Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.04

CHARACTERIZATION OF PUTATIVE UPSTREAM REGULATORS OF BKN3, THE BARLEY HOMEOBOX GENE RESPONSIBLE FOR HOODED MUTATION

M.R. STILE*, L. SANTI**, L. ROSSINI***, C. POZZI****, M. OSNATO***, Y. LIU**, E. FILIPPONE*, F. SALAMINI**

*) Department of Soil, Plant and Environmental Sciences–School of Biotechnological Sciences, University of Naples, Portici, Italy

**) Max-Planck-Institute for Plant Breeding Research, Carl-von-Linnè-weg 10, 50827 Colo gne, Germany

***) University of Milan, Dipartimento di Produzione Vegetale, Via Celoria 2, 20133 Milan, Italy

****) Fondazione Parco Tecnologico Padano, V. Haussmann 11, 26900 Lodi, Italy

barley mutant, Shoot Apical Meristem, Knox genes

Molecular and genetic analyses have revealed a subfamily of homeobox-containing genes, Knox (Knotted1-like homeobox) genes that play a vital role in meristem function. Knox activity has been proposed to maintain an indeterminate state in the cells of the shoot apical meristem (SAM). In agreement with this model, Knox genes are specifically expressed in the central region of the SAM and are repressed in lateral organ primordia.

Knox gene function may require the cooperation of, or be inhibited by, other factors that are expressed in a spatially or temporally restricted pattern during development. Loci that regulate Knox expression have been isolated and characterized in both dicot and monocot species, e.g. phantastica (phan) in Antirrhinum (Waites e Hudson, 1995) and rough-sheath2 (rs2) in maize (Schneeberger et al., 1998). The phenotypes of phan and rs2 recessive mutants resemble those conditioned by gain-of-function mutations in Knox genes, suggesting that phan and rs2 act as repressors of Knox gene expression. So far regulators of Knox genes have been characterized by mutant analysis, but no molecular evidence has been reported for the direct role of these proteins in the control of Knox gene expression.

In barley, the dominant Hooded phenotype is caused by the duplication of a 305bp element in the IV intron of Bkn3, a Knox gene. This element has been used as a bait in a yeast one hybrid screen aimed at identifying putative DNA-binding proteins involved in Bkn3 regulation. This approach led to the isolation of four barley cDNAs, B42, B65, B89, and BBR (Barley B Recombinant, Santi et al., 2003).

In order to gather insight into how these proteins interact with the 305bp intron sequence, we have undertaken the molecular characterization of these barley genes. Genomic and full-length cDNA sequences have been determined and all four genes have been positioned on the established AFLP/RFLP/ISTR linkage map (Castiglioni et al., 1998). Transcript levels for the four genes were compared in wild-type and Hooded barley tissues. Yeast two hybrid assays, conducted with partial and full length cDNA sequences, revealed selective interactions between the proteins. RFP fusion constructs of all four proteins exhibited nuclear localization in tobacco protoplasts, consistent with their putative role in the transcriptional regulation of Bkn3.

References:

Castiglioni et al., 1998, Genetics, 149:2039-2056

Santi et al., 2003, Plant Journal, 34:813-826

Schneeberger et al., 1998, Development, 125:2857-2865

Waites e Hudson, 1995, Development, 121:2143-2154.