Annual Progress Report on Malting Barley Research March, 2002

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158 RESULTS Tissue-specific promoters - Ultimately, tissue-specific promoters will be needed to restrict expression to discrete spike tissues. The pericarp epithelium was previously identified as a possible route of Fusarium infection. In the ’01 grant period, a gene preferentially expressed in the pericarp epithelium was cloned and sequenced by Maria Laura Federico. This gene (EpiLTP) was found to be homologous to non-specific lipid transfer protein genes. Northern blot analysis also showed that EpiLTP is also expressed in the coleoptile and in the root and shoot axes of the developing embryo (Fig. 1). The promoter was cloned, sequenced and ligated to the gfp reporter gene in order to test it through transient expression (Fig. 2). Following particle bombardment of pericarps and other tissues, GFP fluorescence occurred strongly in the pericarp. To identify the core promoter region, deletions were made in the promoter from the 5’ and 3’ directions. A relatively short sequence in the 5’ proximal region was found to be necessary and sufficient to drive gfp expression in the pericarp. The promoter is now being used for stable transformation of barley to test its functions in intact plants. If this proves to be functional and specific, it will be used to drive expression of a hordothionin gene. Transformation with hordothionin - We previously found that the Hth1 cDNA clone does not produce protein in a pET/E. coli system. This could explain why our previous transformation of barley with Hth1 did not produce transformants that would synthesize HTH1 protein constitutively. Hth1 has two 5’ methionine codons separated by 18 nucleotides. When the sequence leading to the second methionine codon was deleted to produce an Hth2 clone, high levels of Trx-HTH fusion protein were produced (Fig. 3A). Surprisingly, the fusion transcript was produced constitutively, and higher levels occurred after induction with IPTG (Fig. 3B). When the Hth2 clone was then used to transform barley, leaves produced high levels of Hth2 mRNA but again did not produce HTH protein, even though the mRNA occurred on membrane-bound polyribosomes and was found in the expected size range of polyribosomes (Fig. 4; manuscript submitted to Plant Mol Biol). Expression of this seed-specific gene in leaves appears to be inhibited by a two-fold mechanism, one involving a sequence element that limits mRNA production or stability and one that limits translation product stability. We are presently constructing new Hth genes that may overcome these limitations. HTH antibodies to a 28 amino acid peptide representing the C-terminal half of the mature HTH protein have been produced. The synthetic peptide and antibodies were produced by AnaSpec Corp. (San Jose). Other related research - 1) Tilahun Abebe has used a suppression subtraction hybridization method to clone over 300 genes expressed in the lemma/palea and not in leaves. Fifty of the clones have been sequenced, providing insights into the activities of the lemma and palea organs (manuscript submitted to Plant Physiol). These appear to be highly photosynthetic organs that need to protect themselves from associated free radical damage. However, by limiting free radicals, these organs may forgo the use of antipathogen defensive mechanisms involving oxy radicals. Two genes (1-5 and 2-6) that are specifically and strongly expressed in the lemma/palea were chosen for further study.
159 The promoters were subcloned from BAC clones supplied by Andy Kleinhofs. The 1-5 promoter is currently being ligated to the gfp reporter gene and will be tested using transient expression assays. Clone 1-5 is particularly interesting because it may encode a defensive protein that binds the carbohydrate moiety of fungal cell walls. This could be part of an alternative defensive mechanism suggested by the above study. 2) We have conducted developmental western blot analysis of permatin protein in germinating seeds. Permatin appears to be extremely stable, with no signs of degradation for at least six days. We are now planning to analyze its presence in malt, during brewing, and in hazy beer. PERSONNEL John Herbst, USDA Technician Jianming Fu, Post Doctoral Associate Laura Oesterle, USDA Technician Maria Laura Federico, Ph.D. Student Tillahun Abebe, Post Doctoral Associate PUBLICATIONS Skadsen RW, Sathish P, Federico ML, Fu J, Kaeppler HF. Cloning of the promoter for a novel barley gene, lem1, and its organ-specific promotion of Gfp expression in lemma and palea. Plant Molecular Biology (in press). Federico ML, Fu J, Kaeppler HF, Skadsen RW. Cloning and analysis of the promoter of a barley gene preferentially expressed in the pericarp epidermis. Abs. 729, p. 151. Am Soc of Plant Biol, Providence, July, 2001. Fu J, Sathish P, Federico ML, Kaeppler HF, Skadsen RW. Constitutive expression of an antifungal protein alpha-hordothionin in transgenic barley. Abs. P-1012. Congress of the Society for In Vitro Biology, June, 2001. In InVitro 37: 25-A.
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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
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97 Results Results from field and g
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99 DEVELOPING BARLEY TISSUE CULTURE
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101 trials of plants regenerated fr
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103 Table 2. Agronomic performance
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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
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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 and 154: 145 Purification of a serine endope
Page 155 and 156: 147 STUDIES ON THE PROTEINASES THAT
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: 157 Tissue-specific gene products f
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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