Annual Progress Report on Malting Barley Research March, 2002

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146 Mikola, M., Brinck, O., and Jones, B.L., Characterization of oat endoproteinases that hydrolyze oat avenins. Cereal Chemistry, 78: 55-58, 2001. Personnel Dr. B.L. Jones, Supervisory Research Chemist Ms. L.A. Marinac, Plant Biologist Ms. D. Fontanini, Graduate Student
147 STUDIES ON THE PROTEINASES THAT ARE PRODUCED WHEN FUSARIUM GROWS ON CEREAL PROTEINS AND BARLEY PROTEINS THAT INHIBIT THEM Anja I. Pekkarinen and Berne L. Jones Cereal Crops Research Unit, USDA, ARS, Department of Agronomy, University of Wisconsin, Madison, and VTT Biotechnology, Espoo, Finland Objectives The protein matrix of the wheat grain endosperm is degraded when the kernels are attacked by the disease Fusarium head blight (FHB) or ‘scab’. It seems likely that the same phenomenon also occurs in heavily infected barley kernels and that it may be related to the method whereby Fusarium attacks the barley plant. We have shown that two alkaline serine proteinases are synthesized by F. culmorum when it is cultured on cereal storage proteins, and it seems likely that these enzymes are the ones that degrade the proteins of infected barley grains. The proteinases are not produced when the Fusarium is grown in media that lack protein. If, as seems probable, one function of these proteinases is to help the fungus attack the barley head, then it seemed possible that the grain might have evolved to produce compounds that could inactivate these enzymes and thus retard its infection by the Fusarium. This study was carried out to purify and characterize the Fusarium proteinases, to use the purified enzymes to test for the presence of inhibitors in the barley and to determine whether it might be possible to manipulate the inhibitors to increase the resistance of barleys to Fusarium attack. <strong>Progress</strong> in studies on Fusarium culmorum endoproteinases In the 2001 AMBA <strong>Annual</strong> Research <strong>Progress</strong> <strong>Report</strong>, we described how two alkaline F. culmorum serine proteinases were purified from a culture medium and characterized. These enzymes were denominated proteinase C (chymotrypsin-like) and proteinase T (trypsin-like). At that time, we already had analyzed the N-terminal amino acid sequence of the proteinase T, but our attempts to sequence the N-terminal amino acids of the other protein had failed because the N-terminus of the enzyme was blocked and couldn’t be sequenced by the Edman degradation. To get around this problem, the purified enzyme has been digested into small peptides with a modified trypsin. These peptides were separated and their amino acids were sequenced at the Protein Chemistry Laboratory in the University of Texas Medical Branch (UTMB), Galveston, TX. The amino acids of the peptides were highly homologous with those of several fungal subtilisins, but no completely identical matches were found among the proteins that are listed in the various amino acid sequence databases, indicating that it was a novel enzyme. Hence, it was renamed proteinase S (subtilisin-like). In addition, we demonstrated that both proteinase S and T hydrolyzed some of the barley storage proteins, the C- and D hordeins, in vitro. The description of the purification and characterization of the proteinase S has recently been published in the European Journal of Biochemistry (2002, Vol. 269, p. 798-807). A manuscript covering the characterization of the second enzyme, proteinase T, has been submitted to the Journal of Agricultural and Food Chemistry. Recently, we discovered that the F. culmorum culture medium contained a third proteinase enzyme. Preliminary studies have indicated that it is very different from the
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Annual Pro
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-i- INTRODUCTION The March 2002 <st
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Barley Transformation G.J. Muehlbau
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1 2000/2001 WINTER NURSERY: BARLEY
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3 2001 Barley Stripe Rust Screening
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5 Triumph/Tyra//Arupo, a selection
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7 Washington (Fossum Cereals and Wa
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9 This project is supported by the
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11 ‘Triumph’, ‘Valier’, ‘
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13 Table 2. Agronomic data for sele
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15 Table 4. Agronomic data for sele
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17 Table 8. Malting quality data* f
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19 Six-rowed barley selections of c
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21 Table 14. Agronomic data for sel
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23 Table 16. Agronomic data for win
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25 Table 19. Malting quality data*
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Garnet/98Ab11865 Garnet/98Ab12895 9
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29 rust resistant NSGC accessions o
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31 DEVELOPMENT OF SIX-ROWED MALTING
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33 backcrossing. The selfed lines w
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35 MINNESOTA BARLEY IMPROVEMENT PRO
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37 ADVANCED LINE EVALUATION Varieti
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39 OTHER BARLEY DISEASES In 2001, r
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41 Wingbermuehle, W. J., K.M. Belin
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43 In 2001 winter/spring, over 300
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45 assessments were made by countin
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47 IDENTIFICATION OF NOVEL DISEASE
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49 Unfortunately, disease levels in
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51 BARLEY TRANSFORMATION G.J. Muehl
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53 We have available the sugarcane
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55 Secondly, we tried to grow F. gr
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57 References: Issac, S. and Jennin
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59 Huntley-dryland) with two replic
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61 Table 2. 1992 thru 2001 Overall
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63 performance for Montana farmers.
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65 Blake, T,. Hensleigh P., Boss D.
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67 settling tower cannot accommodat
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Table 3. Seedling reaction to strip
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71 TWO-ROWED BARLEY IMPROVEMENT PRO
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Table 1. Agronomic trait comparison
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75 mutants reduce plant vigor and g
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77 SIX-ROWED BARLEY IMPROVEMENT PRO
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79 this variety has decreased over
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Table 7. Malt quality comparisons o
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83 • Management studies for malt
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85 STUDIES ON BARLEY DISEASES AND T
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87 With funding from the US Wheat a
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89 MALTING AND BREWING QUALITY OF B
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91 β-glucans, it might be most ins
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10 9 8 7 6 5 4 3 2 1 0 93 Figure 1.
Page 103 and 104: 95 Bolin, P., Schwarz, P., Jones, B
Page 105 and 106: 97 Results Results from field and g
Page 107 and 108: 99 DEVELOPING BARLEY TISSUE CULTURE
Page 109 and 110: 101 trials of plants regenerated fr
Page 111 and 112: 103 Table 2. Agronomic performance
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Page 115 and 116: 107 involving two susceptible backg
Page 117 and 118: 109 THE OREGON BARLEY IMPROVEMENT P
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Page 121 and 122: Germplasm Development: 113 Crosses:
Page 123 and 124: 115 • All lines in spring yield t
Page 125 and 126: 117 Table 2. Agronomic data for Ore
Page 127 and 128: 119 Table 6. Malting quality of win
Page 129 and 130: 121 Table 7. Across location summar
Page 131 and 132: 123 MOLECULAR MARKER ASSISTED MODIF
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Page 135 and 136: 127 exchanges. Spring and winter ba
Page 137 and 138: 129 Table 2. 2000-2001 Agronomic Pe
Page 139 and 140: 131 Table 7. Spring 2- and 6-Row #
Page 141 and 142: 133 2. Direct Seeding Cropping Syst
Page 143 and 144: 135 D.M. Wesenberg - spring and win
Page 145 and 146: 137 A STUDY OF THE MALTING QUALITY
Page 147 and 148: 139 Methods. Our malting schedule.
Page 149 and 150: 141 modification measures indicated
Page 151 and 152: 143 CHARACTERIZATION OF THE PROTEIN
Page 153: 145 Purification of a serine endope
Page 157 and 158: 149 with the proteinase T will be s
Page 159 and 160: 151 10°C increase in thermostabili
Page 161 and 162: 153 Sissons MJ and MacGregor AW (19
Page 163 and 164: µM Carbohydrate / Unit of α-Gluco
Page 165 and 166: 157 Tissue-specific gene products f
Page 167 and 168: 159 The promoters were subcloned fr
Page 169: 161 Fig. 3. Expression of Trx-Hth f
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Annual Progress Report on Malting Barley Research March, 2002

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