkaloid (QA) legumes, with wild accessions containing up to 11% of QA in seeds. Notwithstanding their clear advantages as a natural protecting system, lupin-breeding programs have selected against QA content without proper understanding of quinolizidine alkaloid biosynthetic pathway. This review summarizes the current status in this field, with focus on the utilization of natural mutations such as the one contained in pauper locus, and more recently the development of molecular markers, which along with the advent of sequencing technology, have facilitated the identification of candidate genes located in the pauper region. New insights for future research are provided, including the utilization of differentially expressed genes located on the pauper locus, as candidates for genome editing. Identification of the main genes involved in the biosynthesis of QA will enable precision breeding of low-alkaloid, high nutrition white lupin. This is important as plant based high quality protein for food and feed is an essential for sustainable agricultural productivity.

Directed-Mutagenesis of Flavobacterium meningosepticum Prolyl-Oligopeptidase and a Glutamine-Specific Endopeptidase From Barley.

Wheat gluten proteins are the known cause of celiac disease. The repetitive tracts of proline and glutamine residues in these proteins make them exceptionally resilient to digestion in the gastrointestinal tract. These indigested peptides trigger immune reactions in susceptible individuals, which could be either an allergic reaction or celiac disease. Gluten exclusion diet is the only approved remedy for such disorders. Recently, a combination of a glutamine specific endoprotease from barley (EP-B2), and a prolyl endopeptidase from Flavobacterium meningosepticum (Fm-PEP), when expressed in the wheat endosperm, were shown to reasonably detoxify immunogenic gluten peptides under simulated gastrointestinal conditions. However useful, these "glutenases" are limited in application due to their denaturation at high temperatures, which most of the food processes require. Variants of these enzymes from thermophilic organisms exist, but cannot be applied directly due to their optimum activity at temperatures higher than 37°C. Though, these enzymes can serve as a reference to guide the evolution of peptidases of mesophilic origin toward thermostability. Therefore, a sequence guided site-saturation mutagenesis approach was used here to introduce mutations in the genes encoding Fm-PEP and EP-B2. A thermostable variant of Fm-PEP capable of surviving temperatures up to 90°C and EP-B2 variant with a thermostability of up 60°C were identified using this approach. However, the level of thermostability achieved is not sufficient; the present study has provided evidence that the thermostability of glutenases can be improved. And this pilot study has paved the way for more detailed structural studies in the future to obtain variants of Fm-PEP and EP-B2 that can survive temperatures ~100°C to allow their packing in grains and use of such grains in the food industry.

Gluten Detection Methods and Their Critical Role in Assuring Safe Diets for Celiac Patients.

Celiac disease, wheat sensitivity, and allergy represent three different reactions, which may occur in genetically predisposed individuals on the ingestion of wheat and derived products with various manifestations. Improvements in the disease diagnostics and understanding of disease etiology unveiled that these disorders are widespread around the globe affecting about 7% of the population. The only known treatment so far is a life-long gluten-free diet, which is almost impossible to follow because of the contamination of allegedly "gluten-free" products. Accidental contamination of inherently gluten-free products could take place at any level from field to shelf because of the ubiquity of these proteins/grains. Gluten contamination of allegedly "gluten-free" products is a constant threat to celiac patients and a major health concern. Several detection procedures have been proposed to determine the level of contamination in products for celiac patients. The present article aims to review the advantages and disadvantages of different gluten detection methods, with emphasis on the recent technology that allows identification of the immunogenic-gluten peptides without the use of antibodies. The possibility to detect gluten contamination by different approaches with similar or better detection efficiency in different raw and processed foods will guarantee the safety of the foods for celiac patients.

The Role of Orange Gene in Carotenoid Accumulation: Manipulating Chromoplasts Toward a Colored Future.

Carotenoids are isoprenoid pigments synthesized in plants, algae, and photosynthetic bacteria and fungus. Their role is essential in light capture, photoprotection, pollinator attraction, and phytohormone production. Furthermore, they can regulate plant development when they are processed as small signaling molecules. Due to their importance for human health, as promoters of the immune system and antioxidant activity, carotenoids have been used in the pharmaceutical, food, and nutraceutical industries. Regulation of carotenoid synthesis and accumulation has been extensively studied. Excellent work has been done unraveling the mode of action of phytoene synthase (PSY), a rate-limiting enzyme of carotenoid biosynthesis pathway, in model species and staple crops. Lately, interest has been turned to Orange protein and its interaction with PSY during carotenoid biosynthesis. Discovered as a dominant mutation in Brassica oleracea, Orange protein regulates carotenoid accumulation by posttranscriptionally regulating PSY, promoting the formation of carotenoid-sequestering structures, and also preventing carotenoid degradation. Furthermore, Orange protein contributes to homeostasis regulation, improving plant tolerance to abiotic stress. In this mini review, the focus is made on recent evidence that elucidates Orange protein mode of action and expression in different plant species. Additionally, strategies are proposed to modify Orange gene by utilization of genome editing techniques. A better understanding of carotenoid biosynthesis and accumulation will lead to a positive impact on the development of healthy food for a growing population.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Development and characterization of InDel markers for Lupinus luteus L. (Fabaceae) and cross-species amplification in other Lupin species

Background: Strong artificial selection and/or natural bottle necks may limit genetic variation in domesticated species. Lupinus luteus, an orphan temperate crop, has suffered diversity reductions during its bitter/sweet alkaloid domestication history, limiting breeding efforts and making molecular marker development a difficult task. The main goal of this research was to generate new polymorphic insertion­deletion (InDel) markers to aid yellow lupin genetics and breeding. By combining genomic reduction libraries and next generation sequencing, several polymorphic InDel markers were developed for L. luteus L. Results: A total of 118 InDel in silico polymorphic markers were identified. Eighteen InDel primer sets were evaluated in a diverse L. luteus core collection, where amplified between 2­3 alleles per locus. Observed heterozygosity (HO; 0.0648 to 0.5564) and polymorphic information content (PIC; 0.06 to 0.48) estimations revealed a moderate level of genetic variation across L. luteus accessions. In addition, ten and nine InDel loci amplified successfully Lupinus hispanicus Boiss & Reut, and Lupinus mutabilis Sweet, respectively, two L. luteus close relatives. PCA analysis identified two L. luteus clusters, most likely explained by the domestication species history. Conclusion: The development of InDel markers will facilitate the study of genetic diversity across L. luteus populations, as well as among closely related species.