Annual Progress Report on Malting Barley Research March, 2007

More documents

Recommendations

Info

germination that is known to stimulate expression of many genes in the aleurone layers surrounding the endosperm. To analyze expression of the proteins encoded by the four abundantly expressed Agl genes, we prepared four antisera. Each antiserum was designed to be specific to only one of the four proteins encoded by these four genes. The specificities of these antisera were determined by testing against extracts of E. coli transformed with one of each of the four Agl genes we identified. Each of these cultures expressed only one of the recombinant isozymes. Each antiserum recognized only the isozyme containing the peptide to which it was designed. Again, in this report we will focus on comparing expression of the proteins α-glucosidase1 and α-glucosidase2, which are encoded by Agl1 with Agl2, respectively. Western blot analyses of germinated seed extracts probed with antibodies that recognized only the α-glucosidase1 protein showed that this antibody reacted with seed proteins having molecular masses of approximately 100, 95, 75 and 65 kD. These are data in agreement with those reported by Tibbot et al., 1998. The anti-α-glucosidse1 probe cross reacted with proteins extracted from seedling leaves with approximate molecular masses of 100, 95 and 80 kD. The Western blots using the anti-α-glucosidase2 probe detected cross reacting proteins of approximately 100 and 80 kD in seedling leaves but no cross reacting proteins were detected in germinated seeds. Possible explanations of why the Agl2 gene is transcribed yet the protein is not detected in the seed extracts include: 1) the protein may be present but below the limits of detection with the antibody; 2) the seed protein may be processed differently from the leaf protein such that the epitope is cleaved from the seed protein; 3) the seed protein may be insoluble possibly via membrane associations or aggregations; or 4) RNA silencing may be occurring in the seeds that is not occurring in the leaves. Purification and characterization of Agl2 demonstrated that it not only had maltase activity (the definitive test for Agl), but also had α-xylosidase activity. Xyloglucans were found to be better substrates than maltose for Agl2. This indicates that although Agl2 can contribute to starch degradation, it could have a major role in cell wall degradation during germination. Xyloglucans are the primary hemicelluloses 120
found in plant cell walls. The degradation of xyloglucans would result in the production of both glucose, which is fermentable, and xylose, which is usually not fermentable. Xylose is a aldopentose that most yeasts are unable to use for fermentation. Maximal expression of both Agl1 and Agl2 occurs at ca. five days of germination, about the time that green malt is kilned. An experiment planned for this study that will soon be conducted it to monitor Agl2 activity during mashing will be conducted using xyloglucans as the substrate and end product detection/quantification will be done using our HPLC equipped with a pulsed amperometric detector. Western blots using the Agl2 specific antibody will be conducted to moniter the stability of the Agl2 protein during mashing. Work conducted thus far to develop a modified diastatic power assay has been to initiate a comparison of using boiled soluble starch, nonboiled soluble starch, and a mixture of boiled malt and adjunct rice as substrates for the production of reducing sugars from malt extracts that have been centrifuged at high speed to remove endogenous starch. This work is continuing. References Coen ES, Romero JM,Soyle S, Elliot R, Murphy G, Carpenter F (1990) floricaula: A homeotic gene required for flower development in Antirrhium majus. Cell 63:1311-1322. Hwang YS, Karrer EE, Thomas BR, Chen L, and Rodriquez RL (1998) Three ciselements required for rice α-amylase Amy3D expression during sugar starvation. Plant Mol. Biol. 36:331-341 Jackson DP (1991) In situ hybridization in plants. In: Molecular Plant Pathology: A practical approach, Bowles DJ, Gurr SJ, and McPherson M, eds. Oxford University Press, England Tibbot BK and Skadsen RW (1996) Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley. Plant Mol. Biol. 30:229-241 Tibbot BK, Henson CA, Skadsen RW (1998) Expression of enzymatically active recombinant α-glucosidase in yeast and immunological detection of α-glucosidase from seed. Plant Mol. Biol. 38:379-391 121
Page 1 and 2:
Annual Pro
Page 3 and 4:
-i- INTRODUCTION The March 2007 <st
Page 5 and 6:
USDA-ARS NORTHERN CROP SCIENCE LABO
Page 7 and 8:
Spring Regional over two years at D
Page 9 and 10:
Table 1. 2006 UCD Two-Rowed Malting
Page 11 and 12:
environments was practiced, 2) elit
Page 13 and 14:
Table 4. Selected six-rowed lines c
Page 15 and 16:
Other Barley Research: Feed Program
Page 17 and 18:
Executive Summary AMBA Prog
Page 19 and 20:
Table 1. Agronomic performance of M
Page 21 and 22:
Lines M122 and M124 were rated sati
Page 23 and 24:
EARLY GENERATION LINES Forty F5 pop
Page 25 and 26:
have evaluated a PCR based marker,
Page 27 and 28:
Edward Schiefelbein, Research Scien
Page 29 and 30:
small grains pathology input to thi
Page 31 and 32:
Project Personnel Small Grains Path
Page 33 and 34:
Investigations on Barley Diseases a
Page 35 and 36:
Minnesota barley disease survey and
Page 37 and 38:
pathotypes at the seedling stage in
Page 39 and 40:
germplasm with the highest level of
Page 41 and 42:
Mirlohi, A., Brueggeman, R., Steffe
Page 43 and 44:
Developing Improved Malt Barley Var
Page 45 and 46:
Table 2. Montana’s Statewide Tria
Page 47 and 48:
As a Barley CAP collaborator, our C
Page 49 and 50:
3.2. A barley nursery composed of d
Page 51 and 52:
5. Related Barley Projects Barley P
Page 53 and 54:
Barley Breeding - Advanced Testing
Page 55 and 56:
Table 3. Agronomic comparisons of N
Page 57 and 58:
Preliminary Malting Barley Yield Tr
Page 59 and 60:
dwarf genotypes with the convention
Page 61 and 62:
STUDIES ON BARLEY DISEASES AND THEI
Page 63 and 64:
inoculated plots. The inoculum used
Page 65 and 66:
much of North Dakota in 2006, few f
Page 67 and 68:
5. Collect isolates of barley patho
Page 69 and 70: oot rot. In collaboration with Scot
Page 71 and 72: 2. United States Wheat and Barley S
Page 73 and 74: 6. Lee, S. and Neate, S.M. (2006) P
Page 75 and 76: Objectives Met in the One-Year Fund
Page 77 and 78: These automated methods have been t
Page 79 and 80: Beta-limit dextrin Alpha-Amylase Al
Page 81 and 82: M w kDa 800 600 400 200 0 0.4 0 2 4
Page 83 and 84: Figure 7. Time course of I2-binding
Page 85 and 86: 10 9 8 7 6 5 4 3 2 1 0 1993 1994 19
Page 87 and 88: 2. Other Research (A) Predicting Se
Page 89 and 90: forms (through enzymolysis during m
Page 91 and 92: PUBLICATIONS (2006-07) Schwarz, P.B
Page 93 and 94: CI9214, PI552963 (Heartland), and N
Page 95 and 96: showing good levels of resistance t
Page 97 and 98: Germplasm Enhancement for RWA Resis
Page 99 and 100: Gary Hein in Colorado and Nebraska.
Page 101 and 102: Kevin Hicks, Research Leader, USDA-
Page 103 and 104: Detailed Report on
Page 105 and 106: Other Barley Research and Future Di
Page 107 and 108: Table 1. 2006/2007 Oregon winter ba
Page 109 and 110: Table 4. Agronomic data for OSU win
Page 111 and 112: MALTING QUALITY ANALYSIS OF NEW BAR
Page 113 and 114: We recently secured an industrial c
Page 115 and 116: 2006-2007 Progress
Page 117 and 118: enzyme it encodes. We found that se
Page 119: horseradish peroxidase-conjugated g
Page 123 and 124: Establishing that sequence variatio
Page 125: Charles Karpelenia, technician Joe
show all

Annual Progress Report on Malting Barley Research March, 2007

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?