Annual Progress Report on Malting Barley Research March, 2002

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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.