Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific ß-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., ß-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low ß-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Exogenous spike-in mouse RNAs for accurate differential gene expression analysis in barley using RT-qPCR.

Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) followed by the 2-ΔΔCt method is the most common way to measure transcript levels for relative gene expression assays. The quality of an RT-qPCR assay is dependent upon the identification and validation of reference genes to normalize gene expression data. The so-called housekeeping genes are commonly used as internal reference genes because they are assumed to be ubiquitously expressed at stable levels. Commonly, researchers do not validate their reference genes but rely on historical reference genes or previously validated genes from an unrelated experiment. Using previously validated reference genes to assess gene expression changes occurring during malting resulted in extensive variability. Therefore, a new method was tested and validated to circumvent the use of internal reference genes. Total mouse RNA was chosen as the external reference RNA and a suite of primer sets to putatively stable mouse genes was created to identify stably expressed genes for use as an external reference gene. cDNA was created by co-amplifying total mouse RNA, as an RNA spike-in, and barley RNA. When using the external reference genes to normalize malting gene expression data, standard deviations were significantly reduced and significant differences in transcript abundance were observed, whereas when using the internal reference genes, standard deviations were larger with no significant differences seen. Furthermore, external reference genes were more accurate at assessing expression levels in malting and developing grains, whereas the internal reference genes overestimated abundance in developing grains and underestimated abundance in malting grains.

Description and functional analysis of the transcriptome from malting barley.

The present study aimed to establish an early model of the malting barley transcriptome, which describes the expression of genes and their ontologies, identify the period during malting with the largest dynamic shift in gene expression for future investigation, and to determine the expression patterns of all starch degrading enzyme genes relevant to the malting and brewing industry. Large dynamic increases in gene expression occurred early in malting with differential expressed genes enriched for cell wall and starch hydrolases amongst many malting related categories. Twenty-five of forty starch degrading enzyme genes were differentially expressed in the malting barley transcriptome including eleven α-amylase genes, six ß-amylase genes, three α-glucosidase genes, and all five starch debranching enzyme genes. Four new or novel α-amylase genes, one ß-amylase gene (Bmy3), three α-glucosidase genes, and two isoamylase genes had appreciable expression that requires further exploration into their potential relevance to the malting and brewing industry.

Comparative gene expression analysis of the ß-amylase and hordein gene families in the developing barley grain.

Expression of hordeins and ß-amylase during barley grain development is important in determining malting quality parameters that are controlled by protein and malt enzyme levels. The relationship between protein and enzyme levels is confounding because, in general, protein and malt enzyme activity are positively correlated and the malting and brewing industries demand relatively low levels of protein and relatively high levels of enzymes. Separation of these traits is desirable because high protein levels are one of the primary causes of barley not meeting malt quality standards. Studies on barley grain development have not resulted in a consensus on the temporal accumulation of hordein and endosperm-specific ß-amylase (Bmy1) and thus, it is unclear whether hordeins and Bmy1 are under control of the same temporal regulator (s). Therefore, temporal expression patterns of hordeins (B- [Hor2], C- [Hor1], D- [Hor3], and γ-hordein [Hor5]) were compared to Bmy1 throughout grain development (5 to 35 days after anthesis (DAA)). Transcript accumulation between hordeins and Bmy1 occurred simultaneously beginning during the pre-storage phase of grain development whereas the B1-hordein protein appeared two days before Bmy1 most likely due to variations in gene copy number. Interestingly, the largest increase in hordein and Bmy1 transcript levels occurred between 5 and 9 (Hor2, Hor2-B1, Hor2-B3, Hor3, Hor5-γ1, and Hor5-γ3) or 9 and 13 DAA (Hor1 and Bmy1). Additionally, ubiquitous ß-amylase (Bmy2) has a novel expression pattern and was the predominant ß-amylase present between 5 and 15 DAA whereas Bmy1 was the predominant ß-amylase present between 17 and 35 DAA.

Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize transcript abundance. The most popular gene targets for RT-qPCR are housekeeping genes because they are thought to maintain a static transcript level among a variety of samples. However, more recent studies have shown, several housekeeping genes are not reliably stable. This is the first study to examine the potential of several reference genes for use in RT-qPCR normalization during barley malting. The process of malting barley mechanizes the imbibition and subsequent germination of barley seeds under controlled conditions. Malt quality is controlled by many pleiotropic genes that are determined by examining the result of physiological changes the barley seed undergoes during the malting process. We compared the stability of 13 reference genes across both two-and six-row malting barleys (Conrad and Legacy, respectfully) throughout the entirety of the malting process. Initially, primer target specificity, amplification efficiency and average Ct values were determined for each of the selected primer pairs. Three statistical programs (geNorm, NormFinder, and BestKeeper) were used to rank the stability of each reference gene. Rankings were similar between the two- and six-row with the exception of BestKeeper's ranking of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A consensus ranking among programs was determined using RefFinder. Our results show that Actin (ACT) and Heat Shock Protein 70 (HSP70) were the most stable throughout micromalting, while GAPDH and Cyclophilin (CYP) were the least stable. Two reference genes are necessary for stable transcript normalization according to geNorm and the best two reference genes (ACT and HSP70) provided a sufficient level of stability.

Differential expression of two ß-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain.

Two barley (Hordeum vulgare L.) ß-amylase genes (Bmy1 and Bmy2) were studied during the late maturation phase of grain development in four genotypes. The Bmy1 and Bmy2 DNA and amino acid sequences are extremely similar. The largest sequence differences are in the introns, seventh exon, and 3' UTR. Accumulation of Bmy2 mRNA was examined in developing grain at 17, 19, and 21 days after anthesis (DAA). One genotype, PI 296897, had significantly higher Bmy2 RNA transcript accumulation than the other three genotypes at all developmental stages. All four genotypes had Bmy2 mRNA levels decrease from 17 to 19 DAA, and remain the same from 19 to 21 DAA. Levels of Bmy1 mRNA were twenty thousand to over one hundred thousand times more than Bmy2 mRNA levels in genotypes Legacy, Harrington, and Ashqelon at all developmental stages and PI 296897 at 19 and 21 DAA. PI 296897 had five thousand times more Bmy1 mRNA than Bmy2 mRNA at 17 DAA. However, Bmy2 protein was not found at 17 DAA in any genotype. The presence of Bmy2 was immunologically detected at 19 DAA and was present in greater amounts at 21 DAA. Also, Bmy2 protein was found to be stored in mature grain and localized in the soluble fraction. However, Bmy1 protein was far more prevalent than Bmy2 at all developmental stages in all genotypes. Thus, the vast majority of ß-amylase activity in developing and mature grain can be attributed to endosperm-specific ß-amylase.

Differential RNA expression of Bmy1 during barley seed development and the association with ß-amylase accumulation, activity, and total protein.

The objective of this study was to determine if developing barley (Hordeum vulgare L.) seeds had differences in ß-amylase 1 (Bmy1) mRNA accumulation, ß-amylase (EC 3.2.1.2) activity, ß-amylase protein accumulation, and total protein levels during late seed development from genotypes with different Bmy1 intron III alleles. Two North American malting barley cultivars (Hordeum vulgare ssp. vulgare) were chosen to represent the Bmy1.a and Bmy1.b alleles and, due to limited Bmy1 intron III allele variation in North American cultivars, two wild barleys (Hordeum vulgare ssp. spontaneum) were chosen to represent the Bmy1.c and Bmy1.d alleles. Wild barleys Ashqelon (Bmy1.c) and PI 296897 (Bmy1.d) had 2.5- to 3-fold higher Bmy1 mRNA levels than cultivars Legacy (Bmy1.a) and Harrington (Bmy1.b). Levels of Bmy1 mRNA were not significantly different between cultivated or between wild genotypes. In all four genotypes Bmy1 mRNA levels increased from 17 to 19 days after anthesis (DAA) and remained constant from 19 to 21 DAA. Ashqelon and PI 296897 had more ß-amylase activity on a fresh weight basis than Legacy and Harrington at all developmental stages. ß-Amylase protein levels increased from 17 DAA to maturity in all genotypes. Total protein in grains from wild genotypes was significantly higher than cultivated genotypes at all developmental stages. Higher levels of total protein in Ashqelon and PI 296897 could explain their higher levels of ß-amylase activity, when expressed on a fresh weight basis. When ß-amylase activities are expressed on a protein basis there are no statistical differences between the wild and cultivated barleys at maturity.