Annual Progress Report on Malting Barley Research March, 2002

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148 two serine proteinases that we had characterized earlier. Unlike the proteinases S and T, this enzyme was not inhibited by a barley extract when tested using the experimental conditions that readily detected the S and T enzyme inhibitions. This was interesting, because it indicates that if this enzyme is produced during FHB-infection, the barley may not be able to stop it from functioning. However, we have not yet determined whether this new enzyme is actually produced by the fungus in infected barley grain. The properties of the enzyme will be studied in the near future, if time allows. Fusarium proteinases are produced in FHB-infested barley. Polyclonal antibodies against the purified S and T serine proteinases were produced in rabbits at the University of Wisconsin Medical School Animal Care Unit. These antibodies have been used to show that the proteinases do occur inside Fusarium infected grain (as opposed to only being present in the Fusarium culture medium, from which they were purified). Both antibodies specifically recognized their respective S and T antigens in extracts that were prepared from artificially inoculated, field grown, barley grain. The tested grain samples were obtained from the VTT Microbiology group in Finland. The enzymes were detected only in material that was heavily Fusarium infested, implying that the fungus produced them only after it was well established within the kernel. On the other hand, this may simply indicate that the sensitivity of the method that we used was not adequate for recognizing very small amounts of the proteinases. To determine exactly where the proteinases are produced within the infected grains, the antibodies are now being used to carry out immunomicroscopic studies. This study is being carried out in collaboration with Dr Salla Marttila, a researcher at the Swedish University of Agricultural Sciences in Alnarp, Sweden. Preliminary immunolocalization experiments have shown that the proteinase T was produced during all of the infection stages when Fusarium was applied to barley plants that were grown in a glass house. Similar studies using the proteinase S antibodies have not yet been carried out. A manuscript reporting the results of studies that we have carried out with infected grain from the field experiment is now being prepared. Inhibitors of the Fusarium proteinases Previously, we reported how we had purified five barley proteins that inhibited the activity of proteinase S. These were purified by size-exclusion and ion exchange chromatographies and reverse phase-HPLC. By improving these purification methods we have been able to isolate several other proteins that also inhibited this enzyme. We have also purified a single protein that inhibited the proteinase T. The N-terminal amino acid sequences of the various purified inhibitor samples were analyzed at the UTMB. The proteinase S inhibitors were the barley α-amylase/subtilisin inhibitor (BASI) and the chymotrypsin/subtilisin inhibitors (CI) 1A, 1B and 2A. Some of the inhibitors were CIs that contained all of their amino acids and some were the same molecules from which various numbers of amino acid residues had been removed. The proteinase T inhibitor was a protein called the Bowman-Birk inhibitor (BBI). All of these inhibitors have been described previously in the literature, but it was not known that they affected any of the Fusarium proteinases. Previous studies of the kinetic properties of the CI inhibitors by other reseachers had shown that they were slow-binding inhibitors that form a tight complex with bovine chymotrypsin or bacterial subtilisin. We have now shown that they use a similar mechanism to inhibit the Fusarium proteinase S. The interaction of the BBI
149 with the proteinase T will be studied shortly. We are producing antibodies against the BBI and CI-2A proteins in rabbits so that we can use immunomicroscopic methods to study their interactions with the Fusarium proteinases during the FHB-infection process. Summary We have shown that two alkaline serine proteinases, called S and T, are produced in Fusarium-infested barley grain and that both are able to hydrolyze the storage proteins of barley grains in vitro. Hence, these enzymes apparently participate in the degradation of the barley endosperm protein matrix after the Fusarium attacks the barley grain. We have also demonstrated that barley contains several small proteins that can bind tightly to these enzymes and, by doing so, prevent them from functioning under experimental conditions. Whether or not these inhibitors and the proteinases really do interact in the grain during fungal invasion will have to be determined by immunomicroscopy studies, which we are preparing to carry out with a collaborator. In addition, we have discovered that there is a third proteinase present in F. culmorum culture medium that contains cereal proteins and that this enzyme may not have a counteracting inhibitor in barley grain. We are very interested in examining whether this third proteinase is produced by Fusarium when it grows in the infected grain, and will examine this question if we have time. Publications Pekkarinen, A.I., Niku-Paavola, M-L. and Jones, B.L. Purification and Properties of an Alkaline Proteinase of Fusarium culmorum. Eur. J. Biochem. 269, 798-807, 2001. Pekkarinen, A.I. and Jones, B.L. Some Characteristics of a Trypsin-like Proteinase of Fusarium culmorum. Submitted to J. Ag Food Chem. Pekkarinen, A.I., Jones, B.L. and Niku-Paavola, M-L. Two Fusarium proteinases that probably hydrolyze the storage proteins of scab-infested barleys. ASBC Newsletter 61: PB10. 2001 and Brewers Digest 76 (3); 32 -33, 2001. (Abstract). Pekkarinen, A.I. and Jones, B.L. Alkaline proteinases of fusarium infected barley. Poster presented at the 2nd General Meeting of the International Proteolysis Society (IPS), associated with the International Conference on Protease Inhibitors (ICPI). 2001, Munich-Friesing. IPS abstracts p110, 2001. (Abstract). Personnel Dr. B.L. Jones, Supervisory Research Chemist Ms. A. I. Pekkarinen, Research Intern* * AMBA supported
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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.
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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
Page 113 and 114: 105 BSMV spread between entries. Ho
Page 115 and 116: 107 involving two susceptible backg
Page 117 and 118: 109 THE OREGON BARLEY IMPROVEMENT P
Page 119 and 120: 111 for traits that are difficult/e
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 and 154: 145 Purification of a serine endope
Page 155: 147 STUDIES ON THE PROTEINASES THAT
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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