Charles P. Moehs

Analysis of carotenoid biosynthetic gene expression during marigold petal development

Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to ¿100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.

Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health

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.

Chromosomal proteins of Arabidopsis thaliana.

In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance of single-copy DNA in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence of a set of H2A and H2B variants. Arabidopsis has at least four H2A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat.The high mobility group (HMG) non-histone chromatin proteins have attracted considerable attention because of the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group of non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as HMG proteins and that cross-react with antisera to HMG proteins of wheat.

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.

Dietary Gluten-Induced Gut Dysbiosis Is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease

The composition of the gut microbiome reflects the overall health status of the host. In this study, stool samples representing the gut microbiomes from 6 gluten-sensitive (GS) captive juvenile rhesus macaques were compared with those from 6 healthy, age- and diet-matched peers. A total of 48 samples representing both groups were studied using V4 16S rRNA gene DNA analysis. Samples from GS macaques were further characterized based on type of diet administered: conventional monkey chow, i.e., wheat gluten-containing diet (GD), gluten-free diet (GFD), barley gluten-derived diet (BOMI) and reduced gluten barley-derived diet (RGB). It was hypothesized that the GD diet would lower the gut microbial diversity in GS macaques. This is the first report illustrating the reduction of gut microbial alpha-diversity (p < 0.05) following the consumption of dietary gluten in GS macaques. Selected bacterial families (e.g., Streptococcaceae and Lactobacillaceae) were enriched in GS macaques while Coriobacteriaceae was enriched in healthy animals. Within several weeks after the replacement of the GD by the GFD diet, the composition (beta-diversity) of gut microbiome in GS macaques started to change (p = 0.011) towards that of a normal macaque. Significance for alpha-diversity however, was not reached by the day 70 when the feeding experiment ended. Several inflammation-associated microRNAs (miR-203, -204, -23a, -23b and -29b) were upregulated (p < 0.05) in jejunum of 4 biopsied GS macaques fed GD with predicted binding sites on 16S ribosomal RNA of Lactobacillus reuteri (accession number: NR_025911), Prevotella stercorea (NR_041364) and Streptococcus luteciae (AJ297218) that were overrepresented in feces. Additionally, claudin-1, a validated tight junction protein target of miR-29b was significantly downregulated in jejunal epithelium of GS macaques. Taken together, we predict that with the introduction of effective treatments in future studies the diversity of gut microbiomes in GS macaques will approach those of healthy individuals. Further studies are needed to elucidate the regulatory pathways of inflammatory miRNAs in intestinal mucosa of GS macaques and to correlate their expression with gut dysbiosis.

Supplementation of Reduced Gluten Barley Diet with Oral Prolyl Endopeptidase Effectively Abrogates Enteropathy-Associated Changes in Gluten-Sensitive Macaques

Celiac disease (CD) is an autoimmune disorder that affects approximately three million people in the United States. Furthermore, non-celiac gluten sensitivity (NCGS) affects an estimated additional 6% of the population, e.g., 20 million in the U.S. The only effective treatment of CD and NCGS requires complete removal of gluten sources from the diet. While required adherence to a gluten-free diet (GFD) is extremely difficult to accomplish, efforts to develop additional supportive treatments are needed. To facilitate these efforts, we developed a gluten-sensitive (GS) rhesus macaque model to study the effects of novel therapies. Recently reported results from phase one of this project suggest that partial improvement—but not remission—of gluten-induced disease can be accomplished by 100-fold reduction of dietary gluten, i.e., 200 ppm—by replacement of conventional dietary sources of gluten with a mutant, reduced gluten (RG) barley (lys3a)-derived source. The main focus of this (phase two) study was to determine if the inflammatory effects of the residual gluten in lys3a mutant barley grain could be further reduced by oral supplementation with a prolylendopeptidase (PE). Results reveal that PE supplementation of RG barley diet induces more complete immunological, histopathological and clinical remission than RG barley diet alone. The combined effects of RG barley diet and PE supplementation resulted in a further decrease of inflammatory mediators IFN-γ and TNF secretion by peripheral lymphocytes, as well as decreased plasma anti-gliadin and anti-intestinal tissue transglutaminase (TG2) antibodies, diminished active caspase production in small intestinal mucosa, and eliminated clinical diarrhea—all comparable with a gluten-free diet induced remission. In summary, the beneficial results of a combined RG barley and PE administration in GS macaques may warrant the investigation of similar synergistic approaches.

The Effects of Reduced Gluten Barley Diet on Humoral and Cell-Mediated Systemic Immune Responses of Gluten-Sensitive Rhesus Macaques

Celiac disease (CD) affects approximately 1% of the general population while an estimated additional 6% suffers from a recently characterized, rapidly emerging, similar disease, referred to as non-celiac gluten sensitivity (NCGS). The only effective treatment of CD and NCGS requires removal of gluten sources from the diet. Since required adherence to a gluten-free diet (GFD) is difficult to accomplish, efforts to develop alternative treatments have been intensifying in recent years. In this study, the non-human primate model of CD/NCGS, e.g., gluten-sensitive rhesus macaque, was utilized with the objective to evaluate the treatment potential of reduced gluten cereals using a reduced gluten (RG; 1% of normal gluten) barley mutant as a model. Conventional and RG barleys were used for the formulation of experimental chows and fed to gluten-sensitive (GS) and control macaques to determine if RG barley causes a remission of dietary gluten-induced clinical and immune responses in GS macaques. The impacts of the RG barley diet were compared with the impacts of the conventional barley-containing chow and the GFD. Although remission of the anti-gliadin antibody (AGA) serum responses and an improvement of clinical diarrhea were noted after switching the conventional to the RG barley diet, production of inflammatory cytokines, e.g., interferon-gamma (IFN-γ), tumor necrosis factor (TNF) and interleukin-8 (IL-8) by peripheral CD4+ T helper lymphocytes, persisted during the RG chow treatment and were partially abolished only upon re-administration of the GFD. It was concluded that the RG barley diet might be used for the partial improvement of gluten-induced disease but its therapeutic value still requires upgrading—by co-administration of additional treatments.

Development of reduced gluten wheat enabled by determination of the genetic basis of the lys3a low hordein barley mutant

Celiac disease is the most common food-induced enteropathy in humans with a prevalence of approximately 1% world-wide [1]. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as “gluten,” found in wheat. Related prolamins are present in barley and rye. Both celiac disease and a related condition called non-celiac gluten sensitivity (NCGS) are increasing in incidence [2] [3]. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here we used BSR-seq (Bulked Segregant RNA-seq) and map-based cloning to identify the genetic lesion underlying a recessive, low prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild type barley gene and then used TILLING (Targeting Induced Local Lesions in Genomes) [4] to identify induced SNPs (Single Nucleotide Polymorphisms) in the three homoeologs of the corresponding wheat gene. Combining inactivating mutations in the three sub-genomes of hexaploid bread wheat in a single wheat line lowered gliadin and low molecular weight glutenin accumulation by 50-60% and increased free and protein-bound lysine by 33%. This is the first report of the combination of mutations in homoeologs of a single gene that reduces gluten in wheat.