Progress 10/01/02 to 09/30/07
Outputs
Alpha-glucosdase is important in intiating starch degradation in germinating seeds and leaves. Four alpha-glucosidases genes(Agl1)from germinating seed and leaves of barley have been sequenced and identified by active site ?W (I/N) DMNE? as members of the glycoside hydrolase family 31. Phylogenetic analysis of Agl2 revealed that it had a high degree of homology with several other plant Agls, although it had not previously been reported in barley. Western blot analysis indicated the Agl2 is not present in germinating seeds but is present in leaves. Purification and partial characterization of Agl2 revealed that it not only had maltase acvity (the definitive test for and Agl) but also had alpha-xylanase activity, suggesting a role in cell wall metabolism. Intron III of beta-amylase1 (Bmy1) has been implicated in regulating the expression of Bmy1 in barley. Bmy1 has also been found to be a large contributor to diastatic power, a major measure of malt quality. Complete
sequencing of barley beta-amylase1 (Bmy1) intron III has been completed for 42 accessions, including 13 wild genotypes. Four different alleles have been identified, one of which only occurs in some wild genotypes. Real time PCR is being conducted for genotypes with each of these alleles to determine how Bmy1 expression is affected. Levels of Bmy1 activity and Bmy1 thermostability have been determined for 321 cultivars of barley and are being compared to diastatic power and other measures of malt quality. Considerable variation in both specific activity and thermostabilty for Bmy1, and diastatic power have been found. Preliminary results indicate no predictable correlations between diastatic power and Bmy1 activity suggesting that the presumed relationship between the two is more complicated that previouly assumed. Beta-amylase2 (Bmy2) has been sequenced and submitted to GenBank (accession number DQ889983). We have found this gene is expressed in germinating barley for the first time.
It has been thought to only be a leaf enzyme. Its relative roles in leaves and germinating seeds are unknown and are being investigated.
Impacts
Knowledge of carbohydrases in germinating barley is important for targeting genes for increasing starch degradation for the production of fermentable sugars. This is especially important to the malting and brewing industries. Alpha-glucosidases are important in initiating starch degradation and beta-amylase is important in degrading fragements of starch produced by alpha-amylase and alpha-glucosidase. The information produced from our studies is designed to aid plant breeders in selecting for elite lines of barley that will have the enzymes necessary to produce malts that can rapidly hydrolyze starch in mashing tuns to produce a wort with a high real degree of fermentation (high levels of fermentable sugars). To do this we are identifying alpha-glucosidases and beta-amylases that are both high in activity and that are thermostable. Such information could also be useful the the fuel ethanol industry in boosting efficiency in starch degradation to fermentable sugars.
Publications
- Tanaka, Y. Henson, C.A., Duke, S.H. 2006 Alpha-glucosidases from the glucoside hydrolase family 31 in germinating seeds and seedling leaves of barley. 23rd International Carbohydrate Symposium, Whistler, Canada, poster 88. (invited presentation)
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Progress 01/01/05 to 12/31/05
Outputs
Four alpha-glucosidase (Agl) genes were identified and sequenced in germinating seeds and seedling leaves of barley. Three of these genes were hitherto unreported. A-Glu2 (Agl2) protein was not detected in seeds and the number of Agl2 mRNA copies was lower than mRNA of Agl1 in seeds. This may result from the absence of a GARE sequence in Agl2. The mechanism causing induction of Agl2 expression in the first 2 days of germination is unknown. Bothe A-glu 1 and A-glu2 proteins in leaves as well as A-glu1 protein in seeds were subjected to post-translational degradation. Unmodified proteins of both A-glus were detected in the basal meristematic section of seedling leaves suggesting that processing of A-glu1 and A-glu2 may take place as a function of leaf growth. Allelic variation in beta-amylases in barley is being determed in 22 cultivars and wild genotypes of barley. We have identified 3 alleles of intron 3 which could be significant because intron 3 has been implicated
in the expression of beta-amylases. Further experiments are being conducted to determine which alleles in barley are affecting thermostability. Thermolstability is important in brewing because of the high temperatures used in the mashing process. To date we have found considerable variation in beta-amylase thermostability associated with the various intron 3 alleles.
Impacts
Knowledge of the carbohydrases in germinating barley is important in targeting genes for manipulation of starch degradation. Starch degradation is a necessary process in the production of fermentable sugars and is especially important in the brewing and ethanol industries. Alpha-glucosidases are involved in the initiation of starch degradation and both alpha-glucosidases and beta-amylases are involved in the degradation of starch fragments produced after the initiation of starch degradation. The information concerning these two enzymes that we have produced will be useful in narrowing targets for maximizing starch degradation for the purposes of brewing and ethanol procuction. Specifically, by maximizing the activity of alpha-glucosidases and increasing the thermostability of beta-amylases the time for brewing and ethanol production can be decreased, increasing rates of production and reducing costs.
Publications
- Tanaka, Y., Henson, C. A., Duke, S. H. 2006 Alpha-glucosidases from the glucoside hydrolase family 31 in germinating seeds and seedling leaves of barley. 23rd International Carbohydrate Symposium,Whistler, British Columbia, poster 88
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