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.

Metabolic changes in Avena sativa crowns recovering from freezing.

Extensive research has been conducted on cold acclimation and freezing tolerance of fall-sown cereal plants due to their economic importance; however, little has been reported on the biochemical changes occurring over time after the freezing conditions are replaced by conditions favorable for recovery and growth such as would occur during spring. In this study, GC-MS was used to detect metabolic changes in the overwintering crown tissue of oat (Avena sativa L.) during a fourteen day time-course after freezing. Metabolomic analysis revealed increases in most amino acids, particularly proline, 5-oxoproline and arginine, which increased greatly in crowns that were frozen compared to controls and correlated very significantly with days after freezing. In contrast, sugar and sugar related metabolites were little changed by freezing, except sucrose and fructose which decreased dramatically. In frozen tissue all TCA cycle metabolites, especially citrate and malate, decreased in relation to unfrozen tissue. Alterations in some amino acid pools after freezing were similar to those observed in cold acclimation whereas most changes in sugar pools after freezing were not. These similarities and differences suggest that there are common as well as unique genetic mechanisms between these two environmental conditions that are crucial to the winter survival of plants.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

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.