Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2005
Publication Date: N/A
Interpretive Summary: The fungal pathogen Fusarium graminearum attacks the tissues surrounding developing barley seeds and deposits toxins which destroy grain quality. It is possible to produce antifungal proteins in these tissues so that the invading fungus will be destroyed. Susceptible tissue of the lemma and palea, leaflike scales that surround the seed and eventually form the husk, need such protection. We have cloned the promoter for a gene, Lem2, that is mainly produced in the lemma and palea. Behind this promoter, we attached a reporter gene, gfp, which encodes the green fluorescent protein. When we introduced this gene combination into barley, the resulting plants produced seeds in which the outer lemma and palea glowed green when viewed with short-wave blue light. This proves that the tissue-specific Lem2 promoter works as expected and can now be coupled with antifungal protein genes. We also learned that the DNA sequence which directs this tissue-specific expression is a relatively short region which occurs just before the protein encoding part of the sequence. This work will aid farmers and the malting and brewing industries by eventually producing Fusarium-resistant barley.
Technical Abstract: The Lem2 gene of barley encodes a lectin-like protein that is strongly upregulated by salicylic acid and is preferentially expressed in lemmas, paleas (lemma/palea) and coleoptiles. Transient expression studies indicated that the proximal -75/+273 region determines organ-specificity. In the present study, Golden Promise barley stably transformed with Morex Lem2 promoter/gfp reporter constructs displayed cell- and developmental-specific expression of gfp. This expression corresponded to expression seen in northern blots of Morex organs. Strong GFP fluorescence was observed in the lemma/palea, glumes, coleoptile, auricle and ligule. Weak GFP fluorescence was also observed in the rachis, tips of young leaves, and the leaf sheath. However, strong expression also occurred in the epicarp. Studies showed that this may be due to incomplete methylation of the introduced Lem2 promoter in this organ. In the lemma/palea, gfp underwent a temporal shift in expression from the mesophyll to specialized epidermal cork cells. Similar to the lemma/palea, expression in the leaf sheath was localized in the cork cells. Progressive 5' deletions of the promoter to nucleotide -75, relative to the transcription start site, gradually reduced the level of gfp expression, but tissue- and cell-specific expression was retained.